專利名稱：含人丙型肝炎病毒全長(zhǎng)基因組的核酸構(gòu)建物、核酸構(gòu)建物轉(zhuǎn)入其中的重組全長(zhǎng)病毒基因 ...的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及含丙型肝炎病毒全長(zhǎng)基因組的核酸構(gòu)建物、生產(chǎn)丙型肝炎病毒顆粒的體外方法和所生產(chǎn)的丙型肝炎病毒顆粒的用途。
背景技術(shù)：
丙型肝炎病毒(HCV)屬于黃病毒科家族，是一種具有單鏈正義RNA基因組的病毒，已知引起丙型肝炎。
HCV通過持續(xù)感染引起慢性肝炎。目前，在世界范圍內(nèi)觀察到的慢性肝炎的重要病因是持續(xù)HCV感染。實(shí)際上，大約50％的持續(xù)感染個(gè)體發(fā)展成慢性肝炎。這些患者約有20％在10-20年間由慢性肝炎轉(zhuǎn)變成肝硬化，這些患者中有一些進(jìn)一步發(fā)展成晚期致死性病癥，例如肝癌。
丙型肝炎目前主要通過使用干擾素-α或干擾素-β的療法或使用干擾素-α和利巴韋林(一種嘌呤-核苷衍生物)組合的療法治療。但是，即使在實(shí)施這些療法時(shí)，在所有受治療患者中也僅有約60％觀察到療效。當(dāng)觀察到療效后終止治療時(shí)，一半以上的患者疾病復(fù)發(fā)。
一個(gè)重要目標(biāo)是開發(fā)有效對(duì)抗丙型肝炎的治療劑或預(yù)防劑。在工業(yè)化國(guó)家丙型肝炎發(fā)病率高，最終引發(fā)嚴(yán)重后果，目前還沒有可用的病因療法。因此，需要開發(fā)HCV-特異性化學(xué)療法和疫苗療法。開發(fā)抗HCV藥物的目標(biāo)可為抑制HCV復(fù)制或抑制HCV感染細(xì)胞。
最近，已制備了HCV亞基因組RNA復(fù)制子系統(tǒng)，作為HCV衍生的自主復(fù)制RNA(參見特許文獻(xiàn)1、2和3，非特許文獻(xiàn)1-4)。在HCV亞基因組RNA復(fù)制子系統(tǒng)中，制備其中HCV基因組結(jié)構(gòu)基因被去除并被藥物選擇性標(biāo)記基因取代的HCV復(fù)制子RNA，并將其導(dǎo)入培養(yǎng)細(xì)胞中，由此使復(fù)制子RNA在細(xì)胞中自主復(fù)制。使用這些系統(tǒng)使得有可能分析HCV的復(fù)制機(jī)制。但是，這是一個(gè)其中僅可在HCV病毒增殖和復(fù)制過程中評(píng)價(jià)病毒RNA復(fù)制的實(shí)驗(yàn)系統(tǒng)，不能分析被感染細(xì)胞中HCV病毒顆粒形成和胞外釋放或感染另一細(xì)胞的過程。
此時(shí)，只能在使用動(dòng)物如黑猩猩的實(shí)驗(yàn)系統(tǒng)中評(píng)價(jià)HCV病毒顆粒形成和胞外釋放以及感染另一細(xì)胞的過程(非特許文獻(xiàn)5)。但是，使用活生物(例如動(dòng)物)的實(shí)驗(yàn)系統(tǒng)很復(fù)雜，非常難分析。因此，為了分析HCV病毒顆粒形成和胞外釋放以及感染另一細(xì)胞的過程，為了生產(chǎn)具有抑制該過程的作用機(jī)制的抗HCV藥物，必須建立重現(xiàn)該過程的高度簡(jiǎn)單化的實(shí)驗(yàn)系統(tǒng)，即在細(xì)胞培養(yǎng)實(shí)驗(yàn)系統(tǒng)中的HCV病毒顆粒生產(chǎn)系統(tǒng)。
此外，一旦可使用細(xì)胞培養(yǎng)系統(tǒng)穩(wěn)定提供HCV病毒顆粒，就有可能使用分子生物學(xué)技術(shù)對(duì)病毒減毒或生產(chǎn)非感染性HCV病毒，這可用于疫苗。
特許文獻(xiàn)1日本特許公開(公報(bào))第2001-17187號(hào)特許文獻(xiàn)2國(guó)際專利申請(qǐng)PCT/JP03/15038特許文獻(xiàn)3JP特許申請(qǐng)第2003-329082號(hào)非特許文獻(xiàn)1Lohmann等，Science，(1999)285，110-113頁(yè)非特許文獻(xiàn)2Blight等，Science，(2000)290，1972-1974頁(yè)非特許文獻(xiàn)3Friebe等，J.Virol.，(2001)75(24)12047-12057頁(yè)非特許文獻(xiàn)4Ikeda等，J.Virol.，(2002)76(6)2997-3006頁(yè)非特許文獻(xiàn)5Kolykhalov等，Science，(1997)277，570-574頁(yè)非特許文獻(xiàn)6Kato等，Gastroenterology，(2003)125，1808-1817頁(yè)非特許文獻(xiàn)7Yanagi等，Proc.Natl.Acad.Sci.，(1997)96(16)8738-8743頁(yè)非特許文獻(xiàn)8Okamoto等，J.Gen.Virol.，(1991)73，2697-26704頁(yè)非特許文獻(xiàn)9Aoyagi等，J.Clin.Microbiol.，(1999)37(6)1802-1808頁(yè)發(fā)明公開本發(fā)明的目標(biāo)是提供含全長(zhǎng)HCV基因組序列的RNA的有效復(fù)制方法和在細(xì)胞培養(yǎng)系統(tǒng)中生產(chǎn)含全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA的HCV病毒顆粒的方法。本發(fā)明的目標(biāo)迄今為止還從未實(shí)現(xiàn)過。
為實(shí)現(xiàn)以上目標(biāo)，本發(fā)明的發(fā)明人深入研究，結(jié)果開發(fā)出一種在細(xì)胞培養(yǎng)系統(tǒng)中生產(chǎn)HCV病毒顆粒的方法。即，本發(fā)明如下所述。
一種復(fù)制子RNA，其包含的核苷酸序列含有基因型2a丙型肝炎病毒基因組RNA的5′非翻譯區(qū)、核心蛋白編碼序列、E1蛋白編碼序列、E2蛋白編碼序列、NS2蛋白編碼序列、NS3蛋白編碼序列、NS4A蛋白編碼序列、NS4B蛋白編碼序列、NS5A蛋白編碼序列、NS5B蛋白編碼序列和3′非翻譯區(qū)，至少一種選擇標(biāo)記基因和/或至少一種報(bào)告基因，以及至少一種IRES序列。
在該復(fù)制子RNA中，優(yōu)選核苷酸序列含在5′-3′方向?yàn)橐韵马樞虻?′非翻譯區(qū)、至少一種選擇標(biāo)記基因和/或至少一種報(bào)告基因，和至少一種IRES序列，以及核心蛋白編碼序列、E1蛋白編碼序列、E2蛋白編碼序列、NS2蛋白編碼序列、NS3蛋白編碼序列、NS4A蛋白編碼序列、NS4B蛋白編碼序列、NS5A蛋白編碼序列、NS5B蛋白編碼序列和3′非翻譯區(qū)。
在此復(fù)制子RNA的更優(yōu)選實(shí)施方案中，基因型2a丙型肝炎病毒基因組RNA是含SEQ ID NO12所示核苷酸序列的RNA。
在此復(fù)制子RNA的再更優(yōu)選實(shí)施方案中，5′非翻譯區(qū)含SEQ IDNO1所示核苷酸序列，核心蛋白編碼序列含SEQ ID NO2所示核苷酸序列，E1蛋白編碼序列含SEQ ID NO3所示核苷酸序列，E2蛋白編碼序列含SEQ ID NO4所示核苷酸序列，NS2蛋白編碼序列含SEQ ID NO5所示核苷酸序列，NS3蛋白編碼序列含SEQ ID NO6所示核苷酸序列，NS4A蛋白編碼序列含SEQ ID NO7所示核苷酸序列，NS4B蛋白編碼序列含SEQ ID NO8所示核苷酸序列，NS5A蛋白編碼序列含SEQ ID NO9所示核苷酸序列，NS5B蛋白編碼序列含SEQ ID NO10所示核苷酸序列，而3′非翻譯區(qū)含SEQ ID NO11所示核苷酸序列。
含以下(a)或(b)RNA的復(fù)制子RNA(a)含SEQ ID NO13所示核苷酸序列的RNA；或(b)含通過缺失、置換或添加1-100個(gè)核苷酸而衍生自SEQ ID NO13所示核苷酸序列的核苷酸序列、并具有自主復(fù)制能力和病毒顆粒生產(chǎn)能力的RNA。
一種生產(chǎn)細(xì)胞的方法，其中所述細(xì)胞復(fù)制復(fù)制子RNA并生產(chǎn)病毒顆粒，該方法包括將上述[1]或[2]中任一項(xiàng)的復(fù)制子RNA導(dǎo)入到細(xì)胞中。
用于該方法的細(xì)胞優(yōu)選為增殖性細(xì)胞。同樣或否則，用于該方法的細(xì)胞優(yōu)選為真核細(xì)胞。
用于該方法的真核細(xì)胞優(yōu)選為人肝源細(xì)胞、人子宮頸源細(xì)胞或人胎腎源細(xì)胞。所述真核細(xì)胞更優(yōu)選為Huh7細(xì)胞、HepG2細(xì)胞、IMY-N9細(xì)胞、HeLa細(xì)胞或293細(xì)胞。
利用上述[3]的方法可獲得的細(xì)胞，其復(fù)制復(fù)制子RNA并生產(chǎn)病毒顆粒。
生產(chǎn)丙型肝炎病毒顆粒的方法，包括培養(yǎng)上述[4]的細(xì)胞，以使該細(xì)胞生產(chǎn)病毒顆粒。
利用上述[5]的方法可獲得的丙型肝炎病毒顆粒。
生產(chǎn)丙型肝炎病毒感染細(xì)胞的方法，包括培養(yǎng)上述[4]的細(xì)胞，并用培養(yǎng)物中的病毒顆粒感染其它細(xì)胞。
通過上述[7]的方法可獲得的丙型肝炎病毒感染細(xì)胞。
篩選抗丙型肝炎病毒物質(zhì)的方法，包括在測(cè)試物質(zhì)存在下培養(yǎng)選自以下(a)、(b)和(c)中的至少一種(a)上述[4]的細(xì)胞，(b)上述[8]的丙型肝炎病毒感染細(xì)胞，和(c)上述[6]的丙型肝炎病毒顆粒和丙型肝炎病毒允許細(xì)胞；并檢測(cè)所獲培養(yǎng)物中的復(fù)制子RNA或病毒顆粒。
丙型肝炎疫苗，包含上述[6]的丙型肝炎病毒顆粒或其部分。
使用上述[6]的丙型肝炎病毒顆粒或其部分作為抗原生產(chǎn)丙型肝炎疫苗的方法。
通過使用上述[1]或[2]的復(fù)制子RNA生產(chǎn)用于基因治療的嗜肝病毒載體的方法。
利用上述[12]的方法可獲得的嗜肝病毒載體。
在細(xì)胞中復(fù)制和/或表達(dá)外源基因的方法，包括將編碼外源基因的RNA插入到上述[1]或[2]中任一項(xiàng)的復(fù)制子RNA中，并將其導(dǎo)入到所述細(xì)胞中。
一種細(xì)胞生產(chǎn)方法，其中所述細(xì)胞復(fù)制RNA并生產(chǎn)病毒顆粒，該方法包括將含SEQ ID NO12所示核苷酸序列的RNA導(dǎo)入到該細(xì)胞中。
生產(chǎn)丙型肝炎病毒顆粒的方法，包括將含SEQ ID NO12所示核苷酸序列的RNA導(dǎo)入到細(xì)胞中，并培養(yǎng)細(xì)胞，使該細(xì)胞生產(chǎn)病毒顆粒。
上述[15]或[16]的方法，其中所述細(xì)胞為增殖性細(xì)胞。
生產(chǎn)含外源基因的病毒載體的方法，包括將編碼外源基因的RNA插入到含SEQ ID NO12所示核苷酸序列的RNA中，將其導(dǎo)入到細(xì)胞中，并培養(yǎng)細(xì)胞，使該細(xì)胞生產(chǎn)病毒顆粒。
抗上述[6]的丙型肝炎病毒顆粒的抗體。
在本申請(qǐng)要求其優(yōu)先權(quán)的日本特許申請(qǐng)第2004-045489號(hào)的說(shuō)明書和附圖中的內(nèi)容結(jié)合到本文中。
附圖簡(jiǎn)述

圖1是一幅示意圖，顯示了構(gòu)建模板DNA的方法，該模板DNA用于制備本發(fā)明的全長(zhǎng)HCV復(fù)制子HCV或全長(zhǎng)HCV基因組RNA。圖1的上部顯示了質(zhì)?？寺JFH1的結(jié)構(gòu)，其通過將全長(zhǎng)HCV基因組插入到T7啟動(dòng)子下游產(chǎn)生。圖1的下部顯示了含全長(zhǎng)HCV基因組序列的質(zhì)粒克隆pFGREP-JFH1的結(jié)構(gòu)，其中含新霉素抗性基因和EMCV IRES的DNA片段插入到pJFH1的T7啟動(dòng)子和5′非翻譯區(qū)下游。該圖中顯示的術(shù)語(yǔ)如下。T7T7RNA啟動(dòng)子，5′UTR5′非翻譯區(qū)，C核心蛋白，E1、E2包膜蛋白。NS2、NS3、NS4A、NS4B、4A、4B非結(jié)構(gòu)蛋白。3′UTR3′非翻譯區(qū)。AgeI、PmeI、XbaI限制酶AgeI、PmeI和XbaI的限制性位點(diǎn)。GDD對(duì)應(yīng)于NS5B蛋白活性中心的氨基酸基序GDD的位點(diǎn)。neo新霉素抗性基因。EMCVIRES腦心肌炎病毒內(nèi)部核糖體進(jìn)入位點(diǎn)；圖2是一幅照片，顯示了RNA印跡分析的結(jié)果，其表明rJFH-1在其中已導(dǎo)入全長(zhǎng)HCV基因組RNA rJFH-1的Huh7細(xì)胞中復(fù)制；圖3顯示了培養(yǎng)基中HCV核心蛋白定量的結(jié)果?？招膱A代表其中已導(dǎo)入rJFH1的細(xì)胞，實(shí)心圓代表其中已導(dǎo)入rJFH1/GND的細(xì)胞；圖4圖示了HCV核心蛋白和全長(zhǎng)HCV基因組RNA的量，以及通過蔗糖密度梯度分級(jí)分離已導(dǎo)入rJFH-1的Huh7細(xì)胞的培養(yǎng)上清液所收集的各個(gè)級(jí)分的比重。實(shí)心圓、空心圓和陰影圓分別代表HCV核心蛋白、全長(zhǎng)HCV基因組RNA和比重。
圖5是一幅照片，顯示了其中已轉(zhuǎn)染入全長(zhǎng)HCV復(fù)制子RNArFGREP-JFH1的Huh7細(xì)胞的集落形成；圖6是一幅照片，顯示了全長(zhǎng)HCV復(fù)制子RNA在全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞克隆中的復(fù)制，其中所述克隆已通過將rFGREP-JFH1轉(zhuǎn)染入Huh7細(xì)胞中建立；
圖7是一幅照片，顯示了使用宿主細(xì)胞的基因組DNA作為模板和新霉素抗性基因特異性引物的PCR擴(kuò)增的結(jié)果，用于證實(shí)新霉素抗性基因整合入基因組DNA中。MDNA大小標(biāo)記物，P陽(yáng)性對(duì)照，NHuh7細(xì)胞；圖8是一幅照片，顯示了蛋白質(zhì)印跡分析的結(jié)果，其表明核心蛋白在其中已導(dǎo)入全長(zhǎng)HCV復(fù)制子RNA rFGREP-JFH1的Huh7細(xì)胞中表達(dá)；圖9是一幅照片，顯示了蛋白質(zhì)印跡分析的結(jié)果，其表明NS3蛋白在其中已導(dǎo)入全長(zhǎng)HCV復(fù)制子RNA rFGREP-JFH1的Huh7細(xì)胞中表達(dá)；圖10是一幅照片，顯示了蛋白質(zhì)印跡分析的結(jié)果，其表明NS5A蛋白在其中已導(dǎo)入全長(zhǎng)HCV復(fù)制子RNA rFGREP-JFH1的Huh7細(xì)胞中表達(dá)；圖11圖示了HCV核心蛋白和全長(zhǎng)HCV復(fù)制子RNA的量，以及通過蔗糖密度梯度分級(jí)分離已導(dǎo)入rFGREP-JFH1的Huh7細(xì)胞的培養(yǎng)上清液所收集的各個(gè)級(jí)分的比重。實(shí)心圓、空心圓和陰影圓分別代表HCV核心蛋白、全長(zhǎng)HCV復(fù)制子RNA和比重；圖12是一幅照片，顯示了Huh7細(xì)胞的集落形成，其中已加入全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞培養(yǎng)上清液中的病毒顆粒。
實(shí)施本發(fā)明的最佳方式本發(fā)明如下詳細(xì)闡述。
1.全長(zhǎng)HCV復(fù)制子RNA丙型肝炎病毒(HCV)基因組是單股正鏈RNA，含約9600個(gè)核苷酸。此基因組RNA含5′非翻譯區(qū)(也稱為5′NTR或5′UTR)、由結(jié)構(gòu)區(qū)和非結(jié)構(gòu)區(qū)組成的翻譯區(qū)以及3′非翻譯區(qū)(也稱為3′NTR或3′UTR)。HCV結(jié)構(gòu)蛋白在結(jié)構(gòu)區(qū)中編碼，多種非結(jié)構(gòu)蛋白在非結(jié)構(gòu)區(qū)中編碼。
這樣的HCV結(jié)構(gòu)蛋白(core、E1蛋白和E2蛋白)和非結(jié)構(gòu)蛋白(NS2、NS3、NS4A、NS4B、NS5A和NS5B)如下產(chǎn)生首先將翻譯區(qū)翻譯成單個(gè)連續(xù)的多蛋白，然后通過蛋白酶限制性切割多蛋白釋放。在這些結(jié)構(gòu)蛋白和非結(jié)構(gòu)蛋白(即HCV的病毒蛋白)中，core是核心蛋白，E1和E2是包膜蛋白。非結(jié)構(gòu)蛋白是參與病毒自身復(fù)制的蛋白，已知NS2具有金屬蛋白酶活性，已知NS3具有絲氨酸蛋白酶活性(在N端側(cè)的1/3)和解旋酶活性(在C端側(cè)的2/3)。此外，NS4A是NS3蛋白酶活性的輔因子，已報(bào)道NS5B具有RNA依賴性RNA聚合酶活性。
本發(fā)明的發(fā)明人使用HVC基因組RNA構(gòu)建了具有自主復(fù)制能力和病毒顆粒生產(chǎn)能力的復(fù)制子RNA。
已通過修飾HCV基因組RNA產(chǎn)生的具有自主復(fù)制能力的RNA在本文中叫做“復(fù)制子RNA”或“RNA復(fù)制子”。在本說(shuō)明書中，源自HCV的復(fù)制子RNA還可叫做HCV-RNA復(fù)制子。含全長(zhǎng)HCV基因組RNA的本發(fā)明復(fù)制子RNA在本文叫做“全長(zhǎng)HCV復(fù)制子RNA”。本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA具有生產(chǎn)病毒顆粒的能力。
在本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA的優(yōu)選實(shí)施方案中，丙型肝炎病毒優(yōu)選為但不限于基因型2a丙型肝炎病毒。在本發(fā)明中，“基因型2a丙型肝炎病毒”或“基因型2a HCV”指按照Simmonds等(參見Simmonds，P.等，Hepatology，(1994)10，1321-1324頁(yè))的國(guó)際分類法鑒別為基因型2a的肝炎病毒。在本發(fā)明中，“基因型2a丙型肝炎病毒”或“基因型2a HCV”不僅包括具有天然HCV基因組RNA的病毒，而且包括具有其中天然HCV基因組序列已被人工修飾的基因組RNA的病毒?；蛐?a HCV的具體實(shí)例包括JFH-1毒株(參見日本特許公開(公告)第2002-171978號(hào))。
在本說(shuō)明書中，“丙型肝炎病毒基因組RNA”指含丙型肝炎病毒單鏈正義RNA基因組整個(gè)區(qū)域的核苷酸序列的RNA?；蛐?a丙型肝炎病毒的基因組RNA優(yōu)選為但不限于含SEQ ID NO12所示核苷酸序列的RNA。
本發(fā)明全長(zhǎng)HCV復(fù)制子的一個(gè)實(shí)施方案是包含核苷酸序列的復(fù)制子RNA，該核苷酸序列包含5′非翻譯區(qū)、核心蛋白編碼序列、E1蛋白編碼序列、E2蛋白編碼序列、NS2蛋白編碼序列、NS3蛋白編碼序列、NS4A蛋白編碼序列、NS4B蛋白編碼序列、NS5A蛋白編碼序列、NS5B蛋白編碼序列和3′非翻譯區(qū)、至少一種選擇標(biāo)記基因或報(bào)告基因以及至少一種IRES序列。
本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA不限于但優(yōu)選包含在5′-3′方向?yàn)橐韵马樞虻?′非翻譯區(qū)、至少一種選擇標(biāo)記基因或報(bào)告基因、至少一種IRES序列、核心蛋白編碼序列、E1蛋白編碼序列、E2蛋白編碼序列、NS2蛋白編碼序列、NS3蛋白編碼序列、NS4A蛋白編碼序列、NS4B蛋白編碼序列、NS5A蛋白編碼序列、NS5B蛋白編碼序列和3′非翻譯區(qū)。
在本申請(qǐng)的說(shuō)明書中，“5′非翻譯區(qū)”(5′NTR或5′UTR)、“核心蛋白編碼序列”(核心區(qū)或C區(qū))、“E1蛋白編碼序列”(E1區(qū))、“E2蛋白編碼序列”(E2區(qū))、“NS2蛋白編碼序列”(NS2區(qū))、“NS3蛋白編碼序列”(NS3區(qū))、“NS4A蛋白編碼序列”(NS4A區(qū))、“NS4B蛋白編碼序列”(NS4B區(qū))、“NS5A蛋白編碼序列”(NS5A區(qū))、“NS5B蛋白編碼序列”(NS5B區(qū))和“3′非翻譯區(qū)”(3′NTR或3′UTR)，以及其它特定區(qū)域或位點(diǎn)，以包含基因型2a HCV病毒JFH-1毒株(日本特許公開(公報(bào))第2002-171978號(hào))基因組完整區(qū)域的全長(zhǎng)基因組RNA(SEQ ID NO12)為基準(zhǔn)定義。
另外，本發(fā)明丙型肝炎病毒(HCV)基因組中的部分區(qū)域或位點(diǎn)可以SEQ ID NO1-11所示序列為基準(zhǔn)定義，SEQ ID NO1-11是JFH-1毒株基因組RNA(SEQ ID NO12)的部分核苷酸序列。JFH-1毒株全長(zhǎng)基因組RNA(源自JFH-1克??；SEQ ID NO12)的“5′非翻譯區(qū)”包含SEQ ID NO1所示核苷酸序列。“核心蛋白編碼序列”包含SEQ IDNO2所示核苷酸序列?！癊1蛋白編碼序列”包含SEQ ID NO3所示核苷酸序列?！癊2蛋白編碼序列”包含SEQ ID NO4所示核苷酸序列?！癗S2蛋白編碼序列”包含SEQ ID NO5所示核苷酸序列?！癗S3蛋白編碼序列”包含SEQ ID NO6所示核苷酸序列?！癗S4A蛋白編碼序列”包含SEQ ID NO7所示核苷酸序列?！癗S4B蛋白編碼序列”包含SEQ ID NO8所示核苷酸序列。“NS5A蛋白編碼序列”包含SEQ IDNO9所示核苷酸序列?！癗S5B蛋白編碼序列”包含SEQ ID NO10所示核苷酸序列。“3′非翻譯區(qū)”包含SEQ ID NO11所示核苷酸序列。
例如，HCV源RNA序列中的區(qū)域或位點(diǎn)可由SEQ ID NO1-12的核苷酸序列中的核苷酸編號(hào)限定，核苷酸編號(hào)通過比對(duì)RNA序列和SEQ ID NO1-12所示核苷酸序列來(lái)確定。在比對(duì)中，可存在空位、添加、缺失、置換等。
在本發(fā)明的更優(yōu)選實(shí)施方案中，包含在全長(zhǎng)HCV復(fù)制子RNA中的5′非翻譯區(qū)、核心蛋白編碼序列、E1蛋白編碼序列、E2蛋白編碼序列、NS2蛋白編碼序列、NS3蛋白編碼序列、NS4A蛋白編碼序列、NS4B蛋白編碼序列、NS5A蛋白編碼序列、NS5B蛋白編碼序列和3′非翻譯區(qū)優(yōu)選分別包含SEQ ID NO1-11所示核苷酸序列。
本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA的優(yōu)選實(shí)施方案為含SEQ ID NO1-11所示核苷酸序列、至少一種標(biāo)記基因和/或報(bào)告基因和至少一種IRES序列的復(fù)制子RNA。
本發(fā)明中的“選擇標(biāo)記基因”指賦予細(xì)胞選擇能力的基因，使得只有表達(dá)該基因的細(xì)胞才可被選擇。選擇標(biāo)記基因的一般性實(shí)例包括抗生素抗性基因。本發(fā)明中優(yōu)選的選擇標(biāo)記基因?qū)嵗ㄐ旅顾乜剐曰?、胸苷激酶基因、卡那霉素抗性基因、抗硫胺素抗性基因、腺苷酰轉(zhuǎn)移酶基因、Zeocin抗性基因和嘌呤霉素抗性基因。優(yōu)選新霉素抗性基因和胸苷激酶基因，更優(yōu)選新霉素抗性基因。但是，本發(fā)明中的選擇標(biāo)記基因不限于這些基因。
此外，在本發(fā)明中，“報(bào)告基因”指其編碼基因產(chǎn)物可作為該基因表達(dá)指示劑的標(biāo)記基因。報(bào)告基因的一般性實(shí)例包括催化光發(fā)射反應(yīng)或顯色反應(yīng)的酶的結(jié)構(gòu)基因。本發(fā)明中的報(bào)告基因的優(yōu)選實(shí)例包括源自轉(zhuǎn)座子Tn9的氯霉素乙酰轉(zhuǎn)移酶基因、大腸桿菌源的β-葡糖醛酸酶基因或β-半乳糖苷酶基因、熒光素酶基因、綠色熒光蛋白基因、得自水母的水母發(fā)光蛋白基因和分泌型胎盤堿性磷酸酶(SEAP)基因。但是，本發(fā)明中的報(bào)告基因不限于這些基因。
在全長(zhǎng)復(fù)制子RNA中可包含以上選擇標(biāo)記基因和報(bào)告基因中的僅一種，或者可同時(shí)包含二者。一種或多種選擇標(biāo)記基因或報(bào)告基因可存在于一個(gè)全長(zhǎng)HCV復(fù)制子RNA中。
在本發(fā)明中，“IRES序列”指內(nèi)部核糖體進(jìn)入位點(diǎn)，其允許核糖體通過結(jié)合RNA內(nèi)部啟動(dòng)翻譯。本發(fā)明中的IRES序列的優(yōu)選實(shí)例包括但不限于EMCV IRES(腦心肌炎病毒的內(nèi)部核糖體進(jìn)入位點(diǎn))、FMDV IRES和HCV IRES。更優(yōu)選EMCV IRES和HCV IRES，EMCVIRES是最優(yōu)選的序列。
本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA的再更優(yōu)選實(shí)施方案是含SEQID NO13所示核苷酸序列的RNA。此外，含在SEQ ID NO13所示核苷酸序列中通過缺失、置換或添加1-100個(gè)核苷酸(優(yōu)選1-30個(gè)，更優(yōu)選1-10個(gè)，再更優(yōu)選1-6個(gè)，最優(yōu)選1至若干(2-5)個(gè)核苷酸)而衍生自SEQ ID NO13所示核苷酸序列的核苷酸序列、具有自主復(fù)制能力和病毒顆粒生產(chǎn)能力的復(fù)制子RNA是全長(zhǎng)HCV復(fù)制子RNA的優(yōu)選實(shí)施方案，也包括在本發(fā)明范圍中。
本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA還可包含編碼任選外源基因的RNA，其中所述外源基因要在全長(zhǎng)復(fù)制子RNA已導(dǎo)入其中的細(xì)胞中表達(dá)。編碼外源基因的RNA還可連接在5′非翻譯區(qū)下游，或連接在選擇標(biāo)記基因或報(bào)告基因的上游或下游，或連接在3′非翻譯區(qū)上游。編碼外源基因的RNA可插入在核心蛋白編碼序列、E1蛋白編碼序列、E2蛋白編碼序列、NS2蛋白編碼序列、NS3蛋白編碼序列、NS4A蛋白編碼序列、NS4B蛋白編碼序列、NS5A蛋白編碼序列和NS5B蛋白編碼序列之間的任意位點(diǎn)。
當(dāng)含外源基因編碼RNA的全長(zhǎng)HCV復(fù)制子RNA在已導(dǎo)入其的細(xì)胞中翻譯時(shí)，其可表達(dá)外源基因編碼的基因產(chǎn)物。因此，含外源基因編碼RNA的全長(zhǎng)HCV復(fù)制子RNA還可適用于在細(xì)胞中生產(chǎn)外源基因的基因產(chǎn)物。
本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA可進(jìn)一步包含核酶。核酶連接在選擇標(biāo)記基因和/或報(bào)告基因下游，使得可通過核酶的自切割活性將選擇標(biāo)記基因和/或報(bào)告基因由IRES序列、核心蛋白編碼序列、E1蛋白編碼序列、E2蛋白編碼序列、NS2蛋白編碼序列、NS3蛋白編碼序列、NS4A蛋白編碼序列、NS4B蛋白編碼序列、NS5A蛋白編碼序列和NS5B蛋白編碼序列以及3′非翻譯區(qū)上切下。
在本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA中，上述選擇標(biāo)記基因和/或報(bào)告基因、病毒蛋白編碼序列和外源基因、核酶等被連接在一起，使得它們由全長(zhǎng)HCV復(fù)制子RNA以正確的讀框翻譯。在這些序列中，全長(zhǎng)復(fù)制子RNA編碼的蛋白優(yōu)選通過蛋白酶切割位點(diǎn)等互相連接，使得蛋白被翻譯或表達(dá)為多蛋白，接著由蛋白酶切割成各個(gè)蛋白。
本發(fā)明還涉及編碼本發(fā)明復(fù)制子RNA的DNA載體，優(yōu)選表達(dá)載體。
在本發(fā)明中，RNA的“自主復(fù)制能力”指RNA在導(dǎo)入細(xì)胞中時(shí)能夠自主生長(zhǎng)。RNA的自主復(fù)制能力可通過以下方法證實(shí)，但該方法不是限制性的。用目的RNA轉(zhuǎn)染Huh7細(xì)胞并培養(yǎng)。由所獲得的培養(yǎng)細(xì)胞提取RNA，并使用能夠特異性檢測(cè)導(dǎo)入RNA的探針對(duì)RNA進(jìn)行RNA印跡雜交。目的RNA檢測(cè)證實(shí)自主復(fù)制。在本說(shuō)明書實(shí)施例中有關(guān)集落形成能力實(shí)驗(yàn)、HCV蛋白表達(dá)驗(yàn)證、復(fù)制子RNA檢測(cè)等的描述中闡述了證實(shí)自主復(fù)制能力的具體方法的實(shí)例。
此外，在本發(fā)明中，RNA的“病毒顆粒生產(chǎn)能力”指當(dāng)RNA導(dǎo)入到細(xì)胞(例如培養(yǎng)細(xì)胞，如Huh7細(xì)胞)中時(shí)在細(xì)胞中產(chǎn)生病毒顆粒。病毒顆粒生產(chǎn)能力可通過例如將使用RNA特異性引物的RT-PCR法用于檢測(cè)導(dǎo)入該RNA的細(xì)胞的培養(yǎng)上清液來(lái)證實(shí)。其還可通過對(duì)培養(yǎng)上清液進(jìn)行蔗糖密度梯度法以分離病毒顆粒和通過檢測(cè)HCV蛋白來(lái)證實(shí)。在本說(shuō)明書實(shí)施例中有關(guān)集落形成能力實(shí)驗(yàn)、HCV蛋白表達(dá)驗(yàn)證、復(fù)制子RNA檢測(cè)等的描述中闡述了具體方法的實(shí)例。
2.全長(zhǎng)HCV復(fù)制子RNA的制備可使用本領(lǐng)域技術(shù)人員已知的基因工程技術(shù)制備本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA?？墒褂美绲幌抻贘FH-1毒株作為基因型2a丙型肝炎病毒，利用以下的方法制備全長(zhǎng)HCV復(fù)制子RNA。
首先，常規(guī)地再構(gòu)建對(duì)應(yīng)于JFH-1毒株基因組RNA完整區(qū)域(SEQ ID NO12；該序列在國(guó)際DNA數(shù)據(jù)庫(kù)中在檢索號(hào)AB047639下)的DNA，并將其插入到RNA啟動(dòng)子下游，以便制備DNA克隆。本文使用的“對(duì)應(yīng)于RNA的DNA”指具有通過用T(胸腺嘧啶)取代U(尿嘧啶)而來(lái)源于RNA核苷酸序列的核苷酸序列的DNA。以上RNA啟動(dòng)子優(yōu)選包含在質(zhì)粒克隆中。優(yōu)選RNA啟動(dòng)子的實(shí)例不限于但包括T7RNA啟動(dòng)子、SP6 RNA啟動(dòng)子和SP3 RNA啟動(dòng)子，特別優(yōu)選T7 RNA啟動(dòng)子。
接著，將選擇標(biāo)記基因和/或報(bào)告基因以及IRES序列編碼DNA插入到上述DNA克隆中。優(yōu)選將選擇標(biāo)記基因和/或報(bào)告基因插入到5′非翻譯區(qū)下游，將IRES序列插入到更下游。
隨后，使用如上制備的DNA克隆作為模板，使用RNA聚合酶合成RNA。RNA合成可通過標(biāo)準(zhǔn)方法由5′非翻譯區(qū)開始。當(dāng)DNA克隆為質(zhì)?？寺r(shí)，可用使用限制酶由質(zhì)?？寺∏邢碌腄NA片段作為模板合成RNA。另外，優(yōu)選要合成的RNA的3′端與病毒基因組RNA的3′非翻譯區(qū)的末端具有相同序列，未添加或缺失其它序列。由此合成的RNA是本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA。
3.HCV顆粒的制備可通過將如上所述制備的全長(zhǎng)HCV復(fù)制子RNA導(dǎo)入細(xì)胞中，獲得可復(fù)制全長(zhǎng)HCV復(fù)制子RNA(優(yōu)選持續(xù)復(fù)制(即具有復(fù)制子RNA復(fù)制能力))的重組細(xì)胞。在本說(shuō)明書中，復(fù)制全長(zhǎng)HCV復(fù)制子RNA的重組細(xì)胞被稱為“全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞”。
全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞可生產(chǎn)病毒顆粒。生產(chǎn)的病毒顆粒在由HCV病毒蛋白組成的外殼中含全長(zhǎng)HCV復(fù)制子RNA。因此，本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞生產(chǎn)的病毒顆粒是HCV顆粒。也就是說(shuō)，在本發(fā)明中，可通過培養(yǎng)全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞，在細(xì)胞培養(yǎng)系統(tǒng)中制備HCV顆粒。優(yōu)選地，可通過培養(yǎng)全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞，并收集在培養(yǎng)物(優(yōu)選培養(yǎng)上清液)中產(chǎn)生的病毒顆粒，獲得HCV顆粒。
或者，可利用通過將全長(zhǎng)HCV基因組RNA導(dǎo)入到細(xì)胞中獲得的重組細(xì)胞生產(chǎn)HCV顆粒。全長(zhǎng)HCV基因組RNA在其中導(dǎo)入本發(fā)明的全長(zhǎng)HCV基因組RNA(優(yōu)選來(lái)源于JFH-1克隆的全長(zhǎng)HCV基因組RNA，更優(yōu)選具有SEQ ID NO12所示核苷酸序列的RNA)的細(xì)胞中高效復(fù)制。在本說(shuō)明書中，復(fù)制全長(zhǎng)HCV基因組RNA的細(xì)胞稱為“全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞”。全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞可生產(chǎn)病毒顆粒。全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞生產(chǎn)的病毒顆粒在由HCV病毒蛋白組成的殼中包含全長(zhǎng)HCV基因組RNA。因此，由其中導(dǎo)入本發(fā)明的全長(zhǎng)HCV基因組RNA的細(xì)胞生產(chǎn)的病毒顆粒是HCV顆粒。不限于但優(yōu)選地可通過培養(yǎng)其中導(dǎo)入來(lái)源于JFH-1克隆的全長(zhǎng)HCV基因組RNA(例如具有SEQ ID NO12所示核苷酸序列的RNA)的細(xì)胞，在細(xì)胞培養(yǎng)系統(tǒng)中制備HCV顆粒。例如，可通過培養(yǎng)其中導(dǎo)入全長(zhǎng)HCV基因組RNA(例如具有SEQ IDNO12所示核苷酸序列的RNA)的細(xì)胞，并收集在培養(yǎng)物(優(yōu)選培養(yǎng)上清液)中產(chǎn)生的病毒顆粒，獲得HCV顆粒。
對(duì)于要導(dǎo)入上述全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA的細(xì)胞，可使用任何細(xì)胞，只要其可被傳代培養(yǎng)。這樣的細(xì)胞優(yōu)選為真核細(xì)胞，更優(yōu)選為人細(xì)胞，再更優(yōu)選為人肝源細(xì)胞、人子宮頸源細(xì)胞或人胎腎源細(xì)胞。優(yōu)選可使用增殖性細(xì)胞，包括癌細(xì)胞系、干細(xì)胞系等細(xì)胞，更優(yōu)選使用Huh7細(xì)胞、HepG2細(xì)胞、IMY-N9細(xì)胞、HeLa細(xì)胞和293細(xì)胞等。對(duì)于這些細(xì)胞，可使用市售的細(xì)胞，這些細(xì)胞可得自細(xì)胞保藏機(jī)構(gòu)，或者也可使用由任意細(xì)胞(例如癌細(xì)胞或干細(xì)胞)建立的細(xì)胞系。
可使用本領(lǐng)域技術(shù)人員已知的任何技術(shù)，實(shí)現(xiàn)將全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA導(dǎo)入到細(xì)胞中。這樣的導(dǎo)入方法的實(shí)例包括電穿孔、粒子槍法、脂轉(zhuǎn)染法、磷酸鈣法、微注射法、DEAESepharose法等。特別優(yōu)選使用電穿孔的方法。
全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA可單獨(dú)導(dǎo)入，或者可在與其它核酸混合后導(dǎo)入。為改變?nèi)L(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA的量，同時(shí)將要導(dǎo)入的RNA量保持在一定水平，將需要量的要導(dǎo)入的全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA與由要導(dǎo)入RNA的細(xì)胞提取的總細(xì)胞RNA混合，以使總RNA量達(dá)到一定水平，然后混合物用于導(dǎo)入到細(xì)胞中?？砂凑帐褂玫膶?dǎo)入方法確定用于導(dǎo)入到細(xì)胞中的復(fù)制子RNA的量，其優(yōu)選為1pg-100μg，更優(yōu)選為10pg-10μg。
可利用在全長(zhǎng)HCV復(fù)制子RNA中的選擇標(biāo)記基因或報(bào)告基因的表達(dá)，選擇全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞。具體地說(shuō)，例如，可在培養(yǎng)基中培養(yǎng)這種進(jìn)行了全長(zhǎng)HCV復(fù)制子RNA細(xì)胞導(dǎo)入處理的細(xì)胞，其中細(xì)胞可由于表達(dá)選擇標(biāo)記基因而被選擇?；蛘撸谶M(jìn)行全長(zhǎng)HCV復(fù)制子RNA細(xì)胞導(dǎo)入處理的細(xì)胞培養(yǎng)后，可檢測(cè)報(bào)告基因(例如熒光蛋白)的表達(dá)。
作為實(shí)例，當(dāng)全長(zhǎng)HCV復(fù)制子RNA包含新霉素抗性基因作為選擇標(biāo)記基因時(shí)，將用全長(zhǎng)HCV復(fù)制子RNA進(jìn)行電穿孔法的細(xì)胞接種入培養(yǎng)皿中。在培養(yǎng)12-72小時(shí)(優(yōu)選16-48小時(shí))后，以0.05mg/ml-3.0mg/ml的濃度將G418(新霉素)加入到培養(yǎng)皿中。優(yōu)選在接種后將細(xì)胞連續(xù)培養(yǎng)10-40天，更優(yōu)選14-28天，同時(shí)一周更換培養(yǎng)基兩次，可通過用結(jié)晶紫染色活細(xì)胞，選擇復(fù)制導(dǎo)入的全長(zhǎng)HCV復(fù)制子RNA的細(xì)胞的集落。
可通過標(biāo)準(zhǔn)方法由形成的集落克隆細(xì)胞。由此獲得的復(fù)制全長(zhǎng)HCV復(fù)制子RNA的細(xì)胞克隆在本說(shuō)明書中被稱為“全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞克隆”。本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞包括全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞克隆。
對(duì)于全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞，可通過檢測(cè)復(fù)制的全長(zhǎng)HCV復(fù)制子RNA，確認(rèn)全長(zhǎng)HCV復(fù)制子RNA的選擇標(biāo)記基因或報(bào)告基因沒有整合在宿主基因組DNA中，并進(jìn)一步檢測(cè)HCV蛋白，證實(shí)全長(zhǎng)HCV復(fù)制子RNA在細(xì)胞或細(xì)胞克隆中的實(shí)際復(fù)制。
可按照本領(lǐng)域技術(shù)人員已知的任意RNA檢測(cè)方法，檢測(cè)已復(fù)制的全長(zhǎng)HCV復(fù)制子RNA。例如，可通過使用全長(zhǎng)HCV復(fù)制子RNA特異性DNA片段作為探針的RNA雜交法，檢測(cè)由細(xì)胞提取的總RNA中的全長(zhǎng)HCV復(fù)制子RNA。
而且，可通過例如但不限于對(duì)由細(xì)胞提取出的基因組DNA進(jìn)行PCR，以擴(kuò)增至少一部分選擇標(biāo)記基因或報(bào)告基因，然后確認(rèn)沒有擴(kuò)增產(chǎn)物，來(lái)證實(shí)全長(zhǎng)HCV復(fù)制子RNA中的選擇標(biāo)記基因或報(bào)告基因沒有整合在宿主基因組DNA中。因?yàn)橐话阏J(rèn)為在證實(shí)擴(kuò)增產(chǎn)物的細(xì)胞中選擇標(biāo)記基因或報(bào)告基因可能已整合入宿主基因組中，所以有可能全長(zhǎng)HCV復(fù)制子RNA自身不復(fù)制。在此情況下，可如下所述通過檢測(cè)HCV蛋白進(jìn)一步證實(shí)全長(zhǎng)HCV復(fù)制子RNA的復(fù)制。
可通過例如使抗HCV蛋白(其由導(dǎo)入的全長(zhǎng)HCV復(fù)制子RNA表達(dá))的抗體與提取的細(xì)胞蛋白反應(yīng)，檢測(cè)HCV蛋白。該方法可通過本領(lǐng)域技術(shù)人員已知的任意蛋白檢測(cè)方法進(jìn)行。具體地說(shuō)，例如，可通過將由細(xì)胞提取出的蛋白樣品印跡在硝酸纖維素膜上，使抗HCV蛋白抗體(例如抗-NS3特異性抗體或由丙型肝炎患者收集的抗血清)與硝酸纖維素膜反應(yīng)，并檢測(cè)抗HCV蛋白抗體，檢測(cè)HCV蛋白。如果在提取的細(xì)胞蛋白中檢測(cè)出HCV蛋白，則可推斷該細(xì)胞復(fù)制全長(zhǎng)HCV復(fù)制子RNA，并表達(dá)HCV蛋白。
可通過本領(lǐng)域技術(shù)人員已知的任意病毒檢測(cè)方法，證實(shí)全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞或全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞的病毒顆粒生產(chǎn)能力。例如，通過蔗糖密度梯度分級(jí)分離懷疑生產(chǎn)病毒顆粒的細(xì)胞的培養(yǎng)上清液，并測(cè)定各個(gè)級(jí)分的級(jí)分密度、HCV核心蛋白濃度以及全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA的量。結(jié)果，如果核心蛋白的峰與全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA的峰一致，顯示檢測(cè)峰的級(jí)分的密度(例如1.18-1.20mg)小于分級(jí)分離25％NP40(聚氧乙烯(9)辛基苯基醚)處理的培養(yǎng)上清液所獲得的等同級(jí)分的密度，則可認(rèn)為細(xì)胞具有病毒顆粒生產(chǎn)能力。
例如可使用核心蛋白、E1蛋白或E2蛋白的抗體檢測(cè)培養(yǎng)上清液中釋放的HCV病毒顆粒。另外，可通過使用特異性引物的RT-PCR法擴(kuò)增和檢測(cè)培養(yǎng)上清液中的全長(zhǎng)HCV復(fù)制子RNA，間接檢測(cè)HCV病毒顆粒的存在。
4.用本發(fā)明的HCV顆粒感染另一細(xì)胞本發(fā)明的HCV病毒顆粒具有感染細(xì)胞(優(yōu)選HCV允許細(xì)胞)的能力。本發(fā)明還涉及生產(chǎn)丙型肝炎病毒感染細(xì)胞的方法，包括培養(yǎng)全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞或全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞，并用在由此獲得的培養(yǎng)物(優(yōu)選培養(yǎng)上清液)中的病毒顆粒感染另一細(xì)胞(優(yōu)選HCV允許細(xì)胞)。在本發(fā)明中，HCV允許細(xì)胞指對(duì)HCV敏感的細(xì)胞，優(yōu)選但不限于肝細(xì)胞或淋巴系細(xì)胞。具體地說(shuō)，肝細(xì)胞包括原代肝細(xì)胞、Huh7細(xì)胞、HepG2細(xì)胞、IMY-N9細(xì)胞、HeLa細(xì)胞、203細(xì)胞等。淋巴系細(xì)胞包括但不限于Molt4細(xì)胞、HPB-Ma細(xì)胞、Daudi細(xì)胞等。
當(dāng)用本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞生產(chǎn)的HCV顆粒感染細(xì)胞(例如HCV允許細(xì)胞)時(shí)，全長(zhǎng)HCV復(fù)制子RNA在感染細(xì)胞中復(fù)制，病毒顆粒也在感染細(xì)胞中形成。因?yàn)橛迷谌L(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞中產(chǎn)生的病毒顆粒感染的細(xì)胞表達(dá)選擇標(biāo)記基因和/或報(bào)告基因，所以可利用表達(dá)選擇和/或檢測(cè)感染細(xì)胞。通過用在本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞中產(chǎn)生的病毒顆粒感染細(xì)胞，全長(zhǎng)HCV復(fù)制子RNA在細(xì)胞中復(fù)制，可進(jìn)一步生產(chǎn)病毒顆粒。
更進(jìn)一步，通過用在本發(fā)明的全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞中產(chǎn)生的HCV顆粒感染細(xì)胞(例如HCV允許細(xì)胞)，全長(zhǎng)HCV基因組RNA在感染細(xì)胞中復(fù)制，病毒顆粒也在感染細(xì)胞中形成。通過用在本發(fā)明的全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞中產(chǎn)生的病毒顆粒感染細(xì)胞，全長(zhǎng)HCV基因組RNA在細(xì)胞中復(fù)制，而且可生產(chǎn)病毒顆粒。
在全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞或全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞中產(chǎn)生的HCV病毒顆?？筛腥綡CV允許動(dòng)物，例如黑猩猩等，并在其中誘發(fā)由HCV引起的肝炎。
5.本發(fā)明的其它實(shí)施方案全長(zhǎng)HCV復(fù)制子RNA在本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞中高效復(fù)制。另外，全長(zhǎng)HCV基因組RNA在本發(fā)明的全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞中高效復(fù)制。因此，可使用本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞或全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞高效生產(chǎn)全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA。
在本發(fā)明中，可通過培養(yǎng)全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞，由培養(yǎng)物(培養(yǎng)細(xì)胞和/或培養(yǎng)基)中提取RNA，對(duì)RNA進(jìn)行電泳法，并分離和純化全長(zhǎng)HCV復(fù)制子RNA，生產(chǎn)全長(zhǎng)HCV復(fù)制子RNA。還可通過相似的方法，使用全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞生產(chǎn)全長(zhǎng)HCV基因組RNA。通過這樣的方法生產(chǎn)的RNA包含丙型肝炎病毒的全長(zhǎng)基因組序列。在此情況下，可通過選擇標(biāo)記基因和/或報(bào)告基因和IRES序列中斷丙型肝炎病毒的全長(zhǎng)基因組序列。借助于包含所提供的丙型肝炎病毒全長(zhǎng)基因組序列的RNA的生產(chǎn)方法，使得有可能更詳細(xì)地分析丙型肝炎病毒基因組。
再者，本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞或全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞可適用于生產(chǎn)HCV蛋白。HCV蛋白可利用本領(lǐng)域技術(shù)人員已知的任何方法生產(chǎn)。例如，可通過將全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA導(dǎo)入到細(xì)胞中，培養(yǎng)重組細(xì)胞，并利用已知方法由如此獲得的培養(yǎng)物(培養(yǎng)細(xì)胞和/或培養(yǎng)基)中收集蛋白，生產(chǎn)HCV蛋白。
再者，本發(fā)明的HCV病毒顆?？删哂惺雀涡浴Ｒ虼?，可使用本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA生產(chǎn)嗜肝病毒載體。該病毒載體適用于基因治療。在本發(fā)明中，通過將外源基因編碼RNA整合入全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA中并將整合的RNA導(dǎo)入細(xì)胞中表達(dá)，外源基因可被導(dǎo)入到細(xì)胞中，并在細(xì)胞中復(fù)制。此外，通過制備其中全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA的E1蛋白編碼序列和/或E2蛋白編碼序列被其它生物物種來(lái)源病毒的外殼蛋白編碼序列置換的RNA，使該RNA有可能感染不同的生物物種。還是在此情況下，外源基因整合入全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA中，這可用作在肝細(xì)胞中表達(dá)外源基因的嗜肝病毒載體。
本發(fā)明還涉及生產(chǎn)攜帶外源基因的病毒載體的方法，包括將外源基因編碼RNA插入到含SEQ ID NO12所示核苷酸序列的RNA中，將其導(dǎo)入到細(xì)胞中，并培養(yǎng)細(xì)胞，以生產(chǎn)病毒顆粒。
本發(fā)明提供含本發(fā)明的HCV顆粒或其部分的丙型肝炎疫苗，生產(chǎn)含本發(fā)明的HCV顆?；蚱洳糠值谋透窝滓呙绲姆椒?。
具體地說(shuō)，如上制備的HCV顆?？芍苯佑米饕呙?，或者可在利用本領(lǐng)域已知方法減毒或滅活后使用。例如，可通過使用柱層析、過濾、離心等純化HCV顆粒，獲得HCV疫苗母液?？捎纱薍CV疫苗母液制備減毒活HCV疫苗或滅活HCV疫苗?？赏ㄟ^例如將滅活劑(如福爾馬林、β-丙內(nèi)酯、戊二醛等)加入到病毒懸浮液中并混合，使其與病毒反應(yīng)(Appaiahgari等，Vaccine，(2004)22(27-28)，3669-3675頁(yè))，進(jìn)行病毒滅活。
為生產(chǎn)本發(fā)明的疫苗，有可能使用其中通過使用公開已知的技術(shù)引入突變減弱或消除病原性的HCV復(fù)制子RNA。
本發(fā)明的疫苗制備成溶液或懸浮液給予。還有可能制備為適于溶解或懸浮在液體中的固體物質(zhì)形式。制品可在脂質(zhì)體中乳化或囊化?；钚悦庖咴越M分如HCV顆粒經(jīng)常與藥物可接受的并適于活性成分的賦形劑混合。合適的賦形劑包括例如水、生理鹽水、葡萄糖、甘油、乙醇及其混合物。此外，如果有需要，疫苗可包含少量的助劑(例如增濕劑或乳化劑)、pH緩沖劑和/或用于增強(qiáng)疫苗效力的佐劑。有效佐劑的實(shí)例包括但不限于以下物質(zhì)氫氧化鋁、N-乙酰-胞壁酰-L-蘇氨酰-D-異谷氨酰胺(thr-MDP)、N-乙酰-正-胞壁酰-L-丙氨酰-D-異谷氨酰胺(CGP 11637，nor-MDP)、N-乙酰胞壁酰-L-丙氨酰-D-異谷氨酰胺-L-丙氨酸-2-(1′-2′-二棕櫚酰-sn-甘油-3-羥基磷酰氧基)-乙胺(CGP19835A，稱為MTP-PE)和RIBI。RIBI在2％鯊烯/Tween 80乳濁液中包含三種提取自細(xì)菌的組分，即單磷酰脂質(zhì)A、二分枝菌酸海藻糖和細(xì)胞壁骨架(HPL+TDM+CWS)?？赏ㄟ^檢測(cè)抗免疫原性HCV顆粒的抗體的量測(cè)定佐劑效力，其中所述抗體通過給予由HCV顆粒組成的疫苗產(chǎn)生。
本發(fā)明的疫苗一般胃腸外給予，例如通過注射，如皮下或肌內(nèi)注射。適于其它給予途徑的其它劑型包括栓劑，在某些情況下包括口服制劑。
如果有需要，可將一種或多種具有佐劑活性的上述化合物加入到HCV疫苗中。佐劑為此免疫系統(tǒng)的非特異性刺激因子，在宿主中增強(qiáng)對(duì)HCV疫苗的免疫應(yīng)答。該技術(shù)領(lǐng)域已知佐劑的具體實(shí)例包括完全弗氏佐劑、不完全弗氏佐劑、維生素E、非離子型嵌段共聚物、胞壁酰二肽、皂苷、礦物油、植物油和Carbopol。特別適用于粘膜的佐劑包括例如大腸桿菌不耐熱毒素(LT)和霍亂毒素(CT)。其它合適的佐劑包括例如氫氧化鋁、磷酸鋁或氧化鋁、油乳濁液(例如Bayol(注冊(cè)商標(biāo))或Marcol 52(注冊(cè)商標(biāo)))、皂苷或維生素E增溶物。在優(yōu)選的實(shí)施方案中，本發(fā)明的疫苗包含佐劑。
例如，對(duì)于皮下、皮內(nèi)、肌內(nèi)和靜脈內(nèi)給予的注射，本發(fā)明的HCV疫苗中可包含的藥物可接受的載體和稀釋劑的具體實(shí)例包括穩(wěn)定劑、糖類(例如山梨醇、甘露醇、淀粉、蔗糖、葡萄糖、葡聚糖)、蛋白(例如白蛋白或酪蛋白)、含蛋白的物質(zhì)(例如牛血清白蛋白或脫脂奶)和緩沖液(例如磷酸鹽緩沖液)。
用于栓劑的常規(guī)粘合劑和載體包括例如聚亞烷基二醇或甘油三酯。栓劑可由含0.5％-50％(優(yōu)選1％-20％)活性成分的混合物配制?？诜苿┛砂ǔＪ褂玫馁x形劑。這樣的賦形劑包括例如藥用級(jí)的甘露醇、乳糖、淀粉、硬脂酸鎂、糖精鈉、纖維素、碳酸鎂等。
本發(fā)明的疫苗可以溶液、懸浮液、片劑、丸劑、膠囊、延遲釋放制劑或粉末劑型生產(chǎn)，并可包含10-95％(優(yōu)選25-70％)的活性成分(病毒顆?；蚱洳糠?。
本發(fā)明的疫苗通過適于劑型的方法并以預(yù)防和/或治療有效量給予。劑量為0.01μg-100,000μg，這取決于要治療的患者、患者免疫系統(tǒng)中的抗體形成能力和需要的保護(hù)水平。其還依賴于給予途徑，例如口服、皮下、皮內(nèi)、肌內(nèi)、靜脈內(nèi)等。
該疫苗可通過單次給予程序給予，或優(yōu)選通過復(fù)合給予程序給予。在復(fù)合給予程序中，在給予開始時(shí)進(jìn)行1-10次單獨(dú)給予，接著以維持和/或增強(qiáng)免疫反應(yīng)所需要的間隔給予。例如，可在1-4個(gè)月后進(jìn)行另一類給予，作為第二次給予。必要時(shí)，可在幾個(gè)月后持續(xù)給予。給予方案至少部分地按照個(gè)體患者的需要來(lái)確定，取決于主治醫(yī)師的判斷。
此外，含免疫原性HCV顆粒的疫苗可與其它免疫控制劑(例如免疫球蛋白)共同給予。
HCV顆粒疫苗可通過給予健康個(gè)體，以誘發(fā)對(duì)HCV的免疫應(yīng)答，用于預(yù)防性地對(duì)抗可能的新HCV感染。HCV顆粒疫苗還可通過給予感染HCV的患者并在體內(nèi)誘發(fā)針對(duì)HCV的強(qiáng)免疫應(yīng)答，用作治療性疫苗，以清除HCV。
全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞或全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞或用在這些細(xì)胞中產(chǎn)生的病毒顆粒感染的丙型肝炎病毒感染細(xì)胞，可用作測(cè)試系統(tǒng)，用于篩選促進(jìn)或抑制例如丙型肝炎病毒復(fù)制、病毒顆粒重構(gòu)和病毒顆粒釋放的物質(zhì)(抗丙型肝炎病毒的物質(zhì))。具體地說(shuō)，例如，可通過確定測(cè)試物是否促進(jìn)或抑制全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA的復(fù)制或者病毒顆粒的形成或釋放，在測(cè)試物存在下培養(yǎng)這些細(xì)胞，并檢測(cè)所獲培養(yǎng)物中的全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA或病毒顆粒，篩選促進(jìn)或抑制丙型肝炎病毒生長(zhǎng)的物質(zhì)。在此情況下，可通過測(cè)定由上述細(xì)胞提取的RNA制品中的全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA的量、比率或存在情況，對(duì)培養(yǎng)物中的全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA進(jìn)行檢測(cè)。可通過檢測(cè)培養(yǎng)上清液中HCV蛋白的量、比率或存在情況，對(duì)培養(yǎng)物(主要是培養(yǎng)上清液)中的病毒顆粒進(jìn)行檢測(cè)。
而且，可通過檢測(cè)該培養(yǎng)物中的病毒顆粒，研究由HCV感染患者的血清純化的免疫球蛋白是否可預(yù)防本發(fā)明HCV顆粒的感染。在此測(cè)試中，可使用已用本發(fā)明的HCV病毒顆粒免疫的小鼠、大鼠、兔等的血清?？墒褂貌捎貌糠諬CV蛋白、HCV基因等進(jìn)行的免疫?？梢韵嗨频姆绞綄?duì)其它感染預(yù)防性物質(zhì)進(jìn)行該測(cè)試。
產(chǎn)生的抗本發(fā)明HCV病毒顆粒的本發(fā)明抗體包括多克隆抗體和單克隆抗體。當(dāng)優(yōu)選多克隆抗體時(shí)，第一步用本發(fā)明的HCV顆粒免疫選定的哺乳動(dòng)物(例如小鼠、兔、山羊、綿羊、馬等)。由被免疫動(dòng)物收集血清，并用已知方法加工。如果含針對(duì)HCV表位的多克隆抗體的血清包含針對(duì)其它抗原的抗體，則可通過免疫親和層析純化這些血清。生產(chǎn)多克隆抗血清的方法及其治療方法是本領(lǐng)域已知的。可由已感染HCV的哺乳動(dòng)物分離多克隆抗體。
本領(lǐng)域技術(shù)人員可容易地生產(chǎn)針對(duì)HCV表位的單克隆抗體。已知的常用方法是生產(chǎn)產(chǎn)生單克隆抗體的雜交瘤。例如，可使用在Current Protocols in Immunology(John Wiley & Sons，Inc.)中描述的方法。
可通過細(xì)胞融合或其它方法，例如用腫瘤基因DNA直接轉(zhuǎn)化B淋巴細(xì)胞或用EB病毒轉(zhuǎn)導(dǎo)，生產(chǎn)產(chǎn)生單克隆抗體的細(xì)胞系。
通過這些方法獲得的單克隆抗體和多克隆抗體用于診斷、治療和預(yù)防HCV。
使用本發(fā)明的HCV顆粒生產(chǎn)的抗體可與藥物可接受的增溶劑、添加劑、穩(wěn)定劑、緩沖劑等一起給予。可選擇任何給予途徑，但優(yōu)選皮下、皮內(nèi)和肌內(nèi)給予，更優(yōu)選靜脈內(nèi)給予。
在本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞或全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞中產(chǎn)生的HCV顆粒和HCV允許細(xì)胞可作為測(cè)試系統(tǒng)，用于篩選可刺激或抑制HCV與細(xì)胞結(jié)合的物質(zhì)。具體地說(shuō)，例如，可通過在測(cè)試物存在下培養(yǎng)本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞中產(chǎn)生的HCV顆粒和HCV允許細(xì)胞，檢測(cè)所獲培養(yǎng)物中的全長(zhǎng)HCV復(fù)制子RNA或病毒顆粒，并確定測(cè)試物是否促進(jìn)或抑制復(fù)制子RNA復(fù)制或病毒顆粒形成，篩選可促進(jìn)或抑制丙型肝炎病毒生長(zhǎng)的物質(zhì)。
可按照上述技術(shù)或以下實(shí)施例對(duì)全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA進(jìn)行這樣的檢測(cè)。上述測(cè)試系統(tǒng)可用于生產(chǎn)和評(píng)價(jià)丙型肝炎病毒感染的預(yù)防劑、治療劑或診斷劑。
具體地說(shuō)，上述本發(fā)明測(cè)試系統(tǒng)的應(yīng)用實(shí)例包括以下幾點(diǎn)(1)篩選抑制HCV生長(zhǎng)和感染的物質(zhì)抑制HCV生長(zhǎng)和感染的物質(zhì)包括例如直接或間接影響HCV生長(zhǎng)和感染的有機(jī)化合物、通過與HCV基因組或其互補(bǔ)鏈中的靶序列雜交而直接或間接影響HCV生長(zhǎng)或HCV蛋白翻譯的反義寡核苷酸等。
(2)評(píng)價(jià)在細(xì)胞培養(yǎng)物中具有抗病毒活性的各種物質(zhì)前述各種物質(zhì)包括通過合理藥物設(shè)計(jì)或高通量篩選獲得的物質(zhì)(例如純化和分離的酶)。
(3)鑒別治療HCV感染患者的新靶標(biāo)例如，本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞或全長(zhǎng)HCV基因組RNA復(fù)制型細(xì)胞可用于鑒別可在HCV生長(zhǎng)中起重要作用的宿主細(xì)胞蛋白。
(4)評(píng)價(jià)HCV對(duì)藥物等的抗性獲得能力，并鑒別與抗性相關(guān)的突變(5)生產(chǎn)病毒蛋白作為抗原，用于開發(fā)、生產(chǎn)和評(píng)價(jià)丙型肝炎病毒感染的診斷劑和治療劑(6)生產(chǎn)病毒蛋白作為抗原，用于開發(fā)、生產(chǎn)和評(píng)價(jià)用于丙型肝炎病毒感染的疫苗和生產(chǎn)減毒HCV(7)生產(chǎn)用于診斷和治療丙型肝炎病毒感染的單克隆或多克隆抗體。
基于以下的實(shí)施例和附圖將更具體地描述本發(fā)明。但本發(fā)明的技術(shù)范圍不限于這些實(shí)施例。
實(shí)施例1得自全長(zhǎng)HCV基因組RNA的全長(zhǎng)HCV復(fù)制子RNA的制備(A)構(gòu)建表達(dá)載體構(gòu)建質(zhì)粒DNA，其中DNA(JFH-1克隆)插入到pUC19質(zhì)粒中T7RNA啟動(dòng)子序列的下游，此DNA(JFH-1克隆)含已由暴發(fā)性肝衰竭患者中分離出的丙型肝炎病毒JFH-1毒株(基因型2a)的全長(zhǎng)基因組cDNA。
具體地說(shuō)，將通過擴(kuò)增JFH-1毒株的病毒RNA所獲得的RT-PCR片段克隆入pGEM-T EASY載體(Promega)中，以獲得質(zhì)粒pGEM1-258、pGEM44-486、pGEM317-849、pGEM617-1323、pGEM1141-2367、pGEM2285-3509、pGEM3471-4665、pGEM4547-5970、pGEM5883-7003、pGEM6950-8035、pGEM7984-8892、pGEM8680-9283、pGEM9231-9634和pGEM9594-9678(參見非特許文獻(xiàn)6)。通過使用PCR法和限制酶，將包含在這些質(zhì)粒中的病毒基因組RNA來(lái)源的cDNA連接在一起，以克隆全長(zhǎng)病毒基因組cDNA。將T7R RNA啟動(dòng)子序列插入到全長(zhǎng)病毒基因組cDNA的上游。在下文中，以此方式構(gòu)建的質(zhì)粒DNA稱為pJFH1(圖1的上部)。上述JFH-1克隆的制備已描述于特許文獻(xiàn)1和非特許文獻(xiàn)3。此外，JFH-1克隆的全長(zhǎng)cDNA的核苷酸序列以檢索號(hào)AB047639登記在國(guó)際DNA數(shù)據(jù)庫(kù)(DDBJ/EMBL/GenBank)中。
接著，通過將EMCV-IRES(腦心肌炎病毒的內(nèi)部核糖體進(jìn)入位點(diǎn))和新霉素抗性基因(neo；也稱為新霉素磷酸轉(zhuǎn)移酶基因)插入到pJFH1質(zhì)粒DNA(圖1的下部)的5′非翻譯區(qū)和核心蛋白區(qū)之間，構(gòu)建質(zhì)粒DNA pFGREP-JFH1。該構(gòu)建方法與先前的公開內(nèi)容(非特許文獻(xiàn)4)一致。此外，通過導(dǎo)入突變制備突變質(zhì)粒克隆pJFH1/GND和pFGREP-JFH1/GND，其中所述突變將對(duì)應(yīng)于RNA聚合酶(由pJFH1和pFGREP-JFH1中的NS5B區(qū)編碼)活性中心的氨基酸基序GDD變?yōu)镚ND。因?yàn)橛赏蛔兛寺JFH1/GND和pFGREP-JFH1/GND編碼的NS5B蛋白活性位點(diǎn)的氨基酸序列被改變，所以不能由突變克隆表達(dá)復(fù)制復(fù)制子RNA所需的活性NS5B蛋白。
此外，通過將熒光素酶基因插入到pFGREP-JFH1的第415-420的MluI位點(diǎn)和第2075-2082的PmeI位點(diǎn)之間，以用熒光素酶基因替代pFGREP-JFH1的新霉素抗性基因，制備pFGREP-JFH1/Luc，作為導(dǎo)入報(bào)告基因的表達(dá)載體。另外，通過將pFGREP-JFH1/Luc第10933位的G突變?yōu)锳，制備突變體pFGREP-JFH1/Luc/GND，其中NS5bRNA聚合酶活性中心的GDD基序被變?yōu)镚ND。
通過將綠色熒光蛋白基因插入到pFGREP-JFH1的第415-420的MluI位點(diǎn)和第1142-1149的PmeI位點(diǎn)之間，制備pFGREP-JFH1/EGFP，其中pFGREP-JFH1的新霉素抗性基因被綠色熒光蛋白基因取代。另外，通過將pFGREP-JFH1/EGFP第10000位的G突變?yōu)锳，制備突變體pFGREP-JFH1/EGFP/GND，其中NS5b RNA聚合酶活性中心的GDD基序被變?yōu)镚ND。
通過將分泌型胎盤堿性磷酸酶基因插入到pFGREP-JFH1的第415-420的MluI位點(diǎn)和第1982-1989的PmeI位點(diǎn)之間，以用分泌型胎盤堿性磷酸酶基因取代pFGREP-JFH1的新霉素抗性基因，制備pFGREP-JFH1/SEAP。另外，通過將pFGREP-JFH1/SEAP第10840位的G突變?yōu)锳，制備突變體pFGREP-JFH1/SEAP/GND，其中NS5bRNA聚合酶活性中心的GDD基序被變?yōu)镚ND。
(B)全長(zhǎng)HCV基因組RNA和全長(zhǎng)HCV復(fù)制子RNA的制備如上構(gòu)建表達(dá)載體，用限制酶XbaI消化pJFH1、pJFH1/GND、pFGREP-JFH1和pFGREP-JFH1/GND，以制備模板DNA，用于合成全長(zhǎng)HCV基因組RNA和全長(zhǎng)HCV復(fù)制子RNA。隨后，通過于30℃溫育30分鐘，用20U綠豆核酸酶的50μl反應(yīng)溶液處理10-20μg各XbaI片段。綠豆核酸酶是這樣一種酶其催化涉及選擇性消化雙鏈DNA的單鏈部分的反應(yīng)。通常，如果使用以上XbaI片段原樣作為模板合成RNA，則合成在3′端具有4個(gè)額外堿基CUGA的復(fù)制子RNA，其為XbaI識(shí)別位點(diǎn)的一部分。因此，在該實(shí)施例中，通過用綠豆核酸酶處理XbaI片段，由XbaI片段去除4堿基CUGA。隨后，對(duì)綠豆核酸酶處理后的含XbaI片段的溶液進(jìn)行標(biāo)準(zhǔn)蛋白去除處理，以獲得無(wú)4堿基CUGA的純化XbaI片段，作為以下使用的模板DNA。
接著，使用T7 RNA聚合酶由該模板體外合成RNA。使用MEGAscript(Ambion Co.)進(jìn)行RNA合成。按照生產(chǎn)商的說(shuō)明書使20μl含0.5-1.0μg模板DNA的反應(yīng)混合物反應(yīng)。
在RNA合成后，將DNA酶(2U)加入到反應(yīng)混合物中，于37℃反應(yīng)15分鐘，然后用酸-苯酚處理提取RNA，以去除模板DNA。以此方式由源自pJFH1、pJFH1/GND、pFGREP-JFH1和pFGREP-JFH1/GND的模板DNA合成的RNA分別稱為rJFH1、rJFH1/GND、rFGREP-JFH1和rFGREP-JFH1/GND。這些RNA的核苷酸序列分別示于SEQ ID NO12、13、14和15的rJFH-1、rFGREP-JFH1、rJFH1/GND和rFGREP-JFH1/GND。rJFH1是本發(fā)明全長(zhǎng)HCV基因組RNA的實(shí)例，與JFH-1毒株的全長(zhǎng)HCV基因組具有相同的序列結(jié)構(gòu)。rFGREP-JFH1是本發(fā)明全長(zhǎng)HCV復(fù)制子RNA的實(shí)例。
隨后，通過分別使用如上制備的表達(dá)載體pFGREP-JFH1/Luc、pFGREP-JFH1/Luc/GND、pFGREP-JFH1/EGFP、pFGREP-JFH1/EGFP/GND、pFGREP-JFH1/S EAP和pFGREP-JFH1/SEAP/GND作為模板，生產(chǎn)為HCV復(fù)制子RNA的rFGR-JFH1/Luc(SEQ ID NO21)、rFGR-JFH1/Luc/GND((SEQ ID NO22)、rFGR-JFH1/EGFP(SEQID NO23)、rFGR-JFH1/EGFP/GND(SEQ ID NO24)、rFGR-JFH1/SEAP(SEQ ID NO25)和rFGR-JFH1/SEAP/GND(SEQ ID NO26)。
實(shí)施例2全長(zhǎng)HCV基因組RNA在細(xì)胞中的復(fù)制和病毒顆粒的產(chǎn)生(C)全長(zhǎng)HCV基因組RNA在細(xì)胞中的復(fù)制和病毒顆粒的產(chǎn)生將如上合成的各種量的全長(zhǎng)HCV基因組RNA(rJFH1或rJFH1/GND)與由Huh7細(xì)胞提取的總RNA混合，使RNA量達(dá)到10μg。隨后，利用電穿孔法將混合的RNA導(dǎo)入到Huh7細(xì)胞中。將進(jìn)行電穿孔處理的Huh7細(xì)胞接種入培養(yǎng)皿中。在溫育12、24、48和72小時(shí)后，收集細(xì)胞，提取RNA，并通過RNA印跡法分析。按照Molecular Cloning，A laboratory Manual，第2版，J.Sambrook，E.F.Fritsch，T.Maniatis，Cold Spring Harbor Laboratory Press(1989)進(jìn)行RNA印跡分析。具體地說(shuō)，對(duì)由溫育后細(xì)胞提取的RNA進(jìn)行變性瓊脂糖凝膠電泳，并在電泳后將RNA轉(zhuǎn)移至帶正電荷的尼龍膜上。使由pJFH1制備的32P標(biāo)記DNA或RNA探針與前述轉(zhuǎn)移至膜上的RNA雜交。漂洗膜，并使膜對(duì)膠片曝光，以檢測(cè)對(duì)JFH-1克隆的全長(zhǎng)HCV基因組RNA特異性的RNA條帶。
如圖2所示，當(dāng)rJFH1/GND轉(zhuǎn)染至細(xì)胞中時(shí)，在轉(zhuǎn)染后4小時(shí)導(dǎo)入RNA的條帶被證實(shí)為弱信號(hào)，但信號(hào)隨著傳代次數(shù)變得更弱，轉(zhuǎn)染后24小時(shí)的條帶信號(hào)幾乎不可檢測(cè)。相比之下，當(dāng)轉(zhuǎn)染rJFH1時(shí)，導(dǎo)入RNA條帶的信號(hào)強(qiáng)度首先變?nèi)?，和rJFH1/GND轉(zhuǎn)染后4-12小時(shí)的情況一樣，但在轉(zhuǎn)染后24小時(shí)證實(shí)了清晰的RNA信號(hào)。證實(shí)的信號(hào)是HCV基因組RNA特異性的。也就是說(shuō)，一般認(rèn)為某些導(dǎo)入的全長(zhǎng)HCV基因組RNA復(fù)制并生長(zhǎng)。對(duì)于其中為RNA復(fù)制酶的NS5B活性基序突變的rJFH1/GND，未觀察到復(fù)制，這表明NS5B活性對(duì)全長(zhǎng)HCV基因組RNA復(fù)制很重要。此外，對(duì)得自丙型肝炎病毒，例如H77毒株(非特許文獻(xiàn)7)、J6毒株(非特許文獻(xiàn)8)和本發(fā)明發(fā)明人由慢性肝炎患者分離的JCH1毒株(非特許文獻(xiàn)6)的全長(zhǎng)基因組RNA(先前已全部被分離)進(jìn)行相同的實(shí)驗(yàn)，但證實(shí)這些毒株的全長(zhǎng)HCV基因組RNA未復(fù)制。
(D)轉(zhuǎn)染細(xì)胞培養(yǎng)物的培養(yǎng)基中HCV病毒顆粒的檢測(cè)將如上所述的電穿孔處理的Huh7細(xì)胞接種在培養(yǎng)皿中，培養(yǎng)12、24、48和72小時(shí)，然后檢測(cè)培養(yǎng)上清液中的HCV核心蛋白。按照Ortho HCV抗原IRMA檢驗(yàn)(非特許文獻(xiàn)9)進(jìn)行檢測(cè)。如圖3所示，在用rJFH1轉(zhuǎn)染后48和72小時(shí)檢測(cè)培養(yǎng)上清液中的核心蛋白。為檢驗(yàn)此核心蛋白是否作為病毒顆粒被分泌，通過蔗糖密度梯度分級(jí)分離rJFH1轉(zhuǎn)染后72小時(shí)的培養(yǎng)基。在離心管中，2ml 60％(重量/重量)蔗糖溶液(溶解在50mM Tris pH 7.5/0.1M NaCl/1mM EDTA中)、1ml 50％蔗糖溶液、1ml 40％蔗糖溶液、1ml 30％蔗糖溶液、1mlof 20％蔗糖溶液和1ml 10％蔗糖溶液分層，4ml樣品培養(yǎng)上清液覆蓋于其上。在Beckman轉(zhuǎn)子SW41 Ti中以400,000RPM于4℃離心16小時(shí)。在離心后，由管底部收集各0.5ml的級(jí)分。測(cè)定各個(gè)級(jí)分中的密度、HCV核心蛋白濃度和全長(zhǎng)HCV基因組RNA的量。按照Takeuchi T，Katsume A，Tanaka T，Abe A，Inoue K，Tsukiyama-KoharaK，Kawaguchi R，Tanaka S，Kohara M，“Real-Time detection system forquantification of Hepatitis C virus genome”，Gastroenterology 116636-642(1999)，通過檢測(cè)全長(zhǎng)HCV基因組RNA的5′非翻譯區(qū)的RNA，用定量RT-PCR法進(jìn)行全長(zhǎng)HCV基因組RNA的檢測(cè)。具體地說(shuō)，使用合成引物R6-130-S175′-CGGGAGAGCCATAGTGG-3′(SEQ IDNO16)、R6-290-R195′-AGTACCACAAGGCCTTTCG-3′(SEQ ID NO17)和TaqMan探針R6-148-S21FT，5′-CTGCGGAACCGGTGAGTACAC-3′(SEQ ID NO18)以及EZ rTthRNA PCR試劑盒，PCR擴(kuò)增由細(xì)胞提取的RNA中包含的全長(zhǎng)HCV基因組RNA，然后通過ABI Prism 7700序列檢測(cè)系統(tǒng)檢測(cè)。
如圖4所示，在級(jí)分11中核心蛋白的峰與全長(zhǎng)HCV基因組RNA的峰一致。該級(jí)分的密度約為1.18mg/ml，其表明比重比迄今為止所報(bào)道的核心蛋白和核酸綴合物的比重低。此外，當(dāng)在用0.25％NP40處理培養(yǎng)上清液后進(jìn)行相似的分級(jí)分離時(shí)，核心蛋白和全長(zhǎng)HCV基因組RNA的峰遷移至約1.28mg/ml的比重。也就是說(shuō)，一般認(rèn)為NP40處理剝落了病毒顆粒的表面膜(其含脂質(zhì)，于是具有較低比重)，產(chǎn)生僅由核酸和核心蛋白組成的核心顆粒，因此比重增加。以上結(jié)果表明，通過將rJFH1轉(zhuǎn)染至Huh7細(xì)胞中，全長(zhǎng)HCV基因組RNA在細(xì)胞中復(fù)制，結(jié)果形成病毒顆粒，并分泌入培養(yǎng)上清液中。
實(shí)施例3(E)全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞的制備和細(xì)胞克隆的建立通過如實(shí)施例2所述，將實(shí)施例1中制備的rFGREP-JFH1和rFGREP-JFH1/GND轉(zhuǎn)染入Huh7細(xì)胞，制備全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞，然后嘗試建立全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞克隆。
首先，在分別將rFGREP-JFH1和rFGREP-JFH1/GND轉(zhuǎn)染入Huh7細(xì)胞中后，將細(xì)胞接種在培養(yǎng)皿中。在培養(yǎng)16-24小時(shí)后，加入不同濃度的G418。繼續(xù)培養(yǎng)，同時(shí)每周更換兩次培養(yǎng)基。在培養(yǎng)21天后，用結(jié)晶紫染色存活細(xì)胞。計(jì)數(shù)染色的集落，計(jì)算獲得的集落數(shù)/用于轉(zhuǎn)染的RNA重量。對(duì)于某些培養(yǎng)皿又繼續(xù)培養(yǎng)，以克隆存活細(xì)胞的集落。分別由克隆細(xì)胞中提取RNA、基因組DNA和蛋白，然后檢測(cè)全長(zhǎng)HCV復(fù)制子RNA，研究新霉素抗性基因在基因組DNA中的整合以及HCV蛋白的表達(dá)。這些結(jié)果在下文詳細(xì)給出。
(F)集落形成能力以上轉(zhuǎn)染的結(jié)果表明，對(duì)于在1.0mg/ml G418濃度下用rFGREP-JFH1轉(zhuǎn)染的Huh7細(xì)胞(圖5的左部)，集落形成能力/1μg用于轉(zhuǎn)染的復(fù)制子RNA是368CFU(集落形成單位)/μg RNA。相反，對(duì)于用rFGREP-JFH1/GND轉(zhuǎn)染的Huh7細(xì)胞(圖5的右部)，未觀測(cè)到集落形成。這表明，用rFGREP-JFH1復(fù)制子RNA轉(zhuǎn)染的Huh7細(xì)胞的集落形成能力依賴于由rFGREP-JFH1表達(dá)的NS5B(RNA聚合酶)的活性。也就是說(shuō)，一般認(rèn)為在集落形成細(xì)胞中，借助于由rFGREP-JFH1表達(dá)的NS5B的作用，rFGREP-JFH1復(fù)制子RNA自主復(fù)制，引起新霉素抗性基因持續(xù)表達(dá)，因此保持了G418抗性，結(jié)果使細(xì)胞生長(zhǎng)成為可能。
(G)在已建立細(xì)胞克隆中的全長(zhǎng)HCV復(fù)制子RNA的檢測(cè)利用酸-苯酚提取法，由全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞克隆中提取總RNA，該細(xì)胞按照以上章節(jié)(E)通過將rFGREP-JFH1轉(zhuǎn)染入Huh7細(xì)胞中而建立。隨后，利用RNA印跡法檢測(cè)此總RNA。在該方法中，使用pFGREP-JFH1特異性探針。以相似的方式由未轉(zhuǎn)染的Huh7細(xì)胞提取的總RNA(在圖6中顯示為“Huh7”)，一個(gè)除由Huh7細(xì)胞提取的總RNA以外還含體外合成的107個(gè)拷貝的復(fù)制子RNA的樣品(在圖6中顯示為“107”)和一個(gè)除由Huh7細(xì)胞提取的總RNA以外還含體外合成的108個(gè)拷貝的復(fù)制子RNA的樣品(在圖6中顯示為“108”)用作對(duì)照。在圖6中，1-4表示細(xì)胞克隆號(hào)。
結(jié)果，用rFGREP-JFH1特異性探針檢測(cè)與rFGREP-JFH1具有相似大小的RNA(圖6)。由該結(jié)果證實(shí)轉(zhuǎn)染的rFGREP-JFH1復(fù)制子RNA在細(xì)胞克隆中復(fù)制和生長(zhǎng)。還表明細(xì)胞克隆中的復(fù)制子RNA的量有差異。如圖6所示，例如，克隆2中的復(fù)制子RNA的量少于其它克隆。
(H)驗(yàn)證新霉素抗性基因有無(wú)整合入基因組DNA中對(duì)于按照(E)所獲得的細(xì)胞克隆1-8(如圖7中的FGR-JFH1/2-1至FGR-JFH1/2-8所示)，使用新霉素抗性基因特異性引物(有義引物NEO-S35′-AACAAGATGGATTGCACGCA-3′(SEQ ID NO19)，反義引物NEO-R5′-CGTCAAGAAGGCGATAGAAG-3′(SEQ ID NO20))，并使用由每個(gè)細(xì)胞克隆提取的宿主基因組DNA作為模板，進(jìn)行PCR擴(kuò)增，以便證實(shí)各個(gè)細(xì)胞克隆抗G418的抗性并非是由于新霉素抗性基因整合入宿主細(xì)胞基因組中。結(jié)果，如圖7所示，未觀測(cè)到顯示擴(kuò)增新霉素抗性基因的陽(yáng)性克隆。
(H)的結(jié)果證實(shí)，全長(zhǎng)HCV復(fù)制子RNA在通過本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA轉(zhuǎn)染建立的細(xì)胞克隆中復(fù)制。
(I)HCV蛋白的檢測(cè)通過標(biāo)準(zhǔn)方法由通過rFGREP-JFH1轉(zhuǎn)染建立的細(xì)胞克隆提取蛋白，然后通過SDS-PAGE和蛋白質(zhì)印跡法分析。在此情況下檢測(cè)的細(xì)胞克隆與以上章節(jié)(G)中使用的克隆相同。通過將制備的全長(zhǎng)HCV基因組RNA瞬時(shí)轉(zhuǎn)染入Huh7細(xì)胞中獲得細(xì)胞提取物，將其用作陽(yáng)性對(duì)照(在圖8、9和10中顯示為JFH-1)。通過轉(zhuǎn)染HCV亞基因組RNA復(fù)制子(SGR-JFH1)獲得的克隆的細(xì)胞提取物用作核心蛋白的陰性對(duì)照，并用作NS3和NS5a蛋白的陽(yáng)性對(duì)照(在圖8、9和10中顯示為SGR-JFH1)。未轉(zhuǎn)染的Huh7細(xì)胞的細(xì)胞提取物用作所有蛋白的陰性對(duì)照(在圖8、9和10中顯示為Huh7)。將由各個(gè)細(xì)胞克隆提取的蛋白樣品印跡在PVDF膜(Immobilon-P，Millipore)上，然后使用抗核心蛋白特異性抗體和抗NS3特異性抗體(由Dr.Moradpour惠贈(zèng)；Wolk B等，J.Virology，2000，742293-2304)檢測(cè)由其中的全長(zhǎng)HCV復(fù)制子RNA編碼的核心蛋白和NS3蛋白。如圖8和9所示，對(duì)于通過rFGREP-JFH1轉(zhuǎn)染建立的細(xì)胞克隆1-4，檢測(cè)各個(gè)蛋白中與陽(yáng)性對(duì)照大小相同的蛋白。因?yàn)樵谖崔D(zhuǎn)染Huh7細(xì)胞中既沒有檢測(cè)到核心蛋白，也沒有檢測(cè)到NS3蛋白，所以證實(shí)在細(xì)胞克隆1-4中已轉(zhuǎn)染的全長(zhǎng)HCV復(fù)制子RNA自主復(fù)制，表達(dá)核心蛋白和NS3蛋白。
此外，對(duì)于每個(gè)細(xì)胞克隆，已如上所述檢驗(yàn)NS3蛋白的表達(dá)，還使用丙型肝炎患者的血清作為抗體檢驗(yàn)由全長(zhǎng)HCV復(fù)制子RNA表達(dá)的NS5A蛋白(圖10)。
由上述(H)和(I)的結(jié)果證實(shí)，在已通過全長(zhǎng)HCV復(fù)制子RNA轉(zhuǎn)染建立的細(xì)胞克隆中，全長(zhǎng)HCV復(fù)制子RNA復(fù)制，病毒蛋白也表達(dá)。
(J)全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞中的病毒顆粒生產(chǎn)按照以上章節(jié)(E)將rFGREP-JFH1轉(zhuǎn)染入Huh7細(xì)胞中，建立全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞克隆2和3(FGR-JFH1/2-3)，然后回收其培養(yǎng)上清液。按照與上述(D)相似的方法檢測(cè)培養(yǎng)上清液中的HCV病毒顆粒。結(jié)果示于圖11。在圖11中，陰影圓代表各個(gè)級(jí)分的比重(g/ml)。實(shí)心圓代表核心蛋白的量(fmol/L)。空心圓代表全長(zhǎng)HCV復(fù)制子RNA的滴度(×0.1拷貝/mL)。
如圖11所示，核心蛋白峰與比重約1.18-1.20mg/ml的級(jí)分中的全長(zhǎng)HCV復(fù)制子RNA的峰一致。還在較輕級(jí)分中發(fā)現(xiàn)小峰。以上結(jié)果表明，全長(zhǎng)HCV復(fù)制子RNA在用rFGREP-JFH11轉(zhuǎn)染的Huh7細(xì)胞中復(fù)制，形成病毒顆粒并分泌入其培養(yǎng)上清液中。
實(shí)施例4(K)采用培養(yǎng)上清液中的病毒顆粒的感染實(shí)驗(yàn)通過將在(H)中使用的細(xì)胞克隆1-8(即FGR-JFH1/2-1、FGR-JFH1/2-2、FGR-JFH1/2-3、FGR-JFH1/2-4、FGR-JFH1/2-5、FGR-JGF1/2-6、FGR-JFH1/2-7、FGR-JFH1/2-8)的各個(gè)培養(yǎng)上清液加入Huh7細(xì)胞中，用在培養(yǎng)上清液中的病毒顆粒感染Huh7細(xì)胞。在第2天，以0.3mg/ml向感染的Huh7細(xì)胞的培養(yǎng)基中加入G418，再培養(yǎng)Huh7細(xì)胞21天。在結(jié)束培養(yǎng)后，固定細(xì)胞，并用結(jié)晶紫染色。對(duì)于分別用FGR-JFH1/2-3、FGR-JFH1/2-5和FGR-JFH1/2-6的培養(yǎng)上清液感染的細(xì)胞，觀測(cè)到集落形成。另一方面，對(duì)于用作對(duì)照的亞基因組復(fù)制子細(xì)胞SGR-JFH1/4-1(描述于非特許文獻(xiàn)6)的培養(yǎng)上清液感染的細(xì)胞，未觀測(cè)到集落形成。圖12顯示了與加入的4ml或8mlFGR-JFH1/2-3或SGR-JFH1/4-1培養(yǎng)上清液培養(yǎng)21天后染色培養(yǎng)皿的照片。在其中接種與4ml或8ml FGR-JFH1/2-3培養(yǎng)上清液混合的細(xì)胞的培養(yǎng)皿中，分別發(fā)現(xiàn)3個(gè)和9個(gè)集落。但是，在其中接種與SGR-JFH1/4-1的培養(yǎng)上清液混合的細(xì)胞的培養(yǎng)皿中，未觀測(cè)到集落。
隨后，克隆分別使用FGR-JFH1/2-3和FGR-JFH1/2-5的培養(yǎng)上清液以丙型肝炎病毒感染形成的集落。由用FGR-JFH1/2-3的培養(yǎng)上清液感染的培養(yǎng)皿建立3個(gè)克隆FGR-JFH1/C2-3-11、FGR-JFH1/C2-3-12和FGR-JFH1/C2-3-13，并由用FGR-JFH1/C2-5的培養(yǎng)上清液感染的培養(yǎng)皿建立2個(gè)克隆FGR-JFH1/C2-5-11和FGR-JFH1/C2-5-12。
當(dāng)用FGR-JFH1/C2-3-11、FGR-JFH1/C2-3-12、FGR-JFH1/C2-3-13、FGR-JFH1/C2-5-11和FGR-JFH1/C2-5-12各個(gè)細(xì)胞克隆的培養(yǎng)上清液感染Huh7細(xì)胞時(shí)，在分別用FGR-JFH1/C2-3-12和FGR-JFH1/C2-5-12的培養(yǎng)上清液感染的培養(yǎng)皿中觀測(cè)到集落形成。由用FGR-JFH1/C2-3-12的培養(yǎng)上清液感染的細(xì)胞建立另兩個(gè)克隆FGR-JFH1/C2-3-12-1和FGR-JFH1/C2-3-12-2。由用FGR-JFH1/C2-5-12的培養(yǎng)上清液感染的細(xì)胞建立另兩個(gè)克隆FGR-JFH1/C2-5-12-1和FGR-JFH1/C2-5-12-2。
已用全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞的培養(yǎng)上清液感染的細(xì)胞建立細(xì)胞，由這些細(xì)胞克隆提取RNA、蛋白和基因組DNA。通過使用基因組DNA作為模板的PCR，檢驗(yàn)新霉素抗性基因在這些細(xì)胞克隆的基因組DNA中的整合，結(jié)果全為陰性。而且，通過使用RNA作為模板的定量PCR可檢測(cè)細(xì)胞中復(fù)制的全長(zhǎng)HCV復(fù)制子RNA。再進(jìn)一步，可檢測(cè)培養(yǎng)上清液中的核心蛋白。這些結(jié)果表明，由本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA復(fù)制型細(xì)胞生產(chǎn)的含全長(zhǎng)HCV復(fù)制子RNA的病毒顆?？筛腥玖硪患?xì)胞。
工業(yè)實(shí)用性按照本發(fā)明的方法，可在細(xì)胞培養(yǎng)系統(tǒng)中制備HCV病毒顆粒。使用本發(fā)明的復(fù)制子RNA，可在細(xì)胞培養(yǎng)系統(tǒng)中有效生產(chǎn)含全長(zhǎng)HCV基因組RNA的RNA。而且，通過使用其中導(dǎo)入本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA的細(xì)胞，全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA可在細(xì)胞培養(yǎng)系統(tǒng)中復(fù)制，本發(fā)明的HCV病毒顆?？稍诩?xì)胞培養(yǎng)系統(tǒng)中連續(xù)生產(chǎn)。其中導(dǎo)入本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA的細(xì)胞還可用作測(cè)試系統(tǒng)，用于篩選影響HCV復(fù)制過程、病毒顆粒形成過程和病毒顆粒胞外釋放過程的各種物質(zhì)。本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA和全長(zhǎng)HCV基因組RNA以及病毒顆粒還用作外源基因的病毒載體。本發(fā)明的病毒顆粒或其部分可包含在疫苗中，作為抗丙型肝炎病毒的疫苗抗原。此外，其中本發(fā)明的病毒顆粒和其它細(xì)胞一起培養(yǎng)的系統(tǒng)可用作測(cè)試系統(tǒng)，用于篩選對(duì)病毒顆粒感染細(xì)胞有影響的各種物質(zhì)。本發(fā)明的全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA用作模板，該模板能使HCV全長(zhǎng)基因組序列簡(jiǎn)單再生。
本文提及的所有出版物、特許和特許申請(qǐng)都全文在此引入作為參考。
無(wú)內(nèi)容序列表SEQ ID NO1代表來(lái)自JFH-1克隆的HCV基因組RNA的5′非翻譯區(qū)序列。
SEQ ID NO2代表來(lái)自JFH-1克隆的HCV基因組RNA的核心蛋白編碼序列。
SEQ ID NO3代表來(lái)自JFH-1克隆的HCV基因組RNA的E1蛋白編碼序列。
SEQ ID NO4代表來(lái)自JFH-1克隆的HCV基因組RNA的E2蛋白編碼序列。
SEQ ID NO5代表來(lái)自JFH-1克隆的HCV基因組RNA的NS2蛋白編碼序列。
SEQ ID NO6代表來(lái)自JFH-1克隆的HCV基因組RNA的NS3蛋白編碼序列。
SEQ ID NO7代表來(lái)自JFH-1克隆的HCV基因組RNA的NS4A蛋白編碼序列。
SEQ ID NO8代表來(lái)自JFH-1克隆的HCV基因組RNA的NS4B蛋白編碼序列。
SEQ ID NO9代表來(lái)自JFH-1克隆的HCV基因組RNA的NS5A蛋白編碼序列。
SEQ ID NO10代表來(lái)自JFH-1克隆的HCV基因組RNA的NS5B蛋白編碼序列。
SEQ ID NO11代表來(lái)自JFH-1克隆的HCV基因組RNA的3′非翻譯區(qū)序列。
SEQ ID NO12代表來(lái)自JFH-1克隆的全長(zhǎng)HCV基因組RNA序列。
SEQ ID NO13代表包含來(lái)自JFH-1克隆的全長(zhǎng)HCV基因組RNA的復(fù)制子RNA序列。
SEQ ID NO14代表來(lái)自JFH-1克隆的全長(zhǎng)HCV基因組RNA序列，其中氨基酸基序GDD已突變?yōu)镚ND。
SEQ ID NO15代表包含來(lái)自JFH-1克隆的全長(zhǎng)HCV基因組RNA的復(fù)制子RNA序列，其中氨基酸基序GDD已突變?yōu)镚ND。
SEQ ID NO16-20代表引物序列。
SEQ ID NO21代表來(lái)源于表達(dá)載體pFGREP-JFH1/Luc的復(fù)制子RNA的序列。
SEQ ID NO22代表來(lái)源于表達(dá)載體pFGREP-JFH1/Luc/GND的復(fù)制子RNA的序列。
SEQ ID NO23代表來(lái)源于表達(dá)載體pFGREP-JFH1/EGFP的復(fù)制子RNA的序列。
SEQ ID NO24代表來(lái)源于表達(dá)載體pFGREP-JFH1/EGFP/GND的復(fù)制子RNA的序列。
SEQ ID NO25代表來(lái)源于表達(dá)載體pFGREP-JFH1/SEAP的復(fù)制子RNA的序列。
SEQ ID NO26代表來(lái)源于表達(dá)載體pFGREP-JFH1/SEAP/GND的復(fù)制子RNA的序列。
序列表<110>東京都醫(yī)學(xué)研究機(jī)構(gòu)日本東麗工業(yè)株式會(huì)社<120>含人丙型肝炎病毒全長(zhǎng)基因組的核酸構(gòu)建物、核酸構(gòu)建物轉(zhuǎn)入其中的重組全長(zhǎng)病毒基因組復(fù)制型細(xì)胞和生產(chǎn)人丙型肝炎病毒顆粒的方法<130>PH-2372-PCT<140>PCT/JP2005/003232<141>2005-02-21<150>JP 2004-045489<151>2004-02-20<160>26<170>PatentIn Ver.2.1<210>1<211>340<212>RNA<213>丙型肝炎病毒<220>
<223>來(lái)自JFH-1克隆的丙型肝炎病毒基因組RNA的5′非翻譯區(qū)<220>
<223>發(fā)明人Wakita，Takaji發(fā)明人Kato，Takanobu發(fā)明人Date，Tomoko發(fā)明人Miyamoto，Michiko發(fā)明人Tanabe，Junichi發(fā)明人Sone，Saburo<400>1accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300cgcuugcgag ugccccggga ggucucguag accgugcacc 340<210>2<211>573
<212>RNA<213>丙型肝炎病毒<220>
<223>來(lái)自JFH-1克隆的丙型肝炎病毒基因組RNA的核心蛋白編碼序列<400>2augagcacaa auccuaaacc ucaaagaaaa accaaaagaa acaccaaccg ucgcccagaa 60gacguuaagu ucccgggcgg cggccagauc guuggcggag uauacuuguu gccgcgcagg 120ggccccaggu ugggugugcg cacgacaagg aaaacuucgg agcgguccca gccacguggg 180agacgccagc ccauccccaa agaucggcgc uccacuggca aggccugggg aaaaccaggu 240cgccccuggc cccuauaugg gaaugaggga cucggcuggg caggauggcu ccuguccccc 300cgaggcucuc gccccuccug gggccccacu gacccccggc auaggucgcg caacgugggu 360aaagucaucg acacccuaac guguggcuuu gccgaccuca ugggguacau ccccgucgua 420ggcgccccgc uuaguggcgc cgccagagcu gucgcgcacg gcgugagagu ccuggaggac 480gggguuaauu augcaacagg gaaccuaccc gguuuccccu uuucuaucuu cuugcuggcc 540cuguuguccu gcaucaccgu uccggucucu gcu573<210>3<211>576<212>RNA<213>丙型肝炎病毒<220>
<223>來(lái)自JFH-1克隆的丙型肝炎病毒基因組RNA的E1蛋白編碼序列<400>3gcccagguga agaauaccag uagcagcuac auggugacca augacugcuc caaugacagc 60aucacuuggc agcucgaggc ugcgguucuc cacguccccg ggugcguccc gugcgagaga 120guggggaaua cgucacggug uugggugcca gucucgccaa acauggcugu gcggcagccc 180ggugcccuca cgcagggucu gcggacgcac aucgauaugg uugugauguc cgccaccuuc 240ugcucugcuc ucuacguggg ggaccucugu ggcgggguga ugcucgcggc ccagguguuc 300aucgucucgc cgcaguacca cugguuugug caagaaugca auugcuccau cuacccuggc 360accaucacug gacaccgcau ggcaugggac augaugauga acuggucgcc cacggccacc 420augauccugg cguacgugau gcgcgucccc gaggucauca uagacaucgu uagcggggcu 480cacuggggcg ucauguucgg cuuggccuac uucucuaugc agggagcgug ggcgaagguc 540auugucaucc uucugcuggc cgcuggggug gacgcg 576<210>4<211>1290<212>RNA<213>丙型肝炎病毒<220>
<223>來(lái)自JFH-1克隆的丙型肝炎病毒基因組RNA的E2蛋白編碼序列<400>4
ggcaccacca ccguuggagg cgcuguugca cguuccacca acgugauugc cggcguguuc 60agccauggcc cucagcagaa cauucagcuc auuaacacca acggcaguug gcacaucaac 120cguacugccu ugaauugcaa ugacuccuug aacaccggcu uucucgcggc cuuguucuac 180accaaccgcu uuaacucguc agggugucca gggcgccugu ccgccugccg caacaucgag 240gcuuuccgga uagggugggg cacccuacag uacgaggaua augucaccaa uccagaggau 300augaggccgu acugcuggca cuacccccca aagccgugug gcguaguccc cgcgaggucu 360guguguggcc caguguacug uuucaccccc agcccgguag uagugggcac gaccgacaga 420cguggagugc ccaccuacac auggggagag aaugagacag augucuuccu acugaacagc 480acccgaccgc cgcagggcuc augguucggc ugcacgugga ugaacuccac ugguuucacc 540aagacuugug gcgcgccacc uugccgcacc agagcugacu ucaacgccag cacggacuug 600uugugcccua cggauuguuu uaggaagcau ccugaugcca cuuauauuaa gugugguucu 660gggcccuggc ucacaccaaa gugccugguc cacuacccuu acagacucug gcauuacccc 720ugcacaguca auuuuaccau cuucaagaua agaauguaug uagggggggu ugagcacagg 780cucacggccg caugcaacuu cacucguggg gaucgcugcg acuuggagga cagggacagg 840agucagcugu cuccucuguu gcacucuacc acggaauggg ccauccugcc cugcaccuac 900ucagacuuac ccgcuuuguc aacuggucuu cuccaccuuc accagaacau cguggacgua 960caauacaugu auggccucuc accugcuauc acaaaauacg ucguucgaug ggagugggug 1020guacucuuau uccugcucuu agcggacgcc agagucugcg ccugcuugug gaugcucauc 1080uuguugggcc aggccgaagc agcauuggag aaguuggucg ucuugcacgc ugcgagugcg 1140gcuaacugcc auggccuccu auauuuugcc aucuucuucg uggcagcuug gcacaucagg 1200ggucgggugg uccccuugac caccuauugc cucacuggcc uauggcccuu cugccuacug 1260cucauggcac ugccccggca ggcuuaugcc1290<210>5<211>651<212>RNA<213>丙型肝炎病毒<220>
<223>來(lái)自JFH-1克隆的丙型肝炎病毒基因組RNA的NS2蛋白編碼序列<400>5uaugacgcac cugugcacgg acagauaggc guggguuugu ugauauugau cacccucuuc 60acacucaccc cgggguauaa gacccuccuc ggccaguguc uguggugguu gugcuaucuc 120cugacccugg gggaagccau gauucaggag uggguaccac ccaugcaggu gcgcggcggc 180cgcgauggca ucgcgugggc cgucacuaua uucugcccgg gugugguguu ugacauuacc 240aaauggcuuu uggcguugcu ugggccugcu uaccucuuaa gggccgcuuu gacacaugug 300ccguacuucg ucagagcuca cgcucugaua aggguaugcg cuuuggugaa gcagcucgcg 360ggggguaggu auguucaggu ggcgcuauug gcccuuggca gguggacugg caccuacauc 420uaugaccacc ucacaccuau gucggacugg gccgcuagcg gccugcgcga cuuagcgguc 480gccguggaac ccaucaucuu caguccgaug gagaagaagg ucaucgucug gggagcggag 540acggcugcau guggggacau ucuacaugga cuucccgugu ccgcccgacu cggccaggag 600auccuccucg gcccagcuga uggcuacacc uccaaggggu ggaagcuccu u651<210>6<211>1893<212>RNA
<213>丙型肝炎病毒<220>
<223>來(lái)自JFH-1克隆的丙型肝炎病毒基因組RNA的NS3蛋白編碼序列<400>6gcucccauca cugcuuaugc ccagcaaaca cgaggccucc ugggcgccau aguggugagu 60augacggggc gugacaggac agaacaggcc ggggaagucc aaauccuguc cacagucucu 120caguccuucc ucggaacaac caucucgggg guuuugugga cuguuuacca cggagcuggc 180aacaagacuc uagccggcuu acgggguccg gucacgcaga uguacucgag ugcugagggg 240gacuugguag gcuggcccag ccccccuggg accaagucuu uggagccgug caagugugga 300gccgucgacc uauaucuggu cacgcggaac gcugauguca ucccggcucg gagacgcggg 360gacaagcggg gagcauugcu cuccccgaga cccauuucga ccuugaaggg guccucgggg 420gggccggugc ucugcccuag gggccacguc guugggcucu uccgagcagc ugugugcucu 480cggggcgugg ccaaauccau cgauuucauc cccguugaga cacucgacgu uguuacaagg 540ucucccacuu ucagugacaa cagcacgcca ccggcugugc cccagaccua ucaggucggg 600uacuugcaug cuccaacugg caguggaaag agcaccaagg ucccugucgc guaugccgcc 660cagggguaca aaguacuagu gcuuaacccc ucgguagcug ccacccuggg guuuggggcg 720uaccuaucca aggcacaugg caucaauccc aacauuagga cuggagucag gaccgugaug 780accggggagg ccaucacgua cuccacauau ggcaaauuuc ucgccgaugg gggcugcgcu 840agcggcgccu augacaucau cauaugcgau gaaugccacg cuguggaugc uaccuccauu 900cucggcaucg gaacgguccu ugaucaagca gagacagccg gggucagacu aacugugcug 960gcuacggcca caccccccgg gucagugaca accccccauc ccgauauaga agagguaggc 1020cucgggcggg agggugagau ccccuucuau gggagggcga uuccccuauc cugcaucaag 1080ggagggagac accugauuuu cugccacuca aagaaaaagu gugacgagcu cgcggcggcc 1140cuucggggca ugggcuugaa ugccguggca uacuauagag gguuggacgu cuccauaaua 1200ccagcucagg gagauguggu ggucgucgcc accgacgccc ucaugacggg guacacugga 1260gacuuugacu ccgugaucga cugcaaugua gcggucaccc aagcugucga cuucagccug 1320gaccccaccu ucacuauaac cacacagacu gucccacaag acgcugucuc acgcagucag 1380cgccgcgggc gcacagguag aggaagacag ggcacuuaua gguauguuuc cacuggugaa 1440cgagccucag gaauguuuga caguguagug cuuugugagu gcuacgacgc aggggcugcg 1500ugguacgauc ucacaccagc ggagaccacc gucaggcuua gagcguauuu caacacgccc 1560ggccuacccg ugugucaaga ccaucuugaa uuuugggagg caguuuucac cggccucaca 1620cacauagacg cccacuuccu cucccaaaca aagcaagcgg gggagaacuu cgcguaccua 1680guagccuacc aagcuacggu gugcgccaga gccaaggccc cucccccguc cugggacgcc 1740auguggaagu gccuggcccg acucaagccu acgcuugcgg gccccacacc ucuccuguac 1800cguuugggcc cuauuaccaa ugaggucacc cucacacacc cugggacgaa guacaucgcc 1860acaugcaugc aagcugaccu ugaggucaug acc1893<210>7<211>162<212>RNA<213>丙型肝炎病毒<220>
<223>來(lái)自JFH-1克隆的丙型肝炎病毒基因組RNA的NS4A蛋白編碼序列<400>7
agcacguggg uccuagcugg aggaguccug gcagccgucg ccgcauauug ccuggcgacu 60ggaugcguuu ccaucaucgg ccgcuugcac gucaaccagc gagucgucgu ugcgccggau 120aaggaggucc uguaugaggc uuuugaugag auggaggaau gc 162<210>8<211>783<212>RNA<213>丙型肝炎病毒<220>
<223>來(lái)自JFH-1克隆的丙型肝炎病毒基因組RNA的NS4B蛋白編碼序列<400>8gccucuaggg cggcucucau cgaagagggg cagcggauag ccgagauguu gaaguccaag 60auccaaggcu ugcugcagca ggccucuaag caggcccagg acauacaacc cgcuaugcag 120gcuucauggc ccaaagugga acaauuuugg gccagacaca uguggaacuu cauuagcggc 180auccaauacc ucgcaggauu gucaacacug ccagggaacc ccgcgguggc uuccaugaug 240gcauucagug ccgcccucac caguccguug ucgaccagua ccaccauccu ucucaacauc 300augggaggcu gguuagcguc ccagaucgca ccacccgcgg gggccaccgg cuuugucguc 360aguggccugg ugggggcugc cgugggcagc auaggccugg guaaggugcu gguggacauc 420cuggcaggau auggugcggg cauuucgggg gcccucgucg cauucaagau caugucuggc 480gagaagcccu cuauggaaga ugucaucaau cuacugccug ggauccuguc uccgggagcc 540cugguggugg gggucaucug cgcggccauu cugcgccgcc acgugggacc gggggagggc 600gcgguccaau ggaugaacag gcuuauugcc uuugcuucca gaggaaacca cgucgccccu 660acucacuacg ugacggaguc ggaugcgucg cagcguguga cccaacuacu uggcucucuu 720acuauaacca gccuacucag aagacuccac aauuggauaa cugaggacug ccccauccca 780ugc 783<210>9<211>1398<212>RNA<213>丙型肝炎病毒<220>
<223>來(lái)自JFH-1克隆的丙型肝炎病毒基因組RNA的NS5A蛋白編碼序列<400>9uccggauccu ggcuccgcga cgugugggac uggguuugca ccaucuugac agacuucaaa 60aauuggcuga ccucuaaauu guuccccaag cugcccggcc uccccuucau cucuugucaa 120aagggguaca agggugugug ggccggcacu ggcaucauga ccacgcgcug cccuugcggc 180gccaacaucu cuggcaaugu ccgccugggc ucuaugagga ucacagggcc uaaaaccugc 240augaacaccu ggcaggggac cuuuccuauc aauugcuaca cggagggcca gugcgcgccg 300aaacccccca cgaacuacaa gaccgccauc uggagggugg cggccucgga guacgcggag 360gugacgcagc augggucgua cuccuaugua acaggacuga ccacugacaa ucugaaaauu 420ccuugccaac uaccuucucc agaguuuuuc uccugggugg acggugugca gauccauagg 480uuugcaccca caccaaagcc guuuuuccgg gaugaggucu cguucugcgu ugggcuuaau 540uccuaugcug ucggguccca gcuucccugu gaaccugagc ccgacgcaga cguauugagg 600
uccaugcuaa cagauccgcc ccacaucacg gcggagacug cggcgcggcg cuuggcacgg 660ggaucaccuc caucugaggc gagcuccuca gugagccagc uaucagcacc gucgcugcgg 720gccaccugca ccacccacag caacaccuau gacguggaca uggucgaugc caaccugcuc 780auggagggcg guguggcuca gacagagccu gaguccaggg ugcccguucu ggacuuucuc 840gagccaaugg ccgaggaaga gagcgaccuu gagcccucaa uaccaucgga gugcaugcuc 900cccaggagcg gguuuccacg ggccuuaccg gcuugggcac ggccugacua caacccgccg 960cucguggaau cguggaggag gccagauuac caaccgccca ccguugcugg uugugcucuc 1020ccccccccca agaaggcccc gacgccuccc ccaaggagac gccggacagu gggucugagc 1080gagagcacca uaucagaagc ccuccagcaa cuggccauca agaccuuugg ccagcccccc 1140ucgagcggug augcaggcuc guccacgggg gcgggcgccg ccgaauccgg cgguccgacg 1200uccccuggug agccggcccc cucagagaca gguuccgccu ccucuaugcc cccccucgag 1260ggggagccug gagauccgga ccuggagucu gaucagguag agcuucaacc ucccccccag 1320gggggggggg uagcucccgg uucgggcucg gggucuuggu cuacuugcuc cgaggaggac 1380gauaccaccg ugugcugc 1398<210>10<211>1773<212>RNA<213>丙型肝炎病毒<220>
<223>來(lái)自JFH-1克隆的丙型肝炎病毒基因組RNA的NS5B蛋白編碼序列<400>10uccaugucau acuccuggac cggggcucua auaacucccu guagccccga agaggaaaag 60uugccaauca acccuuugag uaacucgcug uugcgauacc auaacaaggu guacuguaca 120acaucaaaga gcgccucaca gagggcuaaa aagguaacuu uugacaggac gcaagugcuc 180gacgcccauu augacucagu cuuaaaggac aucaagcuag cggcuuccaa ggucagcgca 240aggcuccuca ccuuggagga ggcgugccag uugacuccac cccauucugc aagauccaag 300uauggauucg gggccaagga gguccgcagc uuguccggga gggccguuaa ccacaucaag 360uccgugugga aggaccuccu ggaagaccca caaacaccaa uucccacaac caucauggcc 420aaaaaugagg uguucugcgu ggaccccgcc aaggggggua agaaaccagc ucgccucauc 480guuuacccug accucggcgu ccgggucugc gagaaaaugg cccucuauga cauuacacaa 540aagcuuccuc aggcgguaau gggagcuucc uauggcuucc aguacucccc ugcccaacgg 600guggaguauc ucuugaaagc augggcggaa aagaaggacc ccauggguuu uucguaugau 660acccgaugcu ucgacucaac cgucacugag agagacauca ggaccgagga guccauauac 720caggccugcu cccugcccga ggaggcccgc acugccauac acucgcugac ugagagacuu 780uacguaggag ggcccauguu caacagcaag ggucaaaccu gcgguuacag acguugccgc 840gccagcgggg ugcuaaccac uagcaugggu aacaccauca caugcuaugu gaaagcccua 900gcggccugca aggcugcggg gauaguugcg cccacaaugc ugguaugcgg cgaugaccua 960guagucaucu cagaaagcca ggggacugag gaggacgagc ggaaccugag agccuucacg 1020gaggccauga ccagguacuc ugccccuccu ggugaucccc ccagaccgga auaugaccug 1080gagcuaauaa cauccuguuc cucaaaugug ucuguggcgu ugggcccgcg gggccgccgc 1140agauacuacc ugaccagaga cccaaccacu ccacucgccc gggcugccug ggaaacaguu 1200agacacuccc cuaucaauuc auggcuggga aacaucaucc aguaugcucc aaccauaugg 1260guucgcaugg uccuaaugac acacuucuuc uccauucuca ugguccaaga cacccuggac 1320cagaaccuca acuuugagau guauggauca guauacuccg ugaauccuuu ggaccuucca 1380gccauaauug agagguuaca cgggcuugac gccuuuucua ugcacacaua cucucaccac 1440
gaacugacgc ggguggcuuc agcccucaga aaacuugggg cgccaccccu cagggugugg 1500aagagucggg cucgcgcagu cagggcgucc cucaucuccc guggagggaa agcggccguu 1560ugcggccgau aucucuucaa uugggcggug aagaccaagc ucaaacucac uccauugccg 1620gaggcgcgcc uacuggacuu auccaguugg uucaccgucg gcgccggcgg gggcgacauu 1680uuucacagcg ugucgcgcgc ccgaccccgc ucauuacucu ucggccuacu ccuacuuuuc 1740guagggguag gccucuuccu acuccccgcu cgg1773<210>11<211>239<212>RNA<213>丙型肝炎病毒<220>
<223>來(lái)自JFH-1克隆的丙型肝炎病毒基因組RNA的3′非翻譯區(qū)<400>11uagagcggca cacacuaggu acacuccaua gcuaacuguu ccuuuuuuuu uuuuuuuuuu 60uuuuuuuuuu uuuuuuuuuu uuuuucuuuu uuuuuuuuuu cccucuuucu ucccuucuca 120ucuuauucua cuuucuuucu ugguggcucc aucuuagccc uagucacggc uagcugugaa 180agguccguga gccgcaugac ugcagagagu gccguaacug gucucucugc agaucaugu239<210>12<211>9707<212>RNA<213>丙型肝炎病毒<220>
<223>來(lái)自JFH-1克隆的全長(zhǎng)丙型肝炎病毒基因組RNA<400>12gaauucuaau acgacucacu auagaccugc cccuaauagg ggcgacacuc cgccaugaau 60cacuccccug ugaggaacua cugucuucac gcagaaagcg ccuagccaug gcguuaguau 120gagugucgua cagccuccag gccccccccu cccgggagag ccauaguggu cugcggaacc 180ggugaguaca ccggaauugc cgggaagacu ggguccuuuc uuggauaaac ccacucuaug 240cccggccauu ugggcgugcc cccgcaagac ugcuagccga guagcguugg guugcgaaag 300gccuuguggu acugccugau agggcgcuug cgagugcccc gggaggucuc guagaccgug 360caccaugagc acaaauccua aaccucaaag aaaaaccaaa agaaacacca accgucgccc 420agaagacguu aaguucccgg gcggcggcca gaucguuggc ggaguauacu uguugccgcg 480caggggcccc agguugggug ugcgcacgac aaggaaaacu ucggagcggu cccagccacg 540ugggagacgc cagcccaucc ccaaagaucg gcgcuccacu ggcaaggccu ggggaaaacc 600aggucgcccc uggccccuau augggaauga gggacucggc ugggcaggau ggcuccuguc 660cccccgaggc ucucgccccu ccuggggccc cacugacccc cggcauaggu cgcgcaacgu 720ggguaaaguc aucgacaccc uaacgugugg cuuugccgac cucauggggu acauccccgu 780cguaggcgcc ccgcuuagug gcgccgccag agcugucgcg cacggcguga gaguccugga 840ggacgggguu aauuaugcaa cagggaaccu acccgguuuc cccuuuucua ucuucuugcu 900ggcccuguug uccugcauca ccguuccggu cucugcugcc caggugaaga auaccaguag 960cagcuacaug gugaccaaug acugcuccaa ugacagcauc acuuggcagc ucgaggcugc 1020
gguucuccac guccccgggu gcgucccgug cgagagagug gggaauacgu cacgguguug 1080ggugccaguc ucgccaaaca uggcugugcg gcagcccggu gcccucacgc agggucugcg 1140gacgcacauc gauaugguug ugauguccgc caccuucugc ucugcucucu acguggggga 1200ccucuguggc ggggugaugc ucgcggccca gguguucauc gucucgccgc aguaccacug 1260guuugugcaa gaaugcaauu gcuccaucua cccuggcacc aucacuggac accgcauggc 1320augggacaug augaugaacu ggucgcccac ggccaccaug auccuggcgu acgugaugcg 1380cguccccgag gucaucauag acaucguuag cggggcucac uggggcguca uguucggcuu 1440ggccuacuuc ucuaugcagg gagcgugggc gaaggucauu gucauccuuc ugcuggccgc 1500ugggguggac gcgggcacca ccaccguugg aggcgcuguu gcacguucca ccaacgugau 1560ugccggcgug uucagccaug gcccucagca gaacauucag cucauuaaca ccaacggcag 1620uuggcacauc aaccguacug ccuugaauug caaugacucc uugaacaccg gcuuucucgc 1680ggccuuguuc uacaccaacc gcuuuaacuc gucagggugu ccagggcgcc uguccgccug 1740ccgcaacauc gaggcuuucc ggauagggug gggcacccua caguacgagg auaaugucac 1800caauccagag gauaugaggc cguacugcug gcacuacccc ccaaagccgu guggcguagu 1860ccccgcgagg ucugugugug gcccagugua cuguuucacc cccagcccgg uaguaguggg 1920cacgaccgac agacguggag ugcccaccua cacaugggga gagaaugaga cagaugucuu 1980ccuacugaac agcacccgac cgccgcaggg cucaugguuc ggcugcacgu ggaugaacuc 2040cacugguuuc accaagacuu guggcgcgcc accuugccgc accagagcug acuucaacgc 2100cagcacggac uuguugugcc cuacggauug uuuuaggaag cauccugaug ccacuuauau 2160uaaguguggu ucugggcccu ggcucacacc aaagugccug guccacuacc cuuacagacu 2220cuggcauuac cccugcacag ucaauuuuac caucuucaag auaagaaugu auguaggggg 2280gguugagcac aggcucacgg ccgcaugcaa cuucacucgu ggggaucgcu gcgacuugga 2340ggacagggac aggagucagc ugucuccucu guugcacucu accacggaau gggccauccu 2400gcccugcacc uacucagacu uacccgcuuu gucaacuggu cuucuccacc uucaccagaa 2460caucguggac guacaauaca uguauggccu cucaccugcu aucacaaaau acgucguucg 2520augggagugg gugguacucu uauuccugcu cuuagcggac gccagagucu gcgccugcuu 2580guggaugcuc aucuuguugg gccaggccga agcagcauug gagaaguugg ucgucuugca 2640cgcugcgagu gcggcuaacu gccauggccu ccuauauuuu gccaucuucu ucguggcagc 2700uuggcacauc aggggucggg ugguccccuu gaccaccuau ugccucacug gccuauggcc 2760cuucugccua cugcucaugg cacugccccg gcaggcuuau gccuaugacg caccugugca 2820cggacagaua ggcguggguu uguugauauu gaucacccuc uucacacuca ccccggggua 2880uaagacccuc cucggccagu gucuguggug guugugcuau cuccugaccc ugggggaagc 2940caugauucag gaguggguac cacccaugca ggugcgcggc ggccgcgaug gcaucgcgug 3000ggccgucacu auauucugcc cggguguggu guuugacauu accaaauggc uuuuggcguu 3060gcuugggccu gcuuaccucu uaagggccgc uuugacacau gugccguacu ucgucagagc 3120ucacgcucug auaaggguau gcgcuuuggu gaagcagcuc gcggggggua gguauguuca 3180gguggcgcua uuggcccuug gcagguggac uggcaccuac aucuaugacc accucacacc 3240uaugucggac ugggccgcua gcggccugcg cgacuuagcg gucgccgugg aacccaucau 3300cuucaguccg auggagaaga aggucaucgu cuggggagcg gagacggcug caugugggga 3360cauucuacau ggacuucccg uguccgcccg acucggccag gagauccucc ucggcccagc 3420ugauggcuac accuccaagg gguggaagcu ccuugcuccc aucacugcuu augcccagca 3480aacacgaggc cuccugggcg ccauaguggu gaguaugacg gggcgugaca ggacagaaca 3540ggccggggaa guccaaaucc uguccacagu cucucagucc uuccucggaa caaccaucuc 3600ggggguuuug uggacuguuu accacggagc uggcaacaag acucuagccg gcuuacgggg 3660uccggucacg cagauguacu cgagugcuga gggggacuug guaggcuggc ccagcccccc 3720ugggaccaag ucuuuggagc cgugcaagug uggagccguc gaccuauauc uggucacgcg 3780gaacgcugau gucaucccgg cucggagacg cggggacaag cggggagcau ugcucucccc 3840gagacccauu ucgaccuuga agggguccuc gggggggccg gugcucugcc cuaggggcca 3900cgucguuggg cucuuccgag cagcugugug cucucggggc guggccaaau ccaucgauuu 3960
cauccccguu gagacacucg acguuguuac aaggucuccc acuuucagug acaacagcac 4020gccaccggcu gugccccaga ccuaucaggu cggguacuug caugcuccaa cuggcagugg 4080aaagagcacc aaggucccug ucgcguaugc cgcccagggg uacaaaguac uagugcuuaa 4140ccccucggua gcugccaccc ugggguuugg ggcguaccua uccaaggcac auggcaucaa 4200ucccaacauu aggacuggag ucaggaccgu gaugaccggg gaggccauca cguacuccac 4260auauggcaaa uuucucgccg augggggcug cgcuagcggc gccuaugaca ucaucauaug 4320cgaugaaugc cacgcugugg augcuaccuc cauucucggc aucggaacgg uccuugauca 4380agcagagaca gccgggguca gacuaacugu gcuggcuacg gccacacccc ccgggucagu 4440gacaaccccc caucccgaua uagaagaggu aggccucggg cgggagggug agauccccuu 4500cuaugggagg gcgauucccc uauccugcau caagggaggg agacaccuga uuuucugcca 4560cucaaagaaa aagugugacg agcucgcggc ggcccuucgg ggcaugggcu ugaaugccgu 4620ggcauacuau agaggguugg acgucuccau aauaccagcu cagggagaug ugguggucgu 4680cgccaccgac gcccucauga cgggguacac uggagacuuu gacuccguga ucgacugcaa 4740uguagcgguc acccaagcug ucgacuucag ccuggacccc accuucacua uaaccacaca 4800gacuguccca caagacgcug ucucacgcag ucagcgccgc gggcgcacag guagaggaag 4860acagggcacu uauagguaug uuuccacugg ugaacgagcc ucaggaaugu uugacagugu 4920agugcuuugu gagugcuacg acgcaggggc ugcgugguac gaucucacac cagcggagac 4980caccgucagg cuuagagcgu auuucaacac gcccggccua cccguguguc aagaccaucu 5040ugaauuuugg gaggcaguuu ucaccggccu cacacacaua gacgcccacu uccucuccca 5100aacaaagcaa gcgggggaga acuucgcgua ccuaguagcc uaccaagcua cggugugcgc 5160cagagccaag gccccucccc cguccuggga cgccaugugg aagugccugg cccgacucaa 5220gccuacgcuu gcgggcccca caccucuccu guaccguuug ggcccuauua ccaaugaggu 5280cacccucaca cacccuggga cgaaguacau cgccacaugc augcaagcug accuugaggu 5340caugaccagc acgugggucc uagcuggagg aguccuggca gccgucgccg cauauugccu 5400ggcgacugga ugcguuucca ucaucggccg cuugcacguc aaccagcgag ucgucguugc 5460gccggauaag gagguccugu augaggcuuu ugaugagaug gaggaaugcg ccucuagggc 5520ggcucucauc gaagaggggc agcggauagc cgagauguug aaguccaaga uccaaggcuu 5580gcugcagcag gccucuaagc aggcccagga cauacaaccc gcuaugcagg cuucauggcc 5640caaaguggaa caauuuuggg ccagacacau guggaacuuc auuagcggca uccaauaccu 5700cgcaggauug ucaacacugc cagggaaccc cgcgguggcu uccaugaugg cauucagugc 5760cgcccucacc aguccguugu cgaccaguac caccauccuu cucaacauca ugggaggcug 5820guuagcgucc cagaucgcac cacccgcggg ggccaccggc uuugucguca guggccuggu 5880gggggcugcc gugggcagca uaggccuggg uaaggugcug guggacaucc uggcaggaua 5940uggugcgggc auuucggggg cccucgucgc auucaagauc augucuggcg agaagcccuc 6000uauggaagau gucaucaauc uacugccugg gauccugucu ccgggagccc uggugguggg 6060ggucaucugc gcggccauuc ugcgccgcca cgugggaccg ggggagggcg cgguccaaug 6120gaugaacagg cuuauugccu uugcuuccag aggaaaccac gucgccccua cucacuacgu 6180gacggagucg gaugcgucgc agcgugugac ccaacuacuu ggcucucuua cuauaaccag 6240ccuacucaga agacuccaca auuggauaac ugaggacugc cccaucccau gcuccggauc 6300cuggcuccgc gacguguggg acuggguuug caccaucuug acagacuuca aaaauuggcu 6360gaccucuaaa uuguucccca agcugcccgg ccuccccuuc aucucuuguc aaaaggggua 6420caagggugug ugggccggca cuggcaucau gaccacgcgc ugcccuugcg gcgccaacau 6480cucuggcaau guccgccugg gcucuaugag gaucacaggg ccuaaaaccu gcaugaacac 6540cuggcagggg accuuuccua ucaauugcua cacggagggc cagugcgcgc cgaaaccccc 6600cacgaacuac aagaccgcca ucuggagggu ggcggccucg gaguacgcgg aggugacgca 6660gcaugggucg uacuccuaug uaacaggacu gaccacugac aaucugaaaa uuccuugcca 6720acuaccuucu ccagaguuuu ucuccugggu ggacggugug cagauccaua gguuugcacc 6780cacaccaaag ccguuuuucc gggaugaggu cucguucugc guugggcuua auuccuaugc 6840ugucgggucc cagcuucccu gugaaccuga gcccgacgca gacguauuga gguccaugcu 6900
aacagauccg ccccacauca cggcggagac ugcggcgcgg cgcuuggcac ggggaucacc 6960uccaucugag gcgagcuccu cagugagcca gcuaucagca ccgucgcugc gggccaccug 7020caccacccac agcaacaccu augacgugga cauggucgau gccaaccugc ucauggaggg 7080cgguguggcu cagacagagc cugaguccag ggugcccguu cuggacuuuc ucgagccaau 7140ggccgaggaa gagagcgacc uugagcccuc aauaccaucg gagugcaugc uccccaggag 7200cggguuucca cgggccuuac cggcuugggc acggccugac uacaacccgc cgcucgugga 7260aucguggagg aggccagauu accaaccgcc caccguugcu gguugugcuc uccccccccc 7320caagaaggcc ccgacgccuc ccccaaggag acgccggaca gugggucuga gcgagagcac 7380cauaucagaa gcccuccagc aacuggccau caagaccuuu ggccagcccc ccucgagcgg 7440ugaugcaggc ucguccacgg gggcgggcgc cgccgaaucc ggcgguccga cguccccugg 7500ugagccggcc cccucagaga cagguuccgc cuccucuaug cccccccucg agggggagcc 7560uggagauccg gaccuggagu cugaucaggu agagcuucaa ccuccccccc aggggggggg 7620gguagcuccc gguucgggcu cggggucuug gucuacuugc uccgaggagg acgauaccac 7680cgugugcugc uccaugucau acuccuggac cggggcucua auaacucccu guagccccga 7740agaggaaaag uugccaauca acccuuugag uaacucgcug uugcgauacc auaacaaggu 7800guacuguaca acaucaaaga gcgccucaca gagggcuaaa aagguaacuu uugacaggac 7860gcaagugcuc gacgcccauu augacucagu cuuaaaggac aucaagcuag cggcuuccaa 7920ggucagcgca aggcuccuca ccuuggagga ggcgugccag uugacuccac cccauucugc 7980aagauccaag uauggauucg gggccaagga gguccgcagc uuguccggga gggccguuaa 8040ccacaucaag uccgugugga aggaccuccu ggaagaccca caaacaccaa uucccacaac 8100caucauggcc aaaaaugagg uguucugcgu ggaccccgcc aaggggggua agaaaccagc 8160ucgccucauc guuuacccug accucggcgu ccgggucugc gagaaaaugg cccucuauga 8220cauuacacaa aagcuuccuc aggcgguaau gggagcuucc uauggcuucc aguacucccc 8280ugcccaacgg guggaguauc ucuugaaagc augggcggaa aagaaggacc ccauggguuu 8340uucguaugau acccgaugcu ucgacucaac cgucacugag agagacauca ggaccgagga 8400guccauauac caggccugcu cccugcccga ggaggcccgc acugccauac acucgcugac 8460ugagagacuu uacguaggag ggcccauguu caacagcaag ggucaaaccu gcgguuacag 8520acguugccgc gccagcgggg ugcuaaccac uagcaugggu aacaccauca caugcuaugu 8580gaaagcccua gcggccugca aggcugcggg gauaguugcg cccacaaugc ugguaugcgg 8640cgaugaccua guagucaucu cagaaagcca ggggacugag gaggacgagc ggaaccugag 8700agccuucacg gaggccauga ccagguacuc ugccccuccu ggugaucccc ccagaccgga 8760auaugaccug gagcuaauaa cauccuguuc cucaaaugug ucuguggcgu ugggcccgcg 8820gggccgccgc agauacuacc ugaccagaga cccaaccacu ccacucgccc gggcugccug 8880ggaaacaguu agacacuccc cuaucaauuc auggcuggga aacaucaucc aguaugcucc 8940aaccauaugg guucgcaugg uccuaaugac acacuucuuc uccauucuca ugguccaaga 9000cacccuggac cagaaccuca acuuugagau guauggauca guauacuccg ugaauccuuu 9060ggaccuucca gccauaauug agagguuaca cgggcuugac gccuuuucua ugcacacaua 9120cucucaccac gaacugacgc ggguggcuuc agcccucaga aaacuugggg cgccaccccu 9180cagggugugg aagagucggg cucgcgcagu cagggcgucc cucaucuccc guggagggaa 9240agcggccguu ugcggccgau aucucuucaa uugggcggug aagaccaagc ucaaacucac 9300uccauugccg gaggcgcgcc uacuggacuu auccaguugg uucaccgucg gcgccggcgg 9360gggcgacauu uuucacagcg ugucgcgcgc ccgaccccgc ucauuacucu ucggccuacu 9420ccuacuuuuc guagggguag gccucuuccu acuccccgcu cgguagagcg gcacacacua 9480gguacacucc auagcuaacu guuccuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 9540uuuuuuuucu uuuuuuuuuu uuucccucuu ucuucccuuc ucaucuuauu cuacuuucuu 9600ucuugguggc uccaucuuag cccuagucac ggcuagcugu gaaagguccg ugagccgcau 9660gacugcagag agugccguaa cuggucucuc ugcagaucau gucuaga 9707
<210>13<211>11111<212>RNA<213>人工序列<220>
<223>人工序列描述含來(lái)自JFH-1克隆的全長(zhǎng)丙型肝炎病毒基因組RNA的復(fù)制子RNA<400>13accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360ucaaagaaaa accaaaagaa acaccaaccg ucgcccaaug auugaacaag auggauugca 420cgcagguucu ccggccgcuu ggguggagag gcuauucggc uaugacuggg cacaacagac 480aaucggcugc ucugaugccg ccguguuccg gcugucagcg caggggcgcc cgguucuuuu 540ugucaagacc gaccuguccg gugcccugaa ugaacugcag gacgaggcag cgcggcuauc 600guggcuggcc acgacgggcg uuccuugcgc agcugugcuc gacguuguca cugaagcggg 660aagggacugg cugcuauugg gcgaagugcc ggggcaggau cuccugucau cucaccuugc 720uccugccgag aaaguaucca ucauggcuga ugcaaugcgg cggcugcaua cgcuugaucc 780ggcuaccugc ccauucgacc accaagcgaa acaucgcauc gagcgagcac guacucggau 840ggaagccggu cuugucgauc aggaugaucu ggacgaagag caucaggggc ucgcgccagc 900cgaacuguuc gccaggcuca aggcgcgcau gcccgacggc gaggaucucg ucgugaccca 960uggcgaugcc ugcuugccga auaucauggu ggaaaauggc cgcuuuucug gauucaucga 1020cuguggccgg cugggugugg cggaccgcua ucaggacaua gcguuggcua cccgugauau 1080ugcugaagag cuuggcggcg aaugggcuga ccgcuuccuc gugcuuuacg guaucgccgc 1140ucccgauucg cagcgcaucg ccuucuaucg ccuucuugac gaguucuucu gaguuuaaac 1200ccucucccuc cccccccccu aacguuacug gccgaagccg cuuggaauaa ggccggugug 1260cguuugucua uauguuauuu uccaccauau ugccgucuuu uggcaaugug agggcccgga 1320aaccuggccc ugucuucuug acgagcauuc cuaggggucu uuccccucuc gccaaaggaa 1380ugcaaggucu guugaauguc gugaaggaag caguuccucu ggaagcuucu ugaagacaaa 1440caacgucugu agcgacccuu ugcaggcagc ggaacccccc accuggcgac aggugccucu 1500gcggccaaaa gccacgugua uaagauacac cugcaaaggc ggcacaaccc cagugccacg 1560uugugaguug gauaguugug gaaagaguca aauggcucuc cucaagcgua uucaacaagg 1620ggcugaagga ugcccagaag guaccccauu guaugggauc ugaucugggg ccucggugca 1680caugcuuuac auguguuuag ucgagguuaa aaaaacgucu aggccccccg aaccacgggg 1740acgugguuuu ccuuugaaaa acacgaugau accaugagca caaauccuaa accucaaaga 1800aaaaccaaaa gaaacaccaa ccgucgccca gaagacguua aguucccggg cggcggccag 1860aucguuggcg gaguauacuu guugccgcgc aggggcccca gguugggugu gcgcacgaca 1920aggaaaacuu cggagcgguc ccagccacgu gggagacgcc agcccauccc caaagaucgg 1980cgcuccacug gcaaggccug gggaaaacca ggucgccccu ggccccuaua ugggaaugag 2040ggacucggcu gggcaggaug gcuccugucc ccccgaggcu cucgccccuc cuggggcccc 2100acugaccccc ggcauagguc gcgcaacgug gguaaaguca ucgacacccu aacguguggc 2160uuugccgacc ucauggggua cauccccguc guaggcgccc cgcuuagugg cgccgccaga 2220gcugucgcgc acggcgugag aguccuggag gacgggguua auuaugcaac agggaaccua 2280cccgguuucc ccuuuucuau cuucuugcug gcccuguugu ccugcaucac cguuccgguc 2340ucugcugccc aggugaagaa uaccaguagc agcuacaugg ugaccaauga cugcuccaau 2400
gacagcauca cuuggcagcu cgaggcugcg guucuccacg uccccgggug cgucccgugc 2460gagagagugg ggaauacguc acgguguugg gugccagucu cgccaaacau ggcugugcgg 2520cagcccggug cccucacgca gggucugcgg acgcacaucg auaugguugu gauguccgcc 2580accuucugcu cugcucucua cgugggggac cucuguggcg gggugaugcu cgcggcccag 2640guguucaucg ucucgccgca guaccacugg uuugugcaag aaugcaauug cuccaucuac 2700ccuggcacca ucacuggaca ccgcauggca ugggacauga ugaugaacug gucgcccacg 2760gccaccauga uccuggcgua cgugaugcgc guccccgagg ucaucauaga caucguuagc 2820ggggcucacu ggggcgucau guucggcuug gccuacuucu cuaugcaggg agcgugggcg 2880aaggucauug ucauccuucu gcuggccgcu gggguggacg cgggcaccac caccguugga 2940ggcgcuguug cacguuccac caacgugauu gccggcgugu ucagccaugg cccucagcag 3000aacauucagc ucauuaacac caacggcagu uggcacauca accguacugc cuugaauugc 3060aaugacuccu ugaacaccgg cuuucucgcg gccuuguucu acaccaaccg cuuuaacucg 3120ucaggguguc cagggcgccu guccgccugc cgcaacaucg aggcuuuccg gauagggugg 3180ggcacccuac aguacgagga uaaugucacc aauccagagg auaugaggcc guacugcugg 3240cacuaccccc caaagccgug uggcguaguc cccgcgaggu cugugugugg cccaguguac 3300uguuucaccc ccagcccggu aguagugggc acgaccgaca gacguggagu gcccaccuac 3360acauggggag agaaugagac agaugucuuc cuacugaaca gcacccgacc gccgcagggc 3420ucaugguucg gcugcacgug gaugaacucc acugguuuca ccaagacuug uggcgcgcca 3480ccuugccgca ccagagcuga cuucaacgcc agcacggacu uguugugccc uacggauugu 3540uuuaggaagc auccugaugc cacuuauauu aagugugguu cugggcccug gcucacacca 3600aagugccugg uccacuaccc uuacagacuc uggcauuacc ccugcacagu caauuuuacc 3660aucuucaaga uaagaaugua uguagggggg guugagcaca ggcucacggc cgcaugcaac 3720uucacucgug gggaucgcug cgacuuggag gacagggaca ggagucagcu gucuccucug 3780uugcacucua ccacggaaug ggccauccug cccugcaccu acucagacuu acccgcuuug 3840ucaacugguc uucuccaccu ucaccagaac aucguggacg uacaauacau guauggccuc 3900ucaccugcua ucacaaaaua cgucguucga ugggaguggg ugguacucuu auuccugcuc 3960uuagcggacg ccagagucug cgccugcuug uggaugcuca ucuuguuggg ccaggccgaa 4020gcagcauugg agaaguuggu cgucuugcac gcugcgagug cggcuaacug ccauggccuc 4080cuauauuuug ccaucuucuu cguggcagcu uggcacauca ggggucgggu gguccccuug 4140accaccuauu gccucacugg ccuauggccc uucugccuac ugcucauggc acugccccgg 4200caggcuuaug ccuaugacgc accugugcac ggacagauag gcguggguuu guugauauug 4260aucacccucu ucacacucac cccgggguau aagacccucc ucggccagug ucuguggugg 4320uugugcuauc uccugacccu gggggaagcc augauucagg aguggguacc acccaugcag 4380gugcgcggcg gccgcgaugg caucgcgugg gccgucacua uauucugccc ggguguggug 4440uuugacauuaccaaauggcu uuuggcguug cuugggccug cuuaccucuu aagggccgcu 4500uugacacaug ugccguacuu cgucagagcu cacgcucuga uaaggguaug cgcuuuggug 4560aagcagcucg cgggggguag guauguucag guggcgcuau uggcccuugg cagguggacu 4620ggcaccuaca ucuaugacca ccucacaccu augucggacu gggccgcuag cggccugcgc 4680gacuuagcgg ucgccgugga acccaucauc uucaguccga uggagaagaa ggucaucguc 4740uggggagcgg agacggcugc auguggggac auucuacaug gacuucccgu guccgcccga 4800cucggccagg agauccuccu cggcccagcu gauggcuaca ccuccaaggg guggaagcuc 4860cuugcuccca ucacugcuua ugcccagcaa acacgaggcc uccugggcgc cauaguggug 4920aguaugacgg ggcgugacag gacagaacag gccggggaag uccaaauccu guccacaguc 4980ucucaguccu uccucggaac aaccaucucg gggguuuugu ggacuguuua ccacggagcu 5040ggcaacaaga cucuagccgg cuuacggggu ccggucacgc agauguacuc gagugcugag 5100ggggacuugg uaggcuggcc cagccccccu gggaccaagu cuuuggagcc gugcaagugu 5160ggagccgucg accuauaucu ggucacgcgg aacgcugaug ucaucccggc ucggagacgc 5220ggggacaagc ggggagcauu gcucuccccg agacccauuu cgaccuugaa gggguccucg 5280ggggggccgg ugcucugccc uaggggccac gucguugggc ucuuccgagc agcugugugc 5340
ucucggggcg uggccaaauc caucgauuuc auccccguug agacacucga cguuguuaca 5400aggucuccca cuuucaguga caacagcacg ccaccggcug ugccccagac cuaucagguc 5460ggguacuugc augcuccaac uggcagugga aagagcacca aggucccugu cgcguaugcc 5520gcccaggggu acaaaguacu agugcuuaac cccucgguag cugccacccu gggguuuggg 5580gcguaccuau ccaaggcaca uggcaucaau cccaacauua ggacuggagu caggaccgug 5640augaccgggg aggccaucac guacuccaca uauggcaaau uucucgccga ugggggcugc 5700gcuagcggcg ccuaugacau caucauaugc gaugaaugcc acgcugugga ugcuaccucc 5760auucucggca ucggaacggu ccuugaucaa gcagagacag ccggggucag acuaacugug 5820cuggcuacgg ccacaccccc cgggucagug acaacccccc aucccgauau agaagaggua 5880ggccucgggc gggaggguga gauccccuuc uaugggaggg cgauuccccu auccugcauc 5940aagggaggga gacaccugau uuucugccac ucaaagaaaa agugugacga gcucgcggcg 6000gcccuucggg gcaugggcuu gaaugccgug gcauacuaua gaggguugga cgucuccaua 6060auaccagcuc agggagaugu gguggucguc gccaccgacg cccucaugac gggguacacu 6120ggagacuuug acuccgugau cgacugcaau guagcgguca cccaagcugu cgacuucagc 6180cuggacccca ccuucacuau aaccacacag acugucccac aagacgcugu cucacgcagu 6240cagcgccgcg ggcgcacagg uagaggaaga cagggcacuu auagguaugu uuccacuggu 6300gaacgagccu caggaauguu ugacagugua gugcuuugug agugcuacga cgcaggggcu 6360gcgugguacg aucucacacc agcggagacc accgucaggc uuagagcgua uuucaacacg 6420cccggccuac ccguguguca agaccaucuu gaauuuuggg aggcaguuuu caccggccuc 6480acacacauag acgcccacuu ccucucccaa acaaagcaag cgggggagaa cuucgcguac 6540cuaguagccu accaagcuac ggugugcgcc agagccaagg ccccuccccc guccugggac 6600gccaugugga agugccuggc ccgacucaag ccuacgcuug cgggccccac accucuccug 6660uaccguuugg gcccuauuac caaugagguc acccucacac acccugggac gaaguacauc 6720gccacaugca ugcaagcuga ccuugagguc augaccagca cguggguccu agcuggagga 6780guccuggcag ccgucgccgc auauugccug gcgacuggau gcguuuccau caucggccgc 6840uugcacguca accagcgagu cgucguugcg ccggauaagg agguccugua ugaggcuuuu 6900gaugagaugg aggaaugcgc cucuagggcg gcucucaucg aagaggggca gcggauagcc 6960gagauguuga aguccaagau ccaaggcuug cugcagcagg ccucuaagca ggcccaggac 7020auacaacccg cuaugcaggc uucauggccc aaaguggaac aauuuugggc cagacacaug 7080uggaacuuca uuagcggcau ccaauaccuc gcaggauugu caacacugcc agggaacccc 7140gcgguggcuu ccaugauggc auucagugcc gcccucacca guccguuguc gaccaguacc 7200accauccuuc ucaacaucau gggaggcugg uuagcguccc agaucgcacc acccgcgggg 7260gccaccggcu uugucgucag uggccuggug ggggcugccg ugggcagcau aggccugggu 7320aaggugcugg uggacauccu ggcaggauau ggugcgggca uuucgggggc ccucgucgca 7380uucaagauca ugucuggcga gaagcccucu auggaagaug ucaucaaucu acugccuggg 7440auccugucuc cgggagcccu gguggugggg gucaucugcg cggccauucu gcgccgccac 7500gugggaccgg gggagggcgc gguccaaugg augaacaggc uuauugccuu ugcuuccaga 7560ggaaaccacg ucgccccuac ucacuacgug acggagucgg augcgucgca gcgugugacc 7620caacuacuug gcucucuuac uauaaccagc cuacucagaa gacuccacaa uuggauaacu 7680gaggacugcc ccaucccaug cuccggaucc uggcuccgcg acguguggga cuggguuugc 7740accaucuuga cagacuucaa aaauuggcug accucuaaau uguuccccaa gcugcccggc 7800cuccccuuca ucucuuguca aaagggguac aagggugugu gggccggcac uggcaucaug 7860accacgcgcu gcccuugcgg cgccaacauc ucuggcaaug uccgccuggg cucuaugagg 7920aucacagggc cuaaaaccug caugaacacc uggcagggga ccuuuccuau caauugcuac 7980acggagggcc agugcgcgcc gaaacccccc acgaacuaca agaccgccau cuggagggug 8040gcggccucgg aguacgcgga ggugacgcag caugggucgu acuccuaugu aacaggacug 8100accacugaca aucugaaaau uccuugccaa cuaccuucuc cagaguuuuu cuccugggug 8160gacggugugc agauccauag guuugcaccc acaccaaagc cguuuuuccg ggaugagguc 8220ucguucugcg uugggcuuaa uuccuaugcu gucggguccc agcuucccug ugaaccugag 8280
cccgacgcag acguauugag guccaugcua acagauccgc cccacaucac ggcggagacu 8340gcggcgcggc gcuuggcacg gggaucaccu ccaucugagg cgagcuccuc agugagccag 8400cuaucagcac cgucgcugcg ggccaccugc accacccaca gcaacaccua ugacguggac 8460auggucgaug ccaaccugcu cauggagggc gguguggcuc agacagagcc ugaguccagg 8520gugcccguuc uggacuuucu cgagccaaug gccgaggaag agagcgaccu ugagcccuca 8580auaccaucgg agugcaugcu ccccaggagc ggguuuccac gggccuuacc ggcuugggca 8640cggccugacu acaacccgcc gcucguggaa ucguggagga ggccagauua ccaaccgccc 8700accguugcug guugugcucu cccccccccc aagaaggccc cgacgccucc cccaaggaga 8760cgccggacag ugggucugag cgagagcacc auaucagaag cccuccagca acuggccauc 8820aagaccuuug gccagccccc cucgagcggu gaugcaggcu cguccacggg ggcgggcgcc 8880gccgaauccg gcgguccgac guccccuggu gagccggccc ccucagagac agguuccgcc 8940uccucuaugc ccccccucga gggggagccu ggagauccgg accuggaguc ugaucaggua 9000gagcuucaac cuccccccca gggggggggg guagcucccg guucgggcuc ggggucuugg 9060ucuacuugcu ccgaggagga cgauaccacc gugugcugcu ccaugucaua cuccuggacc 9120ggggcucuaa uaacucccug uagccccgaa gaggaaaagu ugccaaucaa cccuuugagu 9180aacucgcugu ugcgauacca uaacaaggug uacuguacaa caucaaagag cgccucacag 9240agggcuaaaa agguaacuuu ugacaggacg caagugcucg acgcccauua ugacucaguc 9300uuaaaggaca ucaagcuagc ggcuuccaag gucagcgcaa ggcuccucac cuuggaggag 9360gcgugccagu ugacuccacc ccauucugca agauccaagu auggauucgg ggccaaggag 9420guccgcagcu uguccgggag ggccguuaac cacaucaagu ccguguggaa ggaccuccug 9480gaagacccac aaacaccaau ucccacaacc aucauggcca aaaaugaggu guucugcgug 9540gaccccgcca agggggguaa gaaaccagcu cgccucaucg uuuacccuga ccucggcguc 9600cgggucugcg agaaaauggc ccucuaugac auuacacaaa agcuuccuca ggcgguaaug 9660ggagcuuccu auggcuucca guacuccccu gcccaacggg uggaguaucu cuugaaagca 9720ugggcggaaa agaaggaccc cauggguuuu ucguaugaua cccgaugcuu cgacucaacc 9780gucacugaga gagacaucag gaccgaggag uccauauacc aggccugcuc ccugcccgag 9840gaggcccgca cugccauaca cucgcugacu gagagacuuu acguaggagg gcccauguuc 9900aacagcaagg gucaaaccug cgguuacaga cguugccgcg ccagcggggu gcuaaccacu 9960agcaugggua acaccaucac augcuaugug aaagcccuag cggccugcaa ggcugcgggg 10020auaguugcgc ccacaaugcu gguaugcggc gaugaccuag uagucaucuc agaaagccag 10080gggacugagg aggacgagcg gaaccugaga gccuucacgg aggccaugac cagguacucu 10140gccccuccug gugauccccc cagaccggaa uaugaccugg agcuaauaac auccuguucc 10200ucaaaugugu cuguggcguu gggcccgcgg ggccgccgca gauacuaccu gaccagagac 10260ccaaccacuc cacucgcccg ggcugccugg gaaacaguua gacacucccc uaucaauuca 10320uggcugggaa acaucaucca guaugcucca accauauggg uucgcauggu ccuaaugaca 10380cacuucuucu ccauucucau gguccaagac acccuggacc agaaccucaa cuuugagaug 10440uauggaucag uauacuccgu gaauccuuug gaccuuccag ccauaauuga gagguuacac 10500gggcuugacg ccuuuucuau gcacacauac ucucaccacg aacugacgcg gguggcuuca 10560gcccucagaa aacuuggggc gccaccccuc agggugugga agagucgggc ucgcgcaguc 10620agggcguccc ucaucucccg uggagggaaa gcggccguuu gcggccgaua ucucuucaau 10680ugggcgguga agaccaagcu caaacucacu ccauugccgg aggcgcgccu acuggacuua 10740uccaguuggu ucaccgucgg cgccggcggg ggcgacauuu uucacagcgu gucgcgcgcc 10800cgaccccgcu cauuacucuu cggccuacuc cuacuuuucg uagggguagg ccucuuccua 10860cuccccgcuc gguagagcgg cacacacuag guacacucca uagcuaacug uuccuuuuuu 10920uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuucuu uuuuuuuuuu uucccucuuu 10980cuucccuucu caucuuauuc uacuuucuuu cuugguggcu ccaucuuagc ccuagucacg 11040gcuagcugug aaagguccgu gagccgcaug acugcagaga gugccguaac uggucucucu 11100gcagaucaug u11111
<210>14<211>11111<212>RNA<213>人工序列<220>
<223>人工序列的描述來(lái)自JFH-1克隆的全長(zhǎng)丙型肝炎病毒基因組RNA，其中氨基酸基序GDD已突變?yōu)镚ND<400>14accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360ucaaagaaaa accaaaagaa acaccaaccg ucgcccaaug auugaacaag auggauugca 420cgcagguucu ccggccgcuu ggguggagag gcuauucggc uaugacuggg cacaacagac 480aaucggcugc ucugaugccg ccguguuccg gcugucagcg caggggcgcc cgguucuuuu 540ugucaagacc gaccuguccg gugcccugaa ugaacugcag gacgaggcag cgcggcuauc 600guggcuggcc acgacgggcg uuccuugcgc agcugugcuc gacguuguca cugaagcggg 660aagggacugg cugcuauugg gcgaagugcc ggggcaggau cuccugucau cucaccuugc 720uccugccgag aaaguaucca ucauggcuga ugcaaugcgg cggcugcaua cgcuugaucc 780ggcuaccugc ccauucgacc accaagcgaa acaucgcauc gagcgagcac guacucggau 840ggaagccggu cuugucgauc aggaugaucu ggacgaagag caucaggggc ucgcgccagc 900cgaacuguuc gccaggcuca aggcgcgcau gcccgacggc gaggaucucg ucgugaccca 960uggcgaugcc ugcuugccga auaucauggu ggaaaauggc cgcuuuucug gauucaucga 1020cuguggccgg cugggugugg cggaccgcua ucaggacaua gcguuggcua cccgugauau 1080ugcugaagag cuuggcggcg aaugggcuga ccgcuuccuc gugcuuuacg guaucgccgc 1140ucccgauucg cagcgcaucg ccuucuaucg ccuucuugac gaguucuucu gaguuuaaac 1200ccucucccuc cccccccccu aacguuacug gccgaagccg cuuggaauaa ggccggugug 1260cguuugucua uauguuauuu uccaccauau ugccgucuuu uggcaaugug agggcccgga 1320aaccuggccc ugucuucuug acgagcauuc cuaggggucu uuccccucuc gccaaaggaa 1380ugcaaggucu guugaauguc gugaaggaag caguuccucu ggaagcuucu ugaagacaaa 1440caacgucugu agcgacccuu ugcaggcagc ggaacccccc accuggcgac aggugccucu 1500gcggccaaaa gccacgugua uaagauacac cugcaaaggc ggcacaaccc cagugccacg 1560uugugaguug gauaguugug gaaagaguca aauggcucuc cucaagcgua uucaacaagg 1620ggcugaagga ugcccagaag guaccccauu guaugggauc ugaucugggg ccucggugca 1680caugcuuuac auguguuuag ucgagguuaa aaaaacgucu aggccccccg aaccacgggg 1740acgugguuuu ccuuugaaaa acacgaugau accaugagca caaauccuaa accucaaaga 1800aaaaccaaaa gaaacaccaa ccgucgccca gaagacguua aguucccggg cggcggccag 1860aucguuggcg gaguauacuu guugccgcgc aggggcccca gguugggugu gcgcacgaca 1920aggaaaacuu cggagcgguc ccagccacgu gggagacgcc agcccauccc caaagaucgg 1980cgcuccacug gcaaggccug gggaaaacca ggucgccccu ggccccuaua ugggaaugag 2040ggacucggcu gggcaggaug gcuccugucc ccccgaggcu cucgccccuc cuggggcccc 2100acugaccccc ggcauagguc gcgcaacgug gguaaaguca ucgacacccu aacguguggc 2160uuugccgacc ucauggggua cauccccguc guaggcgccc cgcuuagugg cgccgccaga 2220gcugucgcgc acggcgugag aguccuggag gacgggguua auuaugcaac agggaaccua 2280
cccgguuucc ccuuuucuau cuucuugcug gcccuguugu ccugcaucac cguuccgguc 2340ucugcugccc aggugaagaa uaccaguagc agcuacaugg ugaccaauga cugcuccaau 2400gacagcauca cuuggcagcu cgaggcugcg guucuccacg uccccgggug cgucccgugc 2460gagagagugg ggaauacguc acgguguugg gugccagucu cgccaaacau ggcugugcgg 2520cagcccggug cccucacgca gggucugcgg acgcacaucg auaugguugu gauguccgcc 2580accuucugcu cugcucucua cgugggggac cucuguggcg gggugaugcu cgcggcccag 2640guguucaucg ucucgccgca guaccacugg uuugugcaag aaugcaauug cuccaucuac 2700ccuggcacca ucacuggaca ccgcauggca ugggacauga ugaugaacug gucgcccacg 2760gccaccauga uccuggcgua cgugaugcgc guccccgagg ucaucauaga caucguuagc 2820ggggcucacu ggggcgucau guucggcuug gccuacuucu cuaugcaggg agcgugggcg 2880aaggucauug ucauccuucu gcuggccgcu gggguggacg cgggcaccac caccguugga 2940ggcgcuguug cacguuccac caacgugauu gccggcgugu ucagccaugg cccucagcag 3000aacauucagc ucauuaacac caacggcagu uggcacauca accguacugc cuugaauugc 3060aaugacuccu ugaacaccgg cuuucucgcg gccuuguucu acaccaaccg cuuuaacucg 3120ucaggguguc cagggcgccu guccgccugc cgcaacaucg aggcuuuccg gauagggugg 3180ggcacccuac aguacgagga uaaugucacc aauccagagg auaugaggcc guacugcugg 3240cacuaccccc caaagccgug uggcguaguc cccgcgaggu cugugugugg cccaguguac 3300uguuucaccc ccagcccggu aguagugggc acgaccgaca gacguggagu gcccaccuac 3360acauggggag agaaugagac agaugucuuc cuacugaaca gcacccgacc gccgcagggc 3420ucaugguucg gcugcacgug gaugaacucc acugguuuca ccaagacuug uggcgcgcca 3480ccuugccgca ccagagcuga cuucaacgcc agcacggacu uguugugccc uacggauugu 3540uuuaggaagc auccugaugc cacuuauauu aagugugguu cugggcccug gcucacacca 3600aagugccugg uccacuaccc uuacagacuc uggcauuacc ccugcacagu caauuuuacc 3660aucuucaaga uaagaaugua uguagggggg guugagcaca ggcucacggc cgcaugcaac 3720uucacucgug gggaucgcug cgacuuggag gacagggaca ggagucagcu gucuccucug 3780uugcacucua ccacggaaug ggccauccug cccugcaccu acucagacuu acccgcuuug 3840ucaacugguc uucuccaccu ucaccagaac aucguggacg uacaauacau guauggccuc 3900ucaccugcua ucacaaaaua cgucguucga ugggaguggg ugguacucuu auuccugcuc 3960uuagcggacg ccagagucug cgccugcuug uggaugcuca ucuuguuggg ccaggccgaa 4020gcagcauugg agaaguuggu cgucuugcac gcugcgagug cggcuaacug ccauggccuc 4080cuauauuuug ccaucuucuu cguggcagcu uggcacauca ggggucgggu gguccccuug 4140accaccuauu gccucacugg ccuauggccc uucugccuac ugcucauggc acugccccgg 4200caggcuuaug ccuaugacgc accugugcac ggacagauag gcguggguuu guugauauug 4260aucacccucu ucacacucac cccgggguau aagacccucc ucggccagug ucuguggugg 4320uugugcuauc uccugacccu gggggaagcc augauucagg aguggguacc acccaugcag 4380gugcgcggcg gccgcgaugg caucgcgugg gccgucacua uauucugccc ggguguggug 4440uuugacauua ccaaauggcu uuuggcguug cuugggccug cuuaccucuu aagggccgcu 4500uugacacaug ugccguacuu cgucagagcu cacgcucuga uaaggguaug cgcuuuggug 4560aagcagcucg cgggggguag guauguucag guggcgcuau uggcccuugg cagguggacu 4620ggcaccuaca ucuaugacca ccucacaccu augucggacu gggccgcuag cggccugcgc 4680gacuuagcgg ucgccgugga acccaucauc uucaguccga uggagaagaa ggucaucguc 4740uggggagcgg agacggcugc auguggggac auucuacaug gacuucccgu guccgcccga 4800cucggccagg agauccuccu cggcccagcu gauggcuaca ccuccaaggg guggaagcuc 4860cuugcuccca ucacugcuua ugcccagcaa acacgaggcc uccugggcgc cauaguggug 4920aguaugacgg ggcgugacag gacagaacag gccggggaag uccaaauccu guccacaguc 4980ucucaguccu uccucggaac aaccaucucg gggguuuugu ggacuguuua ccacggagcu 5040ggcaacaaga cucuagccgg cuuacggggu ccggucacgc agauguacuc gagugcugag 5100ggggacuugg uaggcuggcc cagccccccu gggaccaagu cuuuggagcc gugcaagugu 5160ggagccgucg accuauaucu ggucacgcgg aacgcugaug ucaucccggc ucggagacgc 5220
ggggacaagc ggggagcauu gcucuccccg agacccauuu cgaccuugaa gggguccucg 5280ggggggccgg ugcucugccc uaggggccac gucguugggc ucuuccgagc agcugugugc 5340ucucggggcg uggccaaauc caucgauuuc auccccguug agacacucga cguuguuaca 5400aggucuccca cuuucaguga caacagcacg ccaccggcug ugccccagac cuaucagguc 5460ggguacuugc augcuccaac uggcagugga aagagcacca aggucccugu cgcguaugcc 5520gcccaggggu acaaaguacu agugcuuaac cccucgguag cugccacccu gggguuuggg 5580gcguaccuau ccaaggcaca uggcaucaau cccaacauua ggacuggagu caggaccgug 5640augaccgggg aggccaucac guacuccaca uauggcaaau uucucgccga ugggggcugc 5700gcuagcggcg ccuaugacau caucauaugc gaugaaugcc acgcugugga ugcuaccucc 5760auucucggca ucggaacggu ccuugaucaa gcagagacag ccggggucag acuaacugug 5820cuggcuacgg ccacaccccc cgggucagug acaacccccc aucccgauau agaagaggua 5880ggccucgggc gggaggguga gauccccuuc uaugggaggg cgauuccccu auccugcauc 5940aagggaggga gacaccugau uuucugccac ucaaagaaaa agugugacga gcucgcggcg 6000gcccuucggg gcaugggcuu gaaugccgug gcauacuaua gaggguugga cgucuccaua 6060auaccagcuc agggagaugu gguggucguc gccaccgacg cccucaugac gggguacacu 6120ggagacuuug acuccgugau cgacugcaau guagcgguca cccaagcugu cgacuucagc 6180cuggacccca ccuucacuau aaccacacag acugucccac aagacgcugu cucacgcagu 6240cagcgccgcg ggcgcacagg uagaggaaga cagggcacuu auagguaugu uuccacuggu 6300gaacgagccu caggaauguu ugacagugua gugcuuugug agugcuacga cgcaggggcu 6360gcgugguacg aucucacacc agcggagacc accgucaggc uuagagcgua uuucaacacg 6420cccggccuac ccguguguca agaccaucuu gaauuuuggg aggcaguuuu caccggccuc 6480acacacauag acgcccacuu ccucucccaa acaaagcaag cgggggagaa cuucgcguac 6540cuaguagccu accaagcuac ggugugcgcc agagccaagg ccccuccccc guccugggac 6600gccaugugga agugccuggc ccgacucaag ccuacgcuug cgggccccac accucuccug 6660uaccguuugg gcccuauuac caaugagguc acccucacac acccugggac gaaguacauc 6720gccacaugca ugcaagcuga ccuugagguc augaccagca cguggguccu agcuggagga 6780guccuggcag ccgucgccgc auauugccug gcgacuggau gcguuuccau caucggccgc 6840uugcacguca accagcgagu cgucguugcg ccggauaagg agguccugua ugaggcuuuu 6900gaugagaugg aggaaugcgc cucuagggcg gcucucaucg aagaggggca gcggauagcc 6960gagauguuga aguccaagau ccaaggcuug cugcagcagg ccucuaagca ggcccaggac 7020auacaacccg cuaugcaggc uucauggccc aaaguggaac aauuuugggc cagacacaug 7080uggaacuuca uuagcggcau ccaauaccuc gcaggauugu caacacugcc agggaacccc 7140gcgguggcuu ccaugauggc auucagugcc gcccucacca guccguuguc gaccaguacc 7200accauccuuc ucaacaucau gggaggcugg uuagcguccc agaucgcacc acccgcgggg 7260gccaccggcu uugucgucag uggccuggug ggggcugccg ugggcagcau aggccugggu 7320aaggugcugg uggacauccu ggcaggauau ggugcgggca uuucgggggc ccucgucgca 7380uucaagauca ugucuggcga gaagcccucu auggaagaug ucaucaaucu acugccuggg 7440auccugucuc cgggagcccu gguggugggg gucaucugcg cggccauucu gcgccgccac 7500gugggaccgg gggagggcgc gguccaaugg augaacaggc uuauugccuu ugcuuccaga 7560ggaaaccacg ucgccccuac ucacuacgug acggagucgg augcgucgca gcgugugacc 7620caacuacuug gcucucuuac uauaaccagc cuacucagaa gacuccacaa uuggauaacu 7680gaggacugcc ccaucccaug cuccggaucc uggcuccgcg acguguggga cuggguuugc 7740accaucuuga cagacuucaa aaauuggcug accucuaaau uguuccccaa gcugcccggc 7800cuccccuuca ucucuuguca aaagggguac aagggugugu gggccggcac uggcaucaug 7860accacgcgcu gcccuugcgg cgccaacauc ucuggcaaug uccgccuggg cucuaugagg 7920aucacagggc cuaaaaccug caugaacacc uggcagggga ccuuuccuau caauugcuac 7980acggagggcc agugcgcgcc gaaacccccc acgaacuaca agaccgccau cuggagggug 8040gcggccucgg aguacgcgga ggugacgcag caugggucgu acuccuaugu aacaggacug 8100accacugaca aucugaaaau uccuugccaa cuaccuucuc cagaguuuuu cuccugggug 8160
gacggugugc agauccauag guuugcaccc acaccaaagc cguuuuuccg ggaugagguc 8220ucguucugcg uugggcuuaa uuccuaugcu gucggguccc agcuucccug ugaaccugag 8280cccgacgcag acguauugag guccaugcua acagauccgc cccacaucac ggcggagacu 8340gcggcgcggc gcuuggcacg gggaucaccu ccaucugagg cgagcuccuc agugagccag 8400cuaucagcac cgucgcugcg ggccaccugc accacccaca gcaacaccua ugacguggac 8460auggucgaug ccaaccugcu cauggagggc gguguggcuc agacagagcc ugaguccagg 8520gugcccguuc uggacuuucu cgagccaaug gccgaggaag agagcgaccu ugagcccuca 8580auaccaucgg agugcaugcu ccccaggagc ggguuuccac gggccuuacc ggcuugggca 8640cggccugacu acaacccgcc gcucguggaa ucguggagga ggccagauua ccaaccgccc 8700accguugcug guugugcucu cccccccccc aagaaggccc cgacgccucc cccaaggaga 8760cgccggacag ugggucugag cgagagcacc auaucagaag cccuccagca acuggccauc 8820aagaccuuug gccagccccc cucgagcggu gaugcaggcu cguccacggg ggcgggcgcc 8880gccgaauccg gcgguccgac guccccuggu gagccggccc ccucagagac agguuccgcc 8940uccucuaugc ccccccucga gggggagccu ggagauccgg accuggaguc ugaucaggua 9000gagcuucaac cuccccccca gggggggggg guagcucccg guucgggcuc ggggucuugg 9060ucuacuugcu ccgaggagga cgauaccacc gugugcugcu ccaugucaua cuccuggacc 9120ggggcucuaa uaacucccug uagccccgaa gaggaaaagu ugccaaucaa cccuuugagu 9180aacucgcugu ugcgauacca uaacaaggug uacuguacaa caucaaagag cgccucacag 9240agggcuaaaa agguaacuuu ugacaggacg caagugcucg acgcccauua ugacucaguc 9300uuaaaggaca ucaagcuagc ggcuuccaag gucagcgcaa ggcuccucac cuuggaggag 9360gcgugccagu ugacuccacc ccauucugca agauccaagu auggauucgg ggccaaggag 9420guccgcagcu uguccgggag ggccguuaac cacaucaagu ccguguggaa ggaccuccug 9480gaagacccac aaacaccaau ucccacaacc aucauggcca aaaaugaggu guucugcgug 9540gaccccgcca agggggguaa gaaaccagcu cgccucaucg uuuacccuga ccucggcguc 9600cgggucugcg agaaaauggc ccucuaugac auuacacaaa agcuuccuca ggcgguaaug 9660ggagcuuccu auggcuucca guacuccccu gcccaacggg uggaguaucu cuugaaagca 9720ugggcggaaa agaaggaccc cauggguuuu ucguaugaua cccgaugcuu cgacucaacc 9780gucacugaga gagacaucag gaccgaggag uccauauacc aggccugcuc ccugcccgag 9840gaggcccgca cugccauaca cucgcugacu gagagacuuu acguaggagg gcccauguuc 9900aacagcaagg gucaaaccug cgguuacaga cguugccgcg ccagcggggu gcuaaccacu 9960agcaugggua acaccaucac augcuaugug aaagcccuag cggccugcaa ggcugcgggg 10020auaguugcgc ccacaaugcu gguaugcggc aaugaccuag uagucaucuc agaaagccag 10080gggacugagg aggacgagcg gaaccugaga gccuucacgg aggccaugac cagguacucu 10140gccccuccug gugauccccc cagaccggaa uaugaccugg agcuaauaac auccuguucc 10200ucaaaugugu cuguggcguu gggcccgcgg ggccgccgca gauacuaccu gaccagagac 10260ccaaccacuc cacucgcccg ggcugccugg gaaacaguua gacacucccc uaucaauuca 10320uggcugggaa acaucaucca guaugcucca accauauggg uucgcauggu ccuaaugaca 10380cacuucuucu ccauucucau gguccaagac acccuggacc agaaccucaa cuuugagaug 10440uauggaucag uauacuccgu gaauccuuug gaccuuccag ccauaauuga gagguuacac 10500gggcuugacg ccuuuucuau gcacacauac ucucaccacg aacugacgcg gguggcuuca 10560gcccucagaa aacuuggggc gccaccccuc agggugugga agagucgggc ucgcgcaguc 10620agggcguccc ucaucucccg uggagggaaa gcggccguuu gcggccgaua ucucuucaau 10680ugggcgguga agaccaagcu caaacucacu ccauugccgg aggcgcgccu acuggacuua 10740uccaguuggu ucaccgucgg cgccggcggg ggcgacauuu uucacagcgu gucgcgcgcc 10800cgaccccgcu cauuacucuu cggccuacuc cuacuuuucg uagggguagg ccucuuccua 10860cuccccgcuc gguagagcgg cacacacuag guacacucca uagcuaacug uuccuuuuuu 10920uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuucuu uuuuuuuuuu uucccucuuu 10980cuucccuucu caucuuauuc uacuuucuuu cuugguggcu ccaucuuagc ccuagucacg 11040gcuagcugug aaagguccgu gagccgcaug acugcagaga gugccguaac uggucucucu 11100
gcagaucaug u11111<210>15<211>9707<212>RNA<213>人工序列<220>
<223>人工序列的描述含來(lái)自JFH-1克隆的全長(zhǎng)丙型肝炎病毒基因組RNA的復(fù)制子RNA，其中氨基酸基序GDD已突變?yōu)镚ND<400>15gaauucuaau acgacucacu auagaccugc cccuaauagg ggcgacacuc cgccaugaau 60cacuccccug ugaggaacua cugucuucac gcagaaagcg ccuagccaug gcguuaguau 120gagugucgua cagccuccag gccccccccu cccgggagag ccauaguggu cugcggaacc 180ggugaguaca ccggaauugc cgggaagacu ggguccuuuc uuggauaaac ccacucuaug 240cccggccauu ugggcgugcc cccgcaagac ugcuagccga guagcguugg guugcgaaag 300gccuuguggu acugccugau agggcgcuug cgagugcccc gggaggucuc guagaccgug 360caccaugagc acaaauccu aaaccucaaag aaaaaccaaa agaaacacca accgucgccc 420agaagacguu aaguucccgg gcggcggcca gaucguuggc ggaguauacu uguugccgcg 480caggggcccc agguugggug ugcgcacgac aaggaaaacu ucggagcggu cccagccacg 540ugggagacgc cagcccaucc ccaaagaucg gcgcuccacu ggcaaggccu ggggaaaacc 600aggucgcccc uggccccuau augggaauga gggacucggc ugggcaggau ggcuccuguc 660cccccgaggc ucucgccccu ccuggggccc cacugacccc cggcauaggu cgcgcaacgu 720ggguaaaguc aucgacaccc uaacgugugg cuuugccgac cucauggggu acauccccgu 780cguaggcgcc ccgcuuagug gcgccgccag agcugucgcg cacggcguga gaguccugga 840ggacgggguu aauuaugcaa cagggaaccu acccgguuuc cccuuuucua ucuucuugcu 900ggcccuguug uccugcauca ccguuccggu cucugcugcc caggugaaga auaccaguag 960cagcuacaug gugaccaaug acugcuccaa ugacagcauc acuuggcagc ucgaggcugc 1020gguucuccac guccccgggu gcgucccgug cgagagagug gggaauacgu cacgguguug 1080ggugccaguc ucgccaaaca uggcugugcg gcagcccggu gcccucacgc agggucugcg 1140gacgcacauc gauaugguug ugauguccgc caccuucugc ucugcucucu acguggggga 1200ccucuguggc ggggugaugc ucgcggccca gguguucauc gucucgccgc aguaccacug 1260guuugugcaa gaaugcaauu gcuccaucua cccuggcacc aucacuggac accgcauggc 1320augggacaug augaugaacu ggucgcccac ggccaccaug auccuggcgu acgugaugcg 1380cguccccgag gucaucauag acaucguuag cggggcucac uggggcguca uguucggcuu 1440ggccuacuuc ucuaugcagg gagcgugggc gaaggucauu gucauccuuc ugcuggccgc 1500ugggguggac gcgggcacca ccaccguugg aggcgcuguu gcacguucca ccaacgugau 1560ugccggcgug uucagccaug gcccucagca gaacauucag cucauuaaca ccaacggcag 1620uuggcacauc aaccguacug ccuugaauug caaugacucc uugaacaccg gcuuucucgc 1680ggccuuguuc uacaccaacc gcuuuaacuc gucagggugu ccagggcgcc uguccgccug 1740ccgcaacauc gaggcuuucc ggauagggug gggcacccua caguacgagg auaaugucac 1800caauccagag gauaugaggc cguacugcug gcacuacccc ccaaagccgu guggcguagu 1860ccccgcgagg ucugugugug gcccagugua cuguuucacc cccagcccgg uaguaguggg 1920cacgaccgac agacguggag ugcccaccua cacaugggga gagaaugaga cagaugucuu 1980ccuacugaac agcacccgac cgccgcaggg cucaugguuc ggcugcacgu ggaugaacuc 2040cacugguuuc accaagacuu guggcgcgcc accuugccgc accagagcug acuucaacgc 2100cagcacggac uuguugugcc cuacggauug uuuuaggaag cauccugaug ccacuuauau 2160
uaaguguggu ucugggcccu ggcucacacc aaagugccug guccacuacc cuuacagacu 2220cuggcauuac cccugcacag ucaauuuuac caucuucaag auaagaaugu auguaggggg 2280gguugagcac aggcucacgg ccgcaugcaa cuucacucgu ggggaucgcu gcgacuugga 2340ggacagggac aggagucagc ugucuccucu guugcacucu accacggaau gggccauccu 2400gcccugcacc uacucagacu uacccgcuuu gucaacuggu cuucuccacc uucaccagaa 2460caucguggac guacaauaca uguauggccu cucaccugcu aucacaaaau acgucguucg 2520augggagugg gugguacucu uauuccugcu cuuagcggac gccagagucu gcgccugcuu 2580guggaugcuc aucuuguugg gccaggccga agcagcauug gagaaguugg ucgucuugca 2640cgcugcgagu gcggcuaacu gccauggccu ccuauauuuu gccaucuucu ucguggcagc 2700uuggcacauc aggggucggg ugguccccuu gaccaccuau ugccucacug gccuauggcc 2760cuucugccua cugcucaugg cacugccccg gcaggcuuau gccuaugacg caccugugca 2820cggacagaua ggcguggguu uguugauauu gaucacccuc uucacacuca ccccggggua 2880uaagacccuc cucggccagu gucuguggug guugugcuau cuccugaccc ugggggaagc 2940caugauucag gaguggguac cacccaugca ggugcgcggc ggccgcgaug gcaucgcgug 3000ggccgucacu auauucugcc cggguguggu guuugacauu accaaauggc uuuuggcguu 3060gcuugggccu gcuuaccucu uaagggccgc uuugacacau gugccguacu ucgucagagc 3120ucacgcucug auaaggguau gcgcuuuggu gaagcagcuc gcggggggua gguauguuca 3180gguggcgcua uuggcccuug gcagguggac uggcaccuac aucuaugacc accucacacc 3240uaugucggac ugggccgcua gcggccugcg cgacuuagcg gucgccgugg aacccaucau 3300cuucaguccg auggagaaga aggucaucgu cuggggagcg gagacggcug caugugggga 3360cauucuacau ggacuucccg uguccgcccg acucggccag gagauccucc ucggcccagc 3420ugauggcuac accuccaagg gguggaagcu ccuugcuccc aucacugcuu augcccagca 3480aacacgaggc cuccugggcg ccauaguggu gaguaugacg gggcgugaca ggacagaaca 3540ggccggggaa guccaaaucc uguccacagu cucucagucc uuccucggaa caaccaucuc 3600ggggguuuug uggacuguuu accacggagc uggcaacaag acucuagccg gcuuacgggg 3660uccggucacg cagauguacu cgagugcuga gggggacuug guaggcuggc ccagcccccc 3720ugggaccaag ucuuuggagc cgugcaagug uggagccguc gaccuauauc uggucacgcg 3780gaacgcugau gucaucccgg cucggagacg cggggacaag cggggagcau ugcucucccc 3840gagacccauu ucgaccuuga agggguccuc gggggggccg gugcucugcc cuaggggcca 3900cgucguuggg cucuuccgag cagcugugug cucucggggc guggccaaau ccaucgauuu 3960cauccccguu gagacacucg acguuguuac aaggucuccc acuuucagug acaacagcac 4020gccaccggcu gugccccaga ccuaucaggu cggguacuug caugcuccaa cuggcagugg 4080aaagagcacc aaggucccug ucgcguaugc cgcccagggg uacaaaguac uagugcuuaa 4140ccccucggua gcugccaccc ugggguuugg ggcguaccua uccaaggcac auggcaucaa 4200ucccaacauu aggacuggag ucaggaccgu gaugaccggg gaggccauca cguacuccac 4260auauggcaaa uuucucgccg augggggcug cgcuagcggc gccuaugaca ucaucauaug 4320cgaugaaugc cacgcugugg augcuaccuc cauucucggc aucggaacgg uccuugauca 4380agcagagaca gccgggguca gacuaacugu gcuggcuacg gccacacccc ccgggucagu 4440gacaaccccc caucccgaua uagaagaggu aggccucggg cgggagggug agauccccuu 4500cuaugggagg gcgauucccc uauccugcau caagggaggg agacaccuga uuuucugcca 4560cucaaagaaa aagugugacg agcucgcggc ggcccuucgg ggcaugggcu ugaaugccgu 4620ggcauacuau agaggguugg acgucuccau aauaccagcu cagggagaug ugguggucgu 4680cgccaccgac gcccucauga cgggguacac uggagacuuu gacuccguga ucgacugcaa 4740uguagcgguc acccaagcug ucgacuucag ccuggacccc accuucacua uaaccacaca 4800gacuguccca caagacgcug ucucacgcag ucagcgccgc gggcgcacag guagaggaag 4860acagggcacu uauagguaug uuuccacugg ugaacgagcc ucaggaaugu uugacagugu 4920agugcuuugu gagugcuacg acgcaggggc ugcgugguac gaucucacac cagcggagac 4980caccgucagg cuuagagcgu auuucaacac gcccggccua cccguguguc aagaccaucu 5040ugaauuuugg gaggcaguuu ucaccggccu cacacacaua gacgcccacu uccucuccca 5100
aacaaagcaa gcgggggaga acuucgcgua ccuaguagcc uaccaagcua cggugugcgc 5160cagagccaag gccccucccc cguccuggga cgccaugugg aagugccugg cccgacucaa 5220gccuacgcuu gcgggcccca caccucuccu guaccguuug ggcccuauua ccaaugaggu 5280cacccucaca cacccuggga cgaaguacau cgccacaugc augcaagcug accuugaggu 5340caugaccagc acgugggucc uagcuggagg aguccuggca gccgucgccg cauauugccu 5400ggcgacugga ugcguuucca ucaucggccg cuugcacguc aaccagcgag ucgucguugc 5460gccggauaag gagguccugu augaggcuuu ugaugagaug gaggaaugcg ccucuagggc 5520ggcucucauc gaagaggggc agcggauagc cgagauguug aaguccaaga uccaaggcuu 5580gcugcagcag gccucuaagc aggcccagga cauacaaccc gcuaugcagg cuucauggcc 5640caaaguggaa caauuuuggg ccagacacau guggaacuuc auuagcggca uccaauaccu 5700cgcaggauug ucaacacugc cagggaaccc cgcgguggcu uccaugaugg cauucagugc 5760cgcccucacc aguccguugu cgaccaguac caccauccuu cucaacauca ugggaggcug 5820guuagcgucc cagaucgcac cacccgcggg ggccaccggc uuugucguca guggccuggu 5880gggggcugcc gugggcagca uaggccuggg uaaggugcug guggacaucc uggcaggaua 5940uggugcgggc auuucggggg cccucgucgc auucaagauc augucuggcg agaagcccuc 6000uauggaagau gucaucaauc uacugccugg gauccugucu ccgggagccc uggugguggg 6060ggucaucugc gcggccauuc ugcgccgcca cgugggaccg ggggagggcg cgguccaaug 6120gaugaacagg cuuauugccu uugcuuccag aggaaaccac gucgccccua cucacuacgu 6180gacggagucg gaugcgucgc agcgugugac ccaacuacuu ggcucucuua cuauaaccag 6240ccuacucaga agacuccaca auuggauaac ugaggacugc cccaucccau gcuccggauc 6300cuggcuccgc gacguguggg acuggguuug caccaucuug acagacuuca aaaauuggcu 6360gaccucuaaa uuguucccca agcugcccgg ccuccccuuc aucucuuguc aaaaggggua 6420caagggugug ugggccggca cuggcaucau gaccacgcgc ugcccuugcg gcgccaacau 6480cucuggcaau guccgccugg gcucuaugag gaucacaggg ccuaaaaccu gcaugaacac 6540cuggcagggg accuuuccua ucaauugcua cacggagggc cagugcgcgc cgaaaccccc 6600cacgaacuac aagaccgcca ucuggagggu ggcggccucg gaguacgcgg aggugacgca 6660gcaugggucg uacuccuaug uaacaggacu gaccacugac aaucugaaaa uuccuugcca 6720acuaccuucu ccagaguuuu ucuccugggu ggacggugug cagauccaua gguuugcacc 6780cacaccaaag ccguuuuucc gggaugaggu cucguucugc guugggcuua auuccuaugc 6840ugucgggucc cagcuucccu gugaaccuga gcccgacgca gacguauuga gguccaugcu 6900aacagauccg ccccacauca cggcggagac ugcggcgcgg cgcuuggcac ggggaucacc 6960uccaucugag gcgagcuccu cagugagcca gcuaucagca ccgucgcugc gggccaccug 7020caccacccac agcaacaccu augacgugga cauggucgau gccaaccugc ucauggaggg 7080cgguguggcu cagacagagc cugaguccag ggugcccguu cuggacuuuc ucgagccaau 7140ggccgaggaa gagagcgacc uugagcccuc aauaccaucg gagugcaugc uccccaggag 7200cggguuucca cgggccuuac cggcuugggc acggccugac uacaacccgc cgcucgugga 7260aucguggagg aggccagauu accaaccgcc caccguugcu gguugugcuc uccccccccc 7320caagaaggcc ccgacgccuc ccccaaggag acgccggaca gugggucuga gcgagagcac 7380cauaucagaa gcccuccagc aacuggccau caagaccuuu ggccagcccc ccucgagcgg 7440ugaugcaggc ucguccacgg gggcgggcgc cgccgaaucc ggcgguccga cguccccugg 7500ugagccggcc cccucagaga cagguuccgc cuccucuaug cccccccucg agggggagcc 7560uggagauccg gaccuggagu cugaucaggu agagcuucaa ccuccccccc aggggggggg 7620gguagcuccc gguucgggcu cggggucuug gucuacuugc uccgaggagg acgauaccac 7680cgugugcugc uccaugucau acuccuggac cggggcucua auaacucccu guagccccga 7740agaggaaaag uugccaauca acccuuugag uaacucgcug uugcgauacc auaacaaggu 7800guacuguaca acaucaaaga gcgccucaca gagggcuaaa aagguaacuu uugacaggac 7860gcaagugcuc gacgcccauu augacucagu cuuaaaggac aucaagcuag cggcuuccaa 7920ggucagcgca aggcuccuca ccuuggagga ggcgugccag uugacuccac cccauucugc 7980aagauccaag uauggauucg gggccaagga gguccgcagc uuguccggga gggccguuaa 8040
ccacaucaag uccgugugga aggaccuccu ggaagaccca caaacaccaa uucccacaac 8100caucauggcc aaaaaugagg uguucugcgu ggaccccgcc aaggggggua agaaaccagc 8160ucgccucauc guuuacccug accucggcgu ccgggucugc gagaaaaugg cccucuauga 8220cauuacacaa aagcuuccuc aggcgguaau gggagcuucc uauggcuucc aguacucccc 8280ugcccaacgg guggaguauc ucuugaaagc augggcggaa aagaaggacc ccauggguuu 8340uucguaugau acccgaugcu ucgacucaac cgucacugag agagacauca ggaccgagga 8400guccauauac caggccugcu cccugcccga ggaggcccgc acugccauac acucgcugac 8460ugagagacuu uacguaggag ggcccauguu caacagcaag ggucaaaccu gcgguuacag 8520acguugccgc gccagcgggg ugcuaaccac uagcaugggu aacaccauca caugcuaugu 8580gaaagcccua gcggccugca aggcugcggg gauaguugcg cccacaaugc ugguaugcgg 8640caaugaccua guagucaucu cagaaagcca ggggacugag gaggacgagc ggaaccugag 8700agccuucacg gaggccauga ccagguacuc ugccccuccu ggugaucccc ccagaccgga 8760auaugaccug gagcuaauaa cauccuguuc cucaaaugug ucuguggcgu ugggcccgcg 8820gggccgccgc agauacuacc ugaccagaga cccaaccacu ccacucgccc gggcugccug 8880ggaaacaguu agacacuccc cuaucaauuc auggcuggga aacaucaucc aguaugcucc 8940aaccauaugg guucgcaugg uccuaaugac acacuucuuc uccauucuca ugguccaaga 9000cacccuggac cagaaccuca acuuugagau guauggauca guauacuccg ugaauccuuu 9060ggaccuucca gccauaauug agagguuaca cgggcuugac gccuuuucua ugcacacaua 9120cucucaccac gaacugacgc ggguggcuuc agcccucaga aaacuugggg cgccaccccu 9180cagggugugg aagagucggg cucgcgcagu cagggcgucc cucaucuccc guggagggaa 9240agcggccguu ugcggccgau aucucuucaa uugggcggug aagaccaagc ucaaacucac 9300uccauugccg gaggcgcgcc uacuggacuu auccaguugg uucaccgucg gcgccggcgg 9360gggcgacauu uuucacagcg ugucgcgcgc ccgaccccgc ucauuacucu ucggccuacu 9420ccuacuuuuc guagggguag gccucuuccu acuccccgcu cgguagagcg gcacacacua 9480gguacacucc auagcuaacu guuccuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 9540uuuuuuuucu uuuuuuuuuu uuucccucuu ucuucccuuc ucaucuuauu cuacuuucuu 9600ucuugguggc uccaucuuag cccuagucac ggcuagcugu gaaagguccg ugagccgcau 9660gacugcagag agugccguaa cuggucucuc ugcagaucau gucuaga 9707<210>16<211>17<212>DNA<213>人工序列<220>
<223>人工序列的描述引物<400>16cgggagagcc atagtgg 17<210>17<211>19<212>DNA<213>人工序列<220>
<223>人工序列的描述引物
<400>17agtaccacaa ggcctttcg19<210>18<211>21<212>DNA<213>人工序列<220>
<223>人工序列的描述引物<400>18ctgcggaacc ggtgagtaca c 21<210>19<211>20<212>DNA<213>人工序列<220>
<223>人工序列的描述引物<400>19aacaagatgg attgcacgca 20<210>20<211>20<212>DNA<213>人工序列<220>
<223>人工序列的描述引物<400>20cgtcaagaag gcgatagaag 20<210>21<211>11969<212>RNA<213>人工序列<220>
<223>人工序列的描述來(lái)源于表達(dá)載體rFGR-JFH1/Luc的復(fù)制子RNA
<400>21accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360ucaaagaaaa accaaaagaa acaccaaccg acgcguaaug gaagacgcca aaaacauaaa 420gaaaggcccg gcgccauucu auccucugga ggauggaacc gcuggagagc aacugcauaa 480ggcuaugaag agauacgccc ugguuccugg aacaauugcu uuuacagaug cacauaucga 540ggugaacauc acguacgcgg aauacuucga aauguccguu cgguuggcag aagcuaugaa 600acgauauggg cugaauacaa aucacagaau cgucguaugc agugaaaacu cucuucaauu 660cuuuaugccg guguugggcg cguuauuuau cggaguugca guugcgcccg cgaacgacau 720uuauaaugaa cgugaauugc ucaacaguau gaacauuucg cagccuaccg uaguguuugu 780uuccaaaaag ggguugcaaa aaauuuugaa cgugcaaaaa aaauuaccaa uaauccagaa 840aauuauuauc auggauucua aaacggauua ccagggauuu cagucgaugu acacguucgu 900cacaucucau cuaccucccg guuuuaauga auacgauuuu guaccagagu ccuuugaucg 960ugacaaaaca auugcacuga uaaugaacuc cucuggaucu acuggguuac cuaagggugu 1020ggcccuuccg cauagaacug ccugcgucag auucucgcau gccagagauc cuauuuuugg 1080caaucaaauc auuccggaua cugcgauuuu aaguguuguu ccauuccauc acgguuuugg 1140aauguuuacu acacucggau auuugauaug uggauuucga gucgucuuaa uguauagauu 1200ugaagaagag cuguuuuuac gaucccuuca ggauuacaaa auucaaagug cguugcuagu 1260accaacccua uuuucauucu ucgccaaaag cacucugauu gacaaauacg auuuaucuaa 1320uuuacacgaa auugcuucug ggggcgcacc ucuuucgaaa gaagucgggg aagcgguugc 1380aaaacgcuuc caucuuccag ggauacgaca aggauauggg cucacugaga cuacaucagc 1440uauucugauu acacccgagg gggaugauaa accgggcgcg gucgguaaag uuguuccauu 1500uuuugaagcg aagguugugg aucuggauac cgggaaaacg cugggcguua aucagagagg 1560cgaauuaugu gucagaggac cuaugauuau guccgguuau guaaacaauc cggaagcgac 1620caacgccuug auugacaagg auggauggcu acauucugga gacauagcuu acugggacga 1680agacgaacac uucuucauag uugaccgcuu gaagucuuua auuaaauaca aaggauauca 1740gguggccccc gcugaauugg aaucgauauu guuacaacac cccaacaucu ucgacgcggg 1800cguggcaggu cuucccgacg augacgccgg ugaacuuccc gccgccguug uuguuuugga 1860gcacggaaag acgaugacgg aaaaagagau cguggauuac gucgccaguc aaguaacaac 1920cgcgaaaaag uugcgcggag gaguuguguu uguggacgaa guaccgaaag gucuuaccgg 1980aaaacucgac gcaagaaaaa ucagagagau ccucauaaag gccaagaagg gcggaaaguc 2040caaauuguaa guuuaaaccc ucucccuccc cccccccuaa cguuacuggc cgaagccgcu 2100uggaauaagg ccggugugcg uuugucuaua uguuauuuuc caccauauug ccgucuuuug 2160gcaaugugag ggcccggaaa ccuggcccug ucuucuugac gagcauuccu aggggucuuu 2220ccccucucgc caaaggaaug caaggucugu ugaaugucgu gaaggaagca guuccucugg 2280aagcuucuug aagacaaaca acgucuguag cgacccuuug caggcagcgg aaccccccac 2340cuggcgacag gugccucugc ggccaaaagc cacguguaua agauacaccu gcaaaggcgg 2400cacaacccca gugccacguu gugaguugga uaguugugga aagagucaaa uggcucuccu 2460caagcguauu caacaagggg cugaaggaug cccagaaggu accccauugu augggaucug 2520aucuggggcc ucggugcaca ugcuuuacau guguuuaguc gagguuaaaa aaacgucuag 2580gccccccgaa ccacggggac gugguuuucc uuugaaaaac acgaugauac caugagcaca 2640aauccuaaac cucaaagaaa aaccaaaaga aacaccaacc gucgcccaga agacguuaag 2700uucccgggcg gcggccagau cguuggcgga guauacuugu ugccgcgcag gggccccagg 2760uugggugugc gcacgacaag gaaaacuucg gagcgguccc agccacgugg gagacgccag 2820cccaucccca aagaucggcg cuccacuggc aaggccuggg gaaaaccagg ucgccccugg 2880
ccccuauaug ggaaugaggg acucggcugg gcaggauggc uccugucccc ccgaggcucu 2940cgccccuccu ggggccccac ugacccccgg cauaggucgc gcaacguggg uaaagucauc 3000gacacccuaa cguguggcuu ugccgaccuc augggguaca uccccgucgu aggcgccccg 3060cuuaguggcg ccgccagagc ugucgcgcac ggcgugagag uccuggagga cgggguuaau 3120uaugcaacag ggaaccuacc cgguuucccc uuuucuaucu ucuugcuggc ccuguugucc 3180ugcaucaccg uuccggucuc ugcugcccag gugaagaaua ccaguagcag cuacauggug 3240accaaugacu gcuccaauga cagcaucacu uggcagcucg aggcugcggu ucuccacguc 3300cccgggugcg ucccgugcga gagagugggg aauacgucac gguguugggu gccagucucg 3360ccaaacaugg cugugcggca gcccggugcc cucacgcagg gucugcggac gcacaucgau 3420augguuguga uguccgccac cuucugcucu gcucucuacg ugggggaccu cuguggcggg 3480gugaugcucg cggcccaggu guucaucguc ucgccgcagu accacugguu ugugcaagaa 3540ugcaauugcu ccaucuaccc uggcaccauc acuggacacc gcauggcaug ggacaugaug 3600augaacuggu cgcccacggc caccaugauc cuggcguacg ugaugcgcgu ccccgagguc 3660aucauagaca ucguuagcgg ggcucacugg ggcgucaugu ucggcuuggc cuacuucucu 3720augcagggag cgugggcgaa ggucauuguc auccuucugc uggccgcugg gguggacgcg 3780ggcaccacca ccguuggagg cgcuguugca cguuccacca acgugauugc cggcguguuc 3840agccauggcc cucagcagaa cauucagcuc auuaacacca acggcaguug gcacaucaac 3900cguacugccu ugaauugcaa ugacuccuug aacaccggcu uucucgcggc cuuguucuac 3960accaaccgcu uuaacucguc agggugucca gggcgccugu ccgccugccg caacaucgag 4020gcuuuccgga uagggugggg cacccuacag uacgaggaua augucaccaa uccagaggau 4080augaggccgu acugcuggca cuacccccca aagccgugug gcguaguccc cgcgaggucu 4140guguguggcc caguguacug uuucaccccc agcccgguag uagugggcac gaccgacaga 4200cguggagugc ccaccuacac auggggagag aaugagacag augucuuccu acugaacagc 4260acccgaccgc cgcagggcuc augguucggc ugcacgugga ugaacuccac ugguuucacc 4320aagacuugug gcgcgccacc uugccgcacc agagcugacu ucaacgccag cacggacuug 4380uugugcccua cggauuguuu uaggaagcau ccugaugcca cuuauauuaa gugugguucu 4440gggcccuggc ucacaccaaa gugccugguc cacuacccuu acagacucug gcauuacccc 4500ugcacaguca auuuuaccau cuucaagaua agaauguaug uagggggggu ugagcacagg 4560cucacggccg caugcaacuu cacucguggg gaucgcugcg acuuggagga cagggacagg 4620agucagcugu cuccucuguu gcacucuacc acggaauggg ccauccugcc cugcaccuac 4680ucagacuuac ccgcuuuguc aacuggucuu cuccaccuuc accagaacau cguggacgua 4740caauacaugu auggccucuc accugcuauc acaaaauacg ucguucgaug ggagugggug 4800guacucuuau uccugcucuu agcggacgcc agagucugcg ccugcuugug gaugcucauc 4860uuguugggcc aggccgaagc agcauuggag aaguuggucg ucuugcacgc ugcgagugcg 4920gcuaacugcc auggccuccu auauuuugcc aucuucuucg uggcagcuug gcacaucagg 4980ggucgggugg uccccuugac caccuauugc cucacuggcc uauggcccuu cugccuacug 5040cucauggcac ugccccggca ggcuuaugcc uaugacgcac cugugcacgg acagauaggc 5100guggguuugu ugauauugau cacccucuuc acacucaccc cgggguauaa gacccuccuc 5160ggccaguguc uguggugguu gugcuaucuc cugacccugg gggaagccau gauucaggag 5220uggguaccac ccaugcaggu gcgcggcggc cgcgauggca ucgcgugggc cgucacuaua 5280uucugcccgg gugugguguu ugacauuacc aaauggcuuu uggcguugcu ugggccugcu 5340uaccucuuaa gggccgcuuu gacacaugug ccguacuucg ucagagcuca cgcucugaua 5400aggguaugcg cuuuggugaa gcagcucgcg ggggguaggu auguucaggu ggcgcuauug 5460gcccuuggca gguggacugg caccuacauc uaugaccacc ucacaccuau gucggacugg 5520gccgcuagcg gccugcgcga cuuagcgguc gccguggaac ccaucaucuu caguccgaug 5580gagaagaagg ucaucgucug gggagcggag acggcugcau guggggacau ucuacaugga 5640cuucccgugu ccgcccgacu cggccaggag auccuccucg gcccagcuga uggcuacacc 5700uccaaggggu ggaagcuccu ugcucccauc acugcuuaug cccagcaaac acgaggccuc 5760cugggcgcca uaguggugag uaugacgggg cgugacagga cagaacaggc cggggaaguc 5820
caaauccugu ccacagucuc ucaguccuuc cucggaacaa ccaucucggg gguuuugugg 5880acuguuuacc acggagcugg caacaagacu cuagccggcu uacggggucc ggucacgcag 5940auguacucga gugcugaggg ggacuuggua ggcuggccca gccccccugg gaccaagucu 6000uuggagccgu gcaagugugg agccgucgac cuauaucugg ucacgcggaa cgcugauguc 6060aucccggcuc ggagacgcgg ggacaagcgg ggagcauugc ucuccccgag acccauuucg 6120accuugaagg gguccucggg ggggccggug cucugcccua ggggccacgu cguugggcuc 6180uuccgagcag cugugugcuc ucggggcgug gccaaaucca ucgauuucau ccccguugag 6240acacucgacg uuguuacaag gucucccacu uucagugaca acagcacgcc accggcugug 6300ccccagaccu aucaggucgg guacuugcau gcuccaacug gcaguggaaa gagcaccaag 6360gucccugucg cguaugccgc ccagggguac aaaguacuag ugcuuaaccc cucgguagcu 6420gccacccugg gguuuggggc guaccuaucc aaggcacaug gcaucaaucc caacauuagg 6480acuggaguca ggaccgugau gaccggggag gccaucacgu acuccacaua uggcaaauuu 6540cucgccgaug ggggcugcgc uagcggcgcc uaugacauca ucauaugcga ugaaugccac 6600gcuguggaug cuaccuccau ucucggcauc ggaacggucc uugaucaagc agagacagcc 6660ggggucagac uaacugugcu ggcuacggcc acaccccccg ggucagugac aaccccccau 6720cccgauauag aagagguagg ccucgggcgg gagggugaga uccccuucua ugggagggcg 6780auuccccuau ccugcaucaa gggagggaga caccugauuu ucugccacuc aaagaaaaag 6840ugugacgagc ucgcggcggc ccuucggggc augggcuuga augccguggc auacuauaga 6900ggguuggacg ucuccauaau accagcucag ggagaugugg uggucgucgc caccgacgcc 6960cucaugacgg gguacacugg agacuuugac uccgugaucg acugcaaugu agcggucacc 7020caagcugucg acuucagccu ggaccccacc uucacuauaa ccacacagac ugucccacaa 7080gacgcugucu cacgcaguca gcgccgcggg cgcacaggua gaggaagaca gggcacuuau 7140agguauguuu ccacugguga acgagccuca ggaauguuug acaguguagu gcuuugugag 7200ugcuacgacg caggggcugc gugguacgau cucacaccag cggagaccac cgucaggcuu 7260agagcguauu ucaacacgcc cggccuaccc gugugucaag accaucuuga auuuugggag 7320gcaguuuuca ccggccucac acacauagac gcccacuucc ucucccaaac aaagcaagcg 7380ggggagaacu ucgcguaccu aguagccuac caagcuacgg ugugcgccag agccaaggcc 7440ccucccccgu ccugggacgc cauguggaag ugccuggccc gacucaagcc uacgcuugcg 7500ggccccacac cucuccugua ccguuugggc ccuauuacca augaggucac ccucacacac 7560ccugggacga aguacaucgc cacaugcaug caagcugacc uugaggucau gaccagcacg 7620uggguccuag cuggaggagu ccuggcagcc gucgccgcau auugccuggc gacuggaugc 7680guuuccauca ucggccgcuu gcacgucaac cagcgagucg ucguugcgcc ggauaaggag 7740guccuguaug aggcuuuuga ugagauggag gaaugcgccu cuagggcggc ucucaucgaa 7800gaggggcagc ggauagccga gauguugaag uccaagaucc aaggcuugcu gcagcaggcc 7860ucuaagcagg cccaggacau acaacccgcu augcaggcuu cauggcccaa aguggaacaa 7920uuuugggcca gacacaugug gaacuucauu agcggcaucc aauaccucgc aggauuguca 7980acacugccag ggaaccccgc gguggcuucc augauggcau ucagugccgc ccucaccagu 8040ccguugucga ccaguaccac cauccuucuc aacaucaugg gaggcugguu agcgucccag 8100aucgcaccac ccgcgggggc caccggcuuu gucgucagug gccugguggg ggcugccgug 8160ggcagcauag gccuggguaa ggugcuggug gacauccugg caggauaugg ugcgggcauu 8220ucgggggccc ucgucgcauu caagaucaug ucuggcgaga agcccucuau ggaagauguc 8280aucaaucuac ugccugggau ccugucuccg ggagcccugg uggugggggu caucugcgcg 8340gccauucugc gccgccacgu gggaccgggg gagggcgcgg uccaauggau gaacaggcuu 8400auugccuuug cuuccagagg aaaccacguc gccccuacuc acuacgugac ggagucggau 8460gcgucgcagc gugugaccca acuacuuggc ucucuuacua uaaccagccu acucagaaga 8520cuccacaauu ggauaacuga ggacugcccc aucccaugcu ccggauccug gcuccgcgac 8580gugugggacu ggguuugcac caucuugaca gacuucaaaa auuggcugac cucuaaauug 8640uuccccaagc ugcccggccu ccccuucauc ucuugucaaa agggguacaa gggugugugg 8700gccggcacug gcaucaugac cacgcgcugc ccuugcggcg ccaacaucuc uggcaauguc 8760
cgccugggcu cuaugaggau cacagggccu aaaaccugca ugaacaccug gcaggggacc 8820uuuccuauca auugcuacac ggagggccag ugcgcgccga aaccccccac gaacuacaag 8880accgccaucu ggaggguggc ggccucggag uacgcggagg ugacgcagca ugggucguac 8940uccuauguaa caggacugac cacugacaau cugaaaauuc cuugccaacu accuucucca 9000gaguuuuucu ccugggugga cggugugcag auccauaggu uugcacccac accaaagccg 9060uuuuuccggg augaggucuc guucugcguu gggcuuaauu ccuaugcugu cgggucccag 9120cuucccugug aaccugagcc cgacgcagac guauugaggu ccaugcuaac agauccgccc 9180cacaucacgg cggagacugc ggcgcggcgc uuggcacggg gaucaccucc aucugaggcg 9240agcuccucag ugagccagcu aucagcaccg ucgcugcggg ccaccugcac cacccacagc 9300aacaccuaug acguggacau ggucgaugcc aaccugcuca uggagggcgg uguggcucag 9360acagagccug aguccagggu gcccguucug gacuuucucg agccaauggc cgaggaagag 9420agcgaccuug agcccucaau accaucggag ugcaugcucc ccaggagcgg guuuccacgg 9480gccuuaccgg cuugggcacg gccugacuac aacccgccgc ucguggaauc guggaggagg 9540ccagauuacc aaccgcccac cguugcuggu ugugcucucc ccccccccaa gaaggccccg 9600acgccucccc caaggagacg ccggacagug ggucugagcg agagcaccau aucagaagcc 9660cuccagcaac uggccaucaa gaccuuuggc cagccccccu cgagcgguga ugcaggcucg 9720uccacggggg cgggcgccgc cgaauccggc gguccgacgu ccccugguga gccggccccc 9780ucagagacag guuccgccuc cucuaugccc ccccucgagg gggagccugg agauccggac 9840cuggagucug aucagguaga gcuucaaccu cccccccagg gggggggggu agcucccggu 9900ucgggcucgg ggucuugguc uacuugcucc gaggaggacg auaccaccgu gugcugcucc 9960augucauacu ccuggaccgg ggcucuaaua acucccugua gccccgaaga ggaaaaguug 10020ccaaucaacc cuuugaguaa cucgcuguug cgauaccaua acaaggugua cuguacaaca 10080ucaaagagcg ccucacagag ggcuaaaaag guaacuuuug acaggacgca agugcucgac 10140gcccauuaug acucagucuu aaaggacauc aagcuagcgg cuuccaaggu cagcgcaagg 10200cuccucaccu uggaggaggc gugccaguug acuccacccc auucugcaag auccaaguau 10260ggauucgggg ccaaggaggu ccgcagcuug uccgggaggg ccguuaacca caucaagucc 10320guguggaagg accuccugga agacccacaa acaccaauuc ccacaaccau cauggccaaa 10380aaugaggugu ucugcgugga ccccgccaag ggggguaaga aaccagcucg ccucaucguu 10440uacccugacc ucggcguccg ggucugcgag aaaauggccc ucuaugacau uacacaaaag 10500cuuccucagg cgguaauggg agcuuccuau ggcuuccagu acuccccugc ccaacgggug 10560gaguaucucu ugaaagcaug ggcggaaaag aaggacccca uggguuuuuc guaugauacc 10620cgaugcuucg acucaaccgu cacugagaga gacaucagga ccgaggaguc cauauaccag 10680gccugcuccc ugcccgagga ggcccgcacu gccauacacu cgcugacuga gagacuuuac 10740guaggagggc ccauguucaa cagcaagggu caaaccugcg guuacagacg uugccgcgcc 10800agcggggugc uaaccacuag cauggguaac accaucacau gcuaugugaa agcccuagcg 10860gccugcaagg cugcggggau aguugcgccc acaaugcugg uaugcggcga ugaccuagua 10920gucaucucag aaagccaggg gacugaggag gacgagcgga accugagagc cuucacggag 10980gccaugacca gguacucugc cccuccuggu gaucccccca gaccggaaua ugaccuggag 11040cuaauaacau ccuguuccuc aaaugugucu guggcguugg gcccgcgggg ccgccgcaga 11100uacuaccuga ccagagaccc aaccacucca cucgcccggg cugccuggga aacaguuaga 11160cacuccccua ucaauucaug gcugggaaac aucauccagu augcuccaac cauauggguu 11220cgcauggucc uaaugacaca cuucuucucc auucucaugg uccaagacac ccuggaccag 11280aaccucaacu uugagaugua uggaucagua uacuccguga auccuuugga ccuuccagcc 11340auaauugaga gguuacacgg gcuugacgcc uuuucuaugc acacauacuc ucaccacgaa 11400cugacgcggg uggcuucagc ccucagaaaa cuuggggcgc caccccucag gguguggaag 11460agucgggcuc gcgcagucag ggcgucccuc aucucccgug gagggaaagc ggccguuugc 11520ggccgauauc ucuucaauug ggcggugaag accaagcuca aacucacucc auugccggag 11580gcgcgccuac uggacuuauc caguugguuc accgucggcg ccggcggggg cgacauuuuu 11640cacagcgugu cgcgcgcccg accccgcuca uuacucuucg gccuacuccu acuuuucgua 11700
gggguaggcc ucuuccuacu ccccgcucgg uagagcggca cacacuaggu acacuccaua 11760gcuaacuguu ccuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuucuuuu 11820uuuuuuuuuu cccucuuucu ucccuucuca ucuuauucua cuuucuuucu ugguggcucc 11880aucuuagccc uagucacggc uagcugugaa agguccguga gccgcaugac ugcagagagu 11940gccguaacug gucucucugc agaucaugu 11969<210>22<211>11969<212>RNA<213>人工序列<220>
<223>人工序列的描述來(lái)源于表達(dá)載體rFGR-JFH1/Luc/GND的復(fù)制子RNA<400>22accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360ucaaagaaaa accaaaagaa acaccaaccg acgcguaaug gaagacgcca aaaacauaaa 420gaaaggcccg gcgccauucu auccucugga ggauggaacc gcuggagagc aacugcauaa 480ggcuaugaag agauacgccc ugguuccugg aacaauugcu uuuacagaug cacauaucga 540ggugaacauc acguacgcgg aauacuucga aauguccguu cgguuggcag aagcuaugaa 600acgauauggg cugaauacaa aucacagaau cgucguaugc agugaaaacu cucuucaauu 660cuuuaugccg guguugggcg cguuauuuau cggaguugca guugcgcccg cgaacgacau 720uuauaaugaa cgugaauugc ucaacaguau gaacauuucg cagccuaccg uaguguuugu 780uuccaaaaag ggguugcaaa aaauuuugaa cgugcaaaaa aaauuaccaa uaauccagaa 840aauuauuauc auggauucua aaacggauua ccagggauuu cagucgaugu acacguucgu 900cacaucucau cuaccucccg guuuuaauga auacgauuuu guaccagagu ccuuugaucg 960ugacaaaaca auugcacuga uaaugaacuc cucuggaucu acuggguuac cuaagggugu 1020ggcccuuccg cauagaacug ccugcgucag auucucgcau gccagagauc cuauuuuugg 1080caaucaaauc auuccggaua cugcgauuuu aaguguuguu ccauuccauc acgguuuugg 1140aauguuuacu acacucggau auuugauaug uggauuucga gucgucuuaa uguauagauu 1200ugaagaagag cuguuuuuac gaucccuuca ggauuacaaa auucaaagug cguugcuagu 1260accaacccua uuuucauucu ucgccaaaag cacucugauu gacaaauacg auuuaucuaa 1320uuuacacgaa auugcuucug ggggcgcacc ucuuucgaaa gaagucgggg aagcgguugc 1380aaaacgcuuc caucuuccag ggauacgaca aggauauggg cucacugaga cuacaucagc 1440uauucugauu acacccgagg gggaugauaa accgggcgcg gucgguaaag uuguuccauu 1500uuuugaagcg aagguugugg aucuggauac cgggaaaacg cugggcguua aucagagagg 1560cgaauuaugu gucagaggac cuaugauuau guccgguuau guaaacaauc cggaagcgac 1620caacgccuug auugacaagg auggauggcu acauucugga gacauagcuu acugggacga 1680agacgaacac uucuucauag uugaccgcuu gaagucuuua auuaaauaca aaggauauca 1740gguggccccc gcugaauugg aaucgauauu guuacaacac cccaacaucu ucgacgcggg 1800cguggcaggu cuucccgacg augacgccgg ugaacuuccc gccgccguug uuguuuugga 1860gcacggaaag acgaugacgg aaaaagagau cguggauuac gucgccaguc aaguaacaac 1920cgcgaaaaag uugcgcggag gaguuguguu uguggacgaa guaccgaaag gucuuaccgg 1980
aaaacucgac gcaagaaaaa ucagagagau ccucauaaag gccaagaagg gcggaaaguc 2040caaauuguaa guuuaaaccc ucucccuccc cccccccuaa cguuacuggc cgaagccgcu 2100uggaauaagg ccggugugcg uuugucuaua uguuauuuuc caccauauug ccgucuuuug 2160gcaaugugag ggcccggaaa ccuggcccug ucuucuugac gagcauuccu aggggucuuu 2220ccccucucgc caaaggaaug caaggucugu ugaaugucgu gaaggaagca guuccucugg 2280aagcuucuug aagacaaaca acgucuguag cgacccuuug caggcagcgg aaccccccac 2340cuggcgacag gugccucugc ggccaaaagc cacguguaua agauacaccu gcaaaggcgg 2400cacaacccca gugccacguu gugaguugga uaguugugga aagagucaaa uggcucuccu 2460caagcguauu caacaagggg cugaaggaug cccagaaggu accccauugu augggaucug 2520aucuggggcc ucggugcaca ugcuuuacau guguuuaguc gagguuaaaa aaacgucuag 2580gccccccgaa ccacggggac gugguuuucc uuugaaaaac acgaugauac caugagcaca 2640aauccuaaac cucaaagaaa aaccaaaaga aacaccaacc gucgcccaga agacguuaag 2700uucccgggcg gcggccagau cguuggcgga guauacuugu ugccgcgcag gggccccagg 2760uugggugugc gcacgacaag gaaaacuucg gagcgguccc agccacgugg gagacgccag 2820cccaucccca aagaucggcg cuccacuggc aaggccuggg gaaaaccagg ucgccccugg 2880ccccuauaug ggaaugaggg acucggcugg gcaggauggc uccugucccc ccgaggcucu 2940cgccccuccu ggggccccac ugacccccgg cauaggucgc gcaacguggg uaaagucauc 3000gacacccuaa cguguggcuu ugccgaccuc augggguaca uccccgucgu aggcgccccg 3060cuuaguggcg ccgccagagc ugucgcgcac ggcgugagag uccuggagga cgggguuaau 3120uaugcaacag ggaaccuacc cgguuucccc uuuucuaucu ucuugcuggc ccuguugucc 3180ugcaucaccg uuccggucuc ugcugcccag gugaagaaua ccaguagcag cuacauggug 3240accaaugacu gcuccaauga cagcaucacu uggcagcucg aggcugcggu ucuccacguc 3300cccgggugcg ucccgugcga gagagugggg aauacgucac gguguugggu gccagucucg 3360ccaaacaugg cugugcggca gcccggugcc cucacgcagg gucugcggac gcacaucgau 3420augguuguga uguccgccac cuucugcucu gcucucuacg ugggggaccu cuguggcggg 3480gugaugcucg cggcccaggu guucaucguc ucgccgcagu accacugguu ugugcaagaa 3540ugcaauugcu ccaucuaccc uggcaccauc acuggacacc gcauggcaug ggacaugaug 3600augaacuggu cgcccacggc caccaugauc cuggcguacg ugaugcgcgu ccccgagguc 3660aucauagaca ucguuagcgg ggcucacugg ggcgucaugu ucggcuuggc cuacuucucu 3720augcagggag cgugggcgaa ggucauuguc auccuucugc uggccgcugg gguggacgcg 3780ggcaccacca ccguuggagg cgcuguugca cguuccacca acgugauugc cggcguguuc 3840agccauggcc cucagcagaa cauucagcuc auuaacacca acggcaguug gcacaucaac 3900cguacugccu ugaauugcaa ugacuccuug aacaccggcu uucucgcggc cuuguucuac 3960accaaccgcu uuaacucguc agggugucca gggcgccugu ccgccugccg caacaucgag 4020gcuuuccgga uagggugggg cacccuacag uacgaggaua augucaccaa uccagaggau 4080augaggccgu acugcuggca cuacccccca aagccgugug gcguaguccc cgcgaggucu 4140guguguggcc caguguacug uuucaccccc agcccgguag uagugggcac gaccgacaga 4200cguggagugc ccaccuacac auggggagag aaugagacag augucuuccu acugaacagc 4260acccgaccgc cgcagggcuc augguucggc ugcacgugga ugaacuccac ugguuucacc 4320aagacuugug gcgcgccacc uugccgcacc agagcugacu ucaacgccag cacggacuug 4380uugugcccua cggauuguuu uaggaagcau ccugaugcca cuuauauuaa gugugguucu 4440gggcccuggc ucacaccaaa gugccugguc cacuacccuu acagacucug gcauuacccc 4500ugcacaguca auuuuaccau cuucaagaua agaauguaug uagggggggu ugagcacagg 4560cucacggccg caugcaacuu cacucguggg gaucgcugcg acuuggagga cagggacagg 4620agucagcugu cuccucuguu gcacucuacc acggaauggg ccauccugcc cugcaccuac 4680ucagacuuac ccgcuuuguc aacuggucuu cuccaccuuc accagaacau cguggacgua 4740caauacaugu auggccucuc accugcuauc acaaaauacg ucguucgaug ggagugggug 4800guacucuuau uccugcucuu agcggacgcc agagucugcg ccugcuugug gaugcucauc 4860uuguugggcc aggccgaagc agcauuggag aaguuggucg ucuugcacgc ugcgagugcg 4920
gcuaacugcc auggccuccu auauuuugcc aucuucuucg uggcagcuug gcacaucagg 4980ggucgggugg uccccuugac caccuauugc cucacuggcc uauggcccuu cugccuacug 5040cucauggcac ugccccggca ggcuuaugcc uaugacgcac cugugcacgg acagauaggc 5100guggguuugu ugauauugau cacccucuuc acacucaccc cgggguauaa gacccuccuc 5160ggccaguguc uguggugguu gugcuaucuc cugacccugg gggaagccau gauucaggag 5220uggguaccac ccaugcaggu gcgcggcggc cgcgauggca ucgcgugggc cgucacuaua 5280uucugcccgg gugugguguu ugacauuacc aaauggcuuu uggcguugcu ugggccugcu 5340uaccucuuaa gggccgcuuu gacacaugug ccguacuucg ucagagcuca cgcucugaua 5400aggguaugcg cuuuggugaa gcagcucgcg ggggguaggu auguucaggu ggcgcuauug 5460gcccuuggca gguggacugg caccuacauc uaugaccacc ucacaccuau gucggacugg 5520gccgcuagcg gccugcgcga cuuagcgguc gccguggaac ccaucaucuu caguccgaug 5580gagaagaagg ucaucgucug gggagcggag acggcugcau guggggacau ucuacaugga 5640cuucccgugu ccgcccgacu cggccaggag auccuccucg gcccagcuga uggcuacacc 5700uccaaggggu ggaagcuccu ugcucccauc acugcuuaug cccagcaaac acgaggccuc 5760cugggcgcca uaguggugag uaugacgggg cgugacagga cagaacaggc cggggaaguc 5820caaauccugu ccacagucuc ucaguccuuc cucggaacaa ccaucucggg gguuuugugg 5880acuguuuacc acggagcugg caacaagacu cuagccggcu uacggggucc ggucacgcag 5940auguacucga gugcugaggg ggacuuggua ggcuggccca gccccccugg gaccaagucu 6000uuggagccgu gcaagugugg agccgucgac cuauaucugg ucacgcggaa cgcugauguc 6060aucccggcuc ggagacgcgg ggacaagcgg ggagcauugc ucuccccgag acccauuucg 6120accuugaagg gguccucggg ggggccggug cucugcccua ggggccacgu cguugggcuc 6180uuccgagcag cugugugcuc ucggggcgug gccaaaucca ucgauuucau ccccguugag 6240acacucgacg uuguuacaag gucucccacu uucagugaca acagcacgcc accggcugug 6300ccccagaccu aucaggucgg guacuugcau gcuccaacug gcaguggaaa gagcaccaag 6360gucccugucg cguaugccgc ccagggguac aaaguacuag ugcuuaaccc cucgguagcu 6420gccacccugg gguuuggggc guaccuaucc aaggcacaug gcaucaaucc caacauuagg 6480acuggaguca ggaccgugau gaccggggag gccaucacgu acuccacaua uggcaaauuu 6540cucgccgaug ggggcugcgc uagcggcgcc uaugacauca ucauaugcga ugaaugccac 6600gcuguggaug cuaccuccau ucucggcauc ggaacggucc uugaucaagc agagacagcc 6660ggggucagac uaacugugcu ggcuacggcc acaccccccg ggucagugac aaccccccau 6720cccgauauag aagagguagg ccucgggcgg gagggugaga uccccuucua ugggagggcg 6780auuccccuau ccugcaucaa gggagggaga caccugauuu ucugccacuc aaagaaaaag 6840ugugacgagc ucgcggcggc ccuucggggc augggcuuga augccguggc auacuauaga 6900ggguuggacg ucuccauaau accagcucag ggagaugugg uggucgucgc caccgacgcc 6960cucaugacgg gguacacugg agacuuugac uccgugaucg acugcaaugu agcggucacc 7020caagcugucg acuucagccu ggaccccacc uucacuauaa ccacacagac ugucccacaa 7080gacgcugucu cacgcaguca gcgccgcggg cgcacaggua gaggaagaca gggcacuuau 7140agguauguuu ccacugguga acgagccuca ggaauguuug acaguguagu gcuuugugag 7200ugcuacgacg caggggcugc gugguacgau cucacaccag cggagaccac cgucaggcuu 7260agagcguauu ucaacacgcc cggccuaccc gugugucaag accaucuuga auuuugggag 7320gcaguuuuca ccggccucac acacauagac gcccacuucc ucucccaaac aaagcaagcg 7380ggggagaacu ucgcguaccu aguagccuac caagcuacgg ugugcgccag agccaaggcc 7440ccucccccgu ccugggacgc cauguggaag ugccuggccc gacucaagcc uacgcuugcg 7500ggccccacac cucuccugua ccguuugggc ccuauuacca augaggucac ccucacacac 7560ccugggacga aguacaucgc cacaugcaug caagcugacc uugaggucau gaccagcacg 7620uggguccuag cuggaggagu ccuggcagcc gucgccgcau auugccuggc gacuggaugc 7680guuuccauca ucggccgcuu gcacgucaac cagcgagucg ucguugcgcc ggauaaggag 7740guccuguaug aggcuuuuga ugagauggag gaaugcgccu cuagggcggc ucucaucgaa 7800gaggggcagc ggauagccga gauguugaag uccaagaucc aaggcuugcu gcagcaggcc 7860
ucuaagcagg cccaggacau acaacccgcu augcaggcuu cauggcccaa aguggaacaa 7920uuuugggcca gacacaugug gaacuucauu agcggcaucc aauaccucgc aggauuguca 7980acacugccag ggaaccccgc gguggcuucc augauggcau ucagugccgc ccucaccagu 8040ccguugucga ccaguaccac cauccuucuc aacaucaugg gaggcugguu agcgucccag 8100aucgcaccac ccgcgggggc caccggcuuu gucgucagug gccugguggg ggcugccgug 8160ggcagcauag gccuggguaa ggugcuggug gacauccugg caggauaugg ugcgggcauu 8220ucgggggccc ucgucgcauu caagaucaug ucuggcgaga agcccucuau ggaagauguc 8280aucaaucuac ugccugggau ccugucuccg ggagcccugg uggugggggu caucugcgcg 8340gccauucugc gccgccacgu gggaccgggg gagggcgcgg uccaauggau gaacaggcuu 8400auugccuuug cuuccagagg aaaccacguc gccccuacuc acuacgugac ggagucggau 8460gcgucgcagc gugugaccca acuacuuggc ucucuuacua uaaccagccu acucagaaga 8520cuccacaauu ggauaacuga ggacugcccc aucccaugcu ccggauccug gcuccgcgac 8580gugugggacu ggguuugcac caucuugaca gacuucaaaa auuggcugac cucuaaauug 8640uuccccaagc ugcccggccu ccccuucauc ucuugucaaa agggguacaa gggugugugg 8700gccggcacug gcaucaugac cacgcgcugc ccuugcggcg ccaacaucuc uggcaauguc 8760cgccugggcu cuaugaggau cacagggccu aaaaccugca ugaacaccug gcaggggacc 8820uuuccuauca auugcuacac ggagggccag ugcgcgccga aaccccccac gaacuacaag 8880accgccaucu ggaggguggc ggccucggag uacgcggagg ugacgcagca ugggucguac 8940uccuauguaa caggacugac cacugacaau cugaaaauuc cuugccaacu accuucucca 9000gaguuuuucu ccugggugga cggugugcag auccauaggu uugcacccac accaaagccg 9060uuuuuccggg augaggucuc guucugcguu gggcuuaauu ccuaugcugu cgggucccag 9120cuucccugug aaccugagcc cgacgcagac guauugaggu ccaugcuaac agauccgccc 9180cacaucacgg cggagacugc ggcgcggcgc uuggcacggg gaucaccucc aucugaggcg 9240agcuccucag ugagccagcu aucagcaccg ucgcugcggg ccaccugcac cacccacagc 9300aacaccuaug acguggacau ggucgaugcc aaccugcuca uggagggcgg uguggcucag 9360acagagccug aguccagggu gcccguucug gacuuucucg agccaauggc cgaggaagag 9420agcgaccuug agcccucaau accaucggag ugcaugcucc ccaggagcgg guuuccacgg 9480gccuuaccgg cuugggcacg gccugacuac aacccgccgc ucguggaauc guggaggagg 9540ccagauuacc aaccgcccac cguugcuggu ugugcucucc ccccccccaa gaaggccccg 9600acgccucccc caaggagacg ccggacagug ggucugagcg agagcaccau aucagaagcc 9660cuccagcaac uggccaucaa gaccuuuggc cagccccccu cgagcgguga ugcaggcucg 9720uccacggggg cgggcgccgc cgaauccggc gguccgacgu ccccugguga gccggccccc 9780ucagagacag guuccgccuc cucuaugccc ccccucgagg gggagccugg agauccggac 9840cuggagucug aucagguaga gcuucaaccu cccccccagg gggggggggu agcucccggu 9900ucgggcucgg ggucuugguc uacuugcucc gaggaggacg auaccaccgu gugcugcucc 9960augucauacu ccuggaccgg ggcucuaaua acucccugua gccccgaaga ggaaaaguug 10020ccaaucaacc cuuugaguaa cucgcuguug cgauaccaua acaaggugua cuguacaaca 10080ucaaagagcg ccucacagag ggcuaaaaag guaacuuuug acaggacgca agugcucgac 10140gcccauuaug acucagucuu aaaggacauc aagcuagcgg cuuccaaggu cagcgcaagg 10200cuccucaccu uggaggaggc gugccaguug acuccacccc auucugcaag auccaaguau 10260ggauucgggg ccaaggaggu ccgcagcuug uccgggaggg ccguuaacca caucaagucc 10320guguggaagg accuccugga agacccacaa acaccaauuc ccacaaccau cauggccaaa 10380aaugaggugu ucugcgugga ccccgccaag ggggguaaga aaccagcucg ccucaucguu 10440uacccugacc ucggcguccg ggucugcgag aaaauggccc ucuaugacau uacacaaaag 10500cuuccucagg cgguaauggg agcuuccuau ggcuuccagu acuccccugc ccaacgggug 10560gaguaucucu ugaaagcaug ggcggaaaag aaggacccca uggguuuuuc guaugauacc 10620cgaugcuucg acucaaccgu cacugagaga gacaucagga ccgaggaguc cauauaccag 10680gccugcuccc ugcccgagga ggcccgcacu gccauacacu cgcugacuga gagacuuuac 10740guaggagggc ccauguucaa cagcaagggu caaaccugcg guuacagacg uugccgcgcc 10800
agcggggugc uaaccacuag cauggguaac accaucacau gcuaugugaa agcccuagcg 10860gccugcaagg cugcggggau aguugcgccc acaaugcugg uaugcggcaa ugaccuagua 10920gucaucucag aaagccaggg gacugaggag gacgagcgga accugagagc cuucacggag 10980gccaugacca gguacucugc cccuccuggu gaucccccca gaccggaaua ugaccuggag 11040cuaauaacau ccuguuccuc aaaugugucu guggcguugg gcccgcgggg ccgccgcaga 11100uacuaccuga ccagagaccc aaccacucca cucgcccggg cugccuggga aacaguuaga 11160cacuccccua ucaauucaug gcugggaaac aucauccagu augcuccaac cauauggguu 11220cgcauggucc uaaugacaca cuucuucucc auucucaugg uccaagacac ccuggaccag 11280aaccucaacu uugagaugua uggaucagua uacuccguga auccuuugga ccuuccagcc 11340auaauugaga gguuacacgg gcuugacgcc uuuucuaugc acacauacuc ucaccacgaa 11400cugacgcggg uggcuucagc ccucagaaaa cuuggggcgc caccccucag gguguggaag 11460agucgggcuc gcgcagucag ggcgucccuc aucucccgug gagggaaagc ggccguuugc 11520ggccgauauc ucuucaauug ggcggugaag accaagcuca aacucacucc auugccggag 11580gcgcgccuac uggacuuauc caguugguuc accgucggcg ccggcggggg cgacauuuuu 11640cacagcgugu cgcgcgcccg accccgcuca uuacucuucg gccuacuccu acuuuucgua 11700gggguaggcc ucuuccuacu ccccgcucgg uagagcggca cacacuaggu acacuccaua 11760gcuaacuguu ccuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuucuuuu 11820uuuuuuuuuu cccucuuucu ucccuucuca ucuuauucua cuuucuuucu ugguggcucc 11880aucuuagccc uagucacggc uagcugugaa agguccguga gccgcaugac ugcagagagu 11940gccguaacug gucucucugc agaucaugu 11969<210>23<211>11036<212>RNA<213>人工序列<220>
<223>人工序列的描述來(lái)源于表達(dá)載體rFGR-JFH1/EGFP的復(fù)制子RNA<400>23accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360ucaaagaaaa accaaaagaa acaccaaccg acgcguaaug gugagcaagg gcgaggagcu 420guucaccggg guggugccca uccuggucga gcuggacggc gacguaaacg gccacaaguu 480cagcgugucc ggcgagggcg agggcgaugc caccuacggc aagcugaccc ugaaguucau 540cugcaccacc ggcaagcugc ccgugcccug gcccacccuc gugaccaccc ugaccuacgg 600cgugcagugc uucagccgcu accccgacca caugaagcag cacgacuucu ucaaguccgc 660caugcccgaa ggcuacgucc aggagcgcac caucuucuuc aaggacgacg gcaacuacaa 720gacccgcgcc gaggugaagu ucgagggcga cacccuggug aaccgcaucg agcugaaggg 780caucgacuuc aaggaggacg gcaacauccu ggggcacaag cuggaguaca acuacaacag 840ccacaacguc uauaucaugg ccgacaagca gaagaacggc aucaagguga acuucaagau 900ccgccacaac aucgaggacg gcagcgugca gcucgccgac cacuaccagc agaacacccc 960caucggcgac ggccccgugc ugcugcccga caaccacuac cugagcaccc aguccgcccu 1020gagcaaagac cccaacgaga agcgcgauca caugguccug cuggaguucg ugaccgccgc 1080
cgggaucacu cucggcaugg acgagcugua caaguaaguu uaaacccucu cccucccccc 1140ccccuaacgu uacuggccga agccgcuugg aauaaggccg gugugcguuu gucuauaugu 1200uauuuuccac cauauugccg ucuuuuggca augugagggc ccggaaaccu ggcccugucu 1260ucuugacgag cauuccuagg ggucuuuccc cucucgccaa aggaaugcaa ggucuguuga 1320augucgugaa ggaagcaguu ccucuggaag cuucuugaag acaaacaacg ucuguagcga 1380cccuuugcag gcagcggaac cccccaccug gcgacaggug ccucugcggc caaaagccac 1440guguauaaga uacaccugca aaggcggcac aaccccagug ccacguugug aguuggauag 1500uuguggaaag agucaaaugg cucuccucaa gcguauucaa caaggggcug aaggaugccc 1560agaagguacc ccauuguaug ggaucugauc uggggccucg gugcacaugc uuuacaugug 1620uuuagucgag guuaaaaaaa cgucuaggcc ccccgaacca cggggacgug guuuuccuuu 1680gaaaaacacg augauaccau gagcacaaau ccuaaaccuc aaagaaaaac caaaagaaac 1740accaaccguc gcccagaaga cguuaaguuc ccgggcggcg gccagaucgu uggcggagua 1800uacuuguugc cgcgcagggg ccccagguug ggugugcgca cgacaaggaa aacuucggag 1860cggucccagc cacgugggag acgccagccc auccccaaag aucggcgcuc cacuggcaag 1920gccuggggaa aaccaggucg ccccuggccc cuauauggga augagggacu cggcugggca 1980ggauggcucc uguccccccg aggcucucgc cccuccuggg gccccacuga cccccggcau 2040aggucgcgca acguggguaa agucaucgac acccuaacgu guggcuuugc cgaccucaug 2100ggguacaucc ccgucguagg cgccccgcuu aguggcgccg ccagagcugu cgcgcacggc 2160gugagagucc uggaggacgg gguuaauuau gcaacaggga accuacccgg uuuccccuuu 2220ucuaucuucu ugcuggcccu guuguccugc aucaccguuc cggucucugc ugcccaggug 2280aagaauacca guagcagcua cauggugacc aaugacugcu ccaaugacag caucacuugg 2340cagcucgagg cugcgguucu ccacgucccc gggugcgucc cgugcgagag aguggggaau 2400acgucacggu guugggugcc agucucgcca aacauggcug ugcggcagcc cggugcccuc 2460acgcaggguc ugcggacgca caucgauaug guugugaugu ccgccaccuu cugcucugcu 2520cucuacgugg gggaccucug uggcggggug augcucgcgg cccagguguu caucgucucg 2580ccgcaguacc acugguuugu gcaagaaugc aauugcucca ucuacccugg caccaucacu 2640ggacaccgca uggcauggga caugaugaug aacuggucgc ccacggccac caugauccug 2700gcguacguga ugcgcguccc cgaggucauc auagacaucg uuagcggggc ucacuggggc 2760gucauguucg gcuuggccua cuucucuaug cagggagcgu gggcgaaggu cauugucauc 2820cuucugcugg ccgcuggggu ggacgcgggc accaccaccg uuggaggcgc uguugcacgu 2880uccaccaacg ugauugccgg cguguucagc cauggcccuc agcagaacau ucagcucauu 2940aacaccaacg gcaguuggca caucaaccgu acugccuuga auugcaauga cuccuugaac 3000accggcuuuc ucgcggccuu guucuacacc aaccgcuuua acucgucagg guguccaggg 3060cgccuguccg ccugccgcaa caucgaggcu uuccggauag gguggggcac ccuacaguac 3120gaggauaaug ucaccaaucc agaggauaug aggccguacu gcuggcacua ccccccaaag 3180ccguguggcg uaguccccgc gaggucugug uguggcccag uguacuguuu cacccccagc 3240ccgguaguag ugggcacgac cgacagacgu ggagugccca ccuacacaug gggagagaau 3300gagacagaug ucuuccuacu gaacagcacc cgaccgccgc agggcucaug guucggcugc 3360acguggauga acuccacugg uuucaccaag acuuguggcg cgccaccuug ccgcaccaga 3420gcugacuuca acgccagcac ggacuuguug ugcccuacgg auuguuuuag gaagcauccu 3480gaugccacuu auauuaagug ugguucuggg cccuggcuca caccaaagug ccugguccac 3540uacccuuaca gacucuggca uuaccccugc acagucaauu uuaccaucuu caagauaaga 3600auguauguag gggggguuga gcacaggcuc acggccgcau gcaacuucac ucguggggau 3660cgcugcgacu uggaggacag ggacaggagu cagcugucuc cucuguugca cucuaccacg 3720gaaugggcca uccugcccug caccuacuca gacuuacccg cuuugucaac uggucuucuc 3780caccuucacc agaacaucgu ggacguacaa uacauguaug gccucucacc ugcuaucaca 3840aaauacgucg uucgauggga guggguggua cucuuauucc ugcucuuagc ggacgccaga 3900gucugcgccu gcuuguggau gcucaucuug uugggccagg ccgaagcagc auuggagaag 3960uuggucgucu ugcacgcugc gagugcggcu aacugccaug gccuccuaua uuuugccauc 4020
uucuucgugg cagcuuggca caucaggggu cggguggucc ccuugaccac cuauugccuc 4080acuggccuau ggcccuucug ccuacugcuc auggcacugc cccggcaggc uuaugccuau 4140gacgcaccug ugcacggaca gauaggcgug gguuuguuga uauugaucac ccucuucaca 4200cucaccccgg gguauaagac ccuccucggc cagugucugu ggugguugug cuaucuccug 4260acccuggggg aagccaugau ucaggagugg guaccaccca ugcaggugcg cggcggccgc 4320gauggcaucg cgugggccgu cacuauauuc ugcccgggug ugguguuuga cauuaccaaa 4380uggcuuuugg cguugcuugg gccugcuuac cucuuaaggg ccgcuuugac acaugugccg 4440uacuucguca gagcucacgc ucugauaagg guaugcgcuu uggugaagca gcucgcgggg 4500gguagguaug uucagguggc gcuauuggcc cuuggcaggu ggacuggcac cuacaucuau 4560gaccaccuca caccuauguc ggacugggcc gcuagcggcc ugcgcgacuu agcggucgcc 4620guggaaccca ucaucuucag uccgauggag aagaagguca ucgucugggg agcggagacg 4680gcugcaugug gggacauucu acauggacuu cccguguccg cccgacucgg ccaggagauc 4740cuccucggcc cagcugaugg cuacaccucc aaggggugga agcuccuugc ucccaucacu 4800gcuuaugccc agcaaacacg aggccuccug ggcgccauag uggugaguau gacggggcgu 4860gacaggacag aacaggccgg ggaaguccaa auccugucca cagucucuca guccuuccuc 4920ggaacaacca ucucgggggu uuuguggacu guuuaccacg gagcuggcaa caagacucua 4980gccggcuuac gggguccggu cacgcagaug uacucgagug cugaggggga cuugguaggc 5040uggcccagcc ccccugggac caagucuuug gagccgugca aguguggagc cgucgaccua 5100uaucugguca cgcggaacgc ugaugucauc ccggcucgga gacgcgggga caagcgggga 5160gcauugcucu ccccgagacc cauuucgacc uugaaggggu ccucgggggg gccggugcuc 5220ugcccuaggg gccacgucgu ugggcucuuc cgagcagcug ugugcucucg gggcguggcc 5280aaauccaucg auuucauccc cguugagaca cucgacguug uuacaagguc ucccacuuuc 5340agugacaaca gcacgccacc ggcugugccc cagaccuauc aggucgggua cuugcaugcu 5400ccaacuggca guggaaagag caccaagguc ccugucgcgu augccgccca gggguacaaa 5460guacuagugc uuaaccccuc gguagcugcc acccuggggu uuggggcgua ccuauccaag 5520gcacauggca ucaaucccaa cauuaggacu ggagucagga ccgugaugac cggggaggcc 5580aucacguacu ccacauaugg caaauuucuc gccgaugggg gcugcgcuag cggcgccuau 5640gacaucauca uaugcgauga augccacgcu guggaugcua ccuccauucu cggcaucgga 5700acgguccuug aucaagcaga gacagccggg gucagacuaa cugugcuggc uacggccaca 5760ccccccgggu cagugacaac cccccauccc gauauagaag agguaggccu cgggcgggag 5820ggugagaucc ccuucuaugg gagggcgauu ccccuauccu gcaucaaggg agggagacac 5880cugauuuucu gccacucaaa gaaaaagugu gacgagcucg cggcggcccu ucggggcaug 5940ggcuugaaug ccguggcaua cuauagaggg uuggacgucu ccauaauacc agcucaggga 6000gauguggugg ucgucgccac cgacgcccuc augacggggu acacuggaga cuuugacucc 6060gugaucgacu gcaauguagc ggucacccaa gcugucgacu ucagccugga ccccaccuuc 6120acuauaacca cacagacugu cccacaagac gcugucucac gcagucagcg ccgcgggcgc 6180acagguagag gaagacaggg cacuuauagg uauguuucca cuggugaacg agccucagga 6240auguuugaca guguagugcu uugugagugc uacgacgcag gggcugcgug guacgaucuc 6300acaccagcgg agaccaccgu caggcuuaga gcguauuuca acacgcccgg ccuacccgug 6360ugucaagacc aucuugaauu uugggaggca guuuucaccg gccucacaca cauagacgcc 6420cacuuccucu cccaaacaaa gcaagcgggg gagaacuucg cguaccuagu agccuaccaa 6480gcuacggugu gcgccagagc caaggccccu cccccguccu gggacgccau guggaagugc 6540cuggcccgac ucaagccuac gcuugcgggc cccacaccuc uccuguaccg uuugggcccu 6600auuaccaaug aggucacccu cacacacccu gggacgaagu acaucgccac augcaugcaa 6660gcugaccuug aggucaugac cagcacgugg guccuagcug gaggaguccu ggcagccguc 6720gccgcauauu gccuggcgac uggaugcguu uccaucaucg gccgcuugca cgucaaccag 6780cgagucgucg uugcgccgga uaaggagguc cuguaugagg cuuuugauga gauggaggaa 6840ugcgccucua gggcggcucu caucgaagag gggcagcgga uagccgagau guugaagucc 6900aagauccaag gcuugcugca gcaggccucu aagcaggccc aggacauaca acccgcuaug 6960
caggcuucau ggcccaaagu ggaacaauuu ugggccagac acauguggaa cuucauuagc 7020ggcauccaau accucgcagg auugucaaca cugccaggga accccgcggu ggcuuccaug 7080auggcauuca gugccgcccu caccaguccg uugucgacca guaccaccau ccuucucaac 7140aucaugggag gcugguuagc gucccagauc gcaccacccg cgggggccac cggcuuuguc 7200gucaguggcc uggugggggc ugccgugggc agcauaggcc uggguaaggu gcugguggac 7260auccuggcag gauauggugc gggcauuucg ggggcccucg ucgcauucaa gaucaugucu 7320ggcgagaagc ccucuaugga agaugucauc aaucuacugc cugggauccu gucuccggga 7380gcccuggugg ugggggucau cugcgcggcc auucugcgcc gccacguggg accgggggag 7440ggcgcggucc aauggaugaa caggcuuauu gccuuugcuu ccagaggaaa ccacgucgcc 7500ccuacucacu acgugacgga gucggaugcg ucgcagcgug ugacccaacu acuuggcucu 7560cuuacuauaa ccagccuacu cagaagacuc cacaauugga uaacugagga cugccccauc 7620ccaugcuccg gauccuggcu ccgcgacgug ugggacuggg uuugcaccau cuugacagac 7680uucaaaaauu ggcugaccuc uaaauuguuc cccaagcugc ccggccuccc cuucaucucu 7740ugucaaaagg gguacaaggg ugugugggcc ggcacuggca ucaugaccac gcgcugcccu 7800ugcggcgcca acaucucugg caauguccgc cugggcucua ugaggaucac agggccuaaa 7860accugcauga acaccuggca ggggaccuuu ccuaucaauu gcuacacgga gggccagugc 7920gcgccgaaac cccccacgaa cuacaagacc gccaucugga ggguggcggc cucggaguac 7980gcggagguga cgcagcaugg gucguacucc uauguaacag gacugaccac ugacaaucug 8040aaaauuccuu gccaacuacc uucuccagag uuuuucuccu ggguggacgg ugugcagauc 8100cauagguuug cacccacacc aaagccguuu uuccgggaug aggucucguu cugcguuggg 8160cuuaauuccu augcugucgg gucccagcuu cccugugaac cugagcccga cgcagacgua 8220uugaggucca ugcuaacaga uccgccccac aucacggcgg agacugcggc gcggcgcuug 8280gcacggggau caccuccauc ugaggcgagc uccucaguga gccagcuauc agcaccgucg 8340cugcgggcca ccugcaccac ccacagcaac accuaugacg uggacauggu cgaugccaac 8400cugcucaugg agggcggugu ggcucagaca gagccugagu ccagggugcc cguucuggac 8460uuucucgagc caauggccga ggaagagagc gaccuugagc ccucaauacc aucggagugc 8520augcucccca ggagcggguu uccacgggcc uuaccggcuu gggcacggcc ugacuacaac 8580ccgccgcucg uggaaucgug gaggaggcca gauuaccaac cgcccaccgu ugcugguugu 8640gcucuccccc cccccaagaa ggccccgacg ccucccccaa ggagacgccg gacagugggu 8700cugagcgaga gcaccauauc agaagcccuc cagcaacugg ccaucaagac cuuuggccag 8760ccccccucga gcggugaugc aggcucgucc acgggggcgg gcgccgccga auccggcggu 8820ccgacguccc cuggugagcc ggcccccuca gagacagguu ccgccuccuc uaugcccccc 8880cucgaggggg agccuggaga uccggaccug gagucugauc agguagagcu ucaaccuccc 8940ccccaggggg gggggguagc ucccgguucg ggcucggggu cuuggucuac uugcuccgag 9000gaggacgaua ccaccgugug cugcuccaug ucauacuccu ggaccggggc ucuaauaacu 9060cccuguagcc ccgaagagga aaaguugcca aucaacccuu ugaguaacuc gcuguugcga 9120uaccauaaca agguguacug uacaacauca aagagcgccu cacagagggc uaaaaaggua 9180acuuuugaca ggacgcaagu gcucgacgcc cauuaugacu cagucuuaaa ggacaucaag 9240cuagcggcuu ccaaggucag cgcaaggcuc cucaccuugg aggaggcgug ccaguugacu 9300ccaccccauu cugcaagauc caaguaugga uucggggcca aggagguccg cagcuugucc 9360gggagggccg uuaaccacau caaguccgug uggaaggacc uccuggaaga cccacaaaca 9420ccaauuccca caaccaucau ggccaaaaau gagguguucu gcguggaccc cgccaagggg 9480gguaagaaac cagcucgccu caucguuuac ccugaccucg gcguccgggu cugcgagaaa 9540auggcccucu augacauuac acaaaagcuu ccucaggcgg uaaugggagc uuccuauggc 9600uuccaguacu ccccugccca acggguggag uaucucuuga aagcaugggc ggaaaagaag 9660gaccccaugg guuuuucgua ugauacccga ugcuucgacu caaccgucac ugagagagac 9720aucaggaccg aggaguccau auaccaggcc ugcucccugc ccgaggaggc ccgcacugcc 9780auacacucgc ugacugagag acuuuacgua ggagggccca uguucaacag caagggucaa 9840accugcgguu acagacguug ccgcgccagc ggggugcuaa ccacuagcau ggguaacacc 9900
aucacaugcu augugaaagc ccuagcggcc ugcaaggcug cggggauagu ugcgcccaca 9960augcugguau gcggcgauga ccuaguaguc aucucagaaa gccaggggac ugaggaggac 10020gagcggaacc ugagagccuu cacggaggcc augaccaggu acucugcccc uccuggugau 10080ccccccagac cggaauauga ccuggagcua auaacauccu guuccucaaa ugugucugug 10140gcguugggcc cgcggggccg ccgcagauac uaccugacca gagacccaac cacuccacuc 10200gcccgggcug ccugggaaac aguuagacac uccccuauca auucauggcu gggaaacauc 10260auccaguaug cuccaaccau auggguucgc augguccuaa ugacacacuu cuucuccauu 10320cucauggucc aagacacccu ggaccagaac cucaacuuug agauguaugg aucaguauac 10380uccgugaauc cuuuggaccu uccagccaua auugagaggu uacacgggcu ugacgccuuu 10440ucuaugcaca cauacucuca ccacgaacug acgcgggugg cuucagcccu cagaaaacuu 10500ggggcgccac cccucagggu guggaagagu cgggcucgcg cagucagggc gucccucauc 10560ucccguggag ggaaagcggc cguuugcggc cgauaucucu ucaauugggc ggugaagacc 10620aagcucaaac ucacuccauu gccggaggcg cgccuacugg acuuauccag uugguucacc 10680gucggcgccg gcgggggcga cauuuuucac agcgugucgc gcgcccgacc ccgcucauua 10740cucuucggcc uacuccuacu uuucguaggg guaggccucu uccuacuccc cgcucgguag 10800agcggcacac acuagguaca cuccauagcu aacuguuccu uuuuuuuuuu uuuuuuuuuu 10860uuuuuuuuuu uuuuuuuuuu uucuuuuuuu uuuuuuuccc ucuuucuucc cuucucaucu 10920uauucuacuu ucuuucuugg uggcuccauc uuagcccuag ucacggcuag cugugaaagg 10980uccgugagcc gcaugacugc agagagugcc guaacugguc ucucugcaga ucaugu 11036<210>24<211>11036<212>RNA<213>人工序列<220>
<223>人工序列的描述來(lái)源于表達(dá)載體rFGR-JFH1/EGFP/GND的復(fù)制子RNA<400>24accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360ucaaagaaaa accaaaagaa acaccaaccg acgcguaaug gugagcaagg gcgaggagcu 420guucaccggg guggugccca uccuggucga gcuggacggc gacguaaacg gccacaaguu 480cagcgugucc ggcgagggcg agggcgaugc caccuacggc aagcugaccc ugaaguucau 540cugcaccacc ggcaagcugc ccgugcccug gcccacccuc gugaccaccc ugaccuacgg 600cgugcagugc uucagccgcu accccgacca caugaagcag cacgacuucu ucaaguccgc 660caugcccgaa ggcuacgucc aggagcgcac caucuucuuc aaggacgacg gcaacuacaa 720gacccgcgcc gaggugaagu ucgagggcga cacccuggug aaccgcaucg agcugaaggg 780caucgacuuc aaggaggacg gcaacauccu ggggcacaag cuggaguaca acuacaacag 840ccacaacguc uauaucaugg ccgacaagca gaagaacggc aucaagguga acuucaagau 900ccgccacaac aucgaggacg gcagcgugca gcucgccgac cacuaccagc agaacacccc 960caucggcgac ggccccgugc ugcugcccga caaccacuac cugagcaccc aguccgcccu 1020gagcaaagac cccaacgaga agcgcgauca caugguccug cuggaguucg ugaccgccgc 1080cgggaucacu cucggcaugg acgagcugua caaguaaguu uaaacccucu cccucccccc 1140
ccccuaacgu uacuggccga agccgcuugg aauaaggccg gugugcguuu gucuauaugu 1200uauuuuccac cauauugccg ucuuuuggca augugagggc ccggaaaccu ggcccugucu 1260ucuugacgag cauuccuagg ggucuuuccc cucucgccaa aggaaugcaa ggucuguuga 1320augucgugaa ggaagcaguu ccucuggaag cuucuugaag acaaacaacg ucuguagcga 1380cccuuugcag gcagcggaac cccccaccug gcgacaggug ccucugcggc caaaagccac 1440guguauaaga uacaccugca aaggcggcac aaccccagug ccacguugug aguuggauag 1500uuguggaaag agucaaaugg cucuccucaa gcguauucaa caaggggcug aaggaugccc 1560agaagguacc ccauuguaug ggaucugauc uggggccucg gugcacaugc uuuacaugug 1620uuuagucgag guuaaaaaaa cgucuaggcc ccccgaacca cggggacgug guuuuccuuu 1680gaaaaacacg augauaccau gagcacaaau ccuaaaccuc aaagaaaaac caaaagaaac 1740accaaccguc gcccagaaga cguuaaguuc ccgggcggcg gccagaucgu uggcggagua 1800uacuuguugc cgcgcagggg ccccagguug ggugugcgca cgacaaggaa aacuucggag 1860cggucccagc cacgugggag acgccagccc auccccaaag aucggcgcuc cacuggcaag 1920gccuggggaa aaccaggucg ccccuggccc cuauauggga augagggacu cggcugggca 1980ggauggcucc uguccccccg aggcucucgc cccuccuggg gccccacuga cccccggcau 2040aggucgcgca acguggguaa agucaucgac acccuaacgu guggcuuugc cgaccucaug 2100ggguacaucc ccgucguagg cgccccgcuu aguggcgccg ccagagcugu cgcgcacggc 2160gugagagucc uggaggacgg gguuaauuau gcaacaggga accuacccgg uuuccccuuu 2220ucuaucuucu ugcuggcccu guuguccugc aucaccguuc cggucucugc ugcccaggug 2280aagaauacca guagcagcua cauggugacc aaugacugcu ccaaugacag caucacuugg 2340cagcucgagg cugcgguucu ccacgucccc gggugcgucc cgugcgagag aguggggaau 2400acgucacggu guugggugcc agucucgcca aacauggcug ugcggcagcc cggugcccuc 2460acgcaggguc ugcggacgca caucgauaug guugugaugu ccgccaccuu cugcucugcu 2520cucuacgugg gggaccucug uggcggggug augcucgcgg cccagguguu caucgucucg 2580ccgcaguacc acugguuugu gcaagaaugc aauugcucca ucuacccugg caccaucacu 2640ggacaccgca uggcauggga caugaugaug aacuggucgc ccacggccac caugauccug 2700gcguacguga ugcgcguccc cgaggucauc auagacaucg uuagcggggc ucacuggggc 2760gucauguucg gcuuggccua cuucucuaug cagggagcgu gggcgaaggu cauugucauc 2820cuucugcugg ccgcuggggu ggacgcgggc accaccaccg uuggaggcgc uguugcacgu 2880uccaccaacg ugauugccgg cguguucagc cauggcccuc agcagaacau ucagcucauu 2940aacaccaacg gcaguuggca caucaaccgu acugccuuga auugcaauga cuccuugaac 3000accggcuuuc ucgcggccuu guucuacacc aaccgcuuua acucgucagg guguccaggg 3060cgccuguccg ccugccgcaa caucgaggcu uuccggauag gguggggcac ccuacaguac 3120gaggauaaug ucaccaaucc agaggauaug aggccguacu gcuggcacua ccccccaaag 3180ccguguggcg uaguccccgc gaggucugug uguggcccag uguacuguuu cacccccagc 3240ccgguaguag ugggcacgac cgacagacgu ggagugccca ccuacacaug gggagagaau 3300gagacagaug ucuuccuacu gaacagcacc cgaccgccgc agggcucaug guucggcugc 3360acguggauga acuccacugg uuucaccaag acuuguggcg cgccaccuug ccgcaccaga 3420gcugacuuca acgccagcac ggacuuguug ugcccuacgg auuguuuuag gaagcauccu 3480gaugccacuu auauuaagug ugguucuggg cccuggcuca caccaaagug ccugguccac 3540uacccuuaca gacucuggca uuaccccugc acagucaauu uuaccaucuu caagauaaga 3600auguauguag gggggguuga gcacaggcuc acggccgcau gcaacuucac ucguggggau 3660cgcugcgacu uggaggacag ggacaggagu cagcugucuc cucuguugca cucuaccacg 3720gaaugggcca uccugcccug caccuacuca gacuuacccg cuuugucaac uggucuucuc 3780caccuucacc agaacaucgu ggacguacaa uacauguaug gccucucacc ugcuaucaca 3840aaauacgucg uucgauggga guggguggua cucuuauucc ugcucuuagc ggacgccaga 3900gucugcgccu gcuuguggau gcucaucuug uugggccagg ccgaagcagc auuggagaag 3960uuggucgucu ugcacgcugc gagugcggcu aacugccaug gccuccuaua uuuugccauc 4020uucuucgugg cagcuuggca caucaggggu cggguggucc ccuugaccac cuauugccuc 4080
acuggccuau ggcccuucug ccuacugcuc auggcacugc cccggcaggc uuaugccuau 4140gacgcaccug ugcacggaca gauaggcgug gguuuguuga uauugaucac ccucuucaca 4200cucaccccgg gguauaagac ccuccucggc cagugucugu ggugguugug cuaucuccug 4260acccuggggg aagccaugau ucaggagugg guaccaccca ugcaggugcg cggcggccgc 4320gauggcaucg cgugggccgu cacuauauuc ugcccgggug ugguguuuga cauuaccaaa 4380uggcuuuugg cguugcuugg gccugcuuac cucuuaaggg ccgcuuugac acaugugccg 4440uacuucguca gagcucacgc ucugauaagg guaugcgcuu uggugaagca gcucgcgggg 4500gguagguaug uucagguggc gcuauuggcc cuuggcaggu ggacuggcac cuacaucuau 4560gaccaccuca caccuauguc ggacugggcc gcuagcggcc ugcgcgacuu agcggucgcc 4620guggaaccca ucaucuucag uccgauggag aagaagguca ucgucugggg agcggagacg 4680gcugcaugug gggacauucu acauggacuu cccguguccg cccgacucgg ccaggagauc 4740cuccucggcc cagcugaugg cuacaccucc aaggggugga agcuccuugc ucccaucacu 4800gcuuaugccc agcaaacacg aggccuccug ggcgccauag uggugaguau gacggggcgu 4860gacaggacag aacaggccgg ggaaguccaa auccugucca cagucucuca guccuuccuc 4920ggaacaaccaucucgggggu uuuguggacu guuuaccacg gagcuggcaa caagacucua 4980gccggcuuac gggguccggu cacgcagaug uacucgagug cugaggggga cuugguaggc 5040uggcccagcc ccccugggac caagucuuug gagccgugca aguguggagc cgucgaccua 5100uaucugguca cgcggaacgc ugaugucauc ccggcucgga gacgcgggga caagcgggga 5160gcauugcucu ccccgagacc cauuucgacc uugaaggggu ccucgggggg gccggugcuc 5220ugcccuaggg gccacgucgu ugggcucuuc cgagcagcug ugugcucucg gggcguggcc 5280aaauccaucg auuucauccc cguugagaca cucgacguug uuacaagguc ucccacuuuc 5340agugacaaca gcacgccacc ggcugugccc cagaccuauc aggucgggua cuugcaugcu 5400ccaacuggca guggaaagag caccaagguc ccugucgcgu augccgccca gggguacaaa 5460guacuagugc uuaaccccuc gguagcugcc acccuggggu uuggggcgua ccuauccaag 5520gcacauggca ucaaucccaa cauuaggacu ggagucagga ccgugaugac cggggaggcc 5580aucacguacu ccacauaugg caaauuucuc gccgaugggg gcugcgcuag cggcgccuau 5640gacaucauca uaugcgauga augccacgcu guggaugcua ccuccauucu cggcaucgga 5700acgguccuug aucaagcaga gacagccggg gucagacuaa cugugcuggc uacggccaca 5760ccccccgggu cagugacaac cccccauccc gauauagaag agguaggccu cgggcgggag 5820ggugagaucc ccuucuaugg gagggcgauu ccccuauccu gcaucaaggg agggagacac 5880cugauuuucu gccacucaaa gaaaaagugu gacgagcucg cggcggcccu ucggggcaug 5940ggcuugaaug ccguggcaua cuauagaggg uuggacgucu ccauaauacc agcucaggga 6000gauguggugg ucgucgccac cgacgcccuc augacggggu acacuggaga cuuugacucc 6060gugaucgacu gcaauguagc ggucacccaa gcugucgacu ucagccugga ccccaccuuc 6120acuauaacca cacagacugu cccacaagac gcugucucac gcagucagcg ccgcgggcgc 6180acagguagag gaagacaggg cacuuauagg uauguuucca cuggugaacg agccucagga 6240auguuugaca guguagugcu uugugagugc uacgacgcag gggcugcgug guacgaucuc 6300acaccagcgg agaccaccgu caggcuuaga gcguauuuca acacgcccgg ccuacccgug 6360ugucaagacc aucuugaauu uugggaggca guuuucaccg gccucacaca cauagacgcc 6420cacuuccucu cccaaacaaa gcaagcgggg gagaacuucg cguaccuagu agccuaccaa 6480gcuacggugu gcgccagagc caaggccccu cccccguccu gggacgccau guggaagugc 6540cuggcccgac ucaagccuac gcuugcgggc cccacaccuc uccuguaccg uuugggcccu 6600auuaccaaug aggucacccu cacacacccu gggacgaagu acaucgccac augcaugcaa 6660gcugaccuug aggucaugac cagcacgugg guccuagcug gaggaguccu ggcagccguc 6720gccgcauauu gccuggcgac uggaugcguu uccaucaucg gccgcuugca cgucaaccag 6780cgagucgucg uugcgccgga uaaggagguc cuguaugagg cuuuugauga gauggaggaa 6840ugcgccucua gggcggcucu caucgaagag gggcagcgga uagccgagau guugaagucc 6900aagauccaag gcuugcugca gcaggccucu aagcaggccc aggacauaca acccgcuaug 6960caggcuucau ggcccaaagu ggaacaauuu ugggccagac acauguggaa cuucauuagc 7020
ggcauccaau accucgcagg auugucaaca cugccaggga accccgcggu ggcuuccaug 7080auggcauuca gugccgcccu caccaguccg uugucgacca guaccaccau ccuucucaac 7140aucaugggag gcugguuagc gucccagauc gcaccacccg cgggggccac cggcuuuguc 7200gucaguggcc uggugggggc ugccgugggc agcauaggcc uggguaaggu gcugguggac 7260auccuggcag gauauggugc gggcauuucg ggggcccucg ucgcauucaa gaucaugucu 7320ggcgagaagc ccucuaugga agaugucauc aaucuacugc cugggauccu gucuccggga 7380gcccuggugg ugggggucau cugcgcggcc auucugcgcc gccacguggg accgggggag 7440ggcgcggucc aauggaugaa caggcuuauu gccuuugcuu ccagaggaaa ccacgucgcc 7500ccuacucacu acgugacgga gucggaugcg ucgcagcgug ugacccaacu acuuggcucu 7560cuuacuauaa ccagccuacu cagaagacuc cacaauugga uaacugagga cugccccauc 7620ccaugcuccg gauccuggcu ccgcgacgug ugggacuggg uuugcaccau cuugacagac 7680uucaaaaauu ggcugaccuc uaaauuguuc cccaagcugc ccggccuccc cuucaucucu 7740ugucaaaagg gguacaaggg ugugugggcc ggcacuggca ucaugaccac gcgcugcccu 7800ugcggcgcca acaucucugg caauguccgc cugggcucua ugaggaucac agggccuaaa 7860accugcauga acaccuggca ggggaccuuu ccuaucaauu gcuacacgga gggccagugc 7920gcgccgaaac cccccacgaa cuacaagacc gccaucugga ggguggcggc cucggaguac 7980gcggagguga cgcagcaugg gucguacucc uauguaacag gacugaccac ugacaaucug 8040aaaauuccuu gccaacuacc uucuccagag uuuuucuccu ggguggacgg ugugcagauc 8100cauagguuug cacccacacc aaagccguuu uuccgggaug aggucucguu cugcguuggg 8160cuuaauuccu augcugucgg gucccagcuu cccugugaac cugagcccga cgcagacgua 8220uugaggucca ugcuaacaga uccgccccac aucacggcgg agacugcggc gcggcgcuug 8280gcacggggau caccuccauc ugaggcgagc uccucaguga gccagcuauc agcaccgucg 8340cugcgggcca ccugcaccac ccacagcaac accuaugacg uggacauggu cgaugccaac 8400cugcucaugg agggcggugu ggcucagaca gagccugagu ccagggugcc cguucuggac 8460uuucucgagc caauggccga ggaagagagc gaccuugagc ccucaauacc aucggagugc 8520augcucccca ggagcggguu uccacgggcc uuaccggcuu gggcacggcc ugacuacaac 8580ccgccgcucg uggaaucgug gaggaggcca gauuaccaac cgcccaccgu ugcugguugu 8640gcucuccccc cccccaagaa ggccccgacg ccucccccaa ggagacgccg gacagugggu 8700cugagcgaga gcaccauauc agaagcccuc cagcaacugg ccaucaagac cuuuggccag 8760ccccccucga gcggugaugc aggcucgucc acgggggcgg gcgccgccga auccggcggu 8820ccgacguccc cuggugagcc ggcccccuca gagacagguu ccgccuccuc uaugcccccc 8880cucgaggggg agccuggaga uccggaccug gagucugauc agguagagcu ucaaccuccc 8940ccccaggggg gggggguagc ucccgguucg ggcucggggu cuuggucuac uugcuccgag 9000gaggacgaua ccaccgugug cugcuccaug ucauacuccu ggaccggggc ucuaauaacu 9060cccuguagcc ccgaagagga aaaguugcca aucaacccuu ugaguaacuc gcuguugcga 9120uaccauaaca agguguacug uacaacauca aagagcgccu cacagagggc uaaaaaggua 9180acuuuugaca ggacgcaagu gcucgacgcc cauuaugacu cagucuuaaa ggacaucaag 9240cuagcggcuu ccaaggucag cgcaaggcuc cucaccuugg aggaggcgug ccaguugacu 9300ccaccccauu cugcaagauc caaguaugga uucggggcca aggagguccg cagcuugucc 9360gggagggccg uuaaccacau caaguccgug uggaaggacc uccuggaaga cccacaaaca 9420ccaauuccca caaccaucau ggccaaaaau gagguguucu gcguggaccc cgccaagggg 9480gguaagaaac cagcucgccu caucguuuac ccugaccucg gcguccgggu cugcgagaaa 9540auggcccucu augacauuac acaaaagcuu ccucaggcgg uaaugggagc uuccuauggc 9600uuccaguacu ccccugccca acggguggag uaucucuuga aagcaugggc ggaaaagaag 9660gaccccaugg guuuuucgua ugauacccga ugcuucgacu caaccgucac ugagagagac 9720aucaggaccg aggaguccau auaccaggcc ugcucccugc ccgaggaggc ccgcacugcc 9780auacacucgc ugacugagag acuuuacgua ggagggccca uguucaacag caagggucaa 9840accugcgguu acagacguug ccgcgccagc ggggugcuaa ccacuagcau ggguaacacc 9900aucacaugcu augugaaagc ccuagcggcc ugcaaggcug cggggauagu ugcgcccaca 9960
augcugguau gcggcaauga ccuaguaguc aucucagaaa gccaggggac ugaggaggac 10020gagcggaacc ugagagccuu cacggaggcc augaccaggu acucugcccc uccuggugau 10080ccccccagac cggaauauga ccuggagcua auaacauccu guuccucaaa ugugucugug 10140gcguugggcc cgcggggccg ccgcagauac uaccugacca gagacccaac cacuccacuc 10200gcccgggcug ccugggaaac aguuagacac uccccuauca auucauggcu gggaaacauc 10260auccaguaug cuccaaccau auggguucgc augguccuaa ugacacacuu cuucuccauu 10320cucauggucc aagacacccu ggaccagaac cucaacuuug agauguaugg aucaguauac 10380uccgugaauc cuuuggaccu uccagccaua auugagaggu uacacgggcu ugacgccuuu 10440ucuaugcaca cauacucuca ccacgaacug acgcgggugg cuucagcccu cagaaaacuu 10500ggggcgccac cccucagggu guggaagagu cgggcucgcg cagucagggc gucccucauc 10560ucccguggag ggaaagcggc cguuugcggc cgauaucucu ucaauugggc ggugaagacc 10620aagcucaaac ucacuccauu gccggaggcg cgccuacugg acuuauccag uugguucacc 10680gucggcgccg gcgggggcga cauuuuucac agcgugucgc gcgcccgacc ccgcucauua 10740cucuucggcc uacuccuacu uuucguaggg guaggccucu uccuacuccc cgcucgguag 10800agcggcacac acuagguaca cuccauagcu aacuguuccu uuuuuuuuuu uuuuuuuuuu 10860uuuuuuuuuu uuuuuuuuuu uucuuuuuuu uuuuuuuccc ucuuucuucc cuucucaucu 10920uauucuacuu ucuuucuugg uggcuccauc uuagcccuag ucacggcuag cugugaaagg 10980uccgugagcc gcaugacugc agagagugcc guaacugguc ucucugcagaucaugu11036<210>25<211>11876<212>RNA<213>人工序列<220>
<223>人工序列的描述來(lái)源于表達(dá)載體rFGR-JFH1/SEAP的復(fù)制子RNA<400>25accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360ucaaagaaaa accaaaagaa acaccaaccg acgcguaaug cugcugcugc ugcugcugcu 420gggccugagg cuacagcucu cccugggcau caucccaguu gaggaggaga acccggacuu 480cuggaaccgc gaggcagccg aggcccuggg ugccgccaag aagcugcagc cugcacagac 540agccgccaag aaccucauca ucuuccuggg cgaugggaug ggggugucua cggugacagc 600ugccaggauc cuaaaagggc agaagaagga caaacugggg ccugagauac cccuggccau 660ggaccgcuuc ccauaugugg cucuguccaa gacauacaau guagacaaac augugccaga 720caguggagcc acagccacgg ccuaccugug cggggucaag ggcaacuucc agaccauugg 780cuugagugca gccgcccgcu uuaaccagug caacacgaca cgcggcaacg aggucaucuc 840cgugaugaau cgggccaaga aagcagggaa gucaguggga gugguaacca ccacacgagu 900gcagcacgcc ucgccagccg gcaccuacgc ccacacggug aaccgcaacu gguacucgga 960cgccgacgug ccugccucgg cccgccagga ggggugccag gacaucgcua cgcagcucau 1020cuccaacaug gacauugacg ugauccuagg uggaggccga aaguacaugu uucgcauggg 1080aaccccagac ccugaguacc cagaugacua cagccaaggu gggaccaggc uggacgggaa 1140gaaucuggu gcaggaauggc uggcgaagcg ccagggugcc cgguaugugu ggaaccgcac 1200
ugagcucaug caggcuuccc uggacccguc ugugacccau cucauggguc ucuuugagcc 1260uggagacaug aaauacgaga uccaccgaga cuccacacug gaccccuccc ugauggagau 1320gacagaggcu gcccugcgcc ugcugagcag gaacccccgc ggcuucuucc ucuucgugga 1380ggguggucgc aucgaccaug gucaucauga aagcagggcu uaccgggcac ugacugagac 1440gaucauguuc gacgacgcca uugagagggc gggccagcuc accagcgagg aggacacgcu 1500gagccucguc acugccgacc acucccacgu cuucuccuuc ggaggcuacc cccugcgagg 1560gagcuccauc uucgggcugg ccccuggcaa ggcccgggac aggaaggccu acacgguccu 1620ccuauacgga aacgguccag gcuaugugcu caaggacggc gcccggccgg auguuaccga 1680gagcgagagc gggagccccg aguaucggca gcagucagca gugccccugg acgaagagac 1740ccacgcaggc gaggacgugg cgguguucgc gcgcggcccg caggcgcacc ugguucacgg 1800cgugcaggag cagaccuuca uagcgcacgu cauggccuuc gccgccugcc uggagcccua 1860caccgccugc gaccuggcgc cccccgccgg caccaccgac gccgcgcacc cggguuacuc 1920uagagucggg gcggccggcc gcuucgagca gacaugaguu uaaacccucu cccucccccc 1980ccccuaacgu uacuggccga agccgcuugg aauaaggccg gugugcguuu gucuauaugu 2040uauuuuccac cauauugccg ucuuuuggca augugagggc ccggaaaccu ggcccugucu 2100ucuugacgag cauuccuagg ggucuuuccc cucucgccaa aggaaugcaa ggucuguuga 2160augucgugaa ggaagcaguu ccucuggaag cuucuugaag acaaacaacg ucuguagcga 2220cccuuugcag gcagcggaac cccccaccug gcgacaggug ccucugcggc caaaagccac 2280guguauaaga uacaccugca aaggcggcac aaccccagug ccacguugug aguuggauag 2340uuguggaaag agucaaaugg cucuccucaa gcguauucaa caaggggcug aaggaugccc 2400agaagguacc ccauuguaug ggaucugauc uggggccucg gugcacaugc uuuacaugug 2460uuuagucgag guuaaaaaaa cgucuaggcc ccccgaacca cggggacgug guuuuccuuu 2520gaaaaacacg augauaccau gagcacaaau ccuaaaccuc aaagaaaaac caaaagaaac 2580accaaccguc gcccagaaga cguuaaguuc ccgggcggcg gccagaucgu uggcggagua 2640uacuuguugc cgcgcagggg ccccagguug ggugugcgca cgacaaggaa aacuucggag 2700cggucccagc cacgugggag acgccagccc auccccaaag aucggcgcuc cacuggcaag 2760gccuggggaa aaccaggucg ccccuggccc cuauauggga augagggacu cggcugggca 2820ggauggcucc uguccccccg aggcucucgc cccuccuggg gccccacuga cccccggcau 2880aggucgcgca acguggguaa agucaucgac acccuaacgu guggcuuugc cgaccucaug 2940ggguacaucc ccgucguagg cgccccgcuu aguggcgccg ccagagcugu cgcgcacggc 3000gugagagucc uggaggacgg gguuaauuau gcaacaggga accuacccgg uuuccccuuu 3060ucuaucuucu ugcuggcccu guuguccugc aucaccguuc cggucucugc ugcccaggug 3120aagaauacca guagcagcua cauggugacc aaugacugcu ccaaugacag caucacuugg 3180cagcucgagg cugcgguucu ccacgucccc gggugcgucc cgugcgagag aguggggaau 3240acgucacggu guugggugcc agucucgcca aacauggcug ugcggcagcc cggugcccuc 3300acgcaggguc ugcggacgca caucgauaug guugugaugu ccgccaccuu cugcucugcu 3360cucuacgugg gggaccucug uggcggggug augcucgcgg cccagguguu caucgucucg 3420ccgcaguacc acugguuugu gcaagaaugc aauugcucca ucuacccugg caccaucacu 3480ggacaccgca uggcauggga caugaugaug aacuggucgc ccacggccac caugauccug 3540gcguacguga ugcgcguccc cgaggucauc auagacaucg uuagcggggc ucacuggggc 3600gucauguucg gcuuggccua cuucucuaug cagggagcgu gggcgaaggu cauugucauc 3660cuucugcugg ccgcuggggu ggacgcgggc accaccaccg uuggaggcgc uguugcacgu 3720uccaccaacg ugauugccgg cguguucagc cauggcccuc agcagaacau ucagcucauu 3780aacaccaacg gcaguuggca caucaaccgu acugccuuga auugcaauga cuccuugaac 3840accggcuuuc ucgcggccuu guucuacacc aaccgcuuua acucgucagg guguccaggg 3900cgccuguccg ccugccgcaa caucgaggcu uuccggauag gguggggcac ccuacaguac 3960gaggauaaug ucaccaaucc agaggauaug aggccguacu gcuggcacua ccccccaaag 4020ccguguggcg uaguccccgc gaggucugug uguggcccag uguacuguuu cacccccagc 4080ccgguaguag ugggcacgac cgacagacgu ggagugccca ccuacacaug gggagagaau 4140
gagacagaug ucuuccuacu gaacagcacc cgaccgccgc agggcucaug guucggcugc 4200acguggauga acuccacugg uuucaccaag acuuguggcg cgccaccuug ccgcaccaga 4260gcugacuuca acgccagcac ggacuuguug ugcccuacgg auuguuuuag gaagcauccu 4320gaugccacuu auauuaagug ugguucuggg cccuggcuca caccaaagug ccugguccac 4380uacccuuaca gacucuggca uuaccccugc acagucaauu uuaccaucuu caagauaaga 4440auguauguag gggggguuga gcacaggcuc acggccgcau gcaacuucac ucguggggau 4500cgcugcgacu uggaggacag ggacaggagu cagcugucuc cucuguugca cucuaccacg 4560gaaugggcca uccugcccug caccuacuca gacuuacccg cuuugucaac uggucuucuc 4620caccuucacc agaacaucgu ggacguacaa uacauguaug gccucucacc ugcuaucaca 4680aaauacgucg uucgauggga guggguggua cucuuauucc ugcucuuagc ggacgccaga 4740gucugcgccu gcuuguggau gcucaucuug uugggccagg ccgaagcagc auuggagaag 4800uuggucgucu ugcacgcugc gagugcggcu aacugccaug gccuccuaua uuuugccauc 4860uucuucgugg cagcuuggca caucaggggu cggguggucc ccuugaccac cuauugccuc 4920acuggccuau ggcccuucug ccuacugcuc auggcacugc cccggcaggc uuaugccuau 4980gacgcaccug ugcacggaca gauaggcgug gguuuguuga uauugaucac ccucuucaca 5040cucaccccgg gguauaagac ccuccucggc cagugucugu ggugguugug cuaucuccug 5100acccuggggg aagccaugau ucaggagugg guaccaccca ugcaggugcg cggcggccgc 5160gauggcaucg cgugggccgu cacuauauuc ugcccgggug ugguguuuga cauuaccaaa 5220uggcuuuugg cguugcuugg gccugcuuac cucuuaaggg ccgcuuugac acaugugccg 5280uacuucguca gagcucacgc ucugauaagg guaugcgcuu uggugaagca gcucgcgggg 5340gguagguaug uucagguggc gcuauuggcc cuuggcaggu ggacuggcac cuacaucuau 5400gaccaccuca caccuauguc ggacugggcc gcuagcggcc ugcgcgacuu agcggucgcc 5460guggaaccca ucaucuucag uccgauggag aagaagguca ucgucugggg agcggagacg 5520gcugcaugug gggacauucu acauggacuu cccguguccg cccgacucgg ccaggagauc 5580cuccucggcc cagcugaugg cuacaccucc aaggggugga agcuccuugc ucccaucacu 5640gcuuaugccc agcaaacacg aggccuccug ggcgccauag uggugaguau gacggggcgu 5700gacaggacag aacaggccgg ggaaguccaa auccugucca cagucucuca guccuuccuc 5760ggaacaacca ucucgggggu uuuguggacu guuuaccacg gagcuggcaa caagacucua 5820gccggcuuac gggguccggu cacgcagaug uacucgagug cugaggggga cuugguaggc 5880uggcccagcc ccccugggac caagucuuug gagccgugca aguguggagc cgucgaccua 5940uaucugguca cgcggaacgc ugaugucauc ccggcucgga gacgcgggga caagcgggga 6000gcauugcucu ccccgagacc cauuucgacc uugaaggggu ccucgggggg gccggugcuc 6060ugcccuaggg gccacgucgu ugggcucuuc cgagcagcug ugugcucucg gggcguggcc 6120aaauccaucg auuucauccc cguugagaca cucgacguug uuacaagguc ucccacuuuc 6180agugacaaca gcacgccacc ggcugugccc cagaccuauc aggucgggua cuugcaugcu 6240ccaacuggca guggaaagag caccaagguc ccugucgcgu augccgccca gggguacaaa 6300guacuagugc uuaaccccuc gguagcugcc acccuggggu uuggggcgua ccuauccaag 6360gcacauggca ucaaucccaa cauuaggacu ggagucagga ccgugaugac cggggaggcc 6420aucacguacu ccacauaugg caaauuucuc gccgaugggg gcugcgcuag cggcgccuau 6480gacaucauca uaugcgauga augccacgcu guggaugcua ccuccauucu cggcaucgga 6540acgguccuug aucaagcaga gacagccggg gucagacuaa cugugcuggc uacggccaca 6600ccccccgggu cagugacaac cccccauccc gauauagaag agguaggccu cgggcgggag 6660ggugagaucc ccuucuaugg gagggcgauu ccccuauccu gcaucaaggg agggagacac 6720cugauuuucu gccacucaaa gaaaaagugu gacgagcucg cggcggcccu ucggggcaug 6780ggcuugaaug ccguggcaua cuauagaggg uuggacgucu ccauaauacc agcucaggga 6840gauguggugg ucgucgccac cgacgcccuc augacggggu acacuggaga cuuugacucc 6900gugaucgacu gcaauguagc ggucacccaa gcugucgacu ucagccugga ccccaccuuc 6960acuauaacca cacagacugu cccacaagac gcugucucac gcagucagcg ccgcgggcgc 7020acagguagag gaagacaggg cacuuauagg uauguuucca cuggugaacg agccucagga 7080
auguuugaca guguagugcu uugugagugc uacgacgcag gggcugcgug guacgaucuc 7140acaccagcgg agaccaccgu caggcuuaga gcguauuuca acacgcccgg ccuacccgug 7200ugucaagacc aucuugaauu uugggaggca guuuucaccg gccucacaca cauagacgcc 7260cacuuccucu cccaaacaaa gcaagcgggg gagaacuucg cguaccuagu agccuaccaa 7320gcuacggugu gcgccagagc caaggccccu cccccguccu gggacgccau guggaagugc 7380cuggcccgac ucaagccuac gcuugcgggc cccacaccuc uccuguaccg uuugggcccu 7440auuaccaaug aggucacccu cacacacccu gggacgaagu acaucgccac augcaugcaa 7500gcugaccuug aggucaugac cagcacgugg guccuagcug gaggaguccu ggcagccguc 7560gccgcauauu gccuggcgac uggaugcguu uccaucaucg gccgcuugca cgucaaccag 7620cgagucgucg uugcgccgga uaaggagguc cuguaugagg cuuuugauga gauggaggaa 7680ugcgccucua gggcggcucu caucgaagag gggcagcgga uagccgagau guugaagucc 7740aagauccaag gcuugcugca gcaggccucu aagcaggccc aggacauaca acccgcuaug 7800caggcuucau ggcccaaagu ggaacaauuu ugggccagac acauguggaa cuucauuagc 7860ggcauccaau accucgcagg auugucaaca cugccaggga accccgcggu ggcuuccaug 7920auggcauuca gugccgcccu caccaguccg uugucgacca guaccaccau ccuucucaac 7980aucaugggag gcugguuagc gucccagauc gcaccacccg cgggggccac cggcuuuguc 8040gucaguggcc uggugggggc ugccgugggc agcauaggcc uggguaaggu gcugguggac 8100auccuggcag gauauggugc gggcauuucg ggggcccucg ucgcauucaa gaucaugucu 8160ggcgagaagc ccucuaugga agaugucauc aaucuacugc cugggauccu gucuccggga 8220gcccuggugg ugggggucau cugcgcggcc auucugcgcc gccacguggg accgggggag 8280ggcgcggucc aauggaugaa caggcuuauu gccuuugcuu ccagaggaaa ccacgucgcc 8340ccuacucacu acgugacgga gucggaugcg ucgcagcgug ugacccaacu acuuggcucu 8400cuuacuauaa ccagccuacu cagaagacuc cacaauugga uaacugagga cugccccauc 8460ccaugcuccg gauccuggcu ccgcgacgug ugggacuggg uuugcaccau cuugacagac 8520uucaaaaauu ggcugaccuc uaaauuguuc cccaagcugc ccggccuccc cuucaucucu 8580ugucaaaagg gguacaaggg ugugugggcc ggcacuggca ucaugaccac gcgcugcccu 8640ugcggcgcca acaucucugg caauguccgc cugggcucua ugaggaucac agggccuaaa 8700accugcauga acaccuggca ggggaccuuu ccuaucaauu gcuacacgga gggccagugc 8760gcgccgaaac cccccacgaa cuacaagacc gccaucugga ggguggcggc cucggaguac 8820gcggagguga cgcagcaugg gucguacucc uauguaacag gacugaccac ugacaaucug 8880aaaauuccuu gccaacuacc uucuccagag uuuuucuccu ggguggacgg ugugcagauc 8940cauagguuug cacccacacc aaagccguuu uuccgggaug aggucucguu cugcguuggg 9000cuuaauuccu augcugucgg gucccagcuu cccugugaac cugagcccga cgcagacgua 9060uugaggucca ugcuaacaga uccgccccac aucacggcgg agacugcggc gcggcgcuug 9120gcacggggau caccuccauc ugaggcgagc uccucaguga gccagcuauc agcaccgucg 9180cugcgggcca ccugcaccac ccacagcaac accuaugacg uggacauggu cgaugccaac 9240cugcucaugg agggcggugu ggcucagaca gagccugagu ccagggugcc cguucuggac 9300uuucucgagc caauggccga ggaagagagc gaccuugagc ccucaauacc aucggagugc 9360augcucccca ggagcggguu uccacgggcc uuaccggcuu gggcacggcc ugacuacaac 9420ccgccgcucg uggaaucgug gaggaggcca gauuaccaac cgcccaccgu ugcugguugu 9480gcucuccccc cccccaagaa ggccccgacg ccucccccaa ggagacgccg gacagugggu 9540cugagcgaga gcaccauauc agaagcccuc cagcaacugg ccaucaagac cuuuggccag 9600ccccccucga gcggugaugc aggcucgucc acgggggcgg gcgccgccga auccggcggu 9660ccgacguccc cuggugagcc ggcccccuca gagacagguu ccgccuccuc uaugcccccc 9720cucgaggggg agccuggaga uccggaccug gagucugauc agguagagcu ucaaccuccc 9780ccccaggggg gggggguagc ucccgguucg ggcucggggu cuuggucuac uugcuccgag 9840gaggacgaua ccaccgugug cugcuccaug ucauacuccu ggaccggggc ucuaauaacu 9900cccuguagcc ccgaagagga aaaguugcca aucaacccuu ugaguaacuc gcuguugcga 9960uaccauaaca agguguacug uacaacauca aagagcgccu cacagagggc uaaaaaggua 10020
acuuuugaca ggacgcaagu gcucgacgcc cauuaugacu cagucuuaaa ggacaucaag 10080cuagcggcuu ccaaggucag cgcaaggcuc cucaccuugg aggaggcgug ccaguugacu 10140ccaccccauu cugcaagauc caaguaugga uucggggcca aggagguccg cagcuugucc 10200gggagggccg uuaaccacau caaguccgug uggaaggacc uccuggaaga cccacaaaca 10260ccaauuccca caaccaucau ggccaaaaau gagguguucu gcguggaccc cgccaagggg 10320gguaagaaac cagcucgccu caucguuuac ccugaccucg gcguccgggu cugcgagaaa 10380auggcccucu augacauuac acaaaagcuu ccucaggcgg uaaugggagc uuccuauggc 10440uuccaguacu ccccugccca acggguggag uaucucuuga aagcaugggc ggaaaagaag 10500gaccccaugg guuuuucgua ugauacccga ugcuucgacu caaccgucac ugagagagac 10560aucaggaccg aggaguccau auaccaggcc ugcucccugc ccgaggaggc ccgcacugcc 10620auacacucgc ugacugagag acuuuacgua ggagggccca uguucaacag caagggucaa 10680accugcgguu acagacguug ccgcgccagc ggggugcuaa ccacuagcau ggguaacacc 10740aucacaugcu augugaaagc ccuagcggcc ugcaaggcug cggggauagu ugcgcccaca 10800augcugguau gcggcgauga ccuaguaguc aucucagaaa gccaggggac ugaggaggac 10860gagcggaacc ugagagccuu cacggaggcc augaccaggu acucugcccc uccuggugau 10920ccccccagac cggaauauga ccuggagcua auaacauccu guuccucaaa ugugucugug 10980gcguugggcc cgcggggccg ccgcagauac uaccugacca gagacccaac cacuccacuc 11040gcccgggcug ccugggaaac aguuagacac uccccuauca auucauggcu gggaaacauc 11100auccaguaug cuccaaccau auggguucgc augguccuaa ugacacacuu cuucuccauu 11160cucauggucc aagacacccu ggaccagaac cucaacuuug agauguaugg aucaguauac 11220uccgugaauc cuuuggaccu uccagccaua auugagaggu uacacgggcu ugacgccuuu 11280ucuaugcaca cauacucuca ccacgaacug acgcgggugg cuucagcccu cagaaaacuu 11340ggggcgccac cccucagggu guggaagagu cgggcucgcg cagucagggc gucccucauc 11400ucccguggag ggaaagcggc cguuugcggc cgauaucucu ucaauugggc ggugaagacc 11460aagcucaaac ucacuccauu gccggaggcg cgccuacugg acuuauccag uugguucacc 11520gucggcgccg gcgggggcga cauuuuucac agcgugucgc gcgcccgacc ccgcucauua 11580cucuucggcc uacuccuacu uuucguaggg guaggccucu uccuacuccc cgcucgguag 11640agcggcacac acuagguaca cuccauagcu aacuguuccu uuuuuuuuuu uuuuuuuuuu 11700uuuuuuuuuu uuuuuuuuuu uucuuuuuuu uuuuuuuccc ucuuucuucc cuucucaucu 11760uauucuacuu ucuuucuugg uggcuccauc uuagcccuag ucacggcuag cugugaaagg 11820uccgugagcc gcaugacugc agagagugcc guaacugguc ucucugcaga ucaugu 11876<210>26<211>11876<212>RNA<213>人工序列<220>
<223>人工序列的描述來(lái)源于表達(dá)載體rFGR-JFH1/sEAP/GND的復(fù)制子RNA<400>26accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu 60cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc 120cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg 180aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg 240caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg 300cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc 360
ucaaagaaaa accaaaagaa acaccaaccg acgcguaaug cugcugcugc ugcugcugcu 420gggccugagg cuacagcucu cccugggcau caucccaguu gaggaggaga acccggacuu 480cuggaaccgc gaggcagccg aggcccuggg ugccgccaag aagcugcagc cugcacagac 540agccgccaag aaccucauca ucuuccuggg cgaugggaug ggggugucua cggugacagc 600ugccaggauc cuaaaagggc agaagaagga caaacugggg ccugagauac cccuggccau 660ggaccgcuuc ccauaugugg cucuguccaa gacauacaau guagacaaac augugccaga 720caguggagcc acagccacgg ccuaccugug cggggucaag ggcaacuucc agaccauugg 780cuugagugca gccgcccgcu uuaaccagug caacacgaca cgcggcaacg aggucaucuc 840cgugaugaau cgggccaaga aagcagggaa gucaguggga gugguaacca ccacacgagu 900gcagcacgcc ucgccagccg gcaccuacgc ccacacggug aaccgcaacu gguacucgga 960cgccgacgug ccugccucgg cccgccagga ggggugccag gacaucgcua cgcagcucau 1020cuccaacaug gacauugacg ugauccuagg uggaggccga aaguacaugu uucgcauggg 1080aaccccagac ccugaguacc cagaugacua cagccaaggu gggaccaggc uggacgggaa 1140gaaucuggug caggaauggc uggcgaagcg ccagggugcc cgguaugugu ggaaccgcac 1200ugagcucaug caggcuuccc uggacccguc ugugacccau cucauggguc ucuuugagcc 1260uggagacaug aaauacgaga uccaccgaga cuccacacug gaccccuccc ugauggagau 1320gacagaggcu gcccugcgcc ugcugagcag gaacccccgc ggcuucuucc ucuucgugga 1380ggguggucgc aucgaccaug gucaucauga aagcagggcu uaccgggcac ugacugagac 1440gaucauguuc gacgacgcca uugagagggc gggccagcuc accagcgagg aggacacgcu 1500gagccucguc acugccgacc acucccacgu cuucuccuuc ggaggcuacc cccugcgagg 1560gagcuccauc uucgggcugg ccccuggcaa ggcccgggac aggaaggccu acacgguccu 1620ccuauacgga aacgguccag gcuaugugcu caaggacggc gcccggccgg auguuaccga 1680gagcgagagc gggagccccg aguaucggca gcagucagca gugccccugg acgaagagac 1740ccacgcaggc gaggacgugg cgguguucgc gcgcggcccg caggcgcacc ugguucacgg 1800cgugcaggag cagaccuuca uagcgcacgu cauggccuuc gccgccugcc uggagcccua 1860caccgccugc gaccuggcgc cccccgccgg caccaccgac gccgcgcacc cggguuacuc 1920uagagucggg gcggccggcc gcuucgagca gacaugaguu uaaacccucu cccucccccc 1980ccccuaacgu uacuggccga agccgcuugg aauaaggccg gugugcguuu gucuauaugu 2040uauuuuccac cauauugccg ucuuuuggca augugagggc ccggaaaccu ggcccugucu 2100ucuugacgag cauuccuagg ggucuuuccc cucucgccaa aggaaugcaa ggucuguuga 2160augucgugaa ggaagcaguu ccucuggaag cuucuugaag acaaacaacg ucuguagcga 2220cccuuugcag gcagcggaac cccccaccug gcgacaggug ccucugcggc caaaagccac 2280guguauaaga uacaccugca aaggcggcac aaccccagug ccacguugug aguuggauag 2340uuguggaaag agucaaaugg cucuccucaa gcguauucaa caaggggcug aaggaugccc 2400agaagguacc ccauuguaug ggaucugauc uggggccucg gugcacaugc uuuacaugug 2460uuuagucgag guuaaaaaaa cgucuaggcc ccccgaacca cggggacgug guuuuccuuu 2520gaaaaacacg augauaccau gagcacaaau ccuaaaccuc aaagaaaaac caaaagaaac 2580accaaccguc gcccagaaga cguuaaguuc ccgggcggcg gccagaucgu uggcggagua 2640uacuuguugc cgcgcagggg ccccagguug ggugugcgca cgacaaggaa aacuucggag 2700cggucccagc cacgugggag acgccagccc auccccaaag aucggcgcuc cacuggcaag 2760gccuggggaa aaccaggucg ccccuggccc cuauauggga augagggacu cggcugggca 2820ggauggcucc uguccccccg aggcucucgc cccuccuggg gccccacuga cccccggcau 2880aggucgcgca acguggguaa agucaucgac acccuaacgu guggcuuugc cgaccucaug 2940ggguacaucc ccgucguagg cgccccgcuu aguggcgccg ccagagcugu cgcgcacggc 3000gugagagucc uggaggacgg gguuaauuau gcaacaggga accuacccgg uuuccccuuu 3060ucuaucuucu ugcuggcccu guuguccugc aucaccguuc cggucucugc ugcccaggug 3120aagaauacca guagcagcua cauggugacc aaugacugcu ccaaugacag caucacuugg 3180cagcucgagg cugcgguucu ccacgucccc gggugcgucc cgugcgagag aguggggaau 3240acgucacggu guugggugcc agucucgcca aacauggcug ugcggcagcc cggugcccuc 3300
acgcaggguc ugcggacgca caucgauaug guugugaugu ccgccaccuu cugcucugcu 3360cucuacgugg gggaccucug uggcggggug augcucgcgg cccagguguu caucgucucg 3420ccgcaguacc acugguuugu gcaagaaugc aauugcucca ucuacccugg caccaucacu 3480ggacaccgca uggcauggga caugaugaug aacuggucgc ccacggccac caugauccug 3540gcguacguga ugcgcguccc cgaggucauc auagacaucg uuagcggggc ucacuggggc 3600gucauguucg gcuuggccua cuucucuaug cagggagcgu gggcgaaggu cauugucauc 3660cuucugcugg ccgcuggggu ggacgcgggc accaccaccg uuggaggcgc uguugcacgu 3720uccaccaacg ugauugccgg cguguucagc cauggcccuc agcagaacau ucagcucauu 3780aacaccaacg gcaguuggca caucaaccgu acugccuuga auugcaauga cuccuugaac 3840accggcuuuc ucgcggccuu guucuacacc aaccgcuuua acucgucagg guguccaggg 3900cgccuguccg ccugccgcaa caucgaggcu uuccggauag gguggggcac ccuacaguac 3960gaggauaaug ucaccaaucc agaggauaug aggccguacu gcuggcacua ccccccaaag 4020ccguguggcg uaguccccgc gaggucugug uguggcccag uguacuguuu cacccccagc 4080ccgguaguag ugggcacgac cgacagacgu ggagugccca ccuacacaug gggagagaau 4140gagacagaug ucuuccuacu gaacagcacc cgaccgccgc agggcucaug guucggcugc 4200acguggauga acuccacugg uuucaccaag acuuguggcg cgccaccuug ccgcaccaga 4260gcugacuuca acgccagcac ggacuuguug ugcccuacgg auuguuuuag gaagcauccu 4320gaugccacuu auauuaagug ugguucuggg cccuggcuca caccaaagug ccugguccac 4380uacccuuaca gacucuggca uuaccccugc acagucaauu uuaccaucuu caagauaaga 4440auguauguag gggggguuga gcacaggcuc acggccgcau gcaacuucac ucguggggau 4500cgcugcgacu uggaggacag ggacaggagu cagcugucuc cucuguugca cucuaccacg 4560gaaugggcca uccugcccug caccuacuca gacuuacccg cuuugucaac uggucuucuc 4620caccuucacc agaacaucgu ggacguacaa uacauguaug gccucucacc ugcuaucaca 4680aaauacgucg uucgauggga guggguggua cucuuauucc ugcucuuagc ggacgccaga 4740gucugcgccu gcuuguggau gcucaucuug uugggccagg ccgaagcagc auuggagaag 4800uuggucgucu ugcacgcugc gagugcggcu aacugccaug gccuccuaua uuuugccauc 4860uucuucgugg cagcuuggca caucaggggu cggguggucc ccuugaccac cuauugccuc 4920acuggccuau ggcccuucug ccuacugcuc auggcacugc cccggcaggc uuaugccuau 4980gacgcaccug ugcacggaca gauaggcgug gguuuguuga uauugaucac ccucuucaca 5040cucaccccgg gguauaagac ccuccucggc cagugucugu ggugguugug cuaucuccug 5100acccuggggg aagccaugau ucaggagugg guaccaccca ugcaggugcg cggcggccgc 5160gauggcaucg cgugggccgu cacuauauuc ugcccgggug ugguguuuga cauuaccaaa 5220uggcuuuugg cguugcuugg gccugcuuac cucuuaaggg ccgcuuugac acaugugccg 5280uacuucguca gagcucacgc ucugauaagg guaugcgcuu uggugaagca gcucgcgggg 5340gguagguaug uucagguggc gcuauuggcc cuuggcaggu ggacuggcac cuacaucuau 5400gaccaccuca caccuauguc ggacugggcc gcuagcggcc ugcgcgacuu agcggucgcc 5460guggaaccca ucaucuucag uccgauggag aagaagguca ucgucugggg agcggagacg 5520gcugcaugug gggacauucu acauggacuu cccguguccg cccgacucgg ccaggagauc 5580cuccucggcc cagcugaugg cuacaccucc aaggggugga agcuccuugc ucccaucacu 5640gcuuaugccc agcaaacacg aggccuccug ggcgccauag uggugaguau gacggggcgu 5700gacaggacag aacaggccgg ggaaguccaa auccugucca cagucucuca guccuuccuc 5760ggaacaacca ucucgggggu uuuguggacu guuuaccacg gagcuggcaa caagacucua 5820gccggcuuac gggguccggu cacgcagaug uacucgagug cugaggggga cuugguaggc 5880uggcccagcc ccccugggac caagucuuug gagccgugca aguguggagc cgucgaccua 5940uaucugguca cgcggaacgc ugaugucauc ccggcucgga gacgcgggga caagcgggga 6000gcauugcucu ccccgagacc cauuucgacc uugaaggggu ccucgggggg gccggugcuc 6060ugcccuaggg gccacgucgu ugggcucuuc cgagcagcug ugugcucucg gggcguggcc 6120aaauccaucg auuucauccc cguugagaca cucgacguug uuacaagguc ucccacuuuc 6180agugacaaca gcacgccacc ggcugugccc cagaccuauc aggucgggua cuugcaugcu 6240
ccaacuggca guggaaagag caccaagguc ccugucgcgu augccgccca gggguacaaa 6300guacuagugc uuaaccccuc gguagcugcc acccuggggu uuggggcgua ccuauccaag 6360gcacauggca ucaaucccaa cauuaggacu ggagucagga ccgugaugac cggggaggcc 6420aucacguacu ccacauaugg caaauuucuc gccgaugggg gcugcgcuag cggcgccuau 6480gacaucauca uaugcgauga augccacgcu guggaugcua ccuccauucu cggcaucgga 6540acgguccuug aucaagcaga gacagccggg gucagacuaa cugugcuggc uacggccaca 6600ccccccgggu cagugacaac cccccauccc gauauagaag agguaggccu cgggcgggag 6660ggugagaucc ccuucuaugg gagggcgauu ccccuauccu gcaucaaggg agggagacac 6720cugauuuucu gccacucaaa gaaaaagugu gacgagcucg cggcggcccu ucggggcaug 6780ggcuugaaug ccguggcaua cuauagaggg uuggacgucu ccauaauacc agcucaggga 6840gauguggugg ucgucgccac cgacgcccuc augacggggu acacuggaga cuuugacucc 6900gugaucgacu gcaauguagc ggucacccaa gcugucgacu ucagccugga ccccaccuuc 6960acuauaacca cacagacugu cccacaagac gcugucucac gcagucagcg ccgcgggcgc 7020acagguagag gaagacaggg cacuuauagg uauguuucca cuggugaacg agccucagga 7080auguuugaca guguagugcu uugugagugc uacgacgcag gggcugcgug guacgaucuc 7140acaccagcgg agaccaccgu caggcuuaga gcguauuuca acacgcccgg ccuacccgug 7200ugucaagacc aucuugaauu uugggaggca guuuucaccg gccucacaca cauagacgcc 7260cacuuccucu cccaaacaaa gcaagcgggg gagaacuucg cguaccuagu agccuaccaa 7320gcuacggugu gcgccagagc caaggccccu cccccguccu gggacgccau guggaagugc 7380cuggcccgac ucaagccuac gcuugcgggc cccacaccuc uccuguaccg uuugggcccu 7440auuaccaaug aggucacccu cacacacccu gggacgaagu acaucgccac augcaugcaa 7500gcugaccuug aggucaugac cagcacgugg guccuagcug gaggaguccu ggcagccguc 7560gccgcauauu gccuggcgac uggaugcguu uccaucaucg gccgcuugca cgucaaccag 7620cgagucgucg uugcgccgga uaaggagguc cuguaugagg cuuuugauga gauggaggaa 7680ugcgccucua gggcggcucu caucgaagag gggcagcgga uagccgagau guugaagucc 7740aagauccaag gcuugcugca gcaggccucu aagcaggccc aggacauaca acccgcuaug 7800caggcuucau ggcccaaagu ggaacaauuu ugggccagac acauguggaa cuucauuagc 7860ggcauccaau accucgcagg auugucaaca cugccaggga accccgcggu ggcuuccaug 7920auggcauuca gugccgcccu caccaguccg uugucgacca guaccaccau ccuucucaac 7980aucaugggag gcugguuagc gucccagauc gcaccacccg cgggggccac cggcuuuguc 8040gucaguggcc uggugggggc ugccgugggc agcauaggcc uggguaaggu gcugguggac 8100auccuggcag gauauggugc gggcauuucg ggggcccucg ucgcauucaa gaucaugucu 8160ggcgagaagc ccucuaugga agaugucauc aaucuacugc cugggauccu gucuccggga 8220gcccuggugg ugggggucau cugcgcggcc auucugcgcc gccacguggg accgggggag 8280ggcgcggucc aauggaugaa caggcuuauu gccuuugcuu ccagaggaaa ccacgucgcc 8340ccuacucacu acgugacgga gucggaugcg ucgcagcgug ugacccaacu acuuggcucu 8400cuuacuauaa ccagccuacu cagaagacuc cacaauugga uaacugagga cugccccauc 8460ccaugcuccg gauccuggcu ccgcgacgug ugggacuggg uuugcaccau cuugacagac 8520uucaaaaauu ggcugaccuc uaaauuguuc cccaagcugc ccggccuccc cuucaucucu 8580ugucaaaagg gguacaaggg ugugugggcc ggcacuggca ucaugaccac gcgcugcccu 8640ugcggcgcca acaucucugg caauguccgc cugggcucua ugaggaucac agggccuaaa 8700accugcauga acaccuggca ggggaccuuu ccuaucaauu gcuacacgga gggccagugc 8760gcgccgaaac cccccacgaa cuacaagacc gccaucugga ggguggcggc cucggaguac 8820gcggagguga cgcagcaugg gucguacucc uauguaacag gacugaccac ugacaaucug 8880aaaauuccuu gccaacuacc uucuccagag uuuuucuccu ggguggacgg ugugcagauc 8940cauagguuug cacccacacc aaagccguuu uuccgggaug aggucucguu cugcguuggg 9000cuuaauuccu augcugucgg gucccagcuu cccugugaac cugagcccga cgcagacgua 9060uugaggucca ugcuaacaga uccgccccac aucacggcgg agacugcggc gcggcgcuug 9120gcacggggau caccuccauc ugaggcgagc uccucaguga gccagcuauc agcaccgucg 9180
cugcgggcca ccugcaccac ccacagcaac accuaugacg uggacauggu cgaugccaac 9240cugcucaugg agggcggugu ggcucagaca gagccugagu ccagggugcc cguucuggac 9300uuucucgagc caauggccga ggaagagagc gaccuugagc ccucaauacc aucggagugc 9360augcucccca ggagcggguu uccacgggcc uuaccggcuu gggcacggcc ugacuacaac 9420ccgccgcucg uggaaucgug gaggaggcca gauuaccaac cgcccaccgu ugcugguugu 9480gcucuccccc cccccaagaa ggccccgacg ccucccccaa ggagacgccg gacagugggu 9540cugagcgaga gcaccauauc agaagcccuc cagcaacugg ccaucaagac cuuuggccag 9600ccccccucga gcggugaugc aggcucgucc acgggggcgg gcgccgccga auccggcggu 9660ccgacguccc cuggugagcc ggcccccuca gagacagguu ccgccuccuc uaugcccccc 9720cucgaggggg agccuggaga uccggaccug gagucugauc agguagagcu ucaaccuccc 9780ccccaggggg gggggguagc ucccgguucg ggcucggggu cuuggucuac uugcuccgag 9840gaggacgaua ccaccgugug cugcuccaug ucauacuccu ggaccggggc ucuaauaacu 9900cccuguagcc ccgaagagga aaaguugcca aucaacccuu ugaguaacuc gcuguugcga 9960uaccauaaca agguguacug uacaacauca aagagcgccu cacagagggc uaaaaaggua 10020acuuuugaca ggacgcaagu gcucgacgcc cauuaugacu cagucuuaaa ggacaucaag 10080cuagcggcuu ccaaggucag cgcaaggcuc cucaccuugg aggaggcgug ccaguugacu 10140ccaccccauu cugcaagauc caaguaugga uucggggcca aggagguccg cagcuugucc 10200gggagggccg uuaaccacau caaguccgug uggaaggacc uccuggaaga cccacaaaca 10260ccaauuccca caaccaucau ggccaaaaau gagguguucu gcguggaccc cgccaagggg 10320gguaagaaac cagcucgccu caucguuuac ccugaccucg gcguccgggu cugcgagaaa 10380auggcccucu augacauuac acaaaagcuu ccucaggcgg uaaugggagc uuccuauggc 10440uuccaguacu ccccugccca acggguggag uaucucuuga aagcaugggc ggaaaagaag 10500gaccccaugg guuuuucgua ugauacccga ugcuucgacu caaccgucac ugagagagac 10560aucaggaccg aggaguccau auaccaggcc ugcucccugc ccgaggaggc ccgcacugcc 10620auacacucgc ugacugagag acuuuacgua ggagggccca uguucaacag caagggucaa 10680accugcgguu acagacguug ccgcgccagc ggggugcuaa ccacuagcau ggguaacacc 10740aucacaugcu augugaaagc ccuagcggcc ugcaaggcug cggggauagu ugcgcccaca 10800augcugguau gcggcaauga ccuaguaguc aucucagaaa gccaggggac ugaggaggac 10860gagcggaacc ugagagccuu cacggaggcc augaccaggu acucugcccc uccuggugau 10920ccccccagac cggaauauga ccuggagcua auaacauccu guuccucaaa ugugucugug 10980gcguugggcc cgcggggccg ccgcagauac uaccugacca gagacccaac cacuccacuc 11040gcccgggcug ccugggaaac aguuagacac uccccuauca auucauggcu gggaaacauc 11100auccaguaug cuccaaccau auggguucgc augguccuaa ugacacacuu cuucuccauu 11160cucauggucc aagacacccu ggaccagaac cucaacuuug agauguaugg aucaguauac 11220uccgugaauc cuuuggaccu uccagccaua auugagaggu uacacgggcu ugacgccuuu 11280ucuaugcaca cauacucuca ccacgaacug acgcgggugg cuucagcccu cagaaaacuu 11340ggggcgccac cccucagggu guggaagagu cgggcucgcg cagucagggc gucccucauc 11400ucccguggag ggaaagcggc cguuugcggc cgauaucucu ucaauugggc ggugaagacc 11460aagcucaaac ucacuccauu gccggaggcg cgccuacugg acuuauccag uugguucacc 11520gucggcgccg gcgggggcga cauuuuucac agcgugucgc gcgcccgacc ccgcucauua 11580cucuucggcc uacuccuacu uuucguaggg guaggccucu uccuacuccc cgcucgguag 11640agcggcacac acuagguaca cuccauagcu aacuguuccu uuuuuuuuuu uuuuuuuuuu 11700uuuuuuuuuu uuuuuuuuuu uucuuuuuuu uuuuuuuccc ucuuucuucc cuucucaucu 11760uauucuacuu ucuuucuugg uggcuccauc uuagcccuag ucacggcuag cugugaaagg 11820uccgugagcc gcaugacugc agagagugcc guaacugguc ucucugcaga ucaugu 1187權(quán)利要求
1.一種復(fù)制子RNA，所述RNA含有的核苷酸序列包含基因型2a丙型肝炎病毒基因組RNA的5′非翻譯區(qū)、核心蛋白編碼序列、E1蛋白編碼序列、E2蛋白編碼序列、NS2蛋白編碼序列、NS3蛋白編碼序列、NS4A蛋白編碼序列、NS4B蛋白編碼序列、NS5A蛋白編碼序列、NS5B蛋白編碼序列和3′非翻譯區(qū)，至少一種選擇標(biāo)記基因和/或至少一種報(bào)告基因以及至少一種IRES序列。
2.權(quán)利要求1的復(fù)制子RNA，其中所述核苷酸序列以5′-3′方向按以下順序包含5′非翻譯區(qū)、至少一種選擇標(biāo)記基因和/或至少一種報(bào)告基因、至少一種IRES序列，以及核心蛋白編碼序列、E1蛋白編碼序列、E2蛋白編碼序列、NS2蛋白編碼序列、NS3蛋白編碼序列、NS4A蛋白編碼序列、NS4B蛋白編碼序列、NS5A蛋白編碼序列、NS5B蛋白編碼序列和3′非翻譯區(qū)。
3.權(quán)利要求1或2的復(fù)制子RNA，其中基因型2a丙型肝炎病毒基因組RNA是包含SEQ ID NO12所示核苷酸序列的RNA。
4.權(quán)利要求1-3中任一項(xiàng)的復(fù)制子RNA，其中5′非翻譯區(qū)含SEQID NO1所示核苷酸序列，核心蛋白編碼序列含SEQ ID NO2所示核苷酸序列，E1蛋白編碼序列含SEQ ID NO3所示核苷酸序列，E2蛋白編碼序列含SEQ ID NO4所示核苷酸序列，NS2蛋白編碼序列含SEQ ID NO5所示核苷酸序列，NS3蛋白編碼序列含SEQ ID NO6所示核苷酸序列，NS4A蛋白編碼序列含SEQ ID NO7所示核苷酸序列，NS4B蛋白編碼序列含SEQ ID NO8所示核苷酸序列，NS5A蛋白編碼序列含SEQ ID NO9所示核苷酸序列，NS5B蛋白編碼序列含SEQ ID NO10所示核苷酸序列，而3′非翻譯區(qū)含SEQ ID NO11所示核苷酸序列。
5.一種復(fù)制子RNA，所述RNA包含以下(a)或(b)的RNA(a)含SEQ ID NO13所示核苷酸序列的RNA；或(b)含通過缺失、置換或添加1-100個(gè)核苷酸而衍生自SEQ ID NO13所示核苷酸序列的核苷酸序列、并具有自主復(fù)制能力和病毒顆粒生產(chǎn)能力的RNA。
6.一種生產(chǎn)細(xì)胞的方法，其中所述細(xì)胞復(fù)制復(fù)制子RNA并生產(chǎn)病毒顆粒，所述方法包括將權(quán)利要求1-5中任一項(xiàng)的復(fù)制子RNA導(dǎo)入到細(xì)胞中。
7.權(quán)利要求6的方法，其中所述細(xì)胞為增殖性細(xì)胞。
8.權(quán)利要求6或7的方法，其中所述細(xì)胞為真核細(xì)胞。
9.權(quán)利要求8的方法，其中所述真核細(xì)胞為人肝源細(xì)胞、人子宮頸源細(xì)胞或人胎腎源細(xì)胞。
10.權(quán)利要求8的方法，其中所述真核細(xì)胞為Huh7細(xì)胞、HepG2細(xì)胞、IMY-N9細(xì)胞、HeLa細(xì)胞或293細(xì)胞。
11.權(quán)利要求6-10中任一項(xiàng)的方法可獲得的細(xì)胞，所述細(xì)胞復(fù)制復(fù)制子RNA并生產(chǎn)病毒顆粒。
12.一種生產(chǎn)丙型肝炎病毒顆粒的方法，所述方法包括培養(yǎng)權(quán)利要求11的細(xì)胞，以使該細(xì)胞生產(chǎn)病毒顆粒。
13.一種丙型肝炎病毒顆粒，所述顆?？衫脵?quán)利要求12的方法獲得。
14.一種生產(chǎn)丙型肝炎病毒感染細(xì)胞的方法，所述方法包括培養(yǎng)權(quán)利要求11的細(xì)胞，并周培養(yǎng)物中的病毒顆粒感染其它細(xì)胞。
15.一種丙型肝炎病毒感染細(xì)胞，所述細(xì)胞可利用權(quán)利要求14的方法獲得。
16.一種篩選抗丙型肝炎病毒的物質(zhì)的方法，所述方法包括在測(cè)試物存在下培養(yǎng)選自以下(a)、(b)和(c)中的至少一種(a)權(quán)利要求11的細(xì)胞，(b)權(quán)利要求15的丙型肝炎病毒感染細(xì)胞，(c)權(quán)利要求13的丙型肝炎病毒顆粒和丙型肝炎病毒允許細(xì)胞；并檢測(cè)產(chǎn)生的培養(yǎng)物中的復(fù)制子RNA或病毒顆粒。
17.一種丙型肝炎疫苗，所述疫苗包含權(quán)利要求13的丙型肝炎病毒顆?；蚱洳糠?。
18.一種生產(chǎn)丙型肝炎疫苗的方法，所述方法使用權(quán)利要求13的丙型肝炎病毒顆?；蚱洳糠肿鳛榭乖?。
19.一種生產(chǎn)用于基因治療的嗜肝病毒載體的方法，所述方法使用權(quán)利要求1-5中任一項(xiàng)的復(fù)制子RNA。
20.一種嗜肝病毒載體，所述載體可利用權(quán)利要求18的方法獲得。
21.一種在細(xì)胞中復(fù)制和/或表達(dá)外源基因的方法，所述方法包括將外源基因編碼RNA插入到權(quán)利要求1-5中任一項(xiàng)的復(fù)制子RNA中，并將其導(dǎo)入到所述細(xì)胞中。
22.一種生產(chǎn)細(xì)胞的方法，其中所述細(xì)胞復(fù)制RNA并生產(chǎn)病毒顆粒，所述方法包括將含SEQ ID NO12所示核苷酸序列的RNA導(dǎo)入到細(xì)胞中。
23.一種生產(chǎn)丙型肝炎病毒顆粒的方法，所述方法包括將含SEQID NO12所示核苷酸序列的RNA導(dǎo)入到細(xì)胞中，并培養(yǎng)細(xì)胞，以使該細(xì)胞生產(chǎn)病毒顆粒。
24.權(quán)利要求21或22的方法，其中所述細(xì)胞是增殖性細(xì)胞。
25.一種生產(chǎn)含外源基因的病毒載體的方法，所述方法包括將外源基因編碼RNA插入到含SEQ ID NO12所示核苷酸序列的RNA中，將其導(dǎo)入到細(xì)胞中，并培養(yǎng)細(xì)胞，以使其生產(chǎn)病毒顆粒。
26.一種抗體，所述抗體抗權(quán)利要求13的丙型肝炎病毒顆粒。
全文摘要
本發(fā)明提供有效復(fù)制含全長(zhǎng)HCV基因組序列的RNA的方法和使用細(xì)胞培養(yǎng)系統(tǒng)生產(chǎn)含全長(zhǎng)HCV復(fù)制子RNA或全長(zhǎng)HCV基因組RNA的HCV病毒顆粒的方法。此外，本發(fā)明涉及生產(chǎn)丙型肝炎病毒顆粒的方法，其包括培養(yǎng)其中導(dǎo)入復(fù)制子RNA的細(xì)胞，并在培養(yǎng)基中產(chǎn)生病毒顆粒，其中所述復(fù)制子RNA包含的核苷酸序列含有基因型2a丙型肝炎病毒的全長(zhǎng)基因組RNA序列、至少一種選擇標(biāo)記基因和/或至少一種報(bào)告基因以及至少一種IRES序列，或含有基因型2a丙型肝炎病毒的全長(zhǎng)基因組RNA。再進(jìn)一步，本發(fā)明還涉及丙型肝炎疫苗和抗丙型肝炎病毒顆粒的抗體。
文檔編號(hào)C12N1/21GK1942585SQ20058001151
公開日2007年4月4日 申請(qǐng)日期2005年2月21日 優(yōu)先權(quán)日2004年2月20日
發(fā)明者脅田隆字, 加藤孝宣, 伊達(dá)朋子, 宮本道子, 田邊純一, 曾根三郎 申請(qǐng)人:財(cái)團(tuán)法人東京都醫(yī)學(xué)研究機(jī)構(gòu), 東麗株式會(huì)社