本發(fā)明涉及生物遺傳工程技術(shù)，具體是涉及一種熱穩(wěn)定性提高的頭孢菌素C?；竿蛔凅w及其構(gòu)建方法。

背景技術(shù)：

頭孢菌素類(cephalosporins)抗生素是繼青霉素之后發(fā)現(xiàn)的第二類應(yīng)用于臨床治療的廣譜抗生素，約占全球抗生素市場的40％(Turkish Journal ofBiochemistry,2014,39(1):51-56)。7-氨基頭孢烷酸(7-ACA)作為許多半合成頭孢菌素類抗生素的關(guān)鍵中間體，在醫(yī)藥工業(yè)具有重要價(jià)值。目前，7-ACA主要由頭孢菌素C(Cephalosporin C，CPC)經(jīng)化學(xué)法或酶法合成。相對于化學(xué)法，酶法具有工藝簡單，安全，高效，污染小等優(yōu)勢，因此日益受到人們的重視。

7-ACA酶法生產(chǎn)主要分為兩步酶法和一步酶法兩種。兩步酶法在國內(nèi)外已經(jīng)成功工業(yè)化，并逐步取代化學(xué)法。雖然兩步酶法避免了環(huán)境污染問題，但由于兩步酶法需要采用D-氨基酸氧化酶和戊二酰-7-氨基頭孢烷酸(GL-7-ACA)酰化酶共同作用(Critical reviews in biotechnology,1998,18(1):1-12)，導(dǎo)致大量副產(chǎn)物產(chǎn)生，降低了產(chǎn)率、提高了成本，因此無法滿足工業(yè)生產(chǎn)的進(jìn)一步需求(Applied microbiology and biotechnology,2013,97(6):2341-2355)。一步酶法是一種新興的7-ACA生產(chǎn)方法，它采用CPC?；钢苯右徊锦；疌PC高效獲得7-ACA(Journal ofmicrobiological methods,2003,54(1):131-135)，一步酶法既具有化學(xué)法轉(zhuǎn)化率高和純度高的優(yōu)勢也具有兩步酶法低污染的優(yōu)勢，具有良好的工業(yè)應(yīng)用前景。

來源于Pseudomonassp.SE83的頭孢菌素C酰化酶SE83acyII編碼83kDa左右的多肽前體，并經(jīng)翻譯后自剪切得到25kDaα亞基和58kDaβ亞基組成的成熟蛋白能夠完成從CPC到7-ACA的一步酶法轉(zhuǎn)化(Protein Engineering Design and Selection,2010,23(12):903-909)。韓國學(xué)者Shin等對Pseudomonassp.SE83acyII進(jìn)行了突變研究，得到的多點(diǎn)突變體V122αA/G140αS/F58βN/I75βT/I176βV/S471βC(也被命名為Pseudomonas sp.SE83acyII S12)相比于野生型酶活提高了8.5倍，并且產(chǎn)物抑制也顯著降低(U.S.Patent7,592,168[P].2009-9-22；US Patent20,070,207,519[P].2007)。然而，CPC酰化酶在應(yīng)用中仍存在對熱敏感、穩(wěn)定性不佳等一些問題(Applied and environmental microbiology,1996,62(8):2919-2925；World Journal of Microbiology and Biotechnology,2011,27(4):823-829)，SE83acyII S12最適溫度僅為40℃，生產(chǎn)過程中的發(fā)熱易使CPC酶失活，導(dǎo)致催化效率降低。因此，提高CPC酶熱穩(wěn)定性使其更好的適應(yīng)運(yùn)輸、工業(yè)生產(chǎn)等條件是推動(dòng)其進(jìn)一步工業(yè)應(yīng)用的關(guān)鍵。

本發(fā)明通過分析頭孢菌素C酰化酶結(jié)構(gòu)上的高B因子位置選擇突變熱點(diǎn)，應(yīng)用分子生物學(xué)技術(shù)構(gòu)建定點(diǎn)飽和突變庫，基于pH指示劑法并利用FX^P實(shí)驗(yàn)室自動(dòng)工作站建立高效的篩選程序，快速篩選出穩(wěn)定性提高的突變體，進(jìn)一步推進(jìn)頭孢菌素C?；傅膬?yōu)良改造，為工業(yè)化生產(chǎn)奠定基礎(chǔ)。

技術(shù)實(shí)現(xiàn)要素：

為實(shí)現(xiàn)上述發(fā)明目的，本發(fā)明提供一種熱穩(wěn)定性提高的頭孢菌素C酰化酶突變體及其構(gòu)建方法。

本發(fā)明的技術(shù)方案是提供一種熱穩(wěn)定性提高的頭孢菌素C?；竿蛔凅w，所述頭孢菌素C酰化酶的氨基酸序列如SEQ ID NO:1所示，或者該序列經(jīng)取代、缺失或添加一個(gè)或多個(gè)氨基酸且與SED ID NO:1所示蛋白具有相同功能的衍生氨基酸序列；編碼該酶的核酸序列如SEQ ID NO:2所示，或者為與SEQ ID NO:2所示序列具有90％以上同源性，且編碼具有頭孢菌素C?；富钚缘牡鞍踪|(zhì)的核酸序列；所述頭孢菌素C?；竿蛔凅w的氨基酸序列是由SEQ ID NO:1所示序列上的氨基酸被另一種氨基酸取代而獲得，所述取代位置為SEQ ID NO:1表示的氨基酸序列的第113位谷氨酸、218位精氨酸、226位賴氨酸、334位谷氨酸、454位谷氨酸、547位甘氨酸、632位色氨酸中的任意一個(gè)。

進(jìn)一步地，所述頭孢菌素C酰化酶突變體，其氨基酸序列為所述第113位谷氨酸被天冬酰胺取代而獲得如SEQ ID NO:3所示的氨基酸序列。

進(jìn)一步地，所述頭孢菌素C?；竿蛔凅w，其氨基酸序列為所述第218位精氨酸被蘇氨酸取代而獲得如SEQ ID NO:4所示的氨基酸序列，或者被谷氨酰胺取代而獲得如SEQ ID NO:5所示的氨基酸序列。

進(jìn)一步地，所述頭孢菌素C酰化酶突變體，其氨基酸序列為所述第226位賴氨酸被纈氨酸取代而獲得如SEQ ID NO:6所示的氨基酸序列。

進(jìn)一步地，所述頭孢菌素C酰化酶突變體，其氨基酸序列為所述第334位谷氨酸被精氨酸取代而獲得如SEQ ID NO:7所示的氨基酸序列。

進(jìn)一步地，所述頭孢菌素C?；竿蛔凅w，其氨基酸序列所述第454位谷氨酸被天冬氨酸取代而獲得如SEQ ID NO:8所示的氨基酸序列。

進(jìn)一步地，所述頭孢菌素C?；竿蛔凅w，其氨基酸序列為所述第547位甘氨酸被谷氨酸取代而獲得如SEQ ID NO:9所示的氨基酸序列。

進(jìn)一步地，所述頭孢菌素C?；竿蛔凅w，其氨基酸序列為所述第632位色氨酸被組氨酸取代而獲得如SEQ ID NO:10所示的氨基酸序列。

進(jìn)一步地，通過基因工程手段在所述頭孢菌素C?；竿蛔凅w的N端與C端引入標(biāo)簽，優(yōu)選為組氨酸標(biāo)簽(His-tag)。

本發(fā)明還提供一種熱穩(wěn)定性提高的頭孢菌素C?；竿蛔凅w的構(gòu)建方法，步驟包括：

1)頭孢菌素C?；负怂嵝蛄幸源竽c桿菌為宿主進(jìn)行密碼子優(yōu)化，并由專業(yè)商業(yè)公司設(shè)計(jì)上下游引物；利用上下游引物，通過PCR(聚合酶鏈?zhǔn)椒磻?yīng))擴(kuò)增法，采用一定的PCR反應(yīng)體系和一系列溫度控制，擴(kuò)增頭孢菌素C?；富?；

2)使用常規(guī)的酶切、連接方式克隆基因至大腸桿菌表達(dá)載體；使用常規(guī)的大腸桿菌培養(yǎng)基培養(yǎng)工程菌，IPTG(異丙基-β-D-硫代吡喃半乳糖苷)誘導(dǎo)劑誘導(dǎo)目的蛋白的表達(dá)；后破碎工程菌并分離純化頭孢菌素C?；福?/p>

3)利用專業(yè)軟件計(jì)算頭孢菌素C?；附Y(jié)構(gòu)上各氨基酸殘基B因子值，選取高B因子氨基酸殘基位置作為突變熱點(diǎn)；

4)通過全質(zhì)粒突變法引入定點(diǎn)飽和突變，采用上述1)、2)中的方法進(jìn)行突變基因擴(kuò)增和工程菌的培養(yǎng)，建立頭孢菌素C?；竿蛔儙?；

5)使用常規(guī)培養(yǎng)方法將工程菌放大培養(yǎng)，后破碎分離取得上清液，加入底物，通過檢測單位時(shí)間內(nèi)吸光值的變化來篩選熱穩(wěn)定性提高的突變體；

6)將篩選獲得的突變體通過2)中的方法培養(yǎng)后，測定頭孢菌素C?；富盍?，利用殘余活力百分比的ln值和時(shí)間計(jì)算其在特定溫度下的半衰期，確定不同突變體的最適溫度。

本發(fā)明的最終的核酸序列通?？梢杂肞CR擴(kuò)增法、基因重組或人工合成中的一種或幾種方法獲得。之后再將其克隆入載體，再轉(zhuǎn)入細(xì)胞，然后通過常規(guī)方法從增殖后的宿主細(xì)胞中分離得到有關(guān)序列。

本發(fā)明的頭孢菌素C?；竿蛔凅w用基因重組方法來制備，具體為，通過PCR方法克隆野生型頭孢菌素C酰化酶基因，選用的表達(dá)載體包括各種含有啟動(dòng)子、調(diào)控基因、篩選基因和克隆位點(diǎn)等元件的質(zhì)粒，例如選用質(zhì)粒pET28a(Novagen)，該質(zhì)粒可在市場上獲得；然后將上述表達(dá)質(zhì)粒轉(zhuǎn)化到重組工程細(xì)胞宿主內(nèi)，從而獲得表達(dá)頭孢菌素C?；傅闹亟M工程細(xì)胞。這些重組工程細(xì)胞可以是來源于動(dòng)物細(xì)胞、植物細(xì)胞、昆蟲細(xì)胞、真菌、酵母和細(xì)菌等，質(zhì)粒pET28a適合轉(zhuǎn)化入大腸桿菌中表達(dá)酰化酶，因此優(yōu)選的重組工程細(xì)胞宿主為大腸桿菌，例如選擇大腸桿菌Escherichia coli BL21(DE3)-CodonPlus-RIL。

本文所用的術(shù)語“轉(zhuǎn)化”是指用基因工程領(lǐng)域技術(shù)人員熟知的方法：將含有目的基因的表達(dá)載體導(dǎo)入宿主細(xì)胞內(nèi)。轉(zhuǎn)化方法因宿主細(xì)胞類型而異，通常包括：電轉(zhuǎn)化；采用氯化鈣、DEAE-葡聚糖或其它物質(zhì)的轉(zhuǎn)染；微粒轟擊；脂質(zhì)體轉(zhuǎn)染；感染等。本發(fā)明中較佳的方法是CaCl₂熱擊化學(xué)轉(zhuǎn)化的方法；隨后，在適宜培養(yǎng)條件下繁殖宿主細(xì)胞。

本領(lǐng)域技術(shù)人員根據(jù)常規(guī)試驗(yàn)就能選擇和確定培養(yǎng)基配方、培養(yǎng)溫度、誘導(dǎo)物、誘導(dǎo)劑量和時(shí)間等條件。采用本領(lǐng)域常規(guī)檢測手段，如聚丙烯酰胺凝膠電泳(SDS-PAGE)、酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)等，可以檢測出本發(fā)明?；傅谋磉_(dá)，本發(fā)明采用1％瓊脂糖凝膠電泳檢測。最后，可用常規(guī)的蛋白分離純化技術(shù)，進(jìn)行酰化酶的純化，其包括離心、過濾、層析等過程；具體地，層析方法又包括親和層析、凝膠過濾、離子交換色譜以及疏水層析等；本發(fā)明提供的該?；傅姆蛛x純化方法為Ni-NTA親和層析。

本發(fā)明的優(yōu)點(diǎn)和有益效果：

1、本發(fā)明提供的頭孢菌素C?；竿蛔凅w，在相同條件下與野生型頭孢菌素C?；妇哂邢嗨频母叽呋钚院彤a(chǎn)物抑制趨勢，且其半衰期t_1/2均在72min以上，長于野生型頭孢菌素C?；傅?1.34min，說明突變體穩(wěn)定性高于野生型，在運(yùn)輸和工業(yè)生產(chǎn)過程中，失活程度較低，其中突變體R218Q最適溫度為43℃左右，較野生型提高3℃，半衰期為231.05min，為野生型的3.76倍，該結(jié)果表明突變體能更好地適應(yīng)反應(yīng)溫度，保證生產(chǎn)催化效率。

2、突變體不但具有更好的熱穩(wěn)定性，而且與野生型頭孢菌素C?；赶啾龋瑩碛邢嗨频母叽呋钚院彤a(chǎn)物抑制趨勢，尤其是R218T，R218Q和K226V，其催化效率k_cat/K_m都高于野生型，更適合工業(yè)生產(chǎn)應(yīng)用。

3、本發(fā)明提供了一種頭孢菌素C?；竿蛔凅w構(gòu)建方法，通過分析高同源蛋白各氨基酸殘基B因子，針對性地對頭孢菌素C?；傅慕Y(jié)構(gòu)進(jìn)行改進(jìn)，通過基因工程方法來獲得頭孢菌素C?；竿蛔凅w，所得的突變體純度、穩(wěn)定性以及催化效率等特性更加適應(yīng)工業(yè)化生產(chǎn)。

4、而在目前SE83acyII S12晶體結(jié)構(gòu)尚未清晰無法直接進(jìn)行分析的情況下，提供了同源建模方法，通過分析高同源N176acy結(jié)構(gòu)的B因子來指導(dǎo)確定突變熱點(diǎn)，擴(kuò)大了獲得頭孢菌素C?；竿蛔凅w的來源，并提高了篩選出符合要求的突變體的幾率。

附圖說明

圖1頭孢菌素C?；?N176acy)高B因子示意圖。

圖2頭孢菌素C?；?SE83acyII S12)突變位點(diǎn)示意圖。

圖3溫度對頭孢菌素C?；讣捌渫蛔凅w活性的影響。

圖4不同的7-ACA底物濃度下頭孢菌素C?；福蛔凅wR218T，R218Q，K226V的產(chǎn)物抑制趨勢。

具體實(shí)施方式

下面結(jié)合具體的實(shí)施例，并參照數(shù)據(jù)進(jìn)一步詳細(xì)描述本發(fā)明。應(yīng)理解，這些實(shí)施例只是為了舉例說明本發(fā)明，而非以任何方式限制發(fā)明的范圍。

本發(fā)明生物材料的來源的一般性說明：

1、引物合成：本發(fā)明中所使用的引物均由南京金斯瑞科技有限公司合成。

2、實(shí)驗(yàn)中所使用Dpn I購于美國New England Biolabs公司，PCR產(chǎn)物純化試劑盒、膠回收產(chǎn)物純化試劑盒和質(zhì)粒小量提取試劑盒，購于美國Axygen Scientific公司；DNA限制性內(nèi)切酶，T4DNA ligase和Prestained Protein Ladder，購于Fermentas公司；PrimeStar Max DNA聚合酶，購于日本Takara公司；DNAMarker、MilliporeAmicon Ultrafiltration Centrifuge Tubes、BCAProteinAssay Kit，購于Thermo Fisher Scientific公司；序列測序?yàn)槿A大基因完成。

實(shí)施例1野生型頭孢菌素C?；富虻目寺?/p>

將來源于Pseudomonas sp.SE83的頭孢菌素C?；富蛞源竽c桿菌為宿主進(jìn)行密碼子優(yōu)化，設(shè)計(jì)上游引物和下游引物序列分別如下：

上游引物F：5'-ATATCATATGACGATGGCGGCCAAGACCGATCGCGAGGCCCTGCAGGCGGCGCTGCCGCCGCTTTCCGGCAGCCTCTCCATTCCGGGTTTAAGCGCCCCTG-3'

下游引物R：5'-ATATCTCGAGTTAGGCCGGAACCAGCTCCTGGCTG-3'

其中下劃線分別為NdeI和XhoI酶切位點(diǎn)。PCR反應(yīng)體系：2×PrimeStar Max DNA聚合酶25μL，上下游引物(10μmol/L)各1.0μL，基因模板(50ng/μL)1.0μL，雙蒸水為22μL。PCR反應(yīng)條件為：98℃2min，然后98℃10sec，55℃15sec，72℃30sec，共25個(gè)循環(huán)；最后72℃10min。反應(yīng)結(jié)束后，對PCR擴(kuò)增產(chǎn)物進(jìn)行1％瓊脂糖凝膠電泳檢測，得到2.4kb大小的條帶，與預(yù)期結(jié)果相符。DpnI消化模板，回收，純化該目的片段，將其用限制性內(nèi)切酶NdeI和XhoI進(jìn)行雙酶切后與經(jīng)同樣酶雙酶切的質(zhì)粒pET28a(Novagen)進(jìn)行連接，將連接產(chǎn)物轉(zhuǎn)化大腸桿菌Escherichia coli BL21(DE3)-CodonPlus-RIL感受態(tài)細(xì)胞中，將轉(zhuǎn)化細(xì)胞涂布于含有50μg/mL卡那霉素的LB平板上篩選陽性克隆，提取質(zhì)粒，對其進(jìn)行測序，測序結(jié)果表明克隆的頭孢菌素C?；富蛐蛄姓_，且正確連入pET28a中，將該重組質(zhì)粒命名為pET28a-SE83-S12。

實(shí)施例2野生型頭孢菌素C?；傅谋磉_(dá)、純化

工程菌按體積比1％接種到含100μg/mL Kan的4mL LB培養(yǎng)基試管中，37℃220rpm培養(yǎng)12h。將該4mL菌液轉(zhuǎn)接至含50μg/mL Kan的1L LB培養(yǎng)基搖瓶中，37℃220rpm培養(yǎng)約2.5h，使OD600達(dá)到0.9左右，加入0.5mM IPTG誘導(dǎo)劑，25℃220rpm誘導(dǎo)培養(yǎng)12-16h。將發(fā)酵后收獲的大腸桿菌菌體懸液超聲破碎，再經(jīng)過Ni-NTA親和層析處理，得到95％以上純度的目的蛋白。

實(shí)施例3野生型頭孢菌素C酰化酶同源建模

由于野生型頭孢菌素C?；窼E83acyII S12晶體結(jié)構(gòu)仍未得到解析，難以通過常規(guī)的蛋白質(zhì)理性設(shè)計(jì)手段進(jìn)行操作，因此提供一種以高同源N176acy(PDB ID:4HSR)為模板進(jìn)行同源建模的方法。

1)進(jìn)入SWISS-MODEL數(shù)據(jù)庫主頁(https://www.swissmodel.expasy.org/)，在Target Sequence工具中輸入頭孢菌素C?；窼E83acyII S12的氨基酸序列進(jìn)行搜索。服務(wù)器直接搜索同源性最高的序列4HSR作為模板進(jìn)行序列比對，并得到目的蛋白結(jié)構(gòu)模型。

2)在Protein Data Bank(PDB數(shù)據(jù)庫)中搜索與頭孢菌素C?；窼E83acyII S12同源性最高，來源于Pseudomonas diminutaN176的頭孢菌素C?；?N176acy，PDB ID：4HSR)的結(jié)構(gòu)，利用Discovery studio軟件對SE83acyII S12結(jié)構(gòu)模型與N176acy晶體結(jié)構(gòu)進(jìn)行比對，進(jìn)一步驗(yàn)證兩者結(jié)構(gòu)上的高同源性。

實(shí)施例4野生型頭孢菌素C?；竿蛔儫狳c(diǎn)的選擇

通過B因子分析的方法確定突變熱點(diǎn)。而對于SE83acyII S12，由于其晶體結(jié)構(gòu)未知，因此通過上述同源建模方法，以高同源N176acy(PDB ID:4HSR)晶體結(jié)構(gòu)為指導(dǎo)選擇突變熱點(diǎn)。

1)N176acy B因子分析：在Protein Data Bank(PDB數(shù)據(jù)庫)中搜索與頭孢菌素C?；窼E83acyII S12同源性最高，來源于Pseudomonas diminutaN176的頭孢菌素C酰化酶(N176acy)的結(jié)構(gòu)。N176acy由α鏈，β鏈兩個(gè)亞基組成，使用Discovery studio軟件對N176acy兩個(gè)亞基中每一個(gè)氨基酸按照B因子大小進(jìn)行排序(表1)，選取每個(gè)亞基中B因子較大的殘基作為突變熱點(diǎn)，分別為ARG218,LYS226,GLU113,ASP547,TRP632,ASP548,GLU454,GLU764和ASP334(圖1)。

表1N176acyα，β鏈氨基酸殘基B因子排序

2)突變位點(diǎn)的對應(yīng)：用PyMOL對N176acy晶體結(jié)構(gòu)與SE83acyII S12結(jié)構(gòu)模型進(jìn)行比對，并根據(jù)比對結(jié)果，將N176acy中高B因子的位點(diǎn)對應(yīng)到SE83acyII S12結(jié)構(gòu)模型上，在SE83acyII S12中分別為ARG218,LYS226,GLU113,GLY547,TRP632,SER548,GLU454,GLU764和GLU334(圖2)

實(shí)施例5野生型頭孢菌素C?；竿蛔儙旖⒓巴蛔凅w篩選

本發(fā)明采用簡易全質(zhì)粒PCR突變法(site-directed mutagenesis method)引入定點(diǎn)飽和突變。設(shè)計(jì)引物序列如下：

R218 5'TAGCAGCCCTGNNKCCGGCCGTTGATGCATTACTGAAAGC 3'

5'ATCAACGGCCGGMNNCAGGGCTGCTAAATCGGCTTCCAGG 3'

K226 5'GATGCATTACTGNNKGCCATGGGTGGTGACGCCAGT 3'

5'CCACCCATGGCMNNCAGTAATGCATCAACGGCCGGAC 3'

E113 5'GAAGCCCTGGGCGCANNKGCAAAGGATATGCTGC 3'

5'TATCCTTTGCMNNTGCGCCCAGGGCTTCAAAATCG 3'

G547 5'CCTGGGTATTCAGNNKAGCCTGCCGGCAGAAGAACT 3'

5’CCGGCAGGCTMNNCTGAATACCCAGGGCTTCTAAG 3’

W632 5’GGCATGTTAAAAGGCNNKAGCTGGGATGAGGCCCTGAGCGAAG 3’

5’CTCATCCCAGCTMNNGCCTTTTAACATGCCTGCGTCG 3’

S548 5’GGTATTCAGGGTNNKCTGCCGGCAGAAGAACTGCGCCAGACC 3’

5’CTGCCGGCAGMNNACCCTGAATACCCAGGGCTTCTAAG 3’

E454 5’GGTTGGAGTGGTNNKCACGAATGGCGCGGTTGGATTCCTC 3’

5’AACCGCGCCATTCGTGMNNACCACTCCAACCCGGAACC 3’

E764 5’CGTATCGCAGCCNNKGCCGTGACCAGCCAGGAGC 3’

5’GCTGGTCACGGCMNNGGCTGCGATACGATCCCAGCTATACAGC 3’

E334 5’CACGCTTTGGCAATNNKTTCGAACCTGTGGCCTGG 3’

5’ACAGGTTCGAAMNNATTGCCAAAGCGTGCGGTGCG 3’

PCR反應(yīng)體系(50μL)為：2×PrimeStar Max聚合酶25μL，上下游引物(10μmol/L)各1.0μL，模板(50ng/μL)1.0μL，雙蒸水為22μL。PCR反應(yīng)條件為：98℃2min，然后98℃10sec，55℃15sec，72℃2min，共25個(gè)循環(huán)；最后72℃10min。反應(yīng)結(jié)束后，對PCR擴(kuò)增產(chǎn)物進(jìn)行1％瓊脂糖凝膠電泳檢測，得到9kb大小的條帶，與預(yù)期結(jié)果相符。DpnI消化模板后，使用Axygen PCR clean up試劑盒回收純化PCR產(chǎn)物，轉(zhuǎn)化至BL21(DE3)codon plus-RIL感受態(tài)細(xì)胞，得到飽和突變文庫，且?guī)烊萘看笥?00個(gè)克隆。

在96孔板每孔中加入200μL含100mg/L Kan的LB培養(yǎng)基，用微生物菌落篩選系統(tǒng)Qpix460挑取單菌落于每孔中，放入搖床37℃、220rpm過夜培養(yǎng)，次日在每孔中加入甘油使終濃度為15％，-80℃凍存作為飽和突變文庫母版。吸取10μL母版中的母菌液加入含有140μL，100mg/L Kan的LB培養(yǎng)基中，放入搖床37℃、220rpm培養(yǎng)至菌液OD600為0.6-0.8，再加入混有IPTG誘導(dǎo)劑的LB培養(yǎng)基至200μL，使IPTG終濃度為0.5mM。在25℃、220rpm條件下誘導(dǎo)12-16h后，將裝有菌液的96孔板用高速冷凍離心機(jī)4500rpm，離心1h，取出并棄盡上清液，菌體放入-80℃冰箱過夜凍存。次日將96孔板從-80℃冰箱中取出在室溫下自然融化1h后再次放入冰箱2h，如此反復(fù)凍融3次，使細(xì)胞充分破碎，再加入200μL 5mM Tris-HCl(pH8.0)，放置于震板儀上充分搖勻，用高速冷凍離心機(jī)4500rpm離心15min后，所得上清液即為粗酶液。將上清液分別加入兩塊96孔板，孔中含有100μL粗酶液，將其中一塊置于室溫下，另一塊放置于55℃水浴鍋中加熱10min，F(xiàn)X^P工作站吸取兩塊板各孔70μL的酶液加至130μL含底物的緩沖溶液中，檢測單位時(shí)間內(nèi)吸光值的變化，篩選熱穩(wěn)定性提高的突變體。測序結(jié)果表明，熱穩(wěn)定性提高的突變體為：E113N，R218T，R218Q，K226V，E334R，E454D，G547E及W632H。

實(shí)施例6突變體的純化及性質(zhì)表征和對比

按照實(shí)施例2的方法得到頭孢菌素C?；竿蛔凅wE113N，R218T，R218Q，K226V，E334R，E454D，G547E及W632H的純酶液。

野生型頭孢菌素C?；讣捌渫蛔凅w的活力測定采用pH指示劑法(參見文獻(xiàn)Journal ofindustrial microbiology&biotechnology,2014,41(11):1617-1625)。具體表現(xiàn)為：反應(yīng)總體系200μL，包含5mmol/L pH8.0Tris-HCl，0.0025％苯酚紅，68.6mmol/L CPC，5μL酶液，在556nm測定其動(dòng)力學(xué)曲線。CPC?；该富顔挝坏亩x：每分鐘釋放1μmol質(zhì)子所需的酶量定義為一個(gè)酶活單位U。測定產(chǎn)物抑制時(shí)，反應(yīng)體系內(nèi)分別加入濃度為0,5mmol/L,10mmol/L,15mmol/L,20mmol/L,25mmol/L的底物7-ACA。

根據(jù)上述pH指示劑法測定酶活并計(jì)算野生型和8個(gè)突變體各自的比活力，以及測算野生型和8個(gè)突變體在不同底物濃度(0.5mmol/L，1mmol/L，2mmol/L，5mmol/L，10mmol/L，15mmol/L，20mmol/L，30mmol/L，40mmol/L和80mmol/L)下的動(dòng)力學(xué)常數(shù)K_m和k_cat；將濃度為0.5mg/ml的野生型和8個(gè)突變體的酶液在40℃下保溫不同時(shí)間(分別是15min，30min，45min，60min，90min，120min，150min，180min，240min和300min)，然后置于冰上冷卻10min，再室溫放置10min，測定酶的殘余活性。根據(jù)酶的殘余活性計(jì)算其半衰期，具體方法為：在一定溫度下孵育酶液，在不同處理時(shí)間取樣測定酶活，測算頭孢菌素C?；笟堄嗷盍Π俜直龋灰詺堄嗷盍Π俜直鹊膌n值對時(shí)間t(min)作圖，直線的斜率為失活常數(shù)k_d，由t_1/2＝ln2/k_d得到頭孢菌素C?；冈谠摐囟认碌陌胨テ?。

表2野生型與突變體的性質(zhì)比較

從表2可以看出，突變體的比活力較野生型沒有太大的變化；其中R218T較野生型的k_cat值有所下降，突變體R218Q和K226V的k_cat有所提升，而其余突變體的k_cat值無明顯變化；相對野生型，突變體R218T，R218Q和K226V的K_m值均有所降低，催化效率k_cat/K_m都高于野生型。除上述三個(gè)突變體外，其他五個(gè)突變體的k_cat值和K_m值與野生型相比并沒有大的變化。而根據(jù)半衰期t_1/2可以看出，在相同條件下，突變體較野生型均能夠更長時(shí)間保持酶活力，說明其熱穩(wěn)定性均高于野生型；其中穩(wěn)定性最佳的突變體是R218T，R218Q和K226V，在40℃下，半衰期t_1/2分別為161.2min，231.0min，169.1min，分別為野生型的2.63倍，3.77倍，2.77倍。

在不同溫度下按照上述pH指示劑法測定野生型和3個(gè)穩(wěn)定性最佳的突變體的酶活。結(jié)果如圖3所示，野生型與大部分突變體的最適溫度均為40℃，而突變體R218Q最適溫度為43℃左右，較野生型提高3℃。

CPC?；府a(chǎn)物抑制性評估：如圖4所示，各突變體與野生型(WT)相比，產(chǎn)物抑制趨勢一致，其中突變體K226V在低濃度7-ACA環(huán)境時(shí)，產(chǎn)物抑制較野生型略微減弱。

上述數(shù)據(jù)結(jié)合圖3說明突變體尤其是R218T，R218Q和K226V在保持與野生型頭孢菌素C?；赶嗨频母叽呋钚院彤a(chǎn)物抑制趨勢的前提下，具有更好的熱穩(wěn)定性，更適合工業(yè)生產(chǎn)應(yīng)用。

以上詳細(xì)描述了本發(fā)明的較佳具體實(shí)施例。應(yīng)當(dāng)理解，本領(lǐng)域的普通技術(shù)人員無需創(chuàng)造性勞動(dòng)就可以根據(jù)本發(fā)明的構(gòu)思作出諸多修改和變化。因此，凡本技術(shù)領(lǐng)域中技術(shù)人員依本發(fā)明的構(gòu)思在現(xiàn)有技術(shù)的基礎(chǔ)上通過邏輯分析、推理或者有限的實(shí)驗(yàn)可以得到的技術(shù)方案，皆應(yīng)在由權(quán)利要求書所確定的保護(hù)范圍內(nèi)。

SEQUENCE LISTING

<110> 武漢瀚海新酶生物科技有限公司

<120> 熱穩(wěn)定性提高的頭孢菌素C?；竿蛔凅w及其構(gòu)建方法

<130> 2016

<160> 30

<170> PatentIn version 3.3

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<211> 774

<212> PRT

<213> Pseudomonas sp. SE83

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gcccaggatc gtctgtttca gatggagctg acacgtcgca aagcactggg tcgcgcagcc 240

gaatggctgg gtgcagaggc cgcagaagcc gatatcctgg tgcgccgctt aggcatggag 300

aaagtgtgtc gtcgcgattt tgaagccctg ggcgcagaag caaaggatat gctgcgcgcc 360

tatgcagcag gcgttaacgc cttcttagca agcggtgccc cgttaccgat tgagtatagt 420

ctgctgggcg ccgagccgga gccgtgggaa ccgtggcata gcattgcagt tatgcgtcgc 480

ctgggtctgc tgatgggcag tgtttggttt aaactgtggc gtatgttagc cctgccggtt 540

gtgggcgccg caaatgccct gaaactgcgc tacgatgatg gcggtcagga tctgctgtgc 600

attcctccgg gcgttgaagc agaacgcctg gaagccgatt tagcagccct gcgtccggcc 660

gttgatgcat tactgaaagc catgggtggt gacgccagtg atgcagccgg tggcggtagc 720

aataattggg cagtggcacc tggtcgtaca gccaccggtc gtccgattct ggccggtgat 780

cctcaccgcg tgttcgagat cccgggcatg tatgcacagc accatctggc ctgcgatcgc 840

ttcgatatga ttggcttaac cgtgcctggc gttccgggct ttccgcataa tgcacacaac 900

ggcaaggtgg cctattgtgt gacccatgca tttatggata cacatgatct gtatctggaa 960

cagtttgccg aagacggtcg caccgcacgc tttggcaatg aattcgaacc tgtggcctgg 1020

cgccgcgatc gtattgccgt gcgtggtggc gccgatcgtg aattcgacat tgttgagacc 1080

cgccatggcc cggttatcgc aggtgatccg ttagaaggcg ccgcactgac cctgcgtagc 1140

gttcagtttg cagaaaccga tctgagtttt gattgcctga cccgcatgcc tggcgcaagc 1200

acagtggccc agctgtatga tgccacccgt ggctggggcc tggttgatca taacctggtt 1260

gccggcgatg tggccggcag cattggtcac ctggttcgtg cccgcgttcc tagtcgccct 1320

cgcgaaaatg gttggctgcc ggttccgggt tggagtggtg agcacgaatg gcgcggttgg 1380

attcctcatg aagccatgcc gcgcgttatt gatcctccgg gcggcctgat tgtgaccgcc 1440

aacaatcgtg tggtggccga cgatcatccg gattatctgt gtaccgattg tcatccgccg 1500

tatcgcgccg agcgcattat ggaacgcctg gtggcaagcc cggcatttgc cgtggatgat 1560

gcagccgcaa ttcatgccga caccctgagt ccgcatgttg gcctgctgcg tgcccgctta 1620

gaagccctgg gtattcaggg tagcctgccg gcagaagaac tgcgccagac cctgattgca 1680

tgggatggcc gcatggatgc cggtagccag gccgccagtg cctataacgc atttcgtcgt 1740

gccctgaccc gtctggtgac cgcacgtagc ggtttagaac aggccattgc acatccgttt 1800

gccgccgtgc ctccgggtgt tagtccgcag ggccaagtgt ggtgggccgt tccgaccctg 1860

ctgcgtaatg acgacgcagg catgttaaaa ggctggagct gggatgaggc cctgagcgaa 1920

gccctgagcg ttgcaaccca gaatctgaca ggccgcggtt ggggcgaaga acaccgcccg 1980

cgttttaccc acccgctgag tgcccaattt ccggcatggg cagccctgct gaatcctgtg 2040

agtcgtccga tcggtggtga tggcgatacc gttctggcaa atggcctggt gccgagtgca 2100

ggcccggaag caacatacgg cgccctgtgt cgctatgttt tcgacgtggg caactgggac 2160

aatagccgtt gggtggtgtt tcatggtgca agcggccatc ctgcaagtcc gcattacgcc 2220

gatcagaatg ccccgtggag cgattgcgcc atggttccga tgctgtatag ctgggatcgt 2280

atcgcagccg aagccgtgac cagccaggag ctggttccgg cctaa 2325

<210> 3

<211> 774

<212> PRT

<213> 人工序列

<400> 3

Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu

1 5 10 15

Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val

20 25 30

Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly

35 40 45

Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg

50 55 60

Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala

65 70 75 80

Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg

85 90 95

Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala

100 105 110

Asn Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe

115 120 125

Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala

130 135 140

Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg

145 150 155 160

Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu

165 170 175

Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp

180 185 190

Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu

195 200 205

Arg Leu Glu Ala Asp Leu Ala Ala Leu Arg Pro Ala Val Asp Ala Leu

210 215 220

Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser

225 230 235 240

Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile

245 250 255

Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala

260 265 270

Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val

275 280 285

Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala

290 295 300

Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu

305 310 315 320

Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu

325 330 335

Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp

340 345 350

Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly

355 360 365

Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala

370 375 380

Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser

385 390 395 400

Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp

405 410 415

His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val

420 425 430

Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val

435 440 445

Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu

450 455 460

Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala

465 470 475 480

Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp

485 490 495

Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala

500 505 510

Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr

515 520 525

Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly

530 535 540

Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala

545 550 555 560

Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn

565 570 575

Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu

580 585 590

Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser

595 600 605

Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp

610 615 620

Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu

625 630 635 640

Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu

645 650 655

Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala

660 665 670

Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly

675 680 685

Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala

690 695 700

Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp

705 710 715 720

Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser

725 730 735

Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val

740 745 750

Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser

755 760 765

Gln Glu Leu Val Pro Ala

770

<210> 4

<211> 774

<212> PRT

<213> 人工序列

<400> 4

Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu

1 5 10 15

Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val

20 25 30

Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly

35 40 45

Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg

50 55 60

Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala

65 70 75 80

Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg

85 90 95

Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala

100 105 110

Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe

115 120 125

Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala

130 135 140

Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg

145 150 155 160

Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu

165 170 175

Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp

180 185 190

Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu

195 200 205

Arg Leu Glu Ala Asp Leu Ala Ala Leu Thr Pro Ala Val Asp Ala Leu

210 215 220

Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser

225 230 235 240

Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile

245 250 255

Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala

260 265 270

Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val

275 280 285

Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala

290 295 300

Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu

305 310 315 320

Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu

325 330 335

Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp

340 345 350

Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly

355 360 365

Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala

370 375 380

Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser

385 390 395 400

Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp

405 410 415

His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val

420 425 430

Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val

435 440 445

Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu

450 455 460

Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala

465 470 475 480

Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp

485 490 495

Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala

500 505 510

Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr

515 520 525

Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly

530 535 540

Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala

545 550 555 560

Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn

565 570 575

Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu

580 585 590

Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser

595 600 605

Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp

610 615 620

Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu

625 630 635 640

Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu

645 650 655

Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala

660 665 670

Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly

675 680 685

Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala

690 695 700

Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp

705 710 715 720

Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser

725 730 735

Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val

740 745 750

Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser

755 760 765

Gln Glu Leu Val Pro Ala

770

<210> 5

<211> 774

<212> PRT

<213> 人工序列

<400> 5

Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu

1 5 10 15

Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val

20 25 30

Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly

35 40 45

Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg

50 55 60

Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala

65 70 75 80

Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg

85 90 95

Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala

100 105 110

Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe

115 120 125

Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala

130 135 140

Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg

145 150 155 160

Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu

165 170 175

Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp

180 185 190

Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu

195 200 205

Arg Leu Glu Ala Asp Leu Ala Ala Leu Gln Pro Ala Val Asp Ala Leu

210 215 220

Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser

225 230 235 240

Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile

245 250 255

Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala

260 265 270

Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val

275 280 285

Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala

290 295 300

Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu

305 310 315 320

Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu

325 330 335

Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp

340 345 350

Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly

355 360 365

Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala

370 375 380

Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser

385 390 395 400

Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp

405 410 415

His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val

420 425 430

Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val

435 440 445

Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu

450 455 460

Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala

465 470 475 480

Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp

485 490 495

Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala

500 505 510

Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr

515 520 525

Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly

530 535 540

Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala

545 550 555 560

Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn

565 570 575

Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu

580 585 590

Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser

595 600 605

Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp

610 615 620

Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu

625 630 635 640

Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu

645 650 655

Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala

660 665 670

Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly

675 680 685

Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala

690 695 700

Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp

705 710 715 720

Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser

725 730 735

Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val

740 745 750

Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser

755 760 765

Gln Glu Leu Val Pro Ala

770

<210> 6

<211> 774

<212> PRT

<213> 人工序列

<400> 6

Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu

1 5 10 15

Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val

20 25 30

Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly

35 40 45

Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg

50 55 60

Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala

65 70 75 80

Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg

85 90 95

Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala

100 105 110

Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe

115 120 125

Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala

130 135 140

Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg

145 150 155 160

Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu

165 170 175

Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp

180 185 190

Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu

195 200 205

Arg Leu Glu Ala Asp Leu Ala Ala Leu Arg Pro Ala Val Asp Ala Leu

210 215 220

Leu Val Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser

225 230 235 240

Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile

245 250 255

Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala

260 265 270

Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val

275 280 285

Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala

290 295 300

Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu

305 310 315 320

Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu

325 330 335

Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp

340 345 350

Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly

355 360 365

Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala

370 375 380

Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser

385 390 395 400

Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp

405 410 415

His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val

420 425 430

Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val

435 440 445

Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu

450 455 460

Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala

465 470 475 480

Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp

485 490 495

Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala

500 505 510

Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr

515 520 525

Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly

530 535 540

Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala

545 550 555 560

Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn

565 570 575

Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu

580 585 590

Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser

595 600 605

Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp

610 615 620

Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu

625 630 635 640

Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu

645 650 655

Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala

660 665 670

Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly

675 680 685

Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala

690 695 700

Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp

705 710 715 720

Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser

725 730 735

Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val

740 745 750

Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser

755 760 765

Gln Glu Leu Val Pro Ala

770

<210> 7

<211> 774

<212> PRT

<213> 人工序列

<400> 7

Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu

1 5 10 15

Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val

20 25 30

Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly

35 40 45

Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg

50 55 60

Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala

65 70 75 80

Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg

85 90 95

Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala

100 105 110

Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe

115 120 125

Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala

130 135 140

Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg

145 150 155 160

Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu

165 170 175

Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp

180 185 190

Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu

195 200 205

Arg Leu Glu Ala Asp Leu Ala Ala Leu Arg Pro Ala Val Asp Ala Leu

210 215 220

Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser

225 230 235 240

Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile

245 250 255

Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala

260 265 270

Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val

275 280 285

Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala

290 295 300

Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu

305 310 315 320

Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Arg Phe Glu

325 330 335

Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp

340 345 350

Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly

355 360 365

Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala

370 375 380

Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser

385 390 395 400

Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp

405 410 415

His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val

420 425 430

Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val

435 440 445

Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu

450 455 460

Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala

465 470 475 480

Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp

485 490 495

Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala

500 505 510

Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr

515 520 525

Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly

530 535 540

Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala

545 550 555 560

Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn

565 570 575

Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu

580 585 590

Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser

595 600 605

Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp

610 615 620

Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu

625 630 635 640

Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu

645 650 655

Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala

660 665 670

Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly

675 680 685

Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala

690 695 700

Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp

705 710 715 720

Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser

725 730 735

Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val

740 745 750

Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser

755 760 765

Gln Glu Leu Val Pro Ala

770

<210> 8

<211> 774

<212> PRT

<213> 人工序列

<400> 8

Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu

1 5 10 15

Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val

20 25 30

Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly

35 40 45

Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg

50 55 60

Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala

65 70 75 80

Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg

85 90 95

Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala

100 105 110

Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe

115 120 125

Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala

130 135 140

Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg

145 150 155 160

Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu

165 170 175

Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp

180 185 190

Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu

195 200 205

Arg Leu Glu Ala Asp Leu Ala Ala Leu Arg Pro Ala Val Asp Ala Leu

210 215 220

Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser

225 230 235 240

Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile

245 250 255

Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala

260 265 270

Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val

275 280 285

Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala

290 295 300

Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu

305 310 315 320

Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu

325 330 335

Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp

340 345 350

Arg Asp Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly

355 360 365

Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala

370 375 380

Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser

385 390 395 400

Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp

405 410 415

His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val

420 425 430

Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val

435 440 445

Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu

450 455 460

Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala

465 470 475 480

Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp

485 490 495

Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala

500 505 510

Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr

515 520 525

Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly

530 535 540

Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala

545 550 555 560

Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn

565 570 575

Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu

580 585 590

Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser

595 600 605

Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp

610 615 620

Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu

625 630 635 640

Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu

645 650 655

Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala

660 665 670

Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly

675 680 685

Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala

690 695 700

Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp

705 710 715 720

Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser

725 730 735

Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val

740 745 750

Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser

755 760 765

Gln Glu Leu Val Pro Ala

770

<210> 9

<211> 774

<212> PRT

<213> 人工序列

<400> 9

Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu

1 5 10 15

Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val

20 25 30

Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly

35 40 45

Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg

50 55 60

Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala

65 70 75 80

Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg

85 90 95

Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala

100 105 110

Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe

115 120 125

Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala

130 135 140

Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg

145 150 155 160

Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu

165 170 175

Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp

180 185 190

Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu

195 200 205

Arg Leu Glu Ala Asp Leu Ala Ala Leu Arg Pro Ala Val Asp Ala Leu

210 215 220

Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser

225 230 235 240

Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile

245 250 255

Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala

260 265 270

Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val

275 280 285

Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala

290 295 300

Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu

305 310 315 320

Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu

325 330 335

Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp

340 345 350

Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly

355 360 365

Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala

370 375 380

Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser

385 390 395 400

Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp

405 410 415

His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val

420 425 430

Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val

435 440 445

Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu

450 455 460

Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala

465 470 475 480

Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp

485 490 495

Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala

500 505 510

Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr

515 520 525

Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly

530 535 540

Ile Gln Glu Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala

545 550 555 560

Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn

565 570 575

Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu

580 585 590

Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser

595 600 605

Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp

610 615 620

Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu

625 630 635 640

Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu

645 650 655

Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala

660 665 670

Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly

675 680 685

Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala

690 695 700

Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp

705 710 715 720

Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser

725 730 735

Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val

740 745 750

Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser

755 760 765

Gln Glu Leu Val Pro Ala

770

<210> 10

<211> 774

<212> PRT

<213> 人工序列

<400> 10

Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu

1 5 10 15

Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val

20 25 30

Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly

35 40 45

Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg

50 55 60

Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala

65 70 75 80

Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg

85 90 95

Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala

100 105 110

Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe

115 120 125

Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala

130 135 140

Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg

145 150 155 160

Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu

165 170 175

Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp

180 185 190

Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu

195 200 205

Arg Leu Glu Ala Asp Leu Ala Ala Leu Arg Pro Ala Val Asp Ala Leu

210 215 220

Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser

225 230 235 240

Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile

245 250 255

Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala

260 265 270

Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val

275 280 285

Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala

290 295 300

Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu

305 310 315 320

Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu

325 330 335

Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp

340 345 350

Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly

355 360 365

Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala

370 375 380

Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser

385 390 395 400

Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp

405 410 415

His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val

420 425 430

Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val

435 440 445

Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu

450 455 460

Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala

465 470 475 480

Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp

485 490 495

Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala

500 505 510

Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr

515 520 525

Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly

530 535 540

Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala

545 550 555 560

Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn

565 570 575

Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu

580 585 590

Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser

595 600 605

Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp

610 615 620

Asp Ala Gly Met Leu Lys Gly His Ser Trp Asp Glu Ala Leu Ser Glu

625 630 635 640

Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu

645 650 655

Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala

660 665 670

Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly

675 680 685

Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala

690 695 700

Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp

705 710 715 720

Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser

725 730 735

Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val

740 745 750

Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser

755 760 765

Gln Glu Leu Val Pro Ala

770

<210> 11

<211> 101

<212> DNA

<213> 人工序列

<400> 11

atatcatatg acgatggcgg ccaagaccga tcgcgaggcc ctgcaggcgg cgctgccgcc 60

gctttccggc agcctctcca ttccgggttt aagcgcccct g 101

<210> 12

<211> 35

<212> DNA

<213> 人工序列

<400> 12

atatctcgag ttaggccgga accagctcct ggctg 35

<210> 13

<211> 40

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (12)..(13)

<223> n is a, c, g, or t

<400> 13

tagcagccct gnnkccggcc gttgatgcat tactgaaagc 40

<210> 14

<211> 40

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (14)..(15)

<223> n is a, c, g, or t

<400> 14

atcaacggcc ggmnncaggg ctgctaaatc ggcttccagg 40

<210> 15

<211> 36

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (13)..(14)

<223> n is a, c, g, or t

<400> 15

gatgcattac tgnnkgccat gggtggtgac gccagt 36

<210> 16

<211> 37

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (13)..(14)

<223> n is a, c, g, or t

<400> 16

ccacccatgg cmnncagtaa tgcatcaacg gccggac 37

<210> 17

<211> 34

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (16)..(17)

<223> n is a, c, g, or t

<400> 17

gaagccctgg gcgcannkgc aaaggatatg ctgc 34

<210> 18

<211> 35

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (12)..(13)

<223> n is a, c, g, or t

<400> 18

tatcctttgc mnntgcgccc agggcttcaa aatcg 35

<210> 19

<211> 36

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (14)..(15)

<223> n is a, c, g, or t

<400> 19

cctgggtatt cagnnkagcc tgccggcaga agaact 36

<210> 20

<211> 35

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (12)..(13)

<223> n is a, c, g, or t

<400> 20

ccggcaggct mnnctgaata cccagggctt ctaag 35

<210> 21

<211> 43

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (16)..(17)

<223> n is a, c, g, or t

<400> 21

ggcatgttaa aaggcnnkag ctgggatgag gccctgagcg aag 43

<210> 22

<211> 37

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (14)..(15)

<223> n is a, c, g, or t

<400> 22

ctcatcccag ctmnngcctt ttaacatgcc tgcgtcg 37

<210> 23

<211> 42

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (13)..(14)

<223> n is a, c, g, or t

<400> 23

ggtattcagg gtnnkctgcc ggcagaagaa ctgcgccaga cc 42

<210> 24

<211> 38

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (12)..(13)

<223> n is a, c, g, or t

<400> 24

ctgccggcag mnnaccctga atacccaggg cttctaag 38

<210> 25

<211> 40

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (13)..(14)

<223> n is a, c, g, or t

<400> 25

ggttggagtg gtnnkcacga atggcgcggt tggattcctc 40

<210> 26

<211> 38

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (18)..(19)

<223> n is a, c, g, or t

<400> 26

aaccgcgcca ttcgtgmnna ccactccaac ccggaacc 38

<210> 27

<211> 34

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (13)..(14)

<223> n is a, c, g, or t

<400> 27

cgtatcgcag ccnnkgccgt gaccagccag gagc 34

<210> 28

<211> 43

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (14)..(15)

<223> n is a, c, g, or t

<400> 28

gctggtcacg gcmnnggctg cgatacgatc ccagctatac agc 43

<210> 29

<211> 35

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (15)..(16)

<223> n is a, c, g, or t

<400> 29

cacgctttgg caatnnkttc gaacctgtgg cctgg 35

<210> 30

<211> 35

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (13)..(14)

<223> n is a, c, g, or t

<400> 30

acaggttcga amnnattgcc aaagcgtgcg gtgcg 35