專利名稱：預(yù)防和治療人前列腺癌的重組蛋白疫苗的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及一種基因工程重組蛋白疫苗，特別是涉及一種預(yù)防和治療人前列腺癌的重組蛋白疫苗。本發(fā)明還涉及編碼這兩種疫苗的基因。
人前列腺特異性抗原(Prostate specific antigen，PSA)是表達(dá)在前列腺癌細(xì)胞的一種特異性抗原，已有人在研制治療和預(yù)防人前列腺癌的PSA疫苗方面做了一些工作。如將PSA的基因克隆入哺乳動(dòng)物細(xì)胞表達(dá)質(zhì)粒制成了核酸疫苗，此疫苗在小鼠可誘導(dǎo)針對(duì)PSA的體液和細(xì)胞免疫(Kim JJ et al.Molecular and immunological analysis of genetic prostatespecific antigen(PSA)vaccine.Oncogene 1998 Dec 17；17(24)3125-35)。還有人將重組PSA和類脂A(lipid A)的混合物以脂質(zhì)體為載體制成的疫苗在前列腺癌患者誘導(dǎo)了PSA反應(yīng)性T細(xì)胞(Meidenbauer N et al.Generation of PSA-reactive effector cells after vaccination with a PSA-basedvaccine in patients with prostate cancer.Prostate 2000 May 1；43(2)88-100)。
細(xì)胞毒性T淋巴細(xì)胞(cytotoxic T lymphocyte，CTL)是人體殺傷腫瘤細(xì)胞的最高效的免疫細(xì)胞。任何一種激發(fā)機(jī)體抗腫瘤免疫的疫苗是否有效，決定于這種疫苗是否能激發(fā)腫瘤特異性CFL。外源性的蛋白類腫瘤抗原在免疫人體后，通常被抗原提呈細(xì)胞攝取、加工，進(jìn)入MHC II類提呈途徑，激發(fā)體液免疫反應(yīng)(Heikema A，Agsteribbe E，Wilschut J，Huckriede A.Generation of heat shock protein-based vaccines by intracellularloading of gp96 with antigenic peptides.Immunol Lett 1997 Jun 1；57(1-3)69-74)，但不能有效激活腫瘤特異性的CTL的產(chǎn)生，因而不能產(chǎn)生有效的腫瘤預(yù)防和治療作用。因此，賦予PSA特異性激活CTL的性質(zhì)就成為研究以PSA為抗原的、預(yù)防和治療前列腺癌疫苗的一個(gè)技術(shù)關(guān)鍵。
熱休克蛋白(heat shock protein，HSP)是存在于多種生物體內(nèi)的一個(gè)分子伴侶蛋白質(zhì)家族。在免疫應(yīng)答的過(guò)程中，熱休克蛋白可表現(xiàn)如下三種基本的功能1、協(xié)助抗原性物質(zhì)進(jìn)入包括樹(shù)突狀細(xì)胞在內(nèi)的抗原提呈細(xì)胞；2、在抗原提呈細(xì)胞中和加工處理的抗原物質(zhì)相互作用使之進(jìn)入MHC I類抗原提呈途徑，進(jìn)而激活抗原特異性CTL；3、刺激樹(shù)突狀細(xì)胞表達(dá)協(xié)同刺激分子(如B7等)和分泌細(xì)胞因子。
近年的研究表明，HSP可使非共鍵結(jié)合的腫瘤抗原在抗原提呈細(xì)胞內(nèi)加工后和MHC I類分子結(jié)合并提呈在其表面，有效地激活CTL去高效殺傷表達(dá)特異性腫瘤抗原的腫瘤細(xì)胞。注射從自體腫瘤提取的熱休克蛋白-抗原肽復(fù)合物可抑制荷瘤小鼠原發(fā)腫瘤的生長(zhǎng)、降低腫瘤的轉(zhuǎn)移率，延長(zhǎng)荷瘤小鼠的生存時(shí)間(Tamura Y，Peng P，Liu K，Daou M，SrivastavaPK.Immunotherapy of tumors with autologous tumor-derived heat shockprotein preparations.Science 1997 Oct 3；278(5335)117-20)。用來(lái)源于前列腺腫瘤的熱休克蛋白-肽復(fù)合物對(duì)小鼠的原發(fā)和轉(zhuǎn)移的前列腺癌細(xì)胞有明顯的生長(zhǎng)抑制作用(Yedavelli SP，Guo L，Daou ME，Srivastava PK，Mittelman A，Tiwari RK.Preventive and therapeutic effect of tumor derivedheat shock protein，gp96，in an experimental prostate cancer model.Int J MolMed 1999 Sep；4(3)243-8)。
本發(fā)明的另一個(gè)目的是提供兩種編碼本發(fā)明的疫苗的基因。
本發(fā)明提供了一種重組蛋白疫苗，它是由卡介苗熱休克蛋白65和1至5個(gè)拷貝的人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位相連接而形成的融合蛋白，其中，所述的人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位的單拷貝多肽具有SEQ ID NO2所示的氨基酸序列。在本發(fā)明的重組蛋白疫苗中，卡介苗熱休克蛋白65可以位于該融合蛋白的氨基端，人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位位于該融合蛋白的羧基端。
在本發(fā)明的重組蛋白疫苗中，所述的人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位的拷貝數(shù)可以為2，具有SEQ ID NO4所示的氨基酸序列。
本發(fā)明的重組蛋白疫苗最好具有SEQ ID NO6所示的氨基酸序列，或者SEQ ID NO8所示的氨基酸序列。
本發(fā)明還提供了編碼本發(fā)明的重組蛋白疫苗的基因。該基因可以具有SEQ ID NO5或SEQ ID NO7所示的核苷酸序列。
人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝多肽的氨基酸序列如SEQ ID NO2所示，人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位雙拷貝多肽的氨基酸序列如SEQ ID NO4所示。人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝多肽和人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位雙單拷貝多肽分別連接于卡介苗熱休克蛋白65融合形成的融合蛋白(序列分別如SEQ ID NO6和SEQ ID NO8所不)在進(jìn)入人體后均可刺激人體的細(xì)胞毒性T細(xì)胞(CTL)發(fā)動(dòng)對(duì)表達(dá)PSA的人前列腺癌細(xì)胞的攻擊和殺傷，因而對(duì)人前列腺癌有預(yù)防和治療的作用。由于人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位雙拷貝多肽比人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝多肽可提供更多的前列腺特異性抗原的抗原信息，因而可產(chǎn)生更強(qiáng)的免疫效果。
卡介苗熱休克蛋白65是一種來(lái)源于卡介苗的蛋白質(zhì)，它在和人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位雙拷貝多肽或人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝多肽形成融合蛋白后，可以協(xié)助它們進(jìn)入包括樹(shù)突狀細(xì)胞在內(nèi)的抗原提呈細(xì)胞，并進(jìn)入MHC I類途徑加工提呈，進(jìn)而激活抗原特異性細(xì)胞毒性T淋巴細(xì)胞(cytotoxic Tlymphocyte，CTL)對(duì)表達(dá)前列腺特異性抗原的人前列腺癌細(xì)胞進(jìn)行攻擊和殺傷。此外，卡介苗熱休克蛋白65還可刺激包括樹(shù)突狀細(xì)胞在內(nèi)的抗原提呈細(xì)胞表達(dá)協(xié)同刺激分子(如B7等)和分泌細(xì)胞因子，這些協(xié)同刺激分子(如B7等)和細(xì)胞因子可激活細(xì)胞毒性T淋巴細(xì)胞的腫瘤細(xì)胞殺傷能力。
本發(fā)明的重組蛋白疫苗可通過(guò)已知的方法進(jìn)行生產(chǎn)。
本發(fā)明的重組蛋白疫苗可通過(guò)皮下注射的方式給人接種，接種的劑量為100-500μg。為了加強(qiáng)效果，可進(jìn)行2-3次的加強(qiáng)免疫。時(shí)間間隔可為2周-2月。
提取結(jié)核桿菌基因組DNA的方法參照Molecular Cloning一書(shū)(J.Sambrook，Isolation of high-molecular-weight DNA from mammalian cells，9.16-9.22，Cold Spring Harbor Laboratory Press，Molecular cloning，1989)。
采用PCR方法自結(jié)核桿菌分離熱休克蛋白65(HSP65)結(jié)構(gòu)基因。采用的5′端引物序列為5′CCATG GCC AAG ACA ATT GCG3′(SEQ IDNO9)，3′端引物序列為5′GAAATCCATGCCACCCAT3′(SEQID NO10)。
所述PCR操作程序是在一500(1微量離心管中加入下列試劑模板cDNA 5μl(mmol/L)10(PCR緩沖液(含氯化鎂)5μldNTPs(10mmol/L)1μl5′端和3′端引物(0.01mmol/L)各0.5μlTaq DNA聚合酶(5u/μl)0.25μl加去離子水至終體積50μl混合后加入礦物油3滴反應(yīng)條件94℃，30″；55℃，1′；72℃，2′，30個(gè)循環(huán)周期后，72℃延伸10分鐘。
采用TA克隆方法克隆PCR產(chǎn)物，方法見(jiàn)文獻(xiàn)(于永利，麻彤輝，楊貴貞.TA克隆及雙鏈DNA測(cè)序介紹一種快速克隆及分析PCR產(chǎn)物的方法.中國(guó)免疫學(xué)雜志，1994，10(1)5)。
按常規(guī)方法(J.Sambrook，Polyacrylamide gel electrophoresis 1.21-1.32，Cold Spring Harbor Laboratory Press，Molecular cloning，1989)提取質(zhì)粒，采用雙脫氧末端終止法，對(duì)插入片段進(jìn)行序列測(cè)定。
用NcoI和HindIII在37℃消化PCR產(chǎn)物，時(shí)間為2小時(shí)。
消化產(chǎn)物瓊經(jīng)脂糖凝膠電泳分離。電泳的條件是1％瓊脂糖凝膠，1(TAE緩沖液，150-200mA，電泳0.5-1小時(shí)。20(TAE緩沖液0.8mol/L Tris base0.4mol/L NaOAc0.04mol/L Na2EDTA用冰醋酸調(diào)pH 8.3。
在紫外燈下觀察并切下瓊脂糖凝膠上的DNA電泳帶。將含DNA區(qū)帶的瓊脂糖凝膠切下(多少？)，置-70℃冷凍15分鐘，然后在65℃水浴中使膠融化；加入等倍體積TE-飽合酚，劇烈振蕩30秒鐘，然后-70℃冷凍15分鐘；室溫融化后，12,000rpm離心5分鐘，將上層液相移至另一管中，用2-2.5倍乙醇沉淀、洗滌和干燥DNA。
將回收的PCR產(chǎn)物克隆入經(jīng)限制性內(nèi)切酶NcoI和HindIII消化的原核細(xì)胞表達(dá)載體pET-28a(+)質(zhì)粒(美國(guó)Novagen公司)的6聚組氨酸(histidine)密碼子的上游。
質(zhì)粒DNA的消化反應(yīng)1μg質(zhì)粒DNA1μl10(緩沖液(見(jiàn)Promega Corporation產(chǎn)品說(shuō)明書(shū))。1μl限制性內(nèi)切酶NcoI(10單位/μl)1μl限制性內(nèi)切酶HindIII(10單位/μl)用雙蒸水補(bǔ)齊至10μl混合后37℃溫育30-120分鐘。
連接反應(yīng)質(zhì)粒DNA(0.5μg/μl)2μlDNA插入片段(300ng/μl)5μl10(連接緩沖液(見(jiàn)Protocols and Applications Guide，p57，PromegaCorporation，Second Edition，1991)1μlT4 DNA連接酶1μl用雙蒸水補(bǔ)齊至10μl混合后置14-16℃水浴6-12小時(shí)。
將含HSP-65基因的重組pET-28a(+)質(zhì)粒轉(zhuǎn)化大腸桿菌。
感受態(tài)細(xì)胞的制備方法是將大腸桿菌在LB瓊脂培養(yǎng)基上劃線，37℃培養(yǎng)12-16小時(shí)；次日從瓊脂平板上取一單菌落于2ml LB培養(yǎng)基中，37℃以225rpm速度震蕩培養(yǎng)12-16小時(shí)；取1ml上述培養(yǎng)物接種于100ml LB培養(yǎng)基中，37℃以225rpm的速度震蕩培養(yǎng)直至OD值為0.5左右(大約3小時(shí))；將菌液冰浴2小時(shí)，然后2,500 Xg，4℃離心20分鐘收集菌體；加入100ml冰冷的Trituration緩沖液(100mmol/L CaCl2，70mmol/L MgCl2，40mmol/L醋酸鈉，pH5.5)，混勻，置冰上45分鐘；1,800 Xg，4℃離心10分鐘，棄上清，加入10ml冰冷的Trituration緩沖液懸浮細(xì)胞；按每份200ul分裝，4C可保存1-2周。若需長(zhǎng)期保存，可加甘油至終濃度為15％，置-70℃?zhèn)溆谩?br> 轉(zhuǎn)化的方法是將200ul感受態(tài)細(xì)胞置冰上融化，然后加入3ul DMSO或β-巰基乙醇，混合后，加入3-5μl連接反應(yīng)液(含重組質(zhì)粒)，溫和混勻，置冰上30分鐘；42℃45秒，然后迅速放回冰中1-2分鐘；加入2ml LB培養(yǎng)液，37℃以225rpm的速度搖蕩培養(yǎng)1小時(shí)；4,000 Xg離心10秒鐘，棄上清，用200ul LB培養(yǎng)液重懸菌體；將菌液鋪于含有適當(dāng)抗生素的LB瓊脂培養(yǎng)板上，涂勻，室溫放置20-30分鐘，倒置于37℃孵箱中培養(yǎng)12-16小時(shí)。用限制性內(nèi)切酶消化的方法鑒定重組克隆。
重組質(zhì)粒冰凍保存于-20℃。含重組質(zhì)粒的菌株在含20-50％甘油培養(yǎng)液中保存于-20℃或-70℃。
卡介苗熱休克蛋白65人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝多肽融合蛋白的基因的測(cè)序(采用雙脫氧末端終止法，具體方法見(jiàn)文獻(xiàn)于永利，麻彤輝，楊貴貞.TA克隆及雙鏈DNA測(cè)序介紹一種快速克隆及分析PCR產(chǎn)物的方法。中國(guó)免疫學(xué)雜志，1994，10(1)5)結(jié)果表明，所得到的卡介苗熱休克蛋白65人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝多肽融合蛋白的基因和設(shè)計(jì)的完全一致。實(shí)施例3 構(gòu)建人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位雙拷貝融合蛋白疫苗的基因含EcoR1切點(diǎn)和Bgl II切點(diǎn)人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝基因的構(gòu)建采用5‘端特異性引物[5’GCCGAGAATTCGAGCCTGAAGAGTTCCTGACTCCGAAAAAA3’(SEQ ID NO13)(含EcoR1切點(diǎn))]和3端特異性引物[5’GGGCGAGATCTACACAGCATGAATTTAGTAACTTTCTGCGG3’(SEQ ID NO14)(含BglII切點(diǎn))]，以人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝融合蛋白疫苗的基因?yàn)槟０?，做PCR放大，得到含EcoR1切點(diǎn)和BglII切點(diǎn)人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝基因的DNA片段，其序列是GCCGAGAATT CGAGCCTGAA GAG TTCCTGACTC CGAAAAAACT GCAGTGCGTTGACCTGCACG TTATCTCTAA CGACGTTTGC GCTCAGGTTC ACCCGCAGAAAGTTACTAAA TTCATGCTGT GTAGATCTCGCCC(SEQ ID NO15)含BamH I切點(diǎn)和HindIII切點(diǎn)人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝基因的構(gòu)建采用5‘端特異性引物[5‘GCGTGGATCCGAGCCTGAAGAGTTCCTGACTCCGAAAAAA(SEQ ID NO16)(含BamH I切點(diǎn))]和3端特異性引物[5’CGCCGAAGCTTGCACAGCATGAATTTAGTAACTTTCTGCGG(SEQID NO17)3’(含BglII切點(diǎn))]，以人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝融合蛋白疫苗的基因?yàn)槟０?，做PCR放大，得到含含BamH I切點(diǎn)和HindIII切點(diǎn)人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝基因的DNA片段，其序列是GCGTGGATCCGAGCCTGAAGAG TTCCTGACTCCGAAAAAACTGCAGTGCGTTGACCTGCACG TTATCTCTAA CGACGTTTGC GCTCAGGTTC ACCCGCAGAAAGTTACTAAA TTCATGCTGTGCAAGCTTCGGCG (SEQ ID NO18)將上述兩個(gè)DNA片段分別克隆的到pMD18-T上，一種質(zhì)粒含含EcoR1切點(diǎn)和Bgl II切點(diǎn)人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝基因，命名為pMD18-T-EcoR1 Bgl II，另一種質(zhì)粒含BamH I切點(diǎn)和HindIII切點(diǎn)人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位單拷貝基因，命名為pMD18-T-BamH I HindIII。
將pMD18-T-EcoR1 Bgl II用Bgl II酶切消化，去磷酸化；將pMD18-T-BamH I HindIII用BamH I酶切消化。連接消化物，得到5‘端帶EcoR1切點(diǎn)，3’端帶HindIII切點(diǎn)的人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位雙拷貝融合蛋白疫苗的基因片段，其序列是GAATTCGAGCCTGAAGAG TTCCTGACTC CGAAAAAACT GCAGTGCGTT16801681 GACCTGCACG TTATCTCTAA CGACGTTTGC GCTCAGGTTC ACCCGCAGAA AGTTACTAAA17401741 TTCATGCTGT GTAGATCCGA GCCTGAAGAG TTCCTGACTC CGAAAAAACT GCAGTGCGTT 18001801 GACCTGCACG TTATCTCTAA CGACGTTTGC GCTCAGGTTC ACCCGCAGAA AGTTACTAAA 18601861 TTCATGCTGT GCAAGCTT(SEQ ID N019)用將EcoR1和HindIII消化此片段，將其克隆入用EcoR1和HindIII雙酶切消化的pET28-HSP65-P1質(zhì)粒，此質(zhì)粒的插入片段含卡介苗熱休克蛋白65人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位雙拷貝融合蛋白疫苗的結(jié)構(gòu)基因。將其轉(zhuǎn)化大腸桿菌得克隆菌。
關(guān)于具體的實(shí)驗(yàn)操作和實(shí)施例2的相似。
用臺(tái)式超聲細(xì)胞破碎儀(SANYO，MSE SONIPREP 150)，設(shè)8個(gè)循環(huán)和50％的時(shí)間間隔，在冰浴中超聲90秒破菌，攪拌10-20min。
加DOC，使終濃度為0.2％(即，加約240ul的20％脫氧膽酸鈉(DOC)?；靹?，靜置10min。20％脫氧膽酸鈉(DOC)貯存液應(yīng)提前1天配制，配方為10g無(wú)水DOC，溶于水中，調(diào)體積至50ml。得粗制裂解物(A)。
13000rpm，4℃離心10min。輕輕傾出上清。
加18ml緩沖液A和2ml 20％DOC貯存液于包涵體沉淀。用組織破碎器充分重懸沉淀，室溫下靜置至少10分鐘。將懸液于4℃離心10min，13000rpm，加39.4ml緩沖液A和0.6ml 20％十二烷基肌氨酸鈉(SKL)儲(chǔ)液(SKL凈濃度為0.3％)。劇烈攪動(dòng)使沉淀慢慢溶解。靜置至少30分鐘。十二烷基肌氨酸鈉(SKL)儲(chǔ)液的配制方法是10g無(wú)水十二烷基肌氨酸鈉(SKL)，溶于H2O中，調(diào)體積至50ml。
將懸液于4℃離心10min，13000rpm。用緩沖液A+0.3％SKL稀釋溶解的蛋白質(zhì)使其濃度為1mg/ml。用緩沖液A將溶解的蛋白質(zhì)稀釋10倍，使其濃度為0.1mg/ml，SKL的終濃度為0.03％。
在4℃溶解的蛋白質(zhì)制備物(體積約為400ml)對(duì)緩沖液A(體積約為4000ml)透析8小時(shí)，同時(shí)充分?jǐn)嚢琛Ｈ缓髶Q新鮮緩沖液并重復(fù)。
從透析袋(口徑1cm)中移出經(jīng)透析的蛋白質(zhì)溶液，4℃、8000rpm離心20min，去除所有的的聚集物。留取上清，做鑷-Saphrose-4-B層析分離HSP-65和前列腺特異性抗原(PSA)的T細(xì)胞表位的融合基因融合蛋白。
采用常規(guī)的SDS-PAGE方法(J.Sambrook，Polyacrylamide gelelectrophoresis 6.36-6.49，Cold Spring Harbor Laboratory Press，Molecularcloning，1989)。鑒定HSP-65和前列腺特異性抗原(PSA)的T細(xì)胞表位的基因融合蛋白的純度，純度為98％。
2、實(shí)驗(yàn)動(dòng)物6-8周的雄性C57BL/6小鼠(吉林大學(xué)醫(yī)學(xué)部實(shí)驗(yàn)動(dòng)物中心)，疫苗組和對(duì)照組各20只。
3、實(shí)驗(yàn)方法1)采用DMEM培養(yǎng)基培養(yǎng)C2細(xì)胞和PSA-C2細(xì)胞。在注射之前，用無(wú)血清DMEM將細(xì)胞洗三次。每只小鼠注射2.5(106個(gè)C2細(xì)胞或PSA-C2細(xì)胞，注射部位是小鼠兩肩胛骨間皮下。在6周以后，測(cè)量小鼠腫瘤塊基底部的面積。
2)在接種腫瘤30天前和15天前，疫苗組分別腹腔注射100微克結(jié)核桿菌熱休克蛋白-人前列腺特異性抗原融合蛋白基因工程疫苗；對(duì)照組分別腹腔注射同疫苗組同體積生理鹽水。4、實(shí)驗(yàn)結(jié)果及結(jié)論動(dòng)物腫瘤塊基底部面積的平均值疫苗組為53mm2，對(duì)照組為250mm2。注射后6周測(cè)量。
表明所實(shí)驗(yàn)藥物能明顯抑制自發(fā)性前列腺腫瘤的生長(zhǎng)。二、清除殘存前列腺癌細(xì)胞實(shí)驗(yàn)1、材料C2細(xì)胞2、實(shí)驗(yàn)動(dòng)物6-8周的雄性C57BL/6小鼠，疫苗組和對(duì)照組各20只3、實(shí)驗(yàn)方法1)采用DMEM培養(yǎng)基培養(yǎng)C2細(xì)胞和PSA-C2細(xì)胞。在注射之前，用無(wú)血清DMEM將細(xì)胞洗三次。每只小鼠注射2.5(106個(gè)C2細(xì)胞或PSA-C2細(xì)胞，注射部位是小鼠兩肩胛骨間皮下。在6周以后，用外科手術(shù)的方法切除腫塊。在切除腫塊后的21天處死小鼠，測(cè)量小鼠復(fù)發(fā)腫瘤塊基底部的面積。
2)在手術(shù)后的第4、7和10天，疫苗組分別腹腔注射100微克結(jié)核桿菌熱休克蛋白-人前列腺特異性抗原融合蛋白基因工程疫苗；對(duì)照組分別腹腔注射同疫苗組同體積生理鹽水。4、實(shí)驗(yàn)結(jié)果及結(jié)論動(dòng)物腫瘤塊基底部面積的平均值疫苗組為36mm2，對(duì)照組為252mm2。表明該受試藥物能明顯清除殘存前列腺癌細(xì)胞。
序列表<110>北京迪威華宇生物技術(shù)有限公司<120>預(yù)防和治療人前列腺癌的重組蛋白疫苗<130>I2001290<160>19<170>PatentIn version 3.1<210>1<211>90<212>DNA<213>Homo sapiens<220><221>CDS<222>(1)..(90)<223><400>1ttc ctg act ccg aaa aaa ctg cag tgc gtt gac ctg cac gtt atc tct 48Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser1 5 10 15aac gac gtt tgc gct cag gtt cac ccg cag aaa gtt act aaa 90Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys20 25 30<210>2<211>30<212>PRT<213>Homo sapiens<400>2Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser1 5 10 15Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys20 25 30<210>3<211>210<212>DNA<213>Artificial Sequence<220><223>Recombinant Gene<220><221>CDS<222>(1)..(210)<223><400>3ttc ctg act ccg aaa aaa ctg cag tgc gtt gac ctg cac gtt atc tct 48Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser1 5 10 15aac gac gtt tgc gct cag gtt cac ccg cag aaa gtt act aaa ttc atg 96Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met20 25 30ctg tgt aga tcc gag cct gaa gag ttc ctg act ccg aaa aaa ctg cag 144Leu Cys Arg Ser Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln35 40 45tgc gtt gac ctg cac gtt atc tct aac gac gtt tgc gct cag gtt cac 192Cys Val Asp Leu His Val Ile Ser Asn Asp Val Cys Ala Gln Val His50 55 60ccg cag aaa gtt act aaa 210Pro Gln Lys Val Thr Lys65 70<210>4<211>70<212>PRT<213>Artificial Sequence<220><223>Recombinant Gene<400>4Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser1 5 10 15Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met20 25 30Leu Cys Arg Ser Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln35 40 45Cys Val Asp Leu His Val Ile Ser Asn Asp Val Cys Ala Gln Val His50 55 60Pro Gln Lys Val Thr Lys65 70<210>5<211>1752<212>DNA<213>Artificial Sequence<220><223>Recombinant Gene<220><221>CDS<222>(1)..(1752)<223><400>5atg gcc aag aca att gcg tac gac gaa gag gcc cgt cgc ggc ctc gag 48Met Ala Lys Thr Ile Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu1 5 10 15cgg ggc ttg aac gcc ctc gcc gat gcg gta aag gtg aca ttg ggc ccc 96Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro20 25 30aag ggc cgc aac gtc gtc ctg gaa aag aag tgg ggt gcc ccc acg atc 144Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile35 40 45acc aac gat ggt gtg tcc atc gcc aag gag atc gag ctg gag gat ccg 192Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro50 55 60tac gag aag atc ggc gcc gag ctg gtc aaa gag gta gcc aag aag acc 240Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr65 70 75 80gat gac gtc gcc ggt gac ggc acc acg acg gcc acc gtg ctg gcc cag 288Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln85 90 95gcg ttg gtt cgc gag ggc ctg cgc aac gtc gcg gcc ggc gcc aac ccg 336Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro100 105 110ctc ggt ctc aaa cgc ggc atc gaa aag gcc gtg gag aag gtc acc gag 384Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Glu Lys Val Thr Glu115 120 125acc ctg ctc aag ggc gcc aag gag gtc gag acc aag gag cag att gcg 432Thr Leu Leu Lys Gly Ala Lys Glu Val Glu Thr Lys Glu Gln Ile Ala130 135 140gcc acc gca gcg att tcg gcg ggt gac cag tcc atc ggt gac ctg atc 480Ala Thr Ala Ala Ile Ser Ala Gly Asp Gln Ser Ile Gly Asp Leu Ile145 150 155 160gcc gag gcg atg gac aag gtg ggc aac gag ggc gtc atc acc gtc gag 528Ala Glu Ala Met Asp Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu165 170 175gag tcc aac acc ttt ggg ctg cag ctc gag ctc acc gag ggt atg cgg 576Glu Ser Asn Thr Phe Gly Leu Gln Leu Glu Leu Thr Glu Gly Met Arg180 185 190ttc gac aag ggc tac atc tcg ggg tac ttc gtg acc gac ccg gag cgt 624Phe Asp Lys Gly Tyr Ile Ser Gly Tyr Phe Val Thr Asp Pro Glu Arg195 200 205cag gag gcg gtc ctg gag gac ccc tac atc ctg ctg gtc agc tcc aag 672Gln Glu Ala Val Leu Glu Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys210 215220gtg tcc act gtc aag gat ctg ctg ccg ctg ctc gag aag gtc atc gga 720Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gly225 230 235 240gcc ggt aag ccg ctg ctg atc atc gcc gag gac gtc gag ggc gag gcg 768Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala245 250 255ctg tcc acc ctg gtc gtc aac aag atc cgc ggc acc ttc aag tcg gtg 816Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val260 265 270gcg gtc aag gct ccc ggc ttc ggc gac cgc cgc aag gcg atg ctg cag 864Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln275 280 285gat atg gcc att ctc acc ggt ggt cag gtg atc agc gaa gag gtc ggc 912Asp Met Ala Ile Leu Thr Gly Gly Gln Val Ile Ser Glu Glu Val Gly290 295 300ctg acg ctg gag aac gcc gac ctg tcg ctg cta ggc aag gcc cgc aag 960Leu Thr Leu Glu Asn Ala Asp Leu Ser Leu Leu Gly Lys Ala Arg Lys305 310 315 320gtc gtg gtc acc aag gac gag acc acc atc gtc gag ggc gcc ggt gac 1008Val Val Val Thr Lys Asp Glu Thr Thr Ile Val Glu Gly Ala Gly Asp325 330 335acc gac gcc atc gcc gga cga gtg gcc cag atc cgc cag gag atc gag 1056Thr Asp Ala Ile Ala Gly Arg Val Ala Gln Ile Arg Gln Glu Ile Glu340 345 350aac agc gac tcc gac tac gac cgt gag aag ctg cag gag cgg ctg gcc 1104Asn Ser Asp Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg Leu Ala355 360 365aag ctg gcc ggt ggt gtc gcg gtc atc aag gcc ggt gcc gcc acc gac 1152Lys Leu Ala Gly Gly Val Ala Val Ile Lys Ala Gly Ala Ala Thr Asp370 375 380gtc gaa ctc aag gag cgc aag cac cgc atc gag gat gcg gtt cgc aat 1200Val Glu Leu Lys Glu Arg Lys His Arg Ile Glu Asp Ala Val Arg Asn385 390 395 400gcc aag gcc gcc gtc gag gag ggc atc gtc gcc ggt ggg ggt gtg acg 1248Ala Lys Ala Ala Val Glu Glu Gly Ile Val Ala Gly Gly Gly Val Thr405 410 415ctg ttg caa gcg gcc ccg acc ctg gac gag ctg aag ctc gaa ggc gac 1296Leu Leu Gln Ala Ala Pro Thr Leu Asp Glu Leu Lys Leu Glu Gly Asp420 425 430gag gcg acc ggc gcc aac atc gtg aag gtg gcg ctg gag gcc ccg ctg 1344Glu Ala Thr Gly Ala Asn Ile Val Lys Val Ala Leu Glu Ala Pro Leu435 440 445aag cag atc gcc ttc aac tcc ggg ctg gag ccg ggc gtg gtg gcc gag 1392Lys Gln Ile Ala Phe Asn Ser Gly Leu Glu Pro Gly Val Val Ala Glu450 455 460aag gtg cgc aac ctg ccg gct ggc cac gga ctg aac gct cag acc ggt 1440Lys Val Arg Asn Leu Pro Ala Gly His Gly Leu Asn Ala Gln Thr Gly465 470 475 480gtc tac gag gat ctg ctc gct gcc ggc gtt gct gac ccg gtc aag gtg 1488Val Tyr Glu Asp Leu Leu Ala Ala Gly Val Ala Asp Pro Val Lys Val485 490 495acc cgt tcg gcg ctg cag aat gcg gcg tcc atc gcg ggg ctg ttc ctg 1536Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Ile Ala Gly Leu Phe Leu500 505 510acc acc gag gcc gtc gtt gcc gac aag ccg gaa aag gag aag gct tcc 1584Thr Thr Glu Ala Val Val Ala Asp Lys Pro Glu Lys Glu Lys Ala Ser515 520 525gtt ccc ggt ggc ggc gac atg ggt ggc atg gat ttc ttc ctg act ccg 1632Val Pro Gly Gly Gly Asp Met Gly Gly Met Asp Phe Phe Leu Thr Pro530 535 540aaa aaa ctg cag tgc gtt gac ctg cac gtt atc tct aac gac gtt tgc 1680Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser Asn Asp Val Cys545 550 555 560gct cag gtt cac ccg cag aaa gtt act aaa aag ctt gcg gcc gca ctc 1728Ala Gln Val His Pro Gln Lys Val Thr Lys Lys Leu Ala Ala Ala Leu565 570 575gag cac cac cac cac cac cac tga 1752Glu His His His His His His580<210>6<211>583<212>PRT<213>Artificial Sequence<220><223>Recombinant Gene<400>6Met Ala Lys Thr Ile Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu1 5 10 15Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro20 25 30Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile35 40 45Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro50 55 60Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr65 70 75 80Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln85 90 95Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro100 105 110Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Glu Lys Val Thr Glu115 120 125Thr Leu Leu Lys Gly Ala Lys Glu Val Glu Thr Lys Glu Gln Ile Ala130 135 140Ala Thr Ala Ala Ile Ser Ala Gly Asp Gln Ser Ile Gly Asp Leu Ile145 150 155 160Ala Glu Ala Met Asp Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu165 170 175Glu Ser Asn Thr Phe Gly Leu Gln Leu Glu Leu Thr Glu Gly Met Arg180 185 190Phe Asp Lys Gly Tyr Ile Ser Gly Tyr Phe Val Thr Asp Pro Glu Arg195 200 205Gln Glu Ala Val Leu Glu Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys210 215 220Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gly225 230 235 240Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala245 250 255Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val260 265 270Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln275 280 285Asp Met Ala Ile Leu Thr Gly Gly Gln Val Ile Ser Glu Glu Val Gly290 295 300Leu Thr Leu Glu Asn Ala Asp Leu Ser Leu Leu Gly Lys Ala Arg Lys305 310 315 320Val Val Val Thr Lys Asp Glu Thr Thr Ile Val Glu Gly Ala Gly Asp325 330 335Thr Asp Ala Ile Ala Gly Arg Val Ala Gln Ile Arg Gln Glu Ile Glu340 345 350Asn Ser Asp Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg Leu Ala355 360 365Lys Leu Ala Gly Gly Val Ala Val Ile Lys Ala Gly Ala Ala Thr Asp370 375 380Val Glu Leu Lys Glu Arg Lys His Arg Ile Glu Asp Ala Val Arg Asn385 390 395 400Ala Lys Ala Ala Val Glu Glu Gly Ile Val Ala Gly Gly Gly Val Thr405 410 415Leu Leu Gln Ala Ala Pro Thr Leu Asp Glu Leu Lys Leu Glu Gly Asp420 425 430Glu Ala Thr Gly Ala Asn Ile Val Lys Val Ala Leu Glu Ala Pro Leu435 440 445Lys Gln Ile Ala Phe Asn Ser Gly Leu Glu Pro Gly Val Val Ala Glu450 455 460Lys Val Arg Asn Leu Pro Ala Gly His Gly Leu Asn Ala Gln Thr Gly465 470 475 480Val Tyr Glu Asp Leu Leu Ala Ala Gly Val Ala Asp Pro Val Lys Val485 490 495Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Ile Ala Gly Leu Phe Leu500 505 510Thr Thr Glu Ala Val Val Ala Asp Lys Pro Glu Lys Glu Lys Ala Ser515520 525Val Pro Gly Gly Gly Asp Met Gly Gly Met Asp Phe Phe Leu Thr Pro530 535 540Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser Asn Asp Val Cys545 550 555 560Ala Gln Val His Pro Gln Lys Val Thr Lys Lys Leu Ala Ala Ala Leu565 570 575Glu His His His His His His580<210>7<211>1914<212>DNA<213>Artificial Sequence<220><223>Recombinant Gene<220><221>CDS<222>(1)..(1914)<223><400>7atg gcc aag aca att gcg tac gac gaa gag gcc cgt cgc ggc ctc gag 48Met Ala Lys Thr Ile Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu1 5 10 15cgg ggc ttg aac gcc ctc gcc gat gcg gta aag gtg aca ttg ggc ccc 96Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro20 25 30aag ggc cgc aac gtc gtc ctg gaa aag aag tgg ggt gcc ccc acg atc 144Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile35 40 45acc aac gat ggt gtg tcc atc gcc aag gag atc gag ctg gag gat ccg 192Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro50 55 60tac gag aag atc ggc gcc gag ctg gtc aaa gag gta gcc aag aag acc 240Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr65 70 75 80gat gac gtc gcc ggt gac ggc acc acg acg gcc acc gtg ctg gcc cag 288Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln85 90 95gcg ttg gtt cgc gag ggc ctg cgc aac gtc gcg gcc ggc gcc aac ccg 336Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro100 105 110ctc ggt ctc aaa cgc ggc atc gaa aag gcc gtg gag aag gtc acc gag 384Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Glu Lys Val Thr Glu115 120 125acc ctg ctc aag ggc gcc aag gag gtc gag acc aag gag cag att gcg 432Thr Leu Leu Lys Gly Ala Lys Glu Val Glu Thr Lys Glu Gln Ile Ala130 135 140gcc acc gca gcg att tcg gcg ggt gac cag tcc atc ggt gac ctg atc 480Ala Thr Ala Ala Ile Ser Ala Gly Asp Gln Ser Ile Gly Asp Leu Ile145 150 155 160gcc gag gcg atg gac aag gtg ggc aac gag ggc gtc atc acc gtc gag 528Ala Glu Ala Met Asp Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu165 170 175gag tcc aac acc ttt ggg ctg cag ctc gag ctc acc gag ggt atg cgg 576Glu Ser Asn Thr Phe Gly Leu Gln Leu Glu Leu Thr Glu Gly Met Arg180 185 190ttc gac aag ggc tac atc tcg ggg tac ttc gtg acc gac ccg gag cgt 624Phe Asp Lys Gly Tyr Ile Ser Gly Tyr Phe Val Thr Asp Pro Glu Arg195 200 205cag gag gcg gtc ctg gag gac ccc tac atc ctg ctg gtc agc tcc aag 672Gln Glu Ala Val Leu Glu Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys210 215 220gtg tcc act gtc aag gat ctg ctg ccg ctg ctc gag aag gtc atc gga 720Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gly225 230 235 240gcc ggt aag ccg ctg ctg atc atc gcc gag gac gtc gag ggc gag gcg 768Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala245 250 255ctg tcc acc ctg gtc gtc aac aag atc cgc ggc acc ttc aag tcg gtg 816Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val260 265 270gcg gtc aag gct ccc ggc ttc ggc gac cgc cgc aag gcg atg ctg cag 864Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln275 280 285gat atg gcc att ctc acc ggt ggt cag gtg atc agc gaa gag gtc ggc 912Asp Met Ala Ile Leu Thr Gly Gly Gln Val Ile Ser Glu Glu Val Gly290 295 300ctg acg ctg gag aac gcc gac ctg tcg ctg cta ggc aag gcc cgc aag 960Leu Thr Leu Glu Asn Ala Asp Leu Ser Leu Leu Gly Lys Ala Arg Lys305 310 315 320gtc gtg gtc acc aag gac gag acc acc atc gtc gag ggc gcc ggt gac 1008Val Val Val Thr Lys Asp Glu Thr Thr Ile Val Glu Gly Ala Gly Asp325 330 335acc gac gcc atc gcc gga cga gtg gcc cag atc cgc cag gag atc gag 1056Thr Asp Ala Ile Ala Gly Arg Val Ala Gln Ile Arg Gln Glu Ile Glu340 345 350aac agc gac tcc gac tac gac cgt gag aag ctg cag gag cgg ctg gcc 1104Asn Ser Asp Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg Leu Ala355 360 365aag ctg gcc ggt ggt gtc gcg gtc atc aag gcc ggt gcc gcc acc gac 1152Lys Leu Ala Gly Gly Val Ala Val Ile Lys Ala Gly Ala Ala Thr Asp370 375 380gtc gaa ctc aag gag cgc aag cac cgc atc gag gat gcg gtt cgc aat 1200Val Glu Leu Lys Glu Arg Lys His Arg Ile Glu Asp Ala Val Arg Asn385 390 395 400gcc aag gcc gcc gtc gag gag ggc atc gtc gcc ggt ggg ggt gtg acg 1248Ala Lys Ala Ala Val Glu Glu Gly Ile Val Ala Gly Gly Gly Val Thr405 410 415ctg ttg caa gcg gcc ccg acc ctg gac gag ctg aag ctc gaa ggc gac 1296Leu Leu Gln Ala Ala Pro Thr Leu Asp Glu Leu Lys Leu Glu Gly Asp420 425 430gag gcg acc ggc gcc aac atc gtg aag gtg gcg ctg gag gcc ccg ctg 1344Glu Ala Thr Gly Ala Asn Ile Val Lys Val Ala Leu Glu Ala Pro Leu435 440 445aag cag atc gcc ttc aac tcc ggg ctg gag ccg ggc gtg gtg gcc gag 1392Lys Gln Ile Ala Phe Asn Ser Gly Leu Glu Pro Gly Val Val Ala Glu450 455 460aag gtg cgc aac ctg ccg gct ggc cac gga ctg aac gct cag acc ggt 1440Lys Val Arg Asn Leu Pro Ala Gly His Gly Leu Asn Ala Gln Thr Gly465 470 475 480gtc tac gag gat ctg ctc gct gcc ggc gtt gct gac ccg gtc aag gtg 1488Val Tyr Glu Asp Leu Leu Ala Ala Gly Val Ala Asp Pro Val Lys Val485 490 495acc cgt tcg gcg ctg cag aat gcg gcg tcc atc gcg ggg ctg ttc ctg 1536Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Ile Ala Gly Leu Phe Leu500 505 510acc acc gag gcc gtc gtt gcc gac aag ccg gaa aag gag aag gct tcc 1584Thr Thr Glu Ala Val Val Ala Asp Lys Pro Glu Lys Glu Lys Ala Ser515 520 525gtt ccc ggt ggc ggc gac atg ggt ggc atg gat ttc cat atg gct agc 1632Val Pro Gly Gly Gly Asp Met Gly Gly Met Asp Phe His Met Ala Ser530 535 540gaa ttc gag cct gaa gac ttc ctg act ccg aaa aaa ctg cag tgc gtt 1680Glu Phe Glu Pro Glu Asp Phe Leu Thr Pro Lys Lys Leu Gln Cys Val545 550 555 560gac ctg cac gtt atc tct aac gac gtt tgc gct cag gtt cac ccg cag 1728Asp Leu His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln565 570 575aaa gtt act aaa ttc atg ctg tgt aga tcc gag cct gaa gag ttc ctg 1776Lys Val Thr Lys Phe Met Leu Cys Arg Ser Glu Pro Glu Glu Phe Leu580 585 590act ccg aaa aaa ctg cag tgc gtt gac ctg cac gtt atc tct aac gac 1824Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser Asn Asp595 600 605gtt tgc gct cag gtt cac ccg cag aaa gtt act aaa ttc atg ctg tgc 1872Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys610 615 620aag ctt gcg gcc gca ctc gag cac cac cac cac cac cac tga 1914Lys Leu Ala Ala Ala Leu Glu His His His His His His625 630 635<210>8<211>637<212>PRT<213>Artificial Sequence<220><223>Recombinant Gene<400>8Met Ala Lys Thr Ile Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu1 5 10 15Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro20 25 30Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr lle35 40 45Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro50 55 60Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr65 70 75 80Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln85 90 95Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Prol00 105 110Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Glu Lys Val Thr Glu115 120 125Thr Leu Leu Lys Gly Ala Lys Glu Val Glu Thr Lys Glu Gln Ile Ala130135 140Ala Thr Ala Ala Ile Ser Ala Gly Asp Gln Ser Ile Gly Asp Leu Ile145 150 155 160Ala Glu Ala Met Asp Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu165 170 175Glu Ser Asn Thr Phe Gly Leu Gln Leu Glu Leu Thr Glu Gly Met Arg180 185 190Phe Asp Lys Gly Tyr Ile Ser Gly Tyr Phe Val Thr Asp Pro Glu Arg195 200 205Gln Glu Ala Val Leu Glu Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys210 215 220Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gly225 230 235 240Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala245 250 255Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val260 265 270Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln275 280 285Asp Met Ala Ile Leu Thr Gly Gly Gln Val Ile Ser Glu Glu Val Gly290 295 300Leu Thr Leu Glu Asn Ala Asp Leu Ser Leu Leu Gly Lys Ala Arg Lys305 310 315 320Val Val Val Thr Lys Asp Glu Thr Thr Ile Val Glu Gly Ala Gly Asp325 330 335Thr Asp Ala Ile Ala Gly Arg Val Ala Gln Ile Arg Gln Glu Ile Glu340 345 350Asn Ser Asp Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg Leu Ala355 360 365Lys Leu Ala Gly Gly Val Ala Val Ile Lys Ala Gly Ala Ala Thr Asp370 375 380Val Glu Leu Lys Glu Arg Lys His Arg Ile Glu Asp Ala Val Arg Asn385 390 395 400Ala Lys Ala Ala Val Glu Glu Gly Ile Val Ala Gly Gly Gly Val Thr405 410 415Leu Leu Gln Ala Ala Pro Thr Leu Asp Glu Leu Lys Leu Glu Gly Asp420 425 430Glu Ala Thr Gly Ala Asn Ile Val Lys Val Ala Leu Glu Ala Pro Leu435 440 445Lys Gln Ile Ala Phe Asn Ser Gly Leu Glu Pro Gly Val Val Ala Glu450 455 460Lys Val Arg Asn Leu Pro Ala Gly His Gly Leu Asn Ala Gln Thr Gly465 470 475 480Val Tyr Glu Asp Leu Leu Ala Ala Gly Val Ala Asp Pro Val Lys Val485 490 495Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Ile Ala Gly Leu Phe Leu500 505 510Thr Thr Glu Ala Val Val Ala Asp Lys Pro Glu Lys Glu Lys Ala Ser515 520 525Val Pro Gly Gly Gly Asp Met Gly Gly Met Asp Phe His Met Ala Ser530 535 540Glu Phe Glu Pro Glu Asp Phe Leu Thr Pro Lys Lys Leu Gln Cys Val545 550 555 560Asp Leu His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln565 570 575Lys Val Thr Lys Phe Met Leu Cys Arg Ser Glu Pro Glu Glu Phe Leu580 585 590Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser Asn Asp595 600 605Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys610 615 620Lys Leu Ala Ala Ala Leu Glu His His His His His His625 630 635<210>9<211>20<212>DNA<213>Artificial Sequence<220><223>Primer<400>9ccatggccaa gacaattgcg 20<210>10<211>18<212>DNA<213>Artificial Sequence<220><223>Primer<400>10gaaatccatg ccacccat18<210>11<211>20<212>DNA<213>Artificial Sequence<220><223>Primer<400>11ccatggccaa gacaattgcg 20<210>12<211>117<212>DNA<213>Artificial Sequence<220><223>Primer<400>12aagcttttta gtaactttct gcgggtgaac ctgagcgcaa acgtcgttag agataacgtg 60caggtcaacg cactgcagtt ttttcggagt caggaagaaa tccatgccac ccatgtc117<210>13<211>41<212>DNA<213>Artificial Sequence<220><223>Primer<400>13gccgagaatt cgagcctgaa gagttcctga ctccgaaaaa a 41<210>14<211>41<212>DNA<213>Artificial Sequence<220><223>primer<400>14gggcgagatc tacacagcat gaatttagta actttctgcg g 41<210>15<211>136<212>DNA<213>Artificial<400>15gccgagaatt cgagcctgaa gagttcctga ctccgaaaaa actgcagtgc gttgacctgc 60acgttatctc taacgacgtt tgcgctcagg ttcacccgca gaaagttact aaattcatgc120tgtgtagatc tcgccc136<210>16<211>40<212>DNA<213>Artificial Sequence<220><223>Primer<400>16gcgtggatcc gagcctgaag agttcctgac tccgaaaaaa 40<210>17<211>41<212>DNA<213>Artificial Sequence<220><223>Primer<400>17cgccgaagct tgcacagcat gaatttagta actttctgcg g41<210>18<211>135<212>DNA<213>Artificial<400>18gcgtggatcc gagcctgaag agttcctgac tccgaaaaaa ctgcagtgcg ttgacctgca60cgttatctct aacgacgttt gcgctcaggt tcacccgcag aaagttacta aattcatgct 120gtgcaagctt cggcg135<210>19<211>246<212>DNA<213>Artificial<400>19gaattcgagc ctgaagagtt cctgactccg aaaaaactgc agtgcgttga cctgcacgtt60atctctaacg acgtttgcgc tcaggttcac ccgcagaaag ttactaaatt catgctgtgt 120agatccgagc ctgaagagtt cctgactccg aaaaaactgc agtgcgttga cctgcacgtt 180atctctaacg acgtttgcgc tcaggttcac ccgcagaaag ttactaaatt catgctgtgc 240aagctt 24權(quán)利要求
1.一種重組蛋白疫苗，它是由卡介苗熱休克蛋白65和1至5個(gè)拷貝的人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位相連接而形成的融合蛋白，其中，所述的人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位的單拷貝多肽具有SEQ ID NO2所示的氨基酸序列。
2.按照權(quán)利要求1所述的重組蛋白疫苗，其中，卡介苗熱休克蛋白65位于該融合蛋白的氨基端，人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位位于該融合蛋白的羧基端。
3.按照權(quán)利要求1所述的重組蛋白疫苗，其中，所述的人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位的拷貝數(shù)為2，具有SEQ ID NO4所示的氨基酸序列。
4.按照權(quán)利要求1所述的重組蛋白疫苗，它具有SEQ ID NO6所示的氨基酸序列。
5.按照權(quán)利要求1所述的重組蛋白疫苗，它具有SEQ ID NO8所示的氨基酸序列。
6.編碼權(quán)利要求1至5中任意一項(xiàng)所述的重組蛋白疫苗的基因。
7.按照權(quán)利要求6所述的基因，它們分別具有SEQ ID NO5或SEQ IDNO7所示的核苷酸序列。
全文摘要
本發(fā)明提供了一種重組蛋白疫苗,它是由卡介苗熱休克蛋白65和1至5個(gè)拷貝的人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位相連接而形成的融合蛋白,其中,所述的人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位的單拷貝多肽具有SEQ ID NO:2所示的氨基酸序列,人前列腺特異性抗原細(xì)胞毒性T淋巴細(xì)胞多表位的雙單拷貝多肽具有SEQ ID NO:4所示的氨基酸序列。本發(fā)明的融合蛋白在應(yīng)用于人體后可有效地預(yù)防和治療前列腺癌。本發(fā)明還提供了編碼這兩種重組蛋白疫苗的基因。
文檔編號(hào)C12N15/31GK1362263SQ0113493
公開(kāi)日2002年8月7日 申請(qǐng)日期2001年11月15日 優(yōu)先權(quán)日2001年1月4日
發(fā)明者王麗穎, 李大鵬, 于永利 申請(qǐng)人:北京迪威華宇生物技術(shù)有限公司