專利名稱：：嵌合狂犬病病毒株rLEP333Q-L(H)的構(gòu)建及其生物學(xué)功能的研究的制作方法
技術(shù)領(lǐng)域：
：本發(fā)明涉及重組疫苗領(lǐng)域，具體地涉及一種用于制備弱毒疫苗的嵌合狂犬病病毒株rLEP333Q-L(H)，本發(fā)明還涉及所述病毒株的應(yīng)用。引言狂犬病是一種很重要的人畜共患病，在世界范圍內(nèi)每年約有60，000余人死于狂犬病[1]，引起嚴(yán)重的公共健康問題?？袢〔《緦儆趶棤畈《究?、狂犬病毒屬，為不分節(jié)段單股負(fù)鏈RNA病毒，其基因組長(zhǎng)約12kb，共編碼5個(gè)結(jié)構(gòu)蛋白核蛋白(N)、磷蛋白(P)、基質(zhì)蛋白(M)、糖蛋白(G)和RNA聚合酶(L)。依賴RNA的RNA聚合酶L是狂犬病病毒中最大的結(jié)構(gòu)蛋白，是一多功能酶，在病毒基因組復(fù)制、轉(zhuǎn)錄及轉(zhuǎn)錄后的加工，包括甲基化戴帽和多聚腺苷酸化等方面發(fā)揮作用。1940年，Koprowski等人從病死女孩腦內(nèi)分離到狂犬病毒Flury株，經(jīng)1日齡雛雞腦內(nèi)、雞胚卵黃囊傳代后，再經(jīng)雞胚傳代178代以上，無論神經(jīng)內(nèi)、外接種都對(duì)動(dòng)物喪失致病力，但新生乳鼠和猴在腦內(nèi)接種時(shí)仍具有殘留毒力[2]。在雞胚傳代68代以前的稱為“LEP”(雞胚低代株)，高于136代的稱為“HEP”(雞胚高代株)。LEP腦內(nèi)感染成年小鼠致死，而HEP腦內(nèi)感染成年小鼠不致死。Flury-LEP株具有優(yōu)良的免疫原性，被廣泛用作人或動(dòng)物的狂犬病滅活疫苗種毒株。WHO對(duì)使用狂犬病病毒弱毒活疫苗的要求是小鼠腦內(nèi)感染不致死，因此，有待對(duì)LEP株進(jìn)行改造，以期獲得安全有效的活疫苗推薦株。為獲得安全、高效的狂犬病弱毒活疫苗株，本研究利用反向遺傳技術(shù)構(gòu)建了將LEP的糖蛋白第333位精氨酸(Arg)突變?yōu)楣劝滨０?Gln)，同時(shí)將L基因替換為HEP的L基因的嵌合病毒rLEP333Q-L(H)。我們鑒定了該嵌合病毒株的生物學(xué)特性，并將其與Flury-LEP、HEP進(jìn)行了比較。結(jié)果表明重組病毒rLEP333Q_L(H)具有很好的安全性和免疫原性，適用于作為狂犬病病毒弱毒疫苗的開發(fā)。
發(fā)明內(nèi)容在第一個(gè)方面中，本發(fā)明提供一株嵌合狂犬病病毒株，該嵌合病毒是在親本弱毒疫苗株Flury-LEP基礎(chǔ)上，將G蛋白第333位精氨酸突變?yōu)楣劝滨０?，同時(shí)將LEP的L基因替換為HEP的L基因，并拯救獲得的感染性病毒顆粒。在第一個(gè)方面的一個(gè)實(shí)施方案中，其中構(gòu)建的病毒株為rLEP333Q_L(H)。在第一個(gè)方面的一個(gè)實(shí)施方案中，所述Flury-LEP的全長(zhǎng)基因組cDNA序列如SEQIDNO1所示。在第一個(gè)方面的一個(gè)實(shí)施方案中，其中所述HEP的L基因的序列如SEQIDNO8所顯示。在第二個(gè)方面中，本發(fā)明提供一種構(gòu)建第一方面的病毒株的方法，所述方法包括下列步驟1)構(gòu)建轉(zhuǎn)錄質(zhì)粒，所述轉(zhuǎn)錄質(zhì)粒包含將G蛋白第333位精氨酸突變?yōu)楣劝滨０?，同時(shí)將LEP的L基因替換為HEP的L基因的Flury-LEP全長(zhǎng)基因組cDNA序列；2)構(gòu)建一個(gè)或多個(gè)轉(zhuǎn)錄輔助質(zhì)粒，所述輔助質(zhì)粒包括編碼狂犬病病毒Flury-LEP核蛋白N、磷酸蛋白P及聚合酶蛋白L基因的開放閱讀框架的cDNA序列；3)將所述轉(zhuǎn)錄質(zhì)粒和轉(zhuǎn)錄輔助質(zhì)粒共轉(zhuǎn)染允許狂犬病病毒Flury-LEP弱毒疫苗株復(fù)制的宿主細(xì)胞，培養(yǎng)轉(zhuǎn)染后的宿主細(xì)胞；4)收獲上清液，獲救嵌合病毒株。在第二個(gè)方面的一個(gè)實(shí)施方案中，其中所述轉(zhuǎn)錄質(zhì)粒為pCI-LEP333Q_L(H)。在第二個(gè)方面的一個(gè)實(shí)施方案中，其中所述轉(zhuǎn)錄輔助質(zhì)粒分別為pCAGG-N、pCAGG-P或pCAGG-L。在第二個(gè)方面的一個(gè)實(shí)施方案中，其中所述宿主細(xì)胞為真核細(xì)胞。在第二個(gè)方面的一個(gè)實(shí)施方案中，其中所述真核細(xì)胞為BHK-21細(xì)胞。在第三個(gè)方面中，本發(fā)明提供一種載體，其包含將G蛋白第333位精氨酸突變?yōu)楣劝滨０?，同時(shí)將LEP的L基因替換為HEP的L基因的Flury-LEP全長(zhǎng)基因組cDNA序列。在在第三個(gè)方面的一個(gè)實(shí)施方案中，所述Flury-LEP的全長(zhǎng)基因組cDNA序列如SEQIDNO1所示。在第三方面的一個(gè)實(shí)施方案中，其中所述HEP的L基因的序列如SEQIDN0:8所顯7J\ο在第四個(gè)方面中，本發(fā)明提供第一個(gè)方面的病毒株和第三個(gè)方面的載體用于制備弱毒疫苗的應(yīng)用。圖1是rLEP和rLEP333Q_L(h)的基因組圖示。黑色方框代表LEP株各個(gè)基因的ORF，白色方框代表HEP株L基因的ORF。圖2左圖間接免疫熒光檢測(cè)拯救病毒rLEP333Q_L(h)感染NA細(xì)胞，右圖陰性對(duì)照。圖3LEP、HEP和rLEP333Q_L(H)在NA細(xì)胞上的生長(zhǎng)動(dòng)力學(xué)曲線。圖4每組小鼠的體重變化情況，以接種病毒當(dāng)天體重為100%，觀察21天。圖5HEP、LEP或rLEP333Q_L(h)免疫小鼠后誘導(dǎo)的中和抗體水平以及攻毒結(jié)果。A=IO6FFU的HEP或rLEP333Q_L(h)，或IO4FFU的HEP、LEP或rLEP333Q_L(h)免疫小鼠3周后誘導(dǎo)的中和抗體水平。B免疫后小鼠用街毒GuangXi09株進(jìn)行攻毒試驗(yàn)結(jié)果，觀察期為4周。具體實(shí)施方式本發(fā)明通過具體的實(shí)施例來進(jìn)一步舉例說明，但下述實(shí)施例不應(yīng)被理解為對(duì)本發(fā)明的限制。實(shí)施例1嵌合病毒rLEP333Q_L(H)的構(gòu)建及其生物學(xué)活性鑒定1材料與方法1.1質(zhì)粒、細(xì)胞株和病毒質(zhì)粒pCAGG、pCI_RBz購(gòu)自中國(guó)農(nóng)業(yè)科學(xué)院哈爾濱獸醫(yī)研究所，pBluescriptSK(+)購(gòu)自clonetech。BHK-21細(xì)胞(ATCCCC1-10)，293T細(xì)胞(CRL-11268)購(gòu)自ATCC，培養(yǎng)基均為含10%胎牛血清的DMEM(購(gòu)自GIBC0)。小鼠腦神經(jīng)瘤細(xì)胞NA細(xì)胞來自軍事醫(yī)學(xué)科學(xué)院軍事獸醫(yī)研究所，培養(yǎng)基為含10%胎牛血清的MEM(購(gòu)自GIBCO)?？袢〔《綟lury-LEP和HEP購(gòu)自中國(guó)獸醫(yī)藥品監(jiān)察所中國(guó)獸醫(yī)微生物菌種保藏管理中心，在BHK-21細(xì)胞上擴(kuò)增，-70°C凍存?zhèn)溆谩?.2主要試劑和儀器T4DNA連接酶，限制性內(nèi)切酶NheI，SphI，MluI，EcoRI，KpnI等均購(gòu)自大連TaKaRa公司。Phusion超保真DNA聚合酶，限制性內(nèi)切酶RsrII購(gòu)自NEB公司。RNA提取試劑Trizol，SuperScriptIII反轉(zhuǎn)錄試劑盒，無血清培養(yǎng)基Opti-MEM，轉(zhuǎn)染試劑Lipofectamine2000均購(gòu)自Invitrogen公司。犢牛血清購(gòu)自Gibco公司。LB培養(yǎng)基成分均購(gòu)自O(shè)XOID公司。膠回收試劑盒(GelExtractionMiniKit)及質(zhì)粒小量提取試劑盒(PlasmidMiniKit)均購(gòu)自O(shè)MEGA公司。質(zhì)粒中量提取試劑盒QIAfilterPlasmidMidiKit購(gòu)自QIAGEN公司。鼠抗狂犬病病毒高免血清由本研究室按本領(lǐng)域常規(guī)方法制備。FITC標(biāo)記的羊抗鼠熒光二抗購(gòu)自Sigma公司。普通顯微鏡，購(gòu)自O(shè)LYMPUS公司；熒光顯微鏡，購(gòu)自ZEISS公司。PCR儀購(gòu)自AppliedBiosystem公司。核酸測(cè)序采用BECKMANCEQ8000自動(dòng)測(cè)序儀，所用試劑為BECKMAN公司產(chǎn)品。P2級(jí)生物安全柜購(gòu)LABC0NC0自公司。二氧化碳培養(yǎng)箱購(gòu)自ThermoElectron公司。不同型號(hào)離心機(jī)均購(gòu)自BECKMAN公司。_70°C超低溫冰箱購(gòu)自NBS公司。普通冰箱冰柜購(gòu)自海爾公司。全自動(dòng)凈水機(jī)MILI-Q購(gòu)自BIOCEL公司。各種規(guī)格細(xì)胞培養(yǎng)瓶及細(xì)胞培養(yǎng)板均購(gòu)自CORNING公司。1.3引物參照GeneBank中所提供的Flury-HEP株基因組序列(GeneBank序列號(hào)AB085828)和Flury-LEP基因序列(GeneBank序列號(hào)DQ099524，F(xiàn)lury-LEP全長(zhǎng)基因組cDNA序列見SEQIDNO1)，將Flury-LEP基因組分為3段(即，F(xiàn)l，F(xiàn)2和F3)分別設(shè)計(jì)PCR引物。其中，片段Fl為Flury-LEP基因序列的1-4063核苷酸序列(SEQIDN0:2)，片段F2為Flury-LEP基因序列的4013-8254核苷酸序列(SEQIDNO3)，片段F3為Flury-LEP基因序列的8187-11925核苷酸序列(SEQIDNO:4)。構(gòu)建全長(zhǎng)基因組cDNA所用引物、引入PmeI酶切位點(diǎn)引物、將G蛋白第333位精氨酸突變?yōu)楣劝滨０返囊锖蛯⑻鎿QL基因的引物(表1)，以及構(gòu)建輔助質(zhì)粒所用引物(表2)均由上海英俊生物技術(shù)有限公司合成。表1RT-PCR和全長(zhǎng)基因組cDNA構(gòu)建所用的引物引物名引物序列Jl______________F1-PF5'-TGCGCTAGCTGTTAAGCGTCTGATGAGTCCGTGAGGACGAAACTATAGGAAAGGAATTCCTATAGTCACGClTAACAACAAAACCAAAGAA-3'Fl-PR5'-GGCACGCGTACTCCACATAACTTGAGTTTGC-3'F2-PF5'-AGGCCTGTATAAGTCTTTAAAGGGAGCA-3'F2-PR5'-ATCGGGGTTCCCGGCCTCTTGACACAAC-3'F3-PF5'-TATGCTAGCTCTGGTTAAGCTCCCACGAATC-3'F3-PR5'-ATACGGJCCGCGAGGAGGTGGAGATGCCATGCCGACCCACGCTTAACAAATAAACAATAAAG-3'Pme5'-GACTTGAAGTTTAAACAGGATGACCGGCCTTCACAI-PRGTCTGGT-3'Pme5'-GTCATCCTGTTTAAACTTCAAGTCCCGAGGATAACCI-PFTCC-3'RtoQ-P5'-TCAAACACCCTTTTGAGGGGATGATCTCATTCCAGGRT0GGACAGACTTGTAGTGAGCATCAGCCTCCATCAAGGT-3'RtoQ-PF5'-GTCTGTCC|AA]ACCTGGAATGAGATCATCCCCTCAAAAGGGTGTTTGA-3'5'CLF5'-AGATGCTGGATCCGGGAGAGGTTTA-3'3'CLR5'-GAAGCGGCCGCCCGGGCCCCGCGGG-3'5，CL+F5'-GAGACCAGACTGTGAAGGCCGGTCA-3'3’CL+R5’-TGACTCAATTGGATCAATAGGGTCA-3'注cDNA片斷與圖1相一致。下劃線部分為病毒特異序列，黑體表示酶切位點(diǎn)，錘頭狀核酶(HamRz)和丁肝病毒核酶(HdvRz)用斜體表示；突變堿基用字符邊框表示。表2輔助質(zhì)粒構(gòu)建所用的引物<table>tableseeoriginaldocumentpage7</column></row><table>注下劃線部分為病毒特異序列，黑體表示酶切位點(diǎn)，突變堿基用字符邊框表示，Kozak序列用斜體表示。1.4構(gòu)建Flury-LEP基因組全長(zhǎng)cDNA克隆和嵌合病毒全長(zhǎng)cDNA克隆1.4.1病毒基因組的提取、反轉(zhuǎn)錄和PCR取Flury-LEP病毒液250iiL，加入750iiLTrizol，經(jīng)常規(guī)方法提取基因組RNA。提取的RNA用SUperSCriptTMIII反轉(zhuǎn)錄試劑盒進(jìn)行反轉(zhuǎn)錄反應(yīng)(反轉(zhuǎn)錄引物見表1)按該試劑盒說明書進(jìn)行操作，獲得末端部分重疊的3個(gè)病毒cDNA片段Fl、F2和F3片段。1.4.2RT-PCR產(chǎn)物的克隆整個(gè)基因組分為末端部分重疊的3個(gè)片斷進(jìn)行RT-PCR擴(kuò)增，PCR產(chǎn)物經(jīng)瓊脂糖凝膠電泳，使用膠回收試劑盒(購(gòu)自O(shè)MEGA公司)回收Fl、F2和F3片段，分別克隆至pBluscriptIIKS(+/")(Clontech)的EcoRv位點(diǎn)，轉(zhuǎn)化感受態(tài)細(xì)胞DH5a，選擇并提取陽性質(zhì)粒pBlue-Fl，pBlue-F2和pBlue_F3，測(cè)序確保克隆的基因組片段與病毒基因組RNA序列完全一致(具體方法見《分子克隆試驗(yàn)指南》，J.薩姆布魯克編，2008年第三版，科學(xué)出版社譯)。1.4.3Flury-LEP基因組全長(zhǎng)cDNA克隆和cDNA片段克隆的構(gòu)建利用基因組cDNA片段F1、F2和F3相鄰重疊部分存在的限制酶切位點(diǎn)進(jìn)行連接組裝。具體地，首先將F3段cDNA經(jīng)NheI和RsrII位點(diǎn)克隆進(jìn)pCI-RBz，所得質(zhì)粒命名為PCI-F3，再將F1段經(jīng)NheI和MluI插入到pCI_F3，所得質(zhì)粒命名為pCI_F31。最后將F2段經(jīng)SphI和MluI克隆進(jìn)PCI-F31，至此Flury-LEP全基因組cDNA組裝完成，構(gòu)建成的病毒基因組轉(zhuǎn)錄質(zhì)粒命名為pCI-LEP。pCI-LEP的基礎(chǔ)上，構(gòu)建在G和L基因之間帶有PmeI酶切位點(diǎn)的全長(zhǎng)cDNA克隆，具體方法是以pCI-LEP為模板，利用F2-PF和PmeI-PR為引物，擴(kuò)增出PCR片段PmeA，以F2-PR和PmeI-PF為引物擴(kuò)增出PCR片段PmeB；然后再以PCR片段PmeA和PmeB為模板，以F2-PF和F2-ra為引物，擴(kuò)增出含有PmeI酶切的F2片段，命名為F2-P。將F2-P經(jīng)SphI和MluI克隆進(jìn)PCI-F31，從而構(gòu)建成重組基因組轉(zhuǎn)錄載體pCI-LEP-PmeI。1.4.4G333突變?nèi)L(zhǎng)基因組的構(gòu)建具體方法是以構(gòu)建好pCI-LEP-PmeI為模板，利用F2-PF和RtoQ-PR為引物，擴(kuò)增出片段MuA；以RtoQ-PF和F2-PR為引物，擴(kuò)增出片段MuB；然后再以PCR片段MuA和MuB為模板，以F2-PF和F2-I^R為引物，擴(kuò)增出糖蛋白第333位精氨酸R突變?yōu)楣劝滨０稱的F2片段，命名為F2-Mu。最后將F2-Mu片段經(jīng)SphI和MluI替換至進(jìn)在1.4.3中已構(gòu)建好的PCI-F31，至此Flury-LEPG333突變?nèi)蚪McDNA組裝完成，構(gòu)建成的病毒基因組轉(zhuǎn)錄質(zhì)粒命名為pCI-LEP333Q。1.4.5嵌合病毒全長(zhǎng)cDNA克隆pCI-LEPG333Q-L(H)的構(gòu)建按照1.4.1中的方法提取Flury-HEP病毒基因組RNA,以5，CLF和3，CLR為引物進(jìn)行RT-PCR擴(kuò)增出含有HEP的L基因(SEQIDNO8)的PCR片段LHD。再以pCI_LEP333Q為模板，以5’CL+F和3’CL+R為引物，擴(kuò)增出含有LEP的部分G基因、假基因和部分L基因的片段LHU。然后，以LHD和LHU這2個(gè)PCR產(chǎn)物為模板，以5’CL+F和3’CLR為引物擴(kuò)增出含有HEP的L基因、其余皆為L(zhǎng)EP基因序列的片段LH。將片段LH和pCI_LEP333Q分別用PmeI和RsrII處理后連接，即將LH替換至pCI_LEP333Q的相應(yīng)位置，至此，嵌合病毒全長(zhǎng)cDNA克隆pCI-LEP333Q-L(H)構(gòu)建成功(圖1)。1.5構(gòu)建轉(zhuǎn)錄輔助質(zhì)粒系統(tǒng)以pCI-LEP為模板，PCR擴(kuò)增編碼核蛋白(N)(SEQIDNO5)、磷酸蛋白(P)(SEQIDN0:6)及聚合酶蛋白(L)基因(SEQIDNO7)的開放閱讀框架(ORF)cDNA序列，分別克隆在pCAGG質(zhì)粒的多克隆位點(diǎn)(MCS)。在起始密碼子前引入Kozak序列(GCCACC)，以增強(qiáng)蛋白表達(dá)；在原有終止密碼子后增加一個(gè)終止密碼子，以有效終止輔助質(zhì)粒mRNA的轉(zhuǎn)錄。構(gòu)建成的輔助質(zhì)粒分別命名為pCAGG-N、pCAGG-P和pCAGG_L。1.6病毒拯救在轉(zhuǎn)染前一天，將293T細(xì)胞傳到35mm6孔板上，當(dāng)細(xì)胞密度達(dá)到80%以上時(shí)即可進(jìn)行轉(zhuǎn)染。將口(1-1^卩3330-1^01)4〇八66州4〇八66-卩4〇八66-1^分別以41^、21^、11^、11^的比例加入到250ii1無血清培養(yǎng)基0PTI-MEM中，混勻；取15ylLipofectamine2000溶于250illOPTI-MEM中，混勻后在室溫靜置5min；然后將Lipofectamine2000與轉(zhuǎn)染質(zhì)粒(具體是pCI-LEP333Q-L(H)、pCAGG-N、pCAGG_P、pCAGG_L各4iig、2iig、1iig、1iig)混合均勻，室溫孵育30min。在此期間用0PTI-MEM洗滌293T細(xì)胞兩次，然后每孔加入0PTI-MEMlml，最后將Lipofectamine2000-質(zhì)?；旌弦?00yl加入細(xì)胞培養(yǎng)孔中，置于37°CC02培養(yǎng)箱中培養(yǎng)8h后吸棄轉(zhuǎn)染液，加入10%DMEM繼續(xù)培養(yǎng)。72小時(shí)后，仔細(xì)吹落培養(yǎng)板上的細(xì)胞，將培養(yǎng)液及細(xì)胞混合物全部接種BHK-21細(xì)胞。72h后，在BHK-21細(xì)胞上用間接免疫熒光(IFA)檢測(cè)病毒滴度，簡(jiǎn)言之，即以鼠抗狂犬病病毒高免血清為一抗，相同倍數(shù)稀釋的小鼠陰性血清為對(duì)照，熒光素(FITC)標(biāo)記的羊抗鼠IgG(Sigma)為二抗，最后用熒光顯微鏡觀察結(jié)果。收獲上清液，獲救突變病毒株，獲救病毒株命名為rLEP333Q-L(H)株。1.7種毒的制備及滴定將拯救的病毒在BHK-21細(xì)胞上進(jìn)行擴(kuò)增，10%DMEM37°C培養(yǎng)3天。制備好的種毒分別在NA細(xì)胞和BHK-21細(xì)胞上培養(yǎng)48h后，以IFA(步驟同1.6)確定病毒滴度。病毒滴度用病灶形成單位(focusformingunit)FFU表示。1.8病毒生長(zhǎng)動(dòng)力學(xué)曲線的測(cè)定為測(cè)定病毒生長(zhǎng)動(dòng)力學(xué)曲線，將LEP、HEP和rLEP333Q-L(H)分別以M.0.I.為0.01分別感染單層NA細(xì)胞。感作lh后，用無菌PBS洗2次后，分別加含0.2%FBS的MEM2ml,分別于接種后24h、72h和120h取細(xì)胞培養(yǎng)液上清，并在NA細(xì)胞測(cè)定滴度，繪制病毒生長(zhǎng)動(dòng)力學(xué)曲線。1.9對(duì)小鼠致病力的測(cè)定8將105FFU/30u1的LEP、HEP和rLEP333Q_L(H)分別腦內(nèi)接種或106FFU/100u1小鼠后腿肌肉注射，56周齡的Balb/c雌性小鼠。連續(xù)觀察21天，記錄每組小鼠的平均體重變化和死亡情況。1.10嵌合病毒rLEP333Q_L(H)對(duì)小鼠的免疫保護(hù)試驗(yàn)為了評(píng)價(jià)嵌合病毒rLEP333Q_L(H)對(duì)小鼠的免疫效果，分別將106FFU/100u1的HEP和rLEP333Q-L(H)，或是104FFU/100u1LEP,HEP和rLEP333Q_L(H)后腿肌肉注射4周齡Balb/c雌性小鼠，每組10只。免疫后3周采血、分離血清，用于中和抗體滴度檢測(cè)。對(duì)免疫后的小鼠進(jìn)行攻毒試驗(yàn)，以街毒GuangXi09為攻擊毒株，每只小鼠后腿肌肉注射50LD50/100u1的GuangXi09，每日觀察小鼠，記錄其死亡情況，持續(xù)觀察4周。1.11中和試驗(yàn)血清樣品首先56°C水浴處理30min滅活，隨后連續(xù)5倍至320倍倍比稀釋，同時(shí)設(shè)陰性、標(biāo)準(zhǔn)血清對(duì)照。攻擊病毒采用標(biāo)準(zhǔn)固定毒CVS株，將稀釋好的血清與含100000個(gè)FFU的病毒等體積混合，37°C孵育lh，然后取100ill加到過夜生長(zhǎng)的24孔BHK-21細(xì)胞上。每個(gè)樣品作3個(gè)重復(fù)。感染后48h進(jìn)行IFA檢測(cè)。被檢血清的中和抗體滴度為使熒光灶抑制等于50%的血清最高倍數(shù)。標(biāo)準(zhǔn)血清的起始稀釋效價(jià)為2IU/ml，待測(cè)抗體單位(IU/ml)=待測(cè)血清中和滴度/標(biāo)準(zhǔn)血清中和滴度X2。2.結(jié)果2.1從全長(zhǎng)cDNA克隆拯救嵌合病毒rLEP333Q-L(H)為了從克隆的cDNA中拯救感染性RV，首先以pCI_LEP333Q_L(H)及表達(dá)RVN、P、L蛋白的輔助質(zhì)粒共轉(zhuǎn)染293T細(xì)胞。3天后收獲細(xì)胞液，盲傳一代后IFA檢測(cè)呈陽性結(jié)果(圖2)。對(duì)拯救病毒進(jìn)行RT-PCR及基因組序列分析結(jié)果顯示，獲救病毒基因組cDNA的第4907的堿基為G，第4910的為T，第4912的為A;G蛋白第333位精氨酸R(CGG)已經(jīng)突變?yōu)楣劝滨０稱(CAA)；擴(kuò)增的部分L基因片段與HEP的L基因一致，和預(yù)期完全相符。結(jié)果表明，通過反向遺傳操作技術(shù)，成功拯救出具有感染性的嵌合病毒rLEP333Q-L(H)。2.2嵌合病毒rLEP333Q_L(H)生物學(xué)特性的鑒定2.2.1動(dòng)力學(xué)曲線rLEP333Q-L(H)在神經(jīng)元細(xì)胞NA上的生長(zhǎng)動(dòng)力學(xué)曲線如圖所示(圖3)，rLEP333Q-L(H)在NA細(xì)胞上的生長(zhǎng)水平在各個(gè)時(shí)間點(diǎn)均低于LEP和HEP，到達(dá)的最高水平為2X106FFU/ml，分別較LEP和HEP低約30倍和20倍。2.2.2嗜神經(jīng)指數(shù)狂犬病病毒的體外嗜神經(jīng)指數(shù)是指病毒在NA細(xì)胞相對(duì)于BHK-21細(xì)胞上的感染性比例[6]，嗜神經(jīng)指數(shù)為1即表示病毒在神經(jīng)元細(xì)胞上感染的能力比在非神經(jīng)細(xì)胞上的高10倍。致病性毒株與弱毒株相比通常具有較高的嗜神經(jīng)指數(shù)。致病性毒株的嗜神經(jīng)指數(shù)一般大于1，而弱毒株的嗜神經(jīng)指數(shù)通常接近于0[3’4’5]。LEP和HEP的嗜神經(jīng)指數(shù)分別為0.8和0(表3)，嵌合病毒rLEP333Q-L(H)的嗜神經(jīng)指數(shù)已下降與無毒株HEP—樣為零。表3LEP、rLEP333Q-L(H)和HEP的體外嗜神經(jīng)性<table>tableseeoriginaldocumentpage10</column></row><table>LEP3.7xl085.3xio70.8HEP5.0xl065.0xl060rLEP333Q-L(h)1.0x107_1.0x1070_a同一種毒在NA細(xì)胞和BHK-21細(xì)胞上的滴度b體外嗜神經(jīng)指數(shù)=log(NA上的滴度)-log(BHK-21上的滴度)2.2.3對(duì)小鼠的致病性為了確定LEP、HEP以及rLEP333Q_L(H)對(duì)小鼠的致病性，每只小鼠腦內(nèi)接種病毒105FFU/30iil。感染LEP的小鼠表現(xiàn)出麻痹、興奮，體重減輕等中樞神經(jīng)系統(tǒng)疾病的臨床癥狀，且所有的老鼠在11天內(nèi)全部死亡。而HEP和rLEP333Q-L(H)感染的小鼠只表現(xiàn)出輕微的癥狀如毛微亂、感染初期體重略減，在21天的觀察期內(nèi)沒有小鼠死亡(圖4)?？袢〔《就ㄟ^肌肉注射感染小鼠可以測(cè)定其由外周侵入到CNS的能力即神經(jīng)侵入能力。LEP106FFU/100u1后腿肌肉注射小鼠，所有的小鼠同樣也在11天內(nèi)全部死亡；而HEP和rLEP333Q-L(H)感染的小鼠則體重持續(xù)增長(zhǎng)，沒有出現(xiàn)死亡的情況。結(jié)果表明，嵌合病毒rLEP333Q-L(H)對(duì)成年小鼠不致病，與LEP相比，對(duì)小鼠致病性下降。2.3嵌合病毒rLEP333Q_L(H)對(duì)小鼠的免疫保護(hù)性根據(jù)0.I.E標(biāo)準(zhǔn)測(cè)定不同毒株誘導(dǎo)小鼠中和抗體水平實(shí)驗(yàn)結(jié)果顯示106FFU/100u1的rLEP333Q-L(H)免疫小鼠誘導(dǎo)產(chǎn)生的中和抗體為3.6士0.89IU，顯著高于HEP誘導(dǎo)的量(圖5A)；104FFU/100ul的LEP肌肉注射小鼠是安全的，因此，用104FFU/100u1的LEP、HEP和rLEP333Q_L(H)分別免疫小鼠并進(jìn)行比較，結(jié)果rLEP333Q-L(H)誘導(dǎo)產(chǎn)生的中和抗體為1.9士1.34IU，顯著高于LEP和HEP誘導(dǎo)的量；攻毒試驗(yàn)的結(jié)果表明，104FFU的rLEP333Q-L(H)免疫可以完全保護(hù)小鼠抵抗50LD5(1的街毒的攻擊量，而LEP和HEP免疫小鼠的保護(hù)率分別為60%和80%(圖5B)。以上的這些結(jié)果表明rLEP333Q_L(H)用于活毒疫苗使用時(shí)，與LEP相比，不僅安全性好，而且免疫原性更強(qiáng)，作為弱毒活疫苗開發(fā)具有很好的前景。3討論本研究通過反向遺傳技術(shù)成功拯救嵌合狂犬病病毒rLEP333Q_L(H)株，該嵌合病毒是在親本弱毒疫苗株Flury-LEP株反向遺傳操作系統(tǒng)基礎(chǔ)上，構(gòu)建了將G蛋白第333位精氨酸突變?yōu)楣劝滨０?，同時(shí)將LEP的L基因替換為HEP的L基因的全長(zhǎng)cDNA克隆并拯救獲得感染性病毒顆粒。對(duì)rLEP333Q_L(H)株的生物學(xué)特性分析發(fā)現(xiàn)，該病毒株在神經(jīng)細(xì)胞NA細(xì)胞上的復(fù)制生長(zhǎng)水平均低于LEP株和HEP株，可見聚合酶基因L的替換對(duì)病毒的生長(zhǎng)有著十分重要的影響。rLEP333Q-L(H)的體外嗜神經(jīng)指數(shù)和體內(nèi)的神經(jīng)侵入能力均已降至和HEP—樣，更為重要的是rLEP333Q-L(H)株腦內(nèi)感染成年小鼠時(shí)，對(duì)小鼠不致死，達(dá)到WHO對(duì)于弱毒活疫苗的使用標(biāo)準(zhǔn)。對(duì)小鼠的免疫試驗(yàn)表明，和同樣安全的HEP相比較，嵌合病毒rLEP333Q-L(H)株誘導(dǎo)更高的中和抗體水平，說明rLEP333Q-L(H)株保持了LEP株免疫原性好的特點(diǎn)；和LEP相比較，由于LEP高滴度肌肉注射小鼠致死，我們選擇了對(duì)小鼠安全的劑量，IO4FFU劑量下LEP、HEP和rLEP333Q-L(H)誘導(dǎo)的中和抗體水平和保護(hù)率的比較，發(fā)現(xiàn)rLEP333Q_L(H)誘導(dǎo)的中和抗體水平不僅高于HEP，也同時(shí)高于LEP，這可能與rLEP333Q-L(H)誘導(dǎo)較強(qiáng)的凋亡水平相關(guān)。通過以上的試驗(yàn)結(jié)果表明了，嵌合病毒rLEP333Q_L(H)不僅安全性好，而且可誘導(dǎo)較高的中和抗體水平，是作為用于開發(fā)作為狂犬病病毒弱毒活疫苗的候選毒株。參考文獻(xiàn)[l]MartinezL.Globalinfectiousdiseasesurveillance.IntJInfectDis,2000,4(4):222-8.[2]KoprowskiH,BlackJ,NelsenDJ.Studiesonchick-embryo-adapted-rabiesvirus.VI.Furtherchangesinpathogenicpropertiesfollowingprolongedcultivationinthedevelopingchickembryo.JImmunol,1954,72(1)94-106.[3]MorimotoK,HooperDC,CarbaughH,etal.Rabiesvirusquasispeciesimplicationsforpathogenesis.ProcNatlAcadSciUSA,1998,95(6):3152_6.[4]MorimotoK,FoleyHD,McGettiganJP,etal.Reinvestigationoftheroleoftherabiesvirusglycoproteininviralpathogenesisusingareversegeneticsapproach.JNeurovirol,2000,6(5):373_81·[5]ItoN,TakayamaM,YamadaK,etal.RescueofrabiesvirusfromclonedcDNAandidentificationofthepathogenicity-relatedgene!glycoproteingeneisassociatedwithvirulenceforadultmice.JVirol,2001,75(19):9121_8.SEQUENCELISTING<110>中國(guó)農(nóng)業(yè)科學(xué)院哈爾濱獸醫(yī)研究所<120>嵌合狂犬病病毒株rLEP333Q-L(H)的構(gòu)建<130>IB100516<160>8<170>PatentInversion3.1<210>1<211>11925<212>DNA<213>狂犬病病毒株<400>1acgcttaacaacaaaaccaaagaagaagcagacagcgtcagttgcaaagcaaaaatgtaa60cacccctacaatggatgccgacaagattgtgttcaaagtcaataatcaggtggtctcttt120gaagcccgagattatcgtggatcaatatgagtacaagtaccctgctatcaaagatttgaa180aaagccttgtataaccctagggaaagcccccgatttaaacaaagcttacaaatcagtttt240atcaggcatgaatgccgccaaacttgatcctgatgatgtatgctcctacttggcagcagc300aatgcagttctttgaggggacatgtccggaagactggaccagctatggaatcctgattgc360acgaaaaggagacaagatcaccccagactctctagtggagataaagcgtactgatgtaga420agggaattgggctctgacaggaggcatggaactgacaagggaccccactgtctccgaaca480tgcatctttagtcggtcttctcctgagtctgtacagattgagcaaaatatcaggacaaaa540cactggtaactataagacaaacattgcggatagaatagagcagattttcgagacagcccc600ttttgttaagatcgtggaacaccataccctgatgacaactcacaagatgtgtgctaattg660gagtactataccgaacttcagatttttggccggaacctacgacatgtttttctcacggat720cgagcatctgtattcggcaatcagagtgggcacagttgtcaccgcttatgaagactgctc780aggactggtatcgtttacagggttcataaagcagatcaatctcaccgcaagagaagcaat840actatatttcttccacaagaactttgaagaagagataaggagaatgtttgagccagggca900agagacagctgttcctcactcttatttcattcacttccgttcactaggcttgagtgggaa960gtctccttattcatcaaatgccgtcggtcatgtgttcaatctcattcactttgttggatg1020ctatatgggtcaagtcagatctctaaatgcaacggttattgctgcatgtgcccctcatga1080gatgtctgttctagggggctatttgggagaggagttcttcggaaaagggacatttgaaag1140aaggttcttcagagacgagaaagaacttcaagaatatgaggcggctgaactgacaaagac1200cgacgtggcactggcagatgacggaaccgtcaactccgatgacgaggactacttctccgg1260tgaaaccagaagtccagaagctgtctatactcgaatcatgatgaatggaggtcgactgaa1320gagatcacatatacggagatatgtctcagtcagttccaatcatcaagcccgtccaaactc1380attcgccgaatttttaaacaaaacgtattcgagtgactcataaggagttgaatgacaggg1440tgccagaaatccatagattgtgtatatccatcatgaaaaaaactaacactcctcctttcg1500aaccatcccaagtatgagcaagatctttgttaatccgagtgcaatcagagccggtctggc1560cgatcttgagatggccgaagagactgttgatctgatcaacagaaacatagaagacaatca1620ggctcatctccagggagaacccatagaagtggacaatctccctgaggacatgaggcaatt1680tcacctgggcgatgaaaaattgtccaaccttggtgagatggttagggtgggcgaaggcaa1740gtatcgagaggactttcagatggatgagggagaggaccccaacctcctgttccaatcgta1800cctggacaatgttggagtccaaatagtcagacaaatgaggtcaggagagagattcctcaa1860gatatggtcaca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ggctttgtggatgaaagaggcctgt4020ataagtctctaaagggagcatgcaaactcaagttatgtggagt4063<210>3<211>4242<212>DNA〈213〉狂犬病病毒株〈400>3aggcctgtataagtctctaaagggagcatgcaaactcaagttatgtggagttctcggact60tagacttatggatggaacatgggtcgcgatgcaaacatcagatgagaccaaatggtgccc120.tccaggtcagttggtgaatttgcacgactttcgctcagacgagattgagcatctcgttgt180ggaagagttagtcaagaaaagagaggagtgtctggatgcactagagtccatcatgaccac240caagtcagtgagtttcagacgtctcagtcacttgagaaaacttgtccctgggtttggaaa300agcatataccatattcaacaaaaccttgatggaggctgatgctcactacaagtctgtccg360gacctggaatgagatcatcccctcaaaagggtgtttgagagttggggggaggtgtcatcc420ccatgtgaacggggtgtttttcaatggtataatattagggtctgacggccatgttctaat480cccagagatgcagtcatccctcctccagcaacatatggagttgttggaatcttcagttat540ccccctgatgcaccccttggcagacccttctacagttttcaaagacggtgatgaggttga600ggattttgttgaagttcacctccccgatgtgcatgaacaggtctcaggagttgaactggg660tctcccgaactgggggaagtatgtattgatgattgcaggggccttgattgccctgatgtt720gataattttcctgatgacatgttgcagaagagtcaatcgaccagaatctacgcaaagcag780tcttggagagacagggagaaatgtgtcagtcacttcccaaagcggaaaagtcatatcttc840atgggagtcatataagagtggaggcgagaccagactgtgaaggccggtcatccttttgac900acttcaagtcccgaggataacctcctctcggggttggggggaatcttgggatccagtagt960cctccttgaactccatccaacagggtagatttaagagtcatgagactttcattaatcatc1020tcagttgatcagacatggtcgtgtagattctcataacacgggagatcttctagcagtttc1080agtgaccaacggtgctttcattctccaggaactgataccaaaggttgtggacaagccaag1140gggtgcttcggattactctgtgcttgggcacagaaagaggtcgtagtttgccccttgata1200gcagattcaacatgaattaactaagaaaggcgatctgcctcccatgaaggacataagcaa1260tagttcacaatcatcttgcatctcagtgaagtgtacataactataaagggctgggtcatc1320taagcatttcagtcgagaaaaaaactgtagaccaaaagaacaactaacaacacttctcat1380cccgagacccatatcaagatgctggatccgggagaggtttatgatgaccctattgatcca1440attgagtcagaggctgaacccagaggaacccccactgtccccaacatcttgaggaactct1500gactacaatctcaactctcctttgatagaggactctgccaaactaatgttagaatggttg1560aaaacagggaacagaccttatcggatgactttgacagacaattgctccaggtcttacaaa1620gttttgaaagattatttcaagaaagtagatttgggttctctcaaagtgggaggaactgct1680gcacaatcaatgatttctctctggttgtatggagcccactctgagtcaaacaggagccgg1740agatgtataaccgacttggcccatttctattccaagtcatcccccatagagaagctgttg1800aattgtacgttaggaaacagaggcctgagaatcccaccagagggggtgttaagttgcctt1860gagagggtcgattatgataaggcatttgggaggtatctggccaacacgtattcctcttat1920ctgtttttccatgtaattaccttatacatgaatgccctagactgggaagaggaaaaaacc1980atcctagcattatggaaagatctaacctcagtggataccgggaaggacttggtcaaattc2040aaagatcaaatatggggactgctggttgtgacaaaggactttgtttactctcagagttct2100aactgtctttttgacagaaactatacactgatgctaaaggatcttttcttgtctcgattc2160aactccttgatgattttgctttctccccctgagccccgatactcagatgacttaatatct2220cagctgtgccagctatacatcgctggggatcaagtcttgtccttgtgtgggaactccggc2280tatgaagtcatcaaaatattggagccatatgtcgtgaacagtttggtccagagggcagag2340aagtttaggcctctcatccactccttgggagactttcctatgtttataaaagacaaggtg2400aatcaacttgaagggactttcggtcccagtgcaaaaaggttttttagggttctagatcaa2460ttcgacaacatacatgatctagtatttgtgtatggctgttacagacattgggggcacccc2520tatatagattatcggaagggtctgtcgaaactatatgatcaagttcacattaagaaagta2580atagataagtcctaccaggagtgtttagcaagtgacttggctagaaggatcctcagatgg2640ggatttgacaagtactccaagtggtatctagattcgagattccttgccctagaccacccc2700ttggctccttatatcaagacccaaacatggccacccaaacatatagtagacttggtgggg2760gacacatggcacaagctcccgatcacgcagatctttgagattcctgaatcaatggacccg2820tcagagatactggatgataaatcacattctttcaccagaacaagactagcttcttggctg2880tccgagaaccgaggggggcctgttcctagcgagaaggtcattatcacggccctgtctaag2940ccacctgtcaatccccgagagtttttgaaatctatcgacctcggaggattgccagatgat3000gacttgataattggcctcagaccaaaggaacgggagttgaagattgagggccgattcttc3060gctctaatgtcatggaatctaagattatattttgtcatcaccgagaagctcttggccaac3120tacattttgccactttttgacgcactgactatgacagacaacctgaacaaggtatttaaa3180aagttgatcgacagggtcaccgggcaagggcttttggactattcgagggtcacatacgca3240tttcacctggactatgagaaatggaacaatcatcaaagattggagtcaacggaggatgta3300ttttctgtcctagatcaggtgtttggattgaagagggtgttttctagaacacacgagttt3360tttcagaagtcctggatctattattcagacagatcagacctcattgggttacgggaggat3420cagatatattgcttggatatgtccaatggtccaacctgctggaatggccaagacggcggg3480ctagagggcttacggcagaagggctggagtctagtcagcttattgatgatagatagagaa3540tctcaaaccaggaacacaagaaccaagatactagctcaaggagacaaccaggttctgtgt3600ccgacatacatgttgtcaccgggattgtctcaagaggggcttctctatgagttagagagc3660atatcgaggaatgcactctcaatataccgagctatcgaggaaggagcatctaaactgggg3720ctgatcatcaagaaagaagagaccatgtgtagttatgactttctcatatatgggaagacc3780cccttatttcgaggcaacatattagtacctgaatccaaaagatgggcccgagtctcttgc3840atctctaacgaccaaatagtcaacctcgccaatataatgtctacagtatccaccaatgcg3900ctgacagtggcacaacactctcaatctctgatcaaacctatgagggattttctgctcatg3960tcagtacaggcagttttccactacctgctgtttagcccaatcttaaaaggcagggtttat4020aagattctgagtgctgaaggggagagctttctcctagccatgtcgcggataatctaccta4080gatccttctttgggaggggtgtctggaatgtctctcgggaggttccatatacgtcagttc4140tcagaccctgtctctgaagggttgtcattctggagagagatctggttaagctcccatgaa4200tcctggattcacgcgttgtgtcaagaggccgggaaccccgat4242<210>4<211>3739<212>DNA〈213〉狂犬病病毒株<400>4gagagatctggttaagctcccatgaatcctggattcacgcgttgtgtcaagaggccggga60accccgatcttggagagagaacactagagagcttcactcgccttttagaagatcctacta120ccttaaatatcaaaggaggggccagtcctaccattctactcaaggatgctatcagaaagg180ctctgtacgacgaggtggacaaggtggaaaattcagagtttcgagaggcaatcctgttgt240ccaagacccatagagataactttatactctttttaaaatctgttgagcctctgttccctc300gatttctcagtgaactcttc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cat1260atacggagatatgtctcagtcagttccaatcatcaagcccgtccaaactcattcgccgaa1320tttttaaacaaaacgtattcgagtgactcataa1353<210>6<211>894<212>DNA〈213〉狂犬病病毒株<400>6atgagcaagatctttgttaatccgagtgcaatcagagccggtctggccgatcttgagatg60gccgaagagactgttgatctgatcaacagaaacatagaagacaatcaggctcatctccag120ggagaacccatagaagtggacaatctccctgaggacatgaggcaatttcacctgggcgat180gaaaaattgtccaaccttggtgagatggttagggtgggcgaaggcaagtatcgagaggac240tttcagatggatgagggagaggaccccaacctcctgttccaatcgtacctggacaatgtt300ggagtccaaatagtcagacaaatgaggtcaggagagagattcctcaagatatggtcacag360accgtagaggaaattatatcctatgtcacggtcaactttcctaaccctccaggaaggtct420tcggaggataaatcaacccaaactactggccgggagctcaagaaggagacaacatccact480ctttctcagagagaaagccaaccttcaaaagccggaatggtggctcaagttgcctctggc540cctccatcccttgaatggtctgccaccaatgaagaggatgatctatcagtagaggctgag600atcgctcatcagattgctgaaagcttttccaagaagtacaagtttccctctcgatcttca660ggaatattcttgtataattttgagcaactggagatgaaccttgatgacatagttaaagag720gcaaaaaatgtaccgggcgtgacccgtctggcccatgatggatccaaaatccccctaaga780tgtgtactgggatgggtcgctttggccaactccaagaaattccaattgatagtcgaggcc840gacaagctaagcaaaatcatgcaagatgacttggatcgctacacatcatgctaa894<210>7<211>6384<212>DNA〈213〉狂犬病病毒株<400>7atgctggatccgggagaggtttatgatgaccctattgatccaattgagtcagaggctgaa60cccagaggaacccccactgtccccaacatcttgaggaactctgactacaatctcaactct120cctttgatagaggactctgccaaactaatgttagaatggttgaaaacagggaacagacct180tatcggatgactttgacagacaattgctccaggtcttacaaagttttgaaagattatttc240aagaaagtagatttgggttctctcaaagtgggaggaactgctgcacaatcaatgatttct300ctctggttgtatggagcccactctgagtcaaacaggagccggagatgtataaccgacttg360gcccatttctattccaagtcatcccccatagagaagctgttgaattgtacgttaggaaac420agaggcctgagaatcccaccagagggggtgttaagttgccttgagagggtcgattatgat480aaggcatttgggaggtatctggccaacacgtattcctcttatctgtttttccatgtaatt540accttatacatgaatgccctagactgggaagaggaaaaaaccatcctagcattatggaaa600gatctaacctcagtggataccgggaaggacttggtcaaattcaaagatcaaatatgggga660ctgctggttgtgacaaaggactttgtttactctcagagttctaactgtctttttgacaga720aactatacactgatgctaaaggatcttttcttgtctcgattcaactccttgatgattttg780ctttctccccctgagccccgatactcagatgacttaatatctcagctgtgccagctatac840atcgctggggatcaagtcttgtccttgtgtgggaactccggctatgaagtcatcaaaata900ttggagccatatgtcgtgaacagtttggtccagagggcagagaagtttaggcctctcatc960cactccttgggagactttcctatgtttataaaagacaaggtgaatcaacttgaagggact1020ttcggtcccagtgcaaaaaggttttttagggttctagatcaattcgacaacatacatgat1080ctagtatttgtgtatggctgttacagacattgggggcacccctatatagattatcggaag1140ggtctgtcgaaactatatgatcaagttcacattaagaaagtaatagataagtcctaccag1200gagtgtttagcaagtgacttggctagaaggatcctcagatggggatttgacaagtactcc1260aagtggtatctagattcgagattccttgccctagaccaccccttggctccttatatcaag1320acccaaacatggccacccaaacatatagtagacttggtgggggacacatggcacaagctc1380ccgatcacgcagatctttgagattcctgaatcaatggacccgtcagagatactggatgat1440aaatcacattctttcaccagaacaagactagcttcttggctgtccgagaaccgagggggg1500cctgttcctagcgagaaggtcattatcacggccctgtctaagccacctgtcaatccccga1560gagtttttgaaatctatcgacctcggaggattgccagatgatgacttgataattggcctc1620agaccaaaggaacgggagttgaagattgagggccgattcttcgctctaatgtcatggaat1680ctaagattatattttgtcatcaccgagaagctcttggccaactacattttgccacttttt1740gacgcactgactatgacagacaacctgaacaaggtatttaaaaagttgatcgacagggtc1800accgggcaagggcttttggactattcgagggtcacatacgcatttcacctggactatgag1860aaatggaacaatcatcaaagattggagtcaacggaggatgtattttctgtcctagatcag1920gtgtttggattgaagagggtgttttctagaacacacgagttttttcagaagtcctggatc1980tattattcagacagatcagacctcattgggttacgggaggatcagatatattgcttggat2040atgtccaatggtccaacctgctggaatggccaagacggcgggctagagggcttacggcag2100aagggctggagtctagtcagcttattgatgatagatagagaatctcaaaccaggaacaca2160agaaccaagatactagctcaaggagacaaccaggttctgtgtccgacatacatgttgtca2220ccgggattgtctcaagaggggcttctctatgagttagagagcatatcgaggaatgcactc2280tcaatataccgagctatcgaggaaggagcatctaaactggggctgatcatcaagaaagaa2340gagaccatgtgtagttatgactttctcatatatgggaagacccccttatttcgaggcaac2400atattagtacctgaatccaaaagatgggcccgagtctcttgcatctctaacgaccaaata2460gtcaacctcgccaatataatgtctacagtatccaccaatgcgctgacagtggcacaacac2520tctcaatctctgatcaaacctatgagggattttctgctcatgtcagtacaggcagttttc2580cactacctgctgtttagcccaatcttaaaaggcagggtttataagattctgagtgctgaa2640ggggagagctttctcctagccatgtcgcggataatctacctagatccttctttgggaggg2700gtgtctggaatgtctctcgggaggttccatatacgtcagttctcagaccctgtctctgaa2760gggttgtcattctggagagagatctggttaagctcccatgaatcctggattcacgcgttg2820tgtcaagaggccgggaaccccgatcttggagagagaacactagagagcttcactcgcctt2880ttagaagatcctactaccttaaatatcaaaggaggggccagtcctaccattctactcaag2940gatgctatcagaaaggctctgtacgacgaggtggacaaggtggaaaattcagagtttcga3000gaggcaatcctgttgtccaagacccatagagataactttatactctttttaaaatctgtt3060gagcctctgttccctcgatttctcagtgaactcttcagttcgtccttcttgggaatacca3120gagtcaattattggactgatacaaaactccaggacaataagaaggcagtttagaaagagt3180ctctcaagaactttagaagagtccttctacaactcagagatccacgggattaatcggatg3240acccagacacctcaaagggtcgggagagtgtggccttgctcttcagagagggcagatcta3300cttagggagatctcttggggaaggaaagtggtaggcacgacagttcctcacccttccgag3360atgttggggctgcttccaaaatcctctatttcctgcacttgtggagcaacagggggaggc3420aatcctagagtttctgtatcagtactcccgtccttcgatcagtcatttttttcacgaggt3480cccctaaagggatacttgggctcgtccacctccatgtcaacccagctattccatgcatgg3540gaaaaagtcactaatgttcatgtggtgaaaagggctatatcgttaaaagaatctataaac3600tggttcatcaatagaaattccaatttggctcaaactctaattagaaacatca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列如SEQIDN08所顯示。5.一種構(gòu)建權(quán)利要求1-4中任一項(xiàng)的病毒株的方法，所述方法包括下列步驟1)構(gòu)建轉(zhuǎn)錄質(zhì)粒，所述轉(zhuǎn)錄質(zhì)粒包含將G蛋白第333位精氨酸突變?yōu)楣劝滨０罚瑫r(shí)將LEP的L基因替換為HEP的L基因的Flury-LEP全長(zhǎng)基因組cDNA序列；2)構(gòu)建一個(gè)或多個(gè)轉(zhuǎn)錄輔助質(zhì)粒，所述輔助質(zhì)粒包括編碼狂犬病病毒Flury-LEP核蛋白N、磷酸蛋白P及聚合酶蛋白L基因的開放閱讀框架的cDNA序列；3)將所述轉(zhuǎn)錄質(zhì)粒和轉(zhuǎn)錄輔助質(zhì)粒共轉(zhuǎn)染允許狂犬病病毒Flury-LEP弱毒疫苗株復(fù)制的宿主細(xì)胞，培養(yǎng)轉(zhuǎn)染后的宿主細(xì)胞；4)收獲上清液，獲救嵌合病毒株。6.權(quán)利要求5的方法，其中所述轉(zhuǎn)錄質(zhì)粒為pCI-LEP333Q-L(H)。7.權(quán)利要求5的方法，其中所述轉(zhuǎn)錄輔助質(zhì)粒分別為pCAGG-N、pCAGG-P或pCAGG_L。8.權(quán)利要求5的方法，其中所述宿主細(xì)胞為BHK-21細(xì)胞。9.一種載體，其包含將G蛋白第333位精氨酸突變?yōu)楣劝滨０罚瑫r(shí)將LEP的L基因替換為HEP的L基因的Flury-LEP全長(zhǎng)基因組cDNA序列。10.權(quán)利要求1-4中任一項(xiàng)的病毒株和權(quán)利要求9的載體用于制備弱毒疫苗的應(yīng)用。全文摘要本發(fā)明屬于重組病毒疫苗領(lǐng)域。本發(fā)明涉及一株嵌合狂犬病病毒rLEP333Q-L(H)弱毒疫苗株，其表達(dá)的狂犬病病毒rLEP糖蛋白第333位精氨酸(Arg)突變?yōu)楣劝滨０?Gln)，同時(shí)將L基因替換為HEP的L基因。本發(fā)明還涉及生產(chǎn)所述病毒株的方法，以及所述病毒株的應(yīng)用。文檔編號(hào)A61K39/205GK101818132SQ20101012163公開日2010年9月1日申請(qǐng)日期2010年2月11日優(yōu)先權(quán)日2010年2月11日發(fā)明者步志高,王喜軍,葛金英,陶麗紅申請(qǐng)人:中國(guó)農(nóng)業(yè)科學(xué)院哈爾濱獸醫(yī)研究所