CN107988174A - 一种狂犬病-犬瘟热-犬细小病毒基因重组病毒株、构建方法及其应用 - Google Patents
一种狂犬病-犬瘟热-犬细小病毒基因重组病毒株、构建方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种狂犬病‑犬瘟热‑犬细小病毒基因重组病毒株、构建方法及其应用。本发明通过同源重组、基因替换和基因插入方法对病毒全长基因组进行基因重排，构建带有犬瘟热G基因和犬细小病毒VP2基因的重排狂犬病病毒基因组质粒，命名：狂犬病‑犬瘟热‑犬细小病毒，即pcDSRV9‑NG‑eGFP+CDV N‑PM+VP2‑L基因组质粒；通过反向遗传法拯救出狂犬病‑犬瘟热‑犬细小病毒，即rSRV9+CDV N+VP2基因重组病毒，重组狂犬病病毒的基因核苷酸序列为SEQ ID NO:1。本发明的重组基因病毒可用于犬科动物及其他野生动物狂犬病、犬瘟热、细小病毒三联口服疫苗研制以预防三种传染性疾病。
Description
技术领域
本发明涉及生物病毒疫苗研究领域，尤其涉及一种狂犬病-犬瘟热-犬细小病毒基因重组病毒株、构建方法及其应用。
背景技术
狂犬病毒属于弹状病毒科，其基因组编码有五种结构蛋白：RNA依赖的RNA聚合酶(L)，核蛋白(N)，磷酸蛋白(P)，基质蛋白(M)，外膜蛋白(G)，G蛋白和N蛋白是狂犬病病毒的主要抗原，能够刺激机体产生相应抗体和细胞免疫。狂犬病主要通过动物咬伤或抓伤进行病毒的传播，病毒是沿外围神经传至神经中枢导致细胞功能损伤和感染动物的死亡。目前，疫苗免疫是预防和控制该病最有效的手段，依靠疫苗免疫能够降低狂犬病的发生。
因此，了解和掌握狂犬病毒的相关信息极其重要，其中，为了提高抗原基因G基因的表达量，避免因外源基因的插入而影响狂犬病免疫原性表达。目前对于狂犬病的研究多限于狂犬病一种病毒，而针对由多种抗原基因重组构建的病毒研究相对较少。
发明内容
有鉴于此，本发明实施例提供了一种重组狂犬病毒株、构建方法及其应用，主要目的是构建带有犬瘟热G基因和犬细小病毒VP2基因的重排狂犬病病毒，以用于研制狂犬病-犬瘟热-细小病毒三联口服疫苗。
为达到上述目的，本发明主要提供了如下技术方案：
一方面，本发明实施例提供了一种狂犬病-犬瘟热-犬细小病毒基因重组病毒株，其为带有犬瘟热G基因和犬细小病毒VP2基因的重排狂犬病病毒，命名为：狂犬病-犬瘟热-犬细小病毒基因重组病毒，所述重组病毒的基因核苷酸序列为SEQ ID NO:1。
另一方面，本发明实施例提供了上述狂犬病-犬瘟热-犬细小病毒基因重组病毒株的构建方法，所述方法包括以下步骤：
分别将狂犬病SRV9病毒的N、P、M、G、L基因进行扩增；
采用Gibson Assembly连接法进行重组基因连接，将SRV9病毒的G基因由原基因组序列的第四位转移至病毒基因组的第二位，再将eGFP报告基因通过酶切位点插入到G和P基因之间，构建成pcDSRV9-N-G-eGFP-PML基因重组载体；
通过PCR方法扩增犬瘟热pMD18-CDV N重组质粒中的CDV N基因；
通过酶切方法对pcDSRV9-N-G-eGFP-PML重组载体的eGFP基因末端酶切位点后进行Hpa I酶切，利用Gibson Assembly连接法将犬瘟热CDV N基因构建至pcDSRV9-N-G-eGFP-PML重组载体上，构建成pcDSRV9-NG-eGFP+CDV N-PML重组载体；
通过PCR扩增犬细小病毒pMD19-T-VP2重组质粒中的VP2基因；
通过酶切方法对pcDSRV9-NG-eGFP+CDV N-PML重组载体中M基因末端酶切位点进行BsiWI酶切，将扩增的VP2基因通过Gibson Assembly连接法插入到pcDSRV9-NG-eGFP+CDVN-PML载体M-L基因之间，构建pcDSRV9-NG-eGFP+CDV N-PM+VP2-L基因重组载体；
通过反向遗传法拯救出重组狂犬病-犬瘟热-犬细小病毒，即rSRV9+CDV N+VP2基因重组病毒。
又一方面，本发明实施例提供了上述重组狂犬病毒株在制备预防狂犬病的药物中的应用。
再一方面，本发明实施例提供了一种疫苗组合物，所述疫苗组合物含有上述重组狂犬病毒株。
与现有技术相比，本发明的有益效果是：
本发明采用Gibson Assembly连接法，将狂犬病、犬瘟热和犬细小病毒基因重排，构建了带有犬瘟热N基因和犬细小病毒VP2基因的重排狂犬病病毒基因组质粒，即pcDSRV9-NG-eGFP+CDV N-PM+VP2-L基因重组载体，再通过反向遗传法拯救出狂犬病-犬瘟热-犬细小病毒，即rSRV9+CDV N+VP2基因重组病毒。
本发明所构建的基因重组病毒，可提高狂犬病病毒抗原基因G基因的表达量，避免了因插入外源基因而影响狂犬病免疫原性基因的表达，并可同时表达犬瘟热和犬细小病毒的免疫原性基因。因此，重组病毒的成功构建，可应用于“狂犬病-犬瘟热-犬细小病毒三联基因重组疫苗”和“狂犬病-犬瘟热-犬细小病毒三联基因重组口服疫苗”的研制。用于饲养犬、宠物犬以及特种养殖的犬科动物如：狐、貉等动物的狂犬病、犬瘟热和犬细小病毒疾病的免疫预防；也可用于野生动物的投喂，以预防三种传染性疾病的传播。
附图说明
图1是本发明实施例提供的构建狂犬病-犬瘟热-犬细小病毒的技术步骤框图。
具体实施方式
为更进一步阐述本发明为达成预定发明目的所采取的技术手段及功效,以下以较佳实施例，对依据本发明申请的具体实施方式、技术方案、特征及其功效，详细说明如后。下述说明中的多个实施例中的特定特征、结构、或特点可由任何合适形式组合。
实施例1(构建狂犬病-犬瘟热-犬细小病毒)
狂犬病-犬瘟热-犬细小病毒基因重组质粒的构建，如图1所示：
分别将狂犬病SRV9病毒的N、P、M、G、L基因进行扩增；
采用Gibson Assembly连接法进行重组基因连接，将SRV9病毒的G基因由原基因组序列的第四位转移至病毒基因组的第二位，再将eGFP报告基因通过酶切位点插入到G和P基因之间，构建成pcDSRV9-N-G-eGFP-PML基因重组载体；
通过PCR方法扩增犬瘟热pMD18-CDV N重组质粒中的CDV N基因；
通过酶切方法对pcDSRV9-N-G-eGFP-PML重组载体的eGFP基因末端酶切位点进行Hpa I酶切，利用Gibson Assembly连接法将犬瘟热CDV N基因构建至pcDSRV9-N-G-eGFP-PML重组载体上，构建成pcDSRV9-NG-eGFP+CDV N-PML重组载体；
通过PCR扩增犬细小病毒pMD19-T-VP2重组质粒中的VP2基因；
通过酶切方法对pcDSRV9-NG-eGFP+CDV N-PML重组载体中M基因末端酶切位点进行BsiW I酶切，将扩增的VP2基因通过Gibson Assembly连接法插入到pcDSRV9-NG-eGFP+CDV N-PML载体M-L基因之间，构建pcDSRV9-NG-eGFP+CDV N-PM+VP2-L基因重组载体；
通过反向遗传法拯救出狂犬病-犬瘟热-犬细小病毒，即rSRV9+CDV N+VP2基因重组病毒。
最后通过转化、筛选阳性克隆、酶切、电泳及测序鉴定后得到狂犬病-犬瘟热-犬细小病毒，即所述重组狂犬病毒株，其基因核苷酸序列为SEQ ID NO:1。
实施例2(反向遗传法拯救实施例1的重组病毒)
通过反向遗传学方法将构建成的狂犬病-犬瘟热-犬细小病毒(pcDSRV9-NG-eGFP+CDV N-PM+VP2-L)重组质粒与pcDNA3.1-N、pcDNA3.1-P、pcDNA3.1-M、pcDNA3.1-G及pcDNA3.1-L辅助质粒通过脂质体转染法按一定浓度共同转染至BSR细胞，拯救出狂犬病-犬瘟热-犬细小病毒“rSRV9+CDV N+VP2基因重组病毒”，并进行RT-PC、间接免疫荧光的检测。
(1)狂犬病基因重组病毒犬口服免疫方法
将犬9只随机分组，第一组为市售犬五联灭活疫苗注射对照组，注射剂量1mL/只；第二组为复合佐剂+rSRV9+CDV N+VP2基因重组病毒口服免疫试验组，灌喂剂量犬5mL/只、猫4mL/只(log7.0FFD50)；第三组为复合佐剂+“rSRV9+CDV N+VP2”基因重组病毒皮下注射试验组，注射剂量犬5mL/只。分别在0d、7d、14d口服及皮下注射，在0d和免后7d、14d、28d、58d和118d采血3mL/只，放在室温静置1～2h，5000rpm/min离心5min，吸取血清；同步采取犬的唾液，放至-20℃冰箱保存、备用。
(2)狂犬病基因重组病毒免疫犬血清IgG抗体检测方法
通过犬前肢桡骨前静脉采血5mL，5000rpm/min，离心5min，分离血清。犬血清IgG抗体检测方法严格按照“犬血清狂犬病特异性IgG抗体ELISA定量检测试剂盒”说明书操作。
通过分别对免疫犬的血液特异性抗狂犬病IgG、犬瘟热IgG、犬细小病毒IgG抗体产生水平进行检测和定量分析，并对市售犬五联灭活疫苗的免疫效果进行对比，以确定狂犬病“rSRV9+CDV N+VP2”基因重组病毒的血液抗狂犬病病毒、犬瘟热病毒和犬细小病毒的中和IgG抗体免疫效果。
(3)狂犬病基因重组病毒免疫犬血清IgA抗体检测方法
用口腔试纸从犬嘴角处插入犬口腔中，让犬充分咀嚼试纸头部棉签，待棉签充分湿润后取出，用镊子将棉签去下放入装有800ul PBS缓冲液的离心管中，在振荡器上充分振荡，5000rpm/min，离心5min，吸取上清；犬口腔黏膜IgA抗体检测方法严格按照“犬狂犬病特异性IgA抗体ELISA检测试剂盒”说明书操作。
通过分别对免疫犬的唾液特异性抗狂犬病分泌型SIgA、犬瘟热SIgA、犬细小病毒SIgA抗体产生水平进行检测和定量分析，并对市售犬五联灭活疫苗的免疫效果进行对比，以确定狂犬病“rSRV9+CDV N+VP2”基因重组病毒的唾液抗狂犬病病毒、犬瘟热病毒和犬细小病毒的SIgA抗体免疫效果。
实施例3(实施例1构建的重组病毒生物学特性的鉴定)
将构建的狂犬病“rSRV9+CDV N+VP2”基因重组病毒，通过电子显微镜观察重组病毒的形态特征；通过免疫荧光实验、实时荧光定量PCR技术、LD50实验，对重组病毒的病毒滴度和病毒毒力进行检测；通过SDS聚丙烯酰胺凝胶电泳、BCA蛋白定量法和Western-blot实验对病毒液外源蛋白的抗原性进行定性、定量分析；将重组病毒免疫实验动物(小鼠和犬)通过双抗夹心ELISA、T淋巴细胞转化试验及本体动物攻毒实验，对狂犬病-犬瘟热-犬细小病毒“rSRV9+CDV N+VP2基因重组病毒”的免疫效价及免疫保护力进行检测。
通过上述检测方法，对“rSRV9+CDV N+VP2”基因重组病毒的病毒形态变化，病毒的增殖遗传学特性、重组病毒免疫蛋白基因(狂犬病N基因、犬瘟热N基因、犬细小病毒VP2基因)的表达和产生水平进行定量分析，并对其免疫原性进行验证，以及机体细胞免疫调节水平的变化，以综合评定重组病毒的免疫效果。
实施例4(实施例1构建的重组病毒遗传稳定性的鉴定)
将重组病毒在BHK-21细胞连续培养30代，分别对每个培养代次进行TCID50测定，绘制病毒毒力变化曲线，观察重组病毒连续传代病毒毒力变化情况。
通过30代次的病毒细胞连续传代，对每个代次细胞培养的病毒进行TCID50检测，并将30代次病毒滴度变化情况绘制“病毒滴度变化曲线”，以确定重组病毒在细胞内的遗传稳定性。
实施例5(实施例1构建的重组病毒生物安全性的鉴定)
用构建的狂犬病“rSRV9+CDV N+VP2基因重组病毒”免疫犬，定期采集免疫动物的唾液、粪便及尿液，接种BHK细胞，通过间接免疫荧光试验和PCR特异性扩增方法和免疫组化试验检测免疫动物体外排毒情况，本方法通过预期半年的生物安全性检测试验，对重组病毒口服免疫后，体外排毒、病毒体内分布于代谢途径、生活环境病毒污染等情况进行鉴定，从而鉴定重组病毒使用安全性。
本发明提供了实施例1制备的重组狂犬病毒株在制备预防狂犬病的药物中的应用。
本发明提供了一种疫苗组合物，所述疫苗组合物含有实施例1制备的重组狂犬病毒株。
本发明所构建的基因重组病毒，可提高狂犬病病毒抗原基因G基因的表达量，避免了因插入外源基因而影响狂犬病免疫原性基因的表达，并可同时表达犬瘟热和犬细小病毒的免疫原性基因。因此，重组病毒的成功构建，可应用于“狂犬病-犬瘟热-犬细小病毒三联基因重组疫苗”和“狂犬病-犬瘟热-犬细小病毒三联基因重组口服疫苗”的研制。用于饲养犬、宠物犬以及特种养殖的犬科动物如：狐、貉等动物的狂犬病、犬瘟热和犬细小病毒疾病的免疫预防；也可用于野生动物的投喂，以预防三种传染性疾病的传播。
本发明构建的重组狂犬病毒株的基因核苷酸序列为SEQ ID NO:1，如下：
TGACGTCGACGGATCGGGAGGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGC(Nhe1)TGTTAAGCGTCTGATGAGTCCGTGAGGACGAAACTATAGGAAAGGAATTCCTATAGTC(Ham RZ序列)ACGCTTAACAACCAGATCAAAGAAAAAACA(N基因的转录起始信号)GACATTGTCAATTGCAAAGCAAAAATGTAACACCCCTACAATGGATGCCGACAAGATTGTATTCAAAGTCAATAATCAGGTGGTCTCTTTGAAGCCTGAGATTATCGTGGATCAATATGAGTACAAGTACCCTGCCATCAAAGATTTGAAAAAGCCCTGTATAACCCTAGGAAAGGCTCCCGATTTAAATAAAGCATACAAGTCAGTTTTGTCAGGCATGAGCGCCGCCAAACTTAATCCTGACGATGTATGTTCCTATTTGGCAGCGGCAATGCAGTTTTTTGAGGGGACATGTCCGGAAGACTGGACCAGCTATGGAATTGTGATTGCACGAAAAGGAGATAAGATCACCCCAGGTTCTCTGGTGGAGATAAAACGTACTGATGTAGAAGGGAATTGGGCTCTGACAGGAGGCATGGAACTGACAAGAGACCCCACTGTCCCTGAGCATGCGTCCTTAGTCGGTCTTCTCTTGAGTCTGTATAGGTTGAGCAAAATATCCGGGCAAAACACTGGTAACTATAAGACAAACATTGCAGACAGGATAGAGCAGATTTTTGAGACAGCCCCTTTTGTTAAAATCGTGGAACACCATACTCTAATGACAACTCACAAAATGTGTGCTAATTGGAGTACTATACCAAACTTCAGATTTTTGGCCGGAACCTATGACATGTTTTTCTCCCGGATTGAGCATCTATATTCAGCAATCAGAGTGGGCACAGTTGTCACTGCTTATGAAGACTGTTCAGGACTGGTATCATTTACTGGGTTCATAAAACAAATCAATCTCACCGCTAGAGAGGCAATACTATATTTCTTCCACAAGAACTTTGAGGAAGAGATAAGAAGAATGTTTGAGCCAGGGCAGGAGACAGCTGTTCCTCACTCTTATTTCATCCACTTCCGTTCACTAGGCTTGAGTGGGAAATCTCCTTATTCATCAAATGCTGTTGGTCACGTGTTCAATCTCATTCACTTTGTAGGATGCTATATGGGTCAAGTCAGATCCCTAAATGCAACGGTTATTGCTGCATGTGCTCCTCATGAAATGTCTGTTCTAGGGGGCTATCTGGGAGAGGAATTCTTCGGGAAAGGGACATTTGAAAGAAGATTCTTCAGAGATGAGAAAGAACTTCAAGAATACGAGGCGGCTGAACTGACAAAGACTGACGTAGCACTGGCAGATGATGGAACTGTCAACTCTGACGACGAGGACTACTTTTCAGGTGAAACCAGAAGTCCGGAGGCTGTTTATACTCGAATCATGATGAATGGAGGTCGACTAAAGAGATCTCACATACGGAGATATGTCTCAGTCAGTTCCAATCATCAAGCCCGTCCAAACTCATTCGCCGAGTTTCTAAACAAGACATATTCGAGTGACTCATAAGAAGTTGAATAACAAAATGCCGGAAATCTACGGATTGTGTATATCCATCATGAAAAAAA(N基因的转录终止信号)CT(间隔序列)AACA(G基因的转录起始信号)TCCCTCAAAAGACTCAAGGAAAGATG(G基因起始密码子)GTTCCTCAGGCTCTCCTGTTTGTACCCCTTCTGGTTTTTCCATTGTGTTTTGGGAAATTCCCTATTTACACGATACCAGACAAGCTTGGTCCCTGGAGTCCGATTGACATACATCACCTCAGCTGACCAAACAATTTGGTAGTGGAGGACGAAGGATGCACCAACCTGTCAGGGTTCTCCTACATGGAACTTAAAGTTGGATACATCTTAGCCATAAAAGTGAACGGGTTCACTTGCACAGGCGTTGTGACGGAGGCTGAAACCTACACTAACTTCGTTGGTTATGTCACAACCACGTTCAAAAGAAAGCATTTCCGCCCAACACCAGATGCATGTAGAGCCGCGTACAACTGGAAGATGGCCGGTGACCCCAGATATGAAGAGTCTCTACACAATCCGTACCCTGACTACCGCTGGCTTCGAACTGTAAAAACCACCAAGGAGTCTCTCGTTATCATATCTCCAAGTGTGGCAGATTTGGACCCATATGACAGATCCCTTCACTCGAGGGTCTTCCCTAGCGGGAAGTGCTCAGGAGTAGCGGTGTCTTCTACCTACTGCTCCACTAACCACGATTACACCATTTGGATGCCCGAGAATCCGAGACTAGGGATGTCTTGTGACATTTTTACCAATAGTAGAGGGAAGAGAGCATCCAAAGGGAGTGAGACTTGCGGCTTTGTAGATGAAAGAGGCCTATATAAGTCTTTAAAAGGAGCATGCAAACTCAAGTTATGTGGAGTTCTAGGACTTAGACTTATGGATGGAACATGGGTCTCGATGCAAACATCAAATGAAACCAAATGGTGCCCTCCCGATAAGTTGGTGAACCTGCACGACTTTCGCTCAGACGAAATTGAGCACCTTGTTGTAGAGGAGTTGGTCAGGAAGAGAGAGGAGTGTCTGGATGCACTAGAGTCCATCATGGCAACCAAGTCAGTGAGTTTCAGACGTCTCAGTCATTTAAGAAAACTTGTCCCTGGGTTTGGAAAAGCATATACCATATTCAACAAGACCTTGATGGAAGCCGATGCTCACTACAAGTCAGTCAGCACTTGGAATGAGGTCCTCCCTTCAAAAGGGTGTTTAAGAGTTGGGGGGGAGGTGTCATCCTCATGTGAACGGGGTGTTTTTCAATGGTATAATATTAGGACCTGACGGCAATGTCTTAATCCCAGAGATGCAATCATCCCTCCTCCAGCAACATATGGAGTTGTTGGAATCCTCGGTTATCCCCCTTGTGCACCCCCTGGCAGACCCGTCTACCGTTTTCAAGGACGGTGACGAGGCTGAGGATTTTGTTGAAGTTCACCTTCCCGATGTGCACAATCAGGTCTCAGGAGTTGACTTGGGTCTCCCGAACTGGGGGAAGTATGTATTACTGAGTGCAGGGGCCCTGACTGCCTTGATGTTGATAATTTTCCTGATGACATGTTGTAGATAAGGACTGGCCGTCCTTTCAACGATCCAAGTCCTGAAGATCACCTCCCCTTGGGGGGTTCTTTTTGAAAAAAA(G基因的N基因的转录终止信号)CAGTTCAACACCCCTACA(Egfp转录起始信号)ATG(Egfp起始密码子)GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA(EGFP终止密码子)TGAAAAAAA(Egfp转录终止信号)CTGTTAACACCCCTACA(Cdvn起始转录信号)ATG(CDVN起始密码子)GCTAGCCTTCTCAAGAGCCTCACATTGTTCAAGAGGACTCGGGACCAACCCCCACTTGCCTCGGGCTCCGGAGGAGCAATAAGAGGGATAAAGCATGTCATTATAGTCCTAATCCCGGGTGATTCAAGCATTGTTACAAGGTCTCGACTATTGGATAGACTTGTTAGATTGGTCGGTGATCCGGAAATCAACGGACCTAAATTAACTGGGATTTTAATCAGTATCCTCTCCTTGTTCGTGGAATCCCCTGGACAGTTGATCCAGAGGATCATAGACGACCCTGATGTAAGCATCAAGTTAGTAGAGGTAATCCCAAGCATCGACTCTGTTTGTGGTCTTACATTTGCATCCAGAGGAGCAAGTTTGGATTCTGAGGCAGATGAGTTCTTCAAAATTGTAGACGAAGGGTCGAAAGCTCAAGGACAATTAGGCTGGTTGGAGAATAAGGATATTGTAGACATAGAAGTTGATGATGCTGAGCAATTCAATATATTGCTAGCTTCCATCTTGGCCCAAATTTGGATCCTGCTCGCTAAAGCAGTGACTGCTCCTGATACTGCAGCCGACTCGGAAATGAGGAGATGGATTAAGTATACCCAACAGAGACGTGTGGTCGGGGAATTTAGAATGAACAAAATCTGGCTTGATATTGTTAGAAACAGGATAGCTGAGGACTTATCTTTGAGGCGATTCATGGTGGCACTCATCTTGGACATCAAACGATCCCCAGGGAACAAGCCTAGAATTGCTGAAATGATTTGTGATATAGATAACTACATTGTGGAAGCTGGATTAGCTAGTTTCATCTTAACTATCAAATTTGGCATTGAAACTATGTATCCGGCTCTTGGGTTGCATGAGTTTTCCGGAGAGTTAACAACTATTGAATCCCTTATGATGCTATATCAACAGATGGGTGAAACAGCACCTTACATGGTTATTCTGGAAAATTCTGTTCAGAACAAATTTAGTGCAGGATCCTACCCACTGCTCTGGAGTTATGCTATGGGAGTTGGTGTTGAGCTTGAAAACTCCATGGGAGGGTTAAATTTCGGTAGATCCTACTTCGATCCGGCCTATTTCAGGCTCGGGCAAGAAATGGTGAGAAGATCTGCCGGCAAAGTAAGCTCTGCACTTGCCGCCGAGCTTGGCATCACCAAGGAAGAGGCTCAGCTAGTGTCAGAAATAGCATCCAAGACAACGGAGGACCGGACGATTCGCACTACTGGTCCCAAGCAATCTCAAATCACTTTTCTACACTCAGAAAGGTCCGAAGTCACTAATCAACAACCCCCAACCATCAACAAGAGGTCCGAAAACCAAGGAGGAGACAAATACCCCATCCACTTCAATGATGAACGGTTTCCAGGGTACACCCCTGATGTCAACAGCTCCGAATGGAGTGAATCACGCTATGATACCCAGACCATTCAAGATGATGGAAACGACGATGACCGGAAATCGATGGAAGCAATCGCCAAGATGAGAATGCTTACTAAGATGCTCAGTCAACCTGGGACCAGTGAAGAGAGTTCTCCTGTCTATAATGATAGAGAGCTACTCAATTAA(CDVN终止信号)TGAAAAAAA(CDNV终止转录信号)CTAACACCCC(P基因的转录起始信号)TCCTTTCGAACCATCCCAAACATGAGCAAGATCTTTGTCAATCCTAGTGCTATTAGAGCCGGTCTGGCCGATCTTGAGATGGCTGAAGAAACTGTTGATCTGATCAATAGAAATATCGAAGACAATCAGGCTCATCTCCAAGGGGAACCCATAGAGGTGGACAATCTCCCTGAGGATATGGGGCGACTTCACCTGGATGATGGAAAATCGCCCAACCATGGTGAGATAGCCAAGGTGGGAGAAGGCAAGTATCGAGAGGACTTTCAGATGGATGAAGGAGAGGATCCTAGCTTCCTGTTCCAGTCATACCTGGAAAATGTTGGAGTCCAAATAGTCAGACAAATGAGGTCAGGAGAGAGATTTCTCAAGATATGGTCACAGACCGTAGAAGAGATTATATCCTATGTCGCGGTCAACTTTCCCAACCCTCCAGGAAAGTCTTCAGAGGATAAATCAACCCAGACTACTGGCCGAGAGCTCAAGAAGGAGACAACACCCACTCCTTCTCAGAGAGAAAGCCAATCATCGAAAGCCAGGATGGCGGCTCAAATTGCTTCTGGCCCTCCAGCCCTTGAATGGTCGGCTACCAATGAAGAGGATGATCTATCAGTGGAGGCTGAGATCGCTCACCAGATTGCAGAAAGTTTCTCCAAAAAATATAAGTTTCCCTCTCGATCCTCAGGGATACTCTTGTATAATTTTGAGCAATTGAAAATGAACCTTGATGATATAGTTAAAGAGGCAAAAAATGTACCAGGTGTGACCCGTTTAGCCCATGACGGGTCCAAACTCCCCCTAAGATGTGTACTGGGATGGGTCGCTTTGGCCAACTCTAAGAAATTCCAGTTGTTAGTCGAATCCGACAAGCTGAGTAAAATCATGCAAGATGACTTGAATCGCTATACATCTTGCTAACCGAACCTCTCCCCTCAGTCCCTCTAGACAATAAAATCCGAGATGTCCCAAAGTCAACATGAAAAAAA(P基因转录终止信号)CAGGCAACA(M基因转录起始信号)CCACTGATAAAATG(M基因起始密码子)AACCTCCTACGTAAGATAGTGAAAAACCGCAGGGACGAGGACACTCAAAAATCCTCTCCCGCGTCAGCCCCTCTGGATGACGATGACTTGTGGCTTCCACCCCCTGAATACGTCCCGCTGAAAGAACTTACAGGCAAGAAGAACATGAGGAACTTTTGTATCAACGGAAGGGTTAAAGTGTGTAGCCCGAATGGTTACTCGTTCAGGATCCTGCGGCACATTCTGAAATCATTCGACGAGATATATTCTGGGAATCATAGGATGATCGGGTTAGTCAAAGTGGTTATTGGACTGGCTTTGTCAGGATCTCCAGTCCCTGAGGGCCTGAACTGGGTATACAAATTGAGGAGAACCTTTATCTTCCAGTGGGCTGATTCCAGGGGCCCTCTTGAAGGGGAGGAGTTGGAATACTCTCAGGAGATCACTTGGGATGATGATACTGAGTTCGTCGGATTGCAAATAAGAGTGATTGCAAAACAGTGTCATATCCAGGGCAGAGTCTGGTGTATCAACATGAACCCGAGAGCATGTCAACTATGGTCTGACATGTCTCTTCAGACACAAAGGTCCGAAGAGGACAAAGATTCCTCTCTGCTTCTAGAATAATCAGATTATATCCCGCAAATTTATCACTTGTTTACCTCTGGAGGAGAGAACATATGGGCTCAACTCCAACCCTTGGGAGCAATATAACAAAAAACATGTTATGGTGCCATTAAACCGCTGCATTTCATCAAAGTCAAGTTGATTACCTTTACATTTTGATCCTCTTGGATGTGAAAAAAA(M基因终止转录信号)CTCAACACCCCTACA(VP2转录起始信号)ATG(VP2起始密码子)AGTGATGGAGCAGTTCAACCAGACGGTGGTCAGCCTGCTGTCAGAAATGAAAGAGCTACAGGATCTGGGAACGGGTCTGGAGGCGGGGGTGGTGGTGGTTCTGGGGGTGTGGGGATTTCTACGGGTACTTTCAATAATCAGACGGAATTTAAATTTTTGGAAAACGGATGGGTGGAAATCACAGCAAACTCAAGCAGACTTGTACATTTAAATATGCCAGAAAGTGAAAATTATAGAAGAGTGGTTGTAAATAATTTGGATAAAACTGCAGTTAACGGAAACATGGCTTTAGATGATACCCATGCACAAATTGTAACACCTTGGTCATTGGTTGATGCAAATGCTTGGGGAGTTTGGTTTAATCCAGGAGATTGGCAACTAATTGTTAATACTATGAGTGAGTTGCATTTAGTTAGTTTTGAACAAGAAATTTTTAATGTTGTTTTAAAGACTGTTTCAGAATCTGCTACTCAGCCACCAACTAAAGTTTATAATAATGATTTAACTGCATCATTGATGGTTGCATTAGATAGTAATAATACTATGCCATTTACTCCAGCAGCTATGAGATCTGAGACATTGGGTTTTTATCCATGGAAACCAACCATACCAACTCCATGGAGATATTATTTTCAATGGGATAGAACATTAATACCATCTCATACTGGAACTAGTGGCACACCAACAAATATATACCATGGTACAGATCCAGATGATGTTCAATTTTACACTATTGAAAATTCTGTGCCAGTACACTTACTAAGAACAGGTGATGAATTTGCTACAGGAACATTTTATTTTGATTGTAAACCATGTAGACTAACACACACATGGCAAACAAATAGAGCATTGGGCTTACCACCATTTCTAAATTCTTTGCCTCAAGCTGAAGGAGGTACTAACTTTGGTTATATAGGAGTTCAACAAGATAAAAGACGTGGTGTAACTCAAATGGGAAATACAAACATTATTACTGAAGCTACTATTATGAGACCAGCTGAGGTTGGTTATAGTGCACCATATTATTCTTTTGAGGCGTCTACACAAGGGCCATTTAAAACACCTATTGCAGCAGGACGGGGGGGAGCGCAAACAGATGAAAATCAAGCAGCAGATGGTGATCCAAGATATGCATTTGGTAGACAACATGGTCAAAAAACTACCACAACAGGAGAAACACCTGAGAGATTTACATATATAGCACATCAAGATACAGGAAGATATCCAGAAGGAGATTGGATTCAAAATATTAACTTTAACCTTCCTGTAACAAATGATAATGTATTGCTACCAACAGATCCAATTGGAGGTAAAGCAGGAATTAACTATACTAATATATTTAATACTTATGGTCCTTTAACTGCATTAAATAATGTACCACCAGTTTATCCAAATGGTCAAATTTGGGATAAAGAATTTGATACTGACTTAAAACCAAGACTTCATGTAAATGCACCATTTGTTTGTCAAAATAATTGTCCTGGTCAATTATTTGTAAAAGTTGCGCCTAATTTAACAAATGAATATGATCCTGATGCATCTGCTAATATGTCAAGAATTGTAACTTACTCAGATTTTTGGTGGAAAGGTAAATTAGTATTTAAAGCTAAACTAAGAGCCTCTCATACTTGGAATCCAATTCAACAAATGAGTATTAATGTAGATAACCAATTTAACTATGTACCAAGTAATATTGGAGGTATGAAAATTGTATATGAAAAATCTCAACTAGCACCTAGAAAATTATATTAATGAAAAAAA(VP2基因终止转录信号)CTGCTATTAACACTTCT(L基因的起始转录信号)CAACCTGAGACTTACTTCAAGATG(L基因基因起始密码子)CTCGATCCTGGAGAGGTCTATGATGACCCTATTGACCCAATCGAGTTAGAGGCTGAACCCAGAGGAACCCCCATTGTCCCCAACATCTTGAGGAACTCTGACTACAATCTCAACTCTCCTTTGATAGAAGATCCTGCTAGACTAATGTTAGAATGGTTAAAAACAGGGAATAGACCTTATCGGATGACTCTAACAGACAATTGCTCCAGGTCTTTCAGAGTTTTGAAAGATTATTTCAAGAAGGTAGATTTGGGTTCTCTCAAGGTGGGCGGAATGGCTGCACAGTCAATGATTTCTCTCTGGTTATATGGTGCCCACTCTGAATCCAACAGGAGCCGGAGATGTATAACAGACTTGGCCCATTTCTATTCCAAGTCGTCCCCCATAGAGAAGCTGTTGAATCTCACGCTAGGAAATAGAGGGCTGAGAATCCCCCCAGAGGGAGTGTTAAGTTGCCTTGAGAGGGTTGATTATGATAATGCATTTGGAAGGTATCTTGCCAACACGTATTCCTCTTACTTGTTCTTCCATGTAATCACCTTATACATGAACGCCCTAGACTGGGATGAGGAAAAGACCATCCTAGCATTATGGAAAGATTTAACCTCAGTGGACATCGGGAAGGACTTGGTAAAGTTCAAAGACCAAATATGGGGACTGCTGATCGTGACAAAGGACTTTGTTTACTCCCAAAGTTCCAATTGTCTTTTTGACAGAAACTACACACTTATGCTAAAAGATCTTTTCTTGTCTCGCTTCAACTCCTTAATGGTCTTGCTCTCTCCCCCAGAGCCCCGATACTCAGATGACTTGATATCTCAACTATGCCAGCTGTACATTGCTGGGGATCAAGTCTTGTCTATGTGTGGAAACTCCGGCTATGAAGTCATCAAAATATTGGAGCCATATGTCGTGAATAGTTTAGTCCAGAGAGCAGAAAAGTTTAGGCCTCTCATTCATTCCTTGGGAGACTTTCCTGTATTTATAAAAGACAAGGTAAGTCAACTTGAAGAGACGTTCGGTCCCTGTGCAAGAAGGTTCTTTAGGGCTCTGGATCAATTCGACAACATACATGACTTGGTTTTTGTGTTTGGCTGTTACAGGCATTGGGGGCACCCATATATAGATTATCGAAAGGGTCTGTCAAAACTATATGATCAGGTTCACCTTAAAAAAGTGATAGATAAGTCCTACCAGGAGTGCTTAGCAAGCGACCTAGCCAGGAGGATCCTTAGATGGGGTTTTGATAAGTACTCCAAGTGGTATCTGGATTCAAGATTCCTAGCCCGAGACCACCCCTTGACTCCTTATATCAAAACCCAAACATGGCCACCCAAACATATTGTAGACTTGGTGGGGGATACATGGCACAAGCTCCCGATCACGCAGATCTTTGAGATTCCTGAATCAATGGATCCGTCAGAAATATTGGATGACAAATCACATTCTTTCACCAGAACGAGACTAGCTTCTTGGCTGTCAGAAAACCGAGGGGGGCCTGTTCCTAGCGAAAAAGTTATTATCACGGCCCTGTCTAAGCCGCCTGTCAATCCCCGAGAGTTTCTGAGGTCTATAGACCTCGGAGGATTGCCAGATGAAGACTTGATAATTGGCCTCAAGCCAAAGGAACGGGAATTGAAGATTGAAGGTCGATTCTTTGCTCTAATGTCATGGAATCTAAGATTGTATTTTGTCATCACTGAAAAACTCTTGGCCAACTACATCTTGCCACTTTTTGACGCGCTGACTATGACAGACAACCTGAACAAGGTGTTTAAAAAGCTGATCGACAGGGTCACCGGGCAAGGGCTTTTGGACTATTCAAGGGTCACATATGCATTTCACCTGGACTATGAAAAGTGGAACAACCATCAAAGATTAGAGTCAACAGAGGATGTATTTTCTGTCCTAGATCAAGTGTTTGGATTGAAGAGAGTGTTTTCTAGAACACACGAGTTTTTTCAAAAGGCCTGGATCTATTATTCAGACAGATCAGACCTCATCGGGTTACGGGAGGATCAAATATACTGCTTAGATGCGTCCAACGGCCCAACCTGTTGGAATGGCCAGGATGGCGGGCTAGAAGGCTTACGGCAGAAGGGCTGGAGTCTAGTCAGCTTATTGATGATAGATAGAGAATCTCAAATCAGGAACACAAGAACCAAAATACTAGCTCAAGGAGACAACCAGGTTTTATGTCCGACATACATGTTGTCGCCAGGGCTATCTCAAGAGGGGCTCCTCTATGAATTGGAGAGAATATCAAGGAATGCACTTTCGATATACAGAGCCGTCGAGGAAGGGGCATCTAAGCTAGGGCTGATCATCAAGAAAGAAGAGACCATGTGTAGTTATGACTTCCTCATCTATGGAAAAACCCCTTTGTTTAGAGGTAACATATTGGTGCCTGAGTCCAAAAGATGGGCCAGAGTCTCTTGCGTCTCTAATGACCAAATAGTCAACCTCGCCAATATAATGTCGACAGTGTCCACCAATGCGCTAACAGTGGCACAACACTCTCAATCTTTGATCAAACCGATGAGGGATTTTCTGCTCATGTCAGTACAGGCAGTCTTTCACTACCTGCTATTTAGCCCAATCTTAAAGGGAAGAGTTTACAAGATTCTGAGCGCTGAAGGGGAGAGCTTTCTCCTAGCCATGTCAAGGATAATCTATCTAGATCCTTCTTTGGGAGGGATATCTGGAATGTCCCTCGGAAGATTCCATATACGACAGTTCTCAGACCCTGTCTCTGAAGGGTTATCCTTCTGGAGAGAGATCTGGTTAAGCTCCCAAGAGTCCTGGATTCACGCGTTGTGTCAAGAGGCTGGAAACCCAGATCTTGGAGAGAGAACACTCGAGAGCTTCACTCGCCTTCTAGAAGATCCGACCACCTTAAATATCAGAGGAGGGGCCAGTCCTACCATTCTACTCAAGGATGCAATCAGAAAGGCTTTATATGACGAGGTGGACAAGGTGGAAAATTCAGAGTTTCGAGAGGCAATCCTGTTGTCCAAGACCCATAGAGATAATTTTATACTCTTCTTAATATCTGTTGAGCCTCTGTTTCCTCGATTTCTCAGTGAGCTATTCAGTTCGTCTTTTTTGGGAATCCCCGAGTCAATCATTGGATTGATACAAAACTCCCGAACGATAAGAAGGCAGTTTAGAAAGAGTCTCTCAAAAACTTTAGAAGAATCCTTCTACAACTCAGAGATCCACGGGATTAGTCGGATGACCCAGACACCTCAGAGGGTTGGGGGGGTGTGGCCTTGCTCTTCAGAGAGGGCAGATCAACTTAGGGAGATCTCTTGGGGAAGAAAAGTGGTAGGCACGACAGTTCCTCACCCTTCTGAGATGTTGGGATTACTTCCCAAGTCCTCTATTTCTTGCACTTGTGGAGCAACAGGAGGAGGCAATCCTAGAGTTTCTGTATCAGTACTCCCGTCCTTTGATCAGTCATTTTTTTCACGAGGCCCCCTAAAGGGATACTTGGGCTCGTCCACCTCTATGTCGACCCAGCTATTCCATGCATGGGAAAAAGTCACTAATGTTCATGTGGTGAAGAGAGCTCTATCGTTAAAAGAATCTATAAACTGGTTCATTACTAGAGATTCCAACTTGGCTCAAGCTCTAATTAGGAACATTATGTCTCTGACAGGCCCTGATTTCCCTCTAGAGGAGGCCCCTGTCTTCAAAAGGACGGGGTCAGCCTTGCATAGGTTCAAGTCTGCCAGATACAGCGAAGGAGGGTATTCTTCTGTCTGCCCGAACCTCCTCTCTCATATTTCTGTTAGTACAGACACCATGTCTGATTTGACCCAAGACGGGAAGAACTACGATTTCATGTTCCAGCCATTGATGCTTTATGCACAGACATGGACATCAGAGCTGGTACAGAGAGACACAAGGCTAAGAGACTCTACGTTTCATTGGCACCTCCGATGCAACAGGTGTGTGAGACCCATTGACGACGTGACCCTGGAGACCTCTCAGATCTTCGAGTTTCCGGATGTGTCGAAAAGAATATCCAGAATGGTTTCTGGGGCTGTGCCTCACTTCCAGAGGCTTCCCGATATCCGTCTGAGACCAGGAGATTTTGAATCTCTAAGCGGTAGAGAAAAGTCTCACCATATCGGATCAGCTCAGGGGCTCTTATACTCAATCTTAGTGGCAATTCACGACTCAGGATACAATGATGGAACCATCTTCCCTGTCAACATATACGGCAAGGTTTCCCCTAGAGACTATTTGAGAGGGCTCGCAAGGGGAGTATTGATAGGATCCTCGATTTGCTTCTTGACAAGAATGACAAATATCAATATTAATAGACCTCTTGAATTGGTCTCAGGGGTAATCTCATATATTCTCCTGAGGCTAGATAACCATCCCTCCTTGTACATAATGCTCAGAGAACCGTCTCTTAGAGGAGAGATATTTTCTATCCCTCAGAAAATCCCCGCCGCTTATCCAACCACTATGAAAGAAGGCAACAGATCAATCTTGTGTTATCTCCAACATGTGCTACGCTATGAGCGAGAGATAATCACGGCGTCTCCAGAGAATGACTGGCTATGGATCTTTTCAGACTTTAGAAGTGCCAAAATGACGTACCTATCCCTCATTACTTACCAGTCTCATCTTCTACTCCAGAGGGTTGAGAGAAACCTATCTAAGAGTATGAGAGATAACCTGCGACAATTGAGTTCTTTGATGAGGCAGGTGCTGGGCGGGCACGGAGAAGATACCTTAGAGTCAGACGACAACATTCAACGACTGCTAAAAGACTCTTTACGAAGGACAAGATGGGTGGATCAAGAGGTGCGCCATGCAGCTAGAACCATGACTGGAGATTACAGCCCCAACAAGAAGGTGTCCCGTAAGGTAGGATGTTCAGAATGGGTCTGCTCTGCTCAACAGGTTGCAGTCTCTACCTCAGCAAACCCGGCCCCTGTCTCGGAGCTTGACATAAGGGCCCTCTCTAAGAGGTTCCAGAACCCTTTGATCTCGGGCTTGAGAGTGGTTCAGTGGGCAACCGGTGCTCATTATAAGCTTAAGCCTATTCTAGATGATCTCAATGTTTTCCCATCTCTCTGCCTTGTAGTTGGGGACGGGTCAGGGGGGATATCAAGGGCAGTCCTCAACATGTTTCCAGATGCCAAGCTTGTGTTCAACAGTCTTTTAGAGGTGAATGACCTGATGGCTTCCGGAACACATCCACTGCCTCCTTCAGCAATCATGAGGGGAGGAAATGATATCGTCTCCAGAGTGATAGATCTTGACTCAATCTGGGAAAAACCGTCCGACTTGAGAAACTTGGCAACCTGGAAATACTTCCAGTCAGTCCAAAAGCAGGTCAACATGTCCTATGACCTCATTATTTGCGATGCAGAAGTTACTGACATTGCATCTATCAACCGGATCACCCTGTTAATGTCCGATTTTGCATTGTCTATAGATGGACCACTCTATTTGGTCTTCAAAACTTATGGGACTATGCTAGTAAATCCAAACTACAAGGCTATTCAACACCTGTCAAGAGCGTTCCCCTCGGTCACAGGGTTTATCACCCAAGTAACTTCGTCTTTTTCATCTGAGCTCTACCTCCGATTCTCCAAACGAGGGAAGTTTTTCAGAGATGCTGAGTACTTGACCTCTTCCACCCTTCGAGAAATGAGCCTTGTGTTATTCAATTGTAGCAGCCCCAAGAGTGAGATGCAGAGAGCTCGTTCCTTGAACTATCAGGATCTTGTGAGAGGATTTCCTGAAGAAATCATATCAAATCCTTACAATGAGATGATCATAACTCTGATTGACAGTGATGTAGAATCTTTTCTAGTCCACAAGATGGTTGATGATCTTGAGTTACAGAGGGGAACTCTGTCTAAAGTGGCTATCATTATAGCCATCATGATAGTTTTCTCCAACAGAGTCTTCAACGTTTCCAAACCCCTAACTGACCCCTCGTTCTATCCACCGTCTGATCCCAAAATCCTGAGGCACTTCAACATATGTTGCAGTACTATGATGTATCTATCTACTGCTTTAGGTGACGTCCCTAGCTTCGCAAGACTTCACGACCTGTATAACAGACCTATAACTTATTACTTCAGAAAGCAAGTCATTCGAGGGAACGTTTATCTATCTTGGAGTTGGTCCAACGACACCTCAGTGTTCAAAAGGGTAGCCTGTAATTCTAGCCTGAGTCTGTCATCTCACTGGATCAGGTTGATTTACAAGATAGTGAAGACTACCAGACTCGTTGGCAGCATCAAGGATCTATCCAGAGAAGTGGAAAGACACCTTCATAGGTACAACAGGTGGATCACCCTAGAGGATATCAGATCTAGATCATCCCTACTAGACTACAGTTGCCTGTGA(L基因的终止密码子)ACCGGATACTCCTGGAAGCCTGCCCATGCTAAGACTCTTGTGTGATGTATCTTGAAAAAA A(L基因的终止转录信号)CAAGATCCTAAATCTGAACCTTTGGTTGTTTGATTGTTTTTCTCATTTTTGTTGTTTATTTGTTAAGCGTGGGTCGGCATGGCATCTCCACCTCCTCG(Hdv RZ序列)
本发明实施例中未尽之处，本领域技术人员均可从现有技术中选用。
以上公开的仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以上述权利要求的保护范围为准。
Claims (4)
1.一种狂犬病-犬瘟热-犬细小病毒基因重组病毒株，其特征在于，其为带有犬瘟热G基因和犬细小病毒VP2基因的重排狂犬病病毒，命名为：狂犬病-犬瘟热-犬细小病毒基因重组病毒，所述重组病毒的基因核苷酸序列为SEQ ID NO:1。
2.权利要求1所述的狂犬病-犬瘟热-犬细小病毒基因重组病毒株的构建方法，所述方法包括以下步骤：
分别将狂犬病SRV9病毒的N、P、M、G、L基因进行扩增；
采用Gibson Assembly连接法进行重组基因连接，将SRV9病毒的G基因由原基因组序列的第四位转移至病毒基因组的第二位，再将eGFP报告基因通过酶切位点插入到G和P基因之间，构建成pcDSRV9-N-G-eGFP-PML基因重组载体；
通过PCR方法扩增犬瘟热pMD18-CDV N重组质粒中的CDV N基因；
通过酶切方法对pcDSRV9-N-G-eGFP-PML重组载体的eGFP基因末端酶切位点后进行HpaI酶切，利用Gibson Assembly连接法将犬瘟热CDV N基因构建至pcDSRV9-N-G-eGFP-PML重组载体上，构建成pcDSRV9-NG-eGFP+CDV N-PML重组载体；
通过PCR扩增犬细小病毒pMD19-T-VP2重组质粒中的VP2基因；
通过酶切方法对pcDSRV9-NG-eGFP+CDV N-PML重组载体中M基因末端酶切位点进行BsiWI酶切，将扩增的VP2基因通过Gibson Assembly连接法插入到pcDSRV9-NG-eGFP+CDV N-PML载体M-L基因之间，构建pcDSRV9-NG-eGFP+CDV N-PM+VP2-L基因重组载体；
通过反向遗传法拯救出重组狂犬病-犬瘟热-犬细小病毒，即rSRV9+CDV N+VP2基因重组病毒。
3.权利要求1或2所述的狂犬病-犬瘟热-犬细小病毒基因重组病毒株在制备预防狂犬病的药物中的应用。
4.一种疫苗组合物，其特征在于，所述疫苗组合物含有权利要求1或2所述的所述的狂犬病-犬瘟热-犬细小病毒基因重组病毒株。
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628490A (zh) * | 2019-01-26 | 2019-04-16 | 青岛农业大学 | 一种预防犬瘟热的shRNA重组腺相关病毒 |
| CN110893234A (zh) * | 2019-12-06 | 2020-03-20 | 江苏省农业科学院 | 一种犬瘟热、犬细小病毒病、狂犬病三联亚单位疫苗 |
| CN110951699A (zh) * | 2019-12-17 | 2020-04-03 | 长春西诺生物科技有限公司 | 表达犬瘟热病毒结构蛋白的重组狂犬病病毒及其应用 |
| CN113416750A (zh) * | 2021-07-05 | 2021-09-21 | 青岛农业大学 | 一种表达犬细小病毒2a型VP2的重组犬瘟热病毒 |
| CN115960262A (zh) * | 2022-10-19 | 2023-04-14 | 四川农业大学 | 展示cdv抗原表位的犬细小病毒样颗粒及构建方法和应用 |
| CN116376981A (zh) * | 2023-04-21 | 2023-07-04 | 西北农林科技大学 | 一种重组犬细小病毒假病毒 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1117081A (zh) * | 1995-03-16 | 1996-02-21 | 高云 | 犬瘟热狂犬病细小病毒三联活疫苗、毒种及制造方法 |
| CN1137347A (zh) * | 1995-06-22 | 1996-12-11 | 中国人民解放军第四军医大学 | 犬五联(犬狂犬、犬瘟热、犬细小病毒、犬腺病毒2型、犬副流感)活疫苗 |
| CN107320720A (zh) * | 2016-04-29 | 2017-11-07 | 普莱柯生物工程股份有限公司 | 一种疫苗组合物、试剂盒及应用 |
-
2017
- 2017-12-13 CN CN201711331261.1A patent/CN107988174A/zh active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1117081A (zh) * | 1995-03-16 | 1996-02-21 | 高云 | 犬瘟热狂犬病细小病毒三联活疫苗、毒种及制造方法 |
| CN1137347A (zh) * | 1995-06-22 | 1996-12-11 | 中国人民解放军第四军医大学 | 犬五联(犬狂犬、犬瘟热、犬细小病毒、犬腺病毒2型、犬副流感)活疫苗 |
| CN107320720A (zh) * | 2016-04-29 | 2017-11-07 | 普莱柯生物工程股份有限公司 | 一种疫苗组合物、试剂盒及应用 |
Non-Patent Citations (3)
| Title |
|---|
| 夏婷婷等: "狂犬病病毒结构基因的克隆及犬瘟热病毒N蛋白基因与eGFP基因重组质粒的构建", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 李全等: "犬癌热病毒、犬细小病毒及狂犬病毒H联多表位重组蛋白的构建及其免疫效果研究", 《中国博士学位论文全文数据库农业科技辑》 * |
| 王伟等: "应用Gibson Assembly方法构建狂犬病病毒SRV9株重组感染性cDNA克隆", 《动物医学进展》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109628490A (zh) * | 2019-01-26 | 2019-04-16 | 青岛农业大学 | 一种预防犬瘟热的shRNA重组腺相关病毒 |
| CN110893234A (zh) * | 2019-12-06 | 2020-03-20 | 江苏省农业科学院 | 一种犬瘟热、犬细小病毒病、狂犬病三联亚单位疫苗 |
| CN110893234B (zh) * | 2019-12-06 | 2023-08-15 | 江苏省农业科学院 | 一种犬瘟热、犬细小病毒病、狂犬病三联亚单位疫苗 |
| CN110951699A (zh) * | 2019-12-17 | 2020-04-03 | 长春西诺生物科技有限公司 | 表达犬瘟热病毒结构蛋白的重组狂犬病病毒及其应用 |
| CN110951699B (zh) * | 2019-12-17 | 2023-11-24 | 长春西诺生物科技有限公司 | 表达犬瘟热病毒结构蛋白的重组狂犬病病毒及其应用 |
| CN113416750A (zh) * | 2021-07-05 | 2021-09-21 | 青岛农业大学 | 一种表达犬细小病毒2a型VP2的重组犬瘟热病毒 |
| CN115960262A (zh) * | 2022-10-19 | 2023-04-14 | 四川农业大学 | 展示cdv抗原表位的犬细小病毒样颗粒及构建方法和应用 |
| CN116376981A (zh) * | 2023-04-21 | 2023-07-04 | 西北农林科技大学 | 一种重组犬细小病毒假病毒 |
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