麻疹病毒沪191株反向遗传系统的建立
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摘要
建立麻疹病毒沪191株(MV-_(S191))反向遗传系统。提取MV-_(S191)的RNA,RT-PCR分7段扩增出麻疹病毒反基因组cDNA,通过酶切、连接,拼接出MV-_(S191)全长反基因组,并分别在其3′端和5′端引入丁型肝炎病毒核酶序列(HdvRZ)和锤头核酸酶序列(HamRZ),克隆到pT7-MCS载体,获得pT7MV_(S191)。在MV-_(S191)的P-M基因间区域插入绿色荧光蛋白(GFP),获得pT7MV_(S191)-ATU-GFP,同时构建表达麻疹病毒核蛋白(N)、磷蛋白(P)和大蛋白(L)的3个辅助质粒。将pT7MV_(S191)、pT7MV_(S191)-ATU-GFP分别与辅助质粒通过脂质体介导共转染BSRT7细胞,热休克3h。转染3d后,取上清,感染Vero-SLAM细胞。经RT-PCR、显微镜检测绿色荧光蛋白表达及合胞体形成,在该系统中成功拯救到两种有感染活性的病毒。成功建立了MV-_(S191)的反向遗传系统,为以麻疹病毒为载体进行新疫苗的研发奠定了基础。
A reverse-genetics system for measles virus strain Shanghai-191(MV-_(S191))was constructed.The genomic RNA of MV-_(S191) was extracted.Seven fragments covering full-length genomic cDNA were amplified by reverse transcription-polymerase chain reaction(RT-PCR).A full length anti-genome was designed to fuse the nucleotide of the viral sequence with hammerhead ribozyme(HamRZ)and hepatitis delta virus ribozyme(HdvRZ)sequence,and then pT7MV_(S191) was constructed.Green fluorescent protein(GFP)with additional transcription units(ATU)was introduced into the P-M intergenic region of the viral genome and pT7MV_(S191)-ATU-GFP was constructed.Three helper plasmids respectively coding the nucleoprotein(N),phosphoprotein(P)and polymerase(L)of viral proteins with pT7-MV_(S191) or pT7MV_(S191)-ATU-GFP together were co-transfected into BSRT7 cells with LipofectamineTM2000 to rescue recombinant MV-_(S191).To improve the efficiency of the MV-_(S191)reverse-genetics system,cells were heat-shocked at43.5℃ for 3hours and then returned to 37℃after overnight incubation at 37℃.After 3days of incubation at 37℃,the transfection medium was harvested and co-cultured with Vero-SLAM cells at 37℃.The rescued MV-_(S191) virus was identified by RT-PCR,cytopathic effect(CPE)and fluorescence microscopy.Infectious viruses were recovered.These data show that a reverse-genetics system was constructed for MV_(S191),and could be applicable for development of vaccines and oncolytic virus.
A reverse-genetics system for measles virus strain Shanghai-191(MV-_(S191))was constructed.The genomic RNA of MV-_(S191) was extracted.Seven fragments covering full-length genomic cDNA were amplified by reverse transcription-polymerase chain reaction(RT-PCR).A full length anti-genome was designed to fuse the nucleotide of the viral sequence with hammerhead ribozyme(HamRZ)and hepatitis delta virus ribozyme(HdvRZ)sequence,and then pT7MV_(S191) was constructed.Green fluorescent protein(GFP)with additional transcription units(ATU)was introduced into the P-M intergenic region of the viral genome and pT7MV_(S191)-ATU-GFP was constructed.Three helper plasmids respectively coding the nucleoprotein(N),phosphoprotein(P)and polymerase(L)of viral proteins with pT7-MV_(S191) or pT7MV_(S191)-ATU-GFP together were co-transfected into BSRT7 cells with LipofectamineTM2000 to rescue recombinant MV-_(S191).To improve the efficiency of the MV-_(S191)reverse-genetics system,cells were heat-shocked at43.5℃ for 3hours and then returned to 37℃after overnight incubation at 37℃.After 3days of incubation at 37℃,the transfection medium was harvested and co-cultured with Vero-SLAM cells at 37℃.The rescued MV-_(S191) virus was identified by RT-PCR,cytopathic effect(CPE)and fluorescence microscopy.Infectious viruses were recovered.These data show that a reverse-genetics system was constructed for MV_(S191),and could be applicable for development of vaccines and oncolytic virus.
引文
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[16]Enterlein S,Volchkov V,Weik M,Kolesnikova L,Volchkova V,Klenk H D,Mühlberger E.Rescue of recombinant Marburg virus from cDNA is dependent on nucleocapsid protein VP30[J].J Virol,2006,80:1038-1043.
[17]Gupta G,Giannino V,Rishi N,Glueck R.Immunogenicity of next-generation HPV vaccines in non-human primates:Measles-vectored HPV vaccine versus Pichia pastoris recombinant protein vaccine[J].Vaccine,2016,34(39):4724-4731.
[18]Hu H M,Chen H W,Hsiao Y J,Wu S H,Chung H H,Hsieh C H,Chong P,Leng C H,Pan C H.The successful induction of T-cell and antibody responses by a recombinant measles virus-vectored tetravalent dengue vaccine provides partial protection against dengue-2infection[J].Hum Vaccin Immunother,2016,12(7):1678-1689.
[19]Despres P,Combredet C,Frenkiel M P,Lorin C,Brahic M,Tangy F.Live measles vaccineexpressingthe secreted form of the West Nile virus envelope glycoprotein protects against West Nile virus encephalitis[J].J Infect Dis,2005,191(2):207-214.
[20]Blechacz B,Russell S J.Measles virus as an oncolytic vector platform[J].Curr Gene Ther,2008,8(3):162-175.
[2]YANG Huiqiang,KANG Zhuang,NI Qianzhi,LIU Yuhong,WEN Xueru,LIU Ying,YONG Xuefei,LIU Jie,SUN Yan,WANG Wei,WU Yonglin,LI Yuhua.Genetic stability of measles virus Shanghai-191strain after continuous subculture in chick embryo fibroblasts[J].Chin J Biologicals,2013,26(3):300-303.
[3]Hilleman M.Current overview of the pathogenesis and prophylaxis of measles with focus on practical implications[J].Vaccine,2002,20:651-665.
[4]Kanazawa Y,O hkaw K,Ueda K,Mita E,Takehara T,Sasaki Y,Kasahara A,Hayashi N.Hammerhead ribozyme mediated inhibition of telomerase activity in extracts of human hepatocellular carcinoma cells[J].Biochem Biophys Res Commun,1996,225(2):570-576.
[5]Been M D,Wickham G S.Self-cleaving ribozymes of hepatitis delta virus RNA[J].Eur J Biochem,1997,247(3):741-753.
[6]Parks C L,Lerch R A,Walpita P,Sidhu M S,Udem S A.Enhanced measles virus cDNA rescue and gene expression after heat shock[J].J Virol,1999,73(5):3560-3566.
[7]WANG Jian,CHEN Xiangpeng,SUN Liyuan.A compound or kit,the application and method for rescuing measles virus:CN103160473B[P].(in Chinese)
[8]Fodor E,Devenish L,Engelhardt O G,Palese P,Brownlee G G,Garcia-Sastre.Rescue of influenza A virus from recombinant DNA[J].J Virol,1999,73(11):9679-9682.
[9]Inoue K.,Shoji Y,Kurane I,Iijima T,Sakai T,Morimoto K.An improved method for recovering rabies virus from cloned cDNA[J].J Virol Methods,2003,107:229-236.
[10]Rodriguez M S,Dargemont C,Stutz F.Nuclear export of RNA[J].Biol Cell,2004,96:639-655.
[11]Roberts A,Rose J K.Recovery of negative strand RNA viruses from plasmid DNAs:A positive approach revitalizes a negative Field[J].Virology,1998,247(1):1-6.
[12]HU Kongxin,YU Jianshi,SUN Yulan,MENG Xiangzhi,WANG Xiaofang,ZHANG Quanfu,LI Chuan,LIANG Mifang,LI Dexin.Construction offull length antigenomic cDNA of measles virus strain CC-47and rescue of infectious virus from the cDNA[J].Chin J Virol,2004,20(3):210-213.
[13]Yu Q,Hardy R W,Wertz G W.Functional cDNA clones of the human respiratory syncytial(RS)virus N,P,and L proteins support replication of RS virus genomic RNA analogs and define minimal transacting requirements for RNA replication[J].J Virol,1995,69(4):2412-2419.
[14]XIU Meihong,WANG Qin,TANG Lihua,CAO Shouchun,LI Weihong,WEI Yan,LU Peng,LIANG Mifang,LI Dexin.Rescue of minireplicon by using the cell line stably expressing the T7RNA polymerase[J].Chin J Virol,2007,23(4):326-330.
[15]Radecke F,Spielhofer P,Schneider H,Kaelin K,Huber M,Dotsch C,Christiansen G,Billeter M A.Rescue of measles viruses from cloned DNA[J].EMBO J,1995,14(23):5773-5784.
[16]Enterlein S,Volchkov V,Weik M,Kolesnikova L,Volchkova V,Klenk H D,Mühlberger E.Rescue of recombinant Marburg virus from cDNA is dependent on nucleocapsid protein VP30[J].J Virol,2006,80:1038-1043.
[17]Gupta G,Giannino V,Rishi N,Glueck R.Immunogenicity of next-generation HPV vaccines in non-human primates:Measles-vectored HPV vaccine versus Pichia pastoris recombinant protein vaccine[J].Vaccine,2016,34(39):4724-4731.
[18]Hu H M,Chen H W,Hsiao Y J,Wu S H,Chung H H,Hsieh C H,Chong P,Leng C H,Pan C H.The successful induction of T-cell and antibody responses by a recombinant measles virus-vectored tetravalent dengue vaccine provides partial protection against dengue-2infection[J].Hum Vaccin Immunother,2016,12(7):1678-1689.
[19]Despres P,Combredet C,Frenkiel M P,Lorin C,Brahic M,Tangy F.Live measles vaccineexpressingthe secreted form of the West Nile virus envelope glycoprotein protects against West Nile virus encephalitis[J].J Infect Dis,2005,191(2):207-214.
[20]Blechacz B,Russell S J.Measles virus as an oncolytic vector platform[J].Curr Gene Ther,2008,8(3):162-175.