欧洲类禽H1N1亚型猪流感病毒反向遗传操作平台的建立
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摘要
为了构建猪流感病毒A/swine/Tian Jin/6/2013(H1N1)的反向遗传操作技术平台,通过RT-PCR技术分别扩增了欧洲类禽H1N1亚型猪流感病毒的8个基因片段,并分别连接至双向转录表达载体p BD上。纯化8个重组质粒,共转染到293T细胞。54 h后收集上清,并接种到MDCK细胞。待细胞病变明显时,进行血凝试验检测收集到的上清,测得血凝效价为1∶32。测定拯救病毒的全基因组序列,结果显示,在核苷酸序列上,拯救病毒与野生病毒完全一致。细胞病变情况显示,拯救毒株与野生毒株无明显差别。本研究成功拯救了欧洲类禽H1 N1亚型猪流感病毒,为猪流感病毒的致病机理、基因功能研究以及H1N1亚型猪流感病毒新型疫苗的研发奠定了基础。
To rescue A/swine/Tian Jin/6/2013(H1N1) of Swine influenza virus, the eight gene segments of H1N1 were amplified by RTPCR and cloned into the transcription/expression vector of p BD. The eight recombinant plasmids were purified and cotransfect into 293 T cells. The supernatant of 293 T cells was collected after 54 hours and inoculated into MDCK cells. When cytopathic effects(CPE) appeared in MDCK cells, virus titer was determined at 1 : 32 in hemagglutination test. The full genome of the rescued virus had no nucleic acid change, compared with the wild-type virus through sequence analysis. There were no obvious differences in CPE on MDCK cells between the rescued virus and the wild type virus. The establishment of reverse genetic system of European avian-like H1N1 subtype Swine influenza virus provided the foundation for further studies on gene function and new influenza vaccine.
To rescue A/swine/Tian Jin/6/2013(H1N1) of Swine influenza virus, the eight gene segments of H1N1 were amplified by RTPCR and cloned into the transcription/expression vector of p BD. The eight recombinant plasmids were purified and cotransfect into 293 T cells. The supernatant of 293 T cells was collected after 54 hours and inoculated into MDCK cells. When cytopathic effects(CPE) appeared in MDCK cells, virus titer was determined at 1 : 32 in hemagglutination test. The full genome of the rescued virus had no nucleic acid change, compared with the wild-type virus through sequence analysis. There were no obvious differences in CPE on MDCK cells between the rescued virus and the wild type virus. The establishment of reverse genetic system of European avian-like H1N1 subtype Swine influenza virus provided the foundation for further studies on gene function and new influenza vaccine.
引文
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[2]Park Y W,Katze M G.Translational control by influenza virus.Identification of cis-acting sequences and transacting factors which may regulate selective viral m RNA translation[J].J Biol Chem,1995,270(47):28433-28439
[3]Brown I H.The epidemiology and evolution of influenza viruses in pigs[J].Vet Microbiol,2000,74(1-2):29-46.
[4]Fouchier R A,Munster V,Wallensten A,et al.Characterization of a novel influenza A virushemagglutinin subtype(H16)obtained from black-headed gulls[J].J Virol,2005,79(5):2814-2822.
[5]Yoon S W,Webby R J,Webster R G.Evolution and ecology of influenza A viruses[J].Curr Top Microbiol Immunol,2014,385:359-375.
[6]Cheung T K,Poon L L.Biology of influenza a virus[J].Ann N Y Acad Sci,2007,1102(1):1-25.
[7]Shope R E.Swine influenza:III.Filtration experiments and etiology[J].J Exp Med,1931,54(3):373-385.
[8]Choi Y K,Seo S H,Kim J A,et al.Avian influenza viruses in Korean live poultry markets and their pathogenic phtential[J].Viology,2005,332(2):529-537.
[9]孟沙沙,乔传玲,陈艳,等.一株类禽型H1N1亚型猪流感病毒的反向遗传系统的建立[J],中国预防兽医学报,2013,35(2):91-94.
[10]Qi X,Cui L,Jiao Y,et al.Antigenic andgenetic characterization of a European avian-like H1N1 swine influenza virus from a boy in China in 2011[J].Arch Virol,2012,158(1):39-53.
[11]Hoffmann E,Stech J,Guan Y,et al.Universal primer set for the full-length amplification of all influenza A viruses[J].Arch Virol,2001,146(12):2275-2289.
[12]杨焕良,乔传玲,陈艳,等.猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法的建立[J].中国预防兽医学报,2009,29(9):714-718.
[13]Ito T,Couceiro J N S S,Kelm S,et al.Molecular basis for the generation in pigs of influenza A viruses with pandemic potential[J].J Virol,1998,72(9):7367-7373.
[14]Smith G J,Vijaykrishna D,Bahl J,et al.Origins and evolutionary genomics of the 2009 swine-origin H1N1influenza A epidemic[J].Nature,2009,459(7250):1122-1125.
[15]Brown I H,Manvell R J,Alexander D J,et al.Swine influenza outbreaks in England due to a new H1N1 virus[J].Vet Rec,1993,132(18):461-462.
[16]Kyriakis C S,Brown I H,Foni E,et al.Virological surveillance and preliminary antigenic characterization of influenza virus in pigs in five Europeancountries from2006 to 2008[J].Zoonoses Public Health,2001,58(2):93-101.
[17]Kuntz-Simon G,Madec F.Genetic and antigenic evolution of swine influenza viruses in Europe and evaluation of their zoonotic potential[J].Zoonoses Public Health,2009,56(6-7):310-325.
[18]Yang H,Qiao C,Tang X,et al.Human infection from avian-like influenza A(H1N1)viruses in pigs,China[J].Emerg Infect Dis,2012,18(7):1144-1146.
[19]Yang H,Chen Y,Qiao C,et al.Prevalence,genetics,and transmissibility in ferrets of Eurasian avian-like H1N1swine influenza viruses[J].Proc Natl Acad Sci U S A,2016,113(2):392-397.
[20]李呈军,陈化兰,反向遗传学技术在流感病毒研究和防控中的应用[J].中国科学:生命科学,2015,45(10):1051-1066.