汉滩病毒包膜糖蛋白假病毒的构建及特性研究
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摘要
目的:研究汉滩病毒包膜糖蛋白与宿主细胞作用的分子机制,构建含有汉滩病毒(HTNV)标准株76~118株包膜糖蛋白的假病毒,并对其进行检测.方法:将表达汉滩病毒包膜糖蛋白的质粒和以VSVΔG为骨架的假病毒颗粒,与293T细胞共转染,构建汉滩病毒的假病毒,用中和实验,受体抑制实验,免疫共沉淀和Western Blot对其抗原性、中和反应、受体作用等进行检测.结果:汉滩病毒假病毒与活病毒有相似的抗原特性,汉滩病毒假病毒可以模拟活病毒进入细胞的方式,其糖蛋白是以二聚体形式存在的.结论:成功构建含有汉滩病毒标准株包膜糖蛋白的假病毒,它与活病毒的抗原特性和受体作用的方式相似,这为进一步研究汉滩病毒包膜糖蛋白与宿主细胞作用的分子机制提供了很好的工具.
AIM: To study the molecular mechanism of hantaanvirus( HTNV) envelope glycoprotein with host cell,and constructe pseudovirions of HTNV 76- 118 strains. METHODS: It was constructed by incorporating of HTNV glycoproteins onto replication-defective vesicular stomatitis virus( VSV) cores in which the gene for the surface G protein has been replaced by the reporter gene. Antigenicity,neutral reaction and receptor function were detected by neutralization test,co-immunoprecipitation and flow cytometry. RESULTS: There was good concordance of the antigenicity and the way inside host cells between HTNV and its pseudovirions. Also,the glycoprotein G N and G C of pseudovirions existed as heterodimers. CONCLUSION: The pseudovirions are successfully constructed and the pseudovirions can be used to simulate the way of HTNV entry and antigenicity. This study provides a good stool for study on molecular mechanism of hantaan virus envelope glycoprotein.
AIM: To study the molecular mechanism of hantaanvirus( HTNV) envelope glycoprotein with host cell,and constructe pseudovirions of HTNV 76- 118 strains. METHODS: It was constructed by incorporating of HTNV glycoproteins onto replication-defective vesicular stomatitis virus( VSV) cores in which the gene for the surface G protein has been replaced by the reporter gene. Antigenicity,neutral reaction and receptor function were detected by neutralization test,co-immunoprecipitation and flow cytometry. RESULTS: There was good concordance of the antigenicity and the way inside host cells between HTNV and its pseudovirions. Also,the glycoprotein G N and G C of pseudovirions existed as heterodimers. CONCLUSION: The pseudovirions are successfully constructed and the pseudovirions can be used to simulate the way of HTNV entry and antigenicity. This study provides a good stool for study on molecular mechanism of hantaan virus envelope glycoprotein.
引文
[1]Cifuentes-Muoz N,Salazar-Quiroz N,Tischler ND.Hantavirus Gn and Gc envelope glycoproteins:key structural units for virus cell entry and virus assembly[J].Viruses,2014,6(4):1801-1822.
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[3]Burns JC,Friedmann T,Driever W,et al.Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors:concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells[J].Proc Natl Acad Sci USA,1993,90(17):8033-8037.
[4]Lawson ND,Stillman EA,Whitt MA,et al.Recombinant vesicular stomatitis viruses from DNA[J].Proc Natl Acad Sci USA,1995,92(10):4477-4481.
[5]Schnell MJ,Buonocore L,Kretzschmar E,et al.Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles[J].Proc Natl Acad Sci USA,1996,93(21):11359-11365.
[6]Bácsi A,Ebbesen P,Szabo J,et al.Pseudotypes of vesicular stomatitis virus-bearing envelope antigens of certain HIV-1 strains permissively infect human syncytiotrophoblasts cultured in vitro:implications for in vivo infection of syncytiotrophoblasts by cell-free HIV-1[J].J Med Virol,2001,64(4):387-397.
[7]Maruyama J,Miyamoto H,Kajihara M,et al.Characterization of the envelope glycoprotein of a novel filovirus,lloviu virus[J].J Virol,2014,88(1):99-109.
[8]Higa MM,Petersen J,Hooper J,et al.Efficient production of Hantaan and Puumala pseudovirions for viral tropism and neutralization studies[J].Virology,2012,423(2):134-142.
[9]Ray N,Whidby J,Stewart S,et al.Study of Andes virus entry and neutralization using a pseudovirion system[J].J Virol Methods,2010,163(2):416-423.
[10]Gavrilovskaya IN,Brown EJ,Ginsberg MH,et al.Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins[J].J Virol,1999,73(5):3951-3959.
[2]Ogino M,Ebihara H,Lee BH,et al.Use of vesicular stomatitis virus pseudotypes bearing hantaan or seoul virus envelope proteins in a rapid and safe neutralization test[J].Clin Diagn Lab Immunol,2003,10(1):154-160.
[3]Burns JC,Friedmann T,Driever W,et al.Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors:concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells[J].Proc Natl Acad Sci USA,1993,90(17):8033-8037.
[4]Lawson ND,Stillman EA,Whitt MA,et al.Recombinant vesicular stomatitis viruses from DNA[J].Proc Natl Acad Sci USA,1995,92(10):4477-4481.
[5]Schnell MJ,Buonocore L,Kretzschmar E,et al.Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles[J].Proc Natl Acad Sci USA,1996,93(21):11359-11365.
[6]Bácsi A,Ebbesen P,Szabo J,et al.Pseudotypes of vesicular stomatitis virus-bearing envelope antigens of certain HIV-1 strains permissively infect human syncytiotrophoblasts cultured in vitro:implications for in vivo infection of syncytiotrophoblasts by cell-free HIV-1[J].J Med Virol,2001,64(4):387-397.
[7]Maruyama J,Miyamoto H,Kajihara M,et al.Characterization of the envelope glycoprotein of a novel filovirus,lloviu virus[J].J Virol,2014,88(1):99-109.
[8]Higa MM,Petersen J,Hooper J,et al.Efficient production of Hantaan and Puumala pseudovirions for viral tropism and neutralization studies[J].Virology,2012,423(2):134-142.
[9]Ray N,Whidby J,Stewart S,et al.Study of Andes virus entry and neutralization using a pseudovirion system[J].J Virol Methods,2010,163(2):416-423.
[10]Gavrilovskaya IN,Brown EJ,Ginsberg MH,et al.Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins[J].J Virol,1999,73(5):3951-3959.