Construction of pseudotype retrovirus which integrates hemagglutinin of H5N1 avian influenza virus isolated from human in Shenzhen
10.3760/cma.j.issn.0254-5101.2009.01.013
- VernacularTitle:整合深圳人源H5N1禽流感病毒株血凝素蛋白的假型逆转录病毒的构建
- Author:
Guoliang ZHANG
;
Boping ZHOU
;
Xinchun CHEN
;
Cheguo CAI
;
Jian LU
;
Guilin YANG
;
Guang NIE
;
Baoluo ZHOU
- Publication Type:Journal Article
- Keywords:
H5N1 avian influenza virus;
Hemagglutinin;
Pseudotype virus;
Retrovirus
- From:
Chinese Journal of Microbiology and Immunology
2009;29(1):53-57
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct pseudotype retrovirus which integrates hemagglutinin(HA)of H5N1 avian influenza virus(AIV)isolated from human in Shenzhen.Methods AIV HA gene was amplified bv RT-PCR,then it was ligated with pGEM-T vector,and identified by restriction enzyme digestion and sequenced.HA gene was cloned into CMV/R vector at the site of Sal Ⅰ and BamH Ⅰ.pHR-Luc,pCMV&8.2 and CMV-HA were co-transfected into 293T cell by co-precipitation with calcium phosphate.The pseudotype virus supernatant was harvested 72 h post-transfection and ultracentrifugation,and the HA and P24 expression on the surface of pseudotype virus was analyzed by western blot.Meanwhile.the infection activity of HIV-HA pseudotype virus was identified in different kinds of cell lines,including MDCK,HeLa,CHO and 293T.Results A/Shenzhen/406H/06 belonged to subclade2.3 with open reading frame(ORF)of HA gene encoded 567 amino acides,whose accession number was EF137706 in GenBank.HA gene was cloned into CMV/R successfully.After co-transfection of above vectors,it revealed that HA protein could integrate pseudotype virus by western blot,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Finally.HIV-HA pseudotype virus could infect 4 kinds of cell lines,which indicated its property of infectivity and catholicity.Conclusion The pseudotype retrnvirns wassuccessfully constructed,which can integrate HA protein of A/Shenzhen/406H/06 and had property of infectivity.It call be used in the further research,including selection of neutralizing antibodies and epitope analysis.
