Expression Of The Capsid Protein VP1 Antigen And Construction And Identification Of The Recombinant Adenovirus Shuttle Plasmid Containing The Capsid Protein VP1 Of Enterovirus 71

Enteorviurs71 is the most described serotype of the genus Enteroviurs(family Picornaviridae). It is a common cause of hand,foot,and mouth disease,aseptic meningitis,encephalitis,poliomyelitis-like paralysis and a lot of epidemics correlated with nerve system. Outbreaks of infection with this virus have occurred periodically throughout the world. At present there is no security effective vaccine to prevent the EV71 infection. So the development of rapid diagnose reagent and gene engineering vaccine have extensive foreground.The study contains two parts:Part one: Expression of the recombinant capsid protein VP1 of Enterovirus 71 in E.coliBased on the clone of the full capsid protein VP1 gene of Enterovirus 71 EVGZ06, we cloned part of coding region of the VP1 capsid gene into pET28a(+). The expression vector pET28a(+)/(502～891) VP1 was constructed,transformed into E.coli BL21,and induced by IPTG. Recombinant protein was purified through metal(Ni2+)chelating affinity chromatography. SDS-PAGE analysis showed that we got a recombinant protein of expected size(about 19 KDa). And the protein was further demonstrated by Western Blotting. It could be recognized by His-Tag monoclonal antibody.The recombinant protein was used as antigen to immune the mice,and the serum from the immuned mice also recognized the Enterovirus 71 come from cell cultivate. Our research will contribute to developing diagnose reagents or vaccine of Enterovirus 71.Part two: Construction and identification of the recombinant adenovirus shuttle plasmid containing the capsid protein VP1 of Enterovirus 71On the basis of successful clone of the full capsid protein VP1 gene and sequence analysis of Enterovirus 71 EVGZ06,the VP1 gene of EV71 derived from the plasmid pMD-20/VP1 by using polymerase chain reaction were inserted into the backward position of cytomegalovirus(CMV) immediate early promoter element of shuttle plasmid of adenovirus expression vector, then the recombinant plasmid(E3-pSKE3L-CMV-VP1-SV40-E3R) was obtained. The recombinant plasmid was identified by endonuclease,PCR and sequencing. VP1 gene was expressed transiently with Lipofectamine 2000 coated in Hep-2 cells which were confirmed by ELISA and Western blotting. This should be useful to construct recombinant adenovirus expression vector which can express VP1 gene. Our research will benefit to the construction and exploition of safe,efficient and industrial recombinant bivalent vaccine of human adenovirus type 3 and EV71.