Expression Of Ev71vp1 Gene In Prokaryotic System And The Study Of Its Immunogenicity

Objective:Enterovirus type 71 (EV71) is a chief pathogen of hand-foot and mouth disease (HFMD). Among the serotypes of EV, EV71 is very important. The VP1 is a major capsid protein of EV71, which includes many epitopes of B and T. It can induce the effective immune responses. Although the DNA vaccine constructed with VP1 alone could stimulate the production of humoral and cellullar immunologic responses, the responses were usually too low to protect the host from lethal EV71 challenge, supposing to have something to do with DNA vaccine that are less cell entered or less purpose gene expressed. It was reported that the protein elicits stronger immunogenicity and the immunised animal can produce antibody efficientively. So many people re-try to use the protein as subunit vaccine to raise its immunogenicity. This vaccine is the protein which have immunity function, separated from the pathogen; or protein, polypeptide and synthetic obtained from recombinant DNA technique. This vaccine is a part of the pathogen, so it's called subunit vaccine. With no infection composition, the subunit vaccine doesn't have pathogenicity. It has already cloned FMDV (Foot and mouth disease virus) VP3 gene to E coli and expressed successfully in 1981. Up to now it already have developed out a lot of subunit vaccines against infectious disease that including inclusive of hepatitis B. Method of treating a EV71 affecting patient is mainly good for Symptoms of the disease, and short of some distinctive, high-effection antiviral medicine. There is no universally accepted precaution vaccine at present. The VPl protein is the main neutralizing antigen part of EV71, and a first chosen component of the vaccine studies. In this study, EV71 VP1 protein, expressed in prokaryotic system, was adopted to immun rabbits. The immune effect was then evaluated by specific immune response.Methods:1 EV71 VP1 gene was amplified by RT-PCR and cloned into pGEM-T vector. pGME-T/VP1 was then transformed to E. coli DH5 a and cul tured in medium containing Ampicilin. The plasmids were extracted and identified by restriction endonuleases digestion as well as sequencing.2 VP1 was obtained from pGME-T/VPl by proper endonucleases digestion and then inserted into pET-His vector which was cut by the same endonucleases. As a result, a prokaryotic expressing plasmid of pET-His/VP1 was formed.3 Then the plasmid was transformed into bacteria BL21 (DE3) pLysS. VP1 fusion protein including six His expression tag was expressed after induction. The protein was verified by Western blot analysis against the His and EV71 antibodis. The transformed bacteria were made into pieces by hypersound. Then the purified inclusion was obtained.4 New Zealand White rabbits weighting 2 kilograms were immuned with VP1 fusion protein three times at two weeks interval. The Immunization by protein was together with complete Freund's adjuvant for the first time, and then incomplete Freund's adjuvant, the titers for EV71-specific antibodies of rabbit sera were measured at the fiftth days after the third immunization, and in order to evaluate the titers of blood viruses.Results:1 EV71 VP1 gene was amplified by RT-PCR and cloned into pGEM-T vector. After restriction endonucleases digestion, its length was the same as espected. DNA sequencing showed that VP1 was identical to the sequence recorded in Genbank.2 The prokaryotic expressing plasmid of pET-His/VP1 was constructed successfully.3 VP1 protein was expressed in prokaryotic expression system successfully. Expressed as insolvable form, the protein was verified by Western blot analysis against the His and EV71 antibodis.4 The mean titers of antibody after third immunization in 3 rabbits were 1:81920,1:40960,1:40960. The statistics analysis showed that antibody titers of rabbits were increased after three times of immunization.Conclusion:1 The prokaryotic expressing plasmid pET-his/VPl was constructed successfully.2 The protein of EV71 VP1 is expressed in E. coil BL21 (DE3) pLysS.3 After administration of vaccine to New Zealand White rabbits, the antibody titer increased.