Expression Of Structural Protein Vp1of Encephalomyocarditis Virus And Preparation Of Monoclonal Antibody Against Vp1Protein

Encephalomyocarditis is an important acute zoonosis, clinically characterized by encephalitis, myocarditis and inflammation around the cardiac muscles. Encephalomyocarditis virus (EMCV) naturally infects a wide range of host species including pigs, rodents, primates and some kinds of insects among which pigs are considered to be the most commonly and severely infected domestic animal species. EMCV infection can cause acut myocarditis and sudden death in preweaned piglets and severe reproductive failure in sows. EMCV is classified in the cardiovirus genus of the picornavirus family. It is a single-stranded positive-sense RNA virus with a genome of about7.8kb which is composed of an open reading frame. EMCV particle, without envelope, is composed of four structural proteins including VP1, VP2, VP3and VP4, among which VP1is most antigenic. The VP1protein can induce the production of neutralization antibody and of protective immunity against EMCV infection. In this study, the VP1gene of EMCV was amplified by PCR from recombinant pET32a-VP1and inserted into pET28a vector and expressed in E.coli BL21with33kDa under the condition of IPTG; The BALB/c mice were vaccinated with purified VP1protein containing adjuvant and7monoclonal antibody against EMCV VP1were obtained. The details are as follows:1. Expression of EMCV VP1gene in procaryotic expression system and preparation of polyclonal antibody of VP1proteinThe VP1gene of EMCV was amplified by PCR from recombinant pET32a-VP1and cloned into a prokaryotic expression vector pET28a. After identification of digestion by BamH Ⅰ and Xhol Ⅰ, the recombinant protein VP1was expressed in E.coli with33kDa fused with His-tag under the condition of IPTG. The result of Western blot showed that the recombinant VP1protein could specifitly interact with mice serum against EMCV. After that, the VPl protein was purified and used to vaccinate BALB/c mice to obtain the polyclonal antibody. The Western blot and IFA results showed that the polyclonal antibody could interact with VP1protein and EMCV respectively.2. Preparation of monoclonal antibody against VPl protein of EMCVThe BALB/c mice were immunized by purified recommbinant VP1protein and the splenocytes of immunized mice were fused with murine myeloma cells to produce hybridoma cell line. After testing by ELISA and subcloning by3or4times,7strains of hybridoma cell line steadily secreting monoclonal antibodys against VP1were obtained, named1F3,1G8,2C8,3A9,6E11,7A7,7C9, repectively. The monoclonal antibodys all belong to IgGl. As expected, the results of Western blot and IFA demonstrated the specificity of reaction of the monoclonal antibody with protein VP1or EMCV.In summary, in this study, the recombinant protein pET32a-VPl was expressed in E.coli prokaryotic expression systom and seven strains of hybridoma cell line were obtained by the BALB/c mice immunized with purified recommbinant VP1protein. It provided potential values for structural and functional studies of EMCV structural protein and for developing early diagnosis method of EMCV infection.