Cloning And Prokaryotic Expression Of Partial Fragment Of E2 Gene Of Swine Fever Virus And Preparation Of Their Polyclonal Antibodies

E2 protein is transmembrane glycoprotein of Classical Swine Fever Virus(CSFV), prone to mutation, a kind of immunologic preponderant protein, and can induce infected animals to protective immunity and neutral antibody of CSFV,section A1,B,C is very important for engendering neutral antibody, cooperative neutral reaction is happened when using monoclonal antibody A1 and B or A1 and C. Expression of recombining CSFV Brescia strain E2 gene to Pseudorabies Virus and purification of gene engineering E2 protein by affinity chromatography from insects cells both protect immune pigs from infection of CSFV. In order to prepare antibody against CSFV E2 protein, do research on application of expressed production of E2 gene to immunologic detection and neutral antigen epitopes of E2 protein, partial fragment of CSFV E2 gene was cloned and expressed.Firstly, specific primers for CSFV E2 gene were designed according to the CSFV genomic sequence registered in GeneBank.Using PCR technology, one gene fragment about 622bp was amplified from a recombinant plasmid PGEX-4T-1- PE2 which was conserved in our experiment. The result of endonuclease Dra I digesting PCR product showed that this gene fragment has the same restriction enzyme site as known CSFV E2 gene. Gene fragments were ligated to pMD18-T vector after purification, the recombinant plasmid pMD18-T-TE2 was transformed into Escherichia coli (E.coli)DH5α,the recombinants were analyzed after they were identified by PCR, subsequently. This suggests that the CSFV TE2 gene has been successfully cloned.Secondly, gene-specific primers were designed based on the cloned TE2 nucleotide sequences and multi-clone restriction enzyme sites of prokaryotic expressive vector pGEX-4T-l, one gene fragment which encode major antigenic domains of E2(A,B,C,D) about 622bp was amplified from a recombinant plasmid pMD18-T-TE2 by PCR. Then the cDNA fragment which encode major antigenic domains of E2(A,B,C,D) was cloned into pGEX-4T-l,the recombinant plasmid PGEX-4T-1-TE2 was identified by PCR and endonuclease EcoR I +Sal I digestion. The PGEX-4T-1-TE2 was transformed into E.coli Rosetta BL21 DE3 and GST-TE2 fusion protein was induced to express by isopropylthio-β-D-galactoside(IPTG). The MW of the fusion protein was about 47 000 as analyzed by SDS-PAGE.After optimizing prokaryotic expression conditions, we determined the optimum inducement time and concentration of IPTG. The solubility of GST-TE2 fusion protein was identified and the result indicated that expressed GST-TE2 protein existed as inclusion bodies. The recombinant plasmid was induced to express large-scale GST-TE2 fusion protein under the optimal condition, the induced recombinant bacteria was lysed by freeze-thaw and sonication. We obtained the GST-TE2 inclusion body protein, which could be solubilized by sonication after the detergent lauroylsarcosine was added. The concentration of fusion protein GST-TE2 was about 1.2mg/ml by folin-hydroxybenzene.Finally, using the GST-TE2 fusion protein as antigen, polyclonal antiserum to CSFV E2 was derived from mouse. Results of agar diffusion assay demonstrated that the polyclonal antibody could well react to TE2 protein. ELISA indicated that the immunized rabbits had antibody titers of 1:25600.Western-blotting showed that polyclonal antibody against CSFV GST-TE2 from mouse contain antibody against GST and antibody against GST-TE2 fusion protein.In conclusion, we have successfully cloned,expressed CSFV TE2 gene, and obtained the polyclonal antibody against CSFV TE2.A11 these provide some experimental materials for the future studies on the immunological function of CSFV E2.