The Recombination And Expression Of Porcine HSP Gp96N Gene With E2 Gene Of CSFV And The Immunogenicity Assay Of Recombinant Proteins

Classical swine fever virus E2 protein is the envelope glycoprotein of classical swine fever virus, which can confer protective immunity. It has been the main objective research of the new vaccine. Studies have shown that Gp96 N has a groove structure zone, this region can bind the antigen tightly as the stable Gp96- peptide complexes. Thus, GP96 N has potential value as a good adjuvant.In this study, the Gp96N(26AA-374AA) gene and E2 gene was connected by 2A gene of Foot and mouth disease virus, the eukaryotic and prokaryotic expression systems are used respectively. The immunogenicity of the prokaryotic recombinant GE was assessed to further explore the adjuvant effect of protein Gp96 N.First of all, the alphavirus replicon vector p SFV1 was transformed into the new eukaryotic expression vector p SCA as CMV promoter fragment and SV40 poly A terminator fragment were inserted into the vector p SFV1, the fluorescent protein EGFP gene, E2 gene and the tandem fragment GE gene were inserted into vector p SCA. Then these recombinant vectors were transfected into BHK-21 cells by liposome. The results showed that vector p SCA-EGFP can express effectively and the indirect immunofluorescence(IFA) test showed that p SCA-E2 and p SCA-GE can express, but the expression efficiency of p SCA-E2 and p SCA-GE plasmid is not high and the mean fluorescence intensity is low.Secondly, the E2 gene and tandem fragment GE were constructed into prokaryotic expression vector p ET-32a(+), respectively. Then p ET-E2 and p ET-GE vectors were transformed into the Rosetta(DE3) competent cells. Cultivation conditions were optimized to guarantee the efficient expression of histidine tagged recombinant proteins E2 and GE. The recombinants were crude purified and renatured by gradient method. According to SDS-PAGE and Western Blotting assay, the results showed that the expression productions were inclusion bodies and recombinants can react with antibody specifically.Finally, in order to study the immunogenicity of recombinant E2 and GE in vivo, the BALB/C mice were immunized by recombinant E2 and GE, C-strain vaccine and PBS through subcutaneous injection. The ELISA assay was been done to detect the antibody level in serum and the results showed that high antibody levels were detected by recombinant E2 and GE vaccine group, however, the antibody level of recombinant GE in experimental group is lower than that of E2 recombinant protein. Therefore, Gp96 N can weaken immunogenicity of tandem protein and we speculated that the position relationship between Gp96 N gene and E2 gene may affect the immune effect.