Prokaryotic Expression Of Immunogenic E2 And Repeated E2 Epitope Of Classical Swine Fever Virus And Preparation Of Specific Antiserum

CSF is a disease with acute, febricity and high contagiouis vital viral disease. HCLV vaccine was successfully studied in 1955, its saety and efficacy was perfect, it made lots of contribution to control and prevent CSFV in China and even all of the world. In recent years, the epidemic of CSFV has been changed a lot, the titre of antibody in many vaccined pigs can not resist the infection of CSF wild virus, it had leaded CSFV diverging and epidemic in pig stocks. The numbers of persistent infection have increased. In order to control CSF effectivly, It is very important to carry out antibody surveillance periodic. On the basis of antibody surveillance, carry out special immune programme, reinforce immune antibody titration low pigs and selection immune reaction inferior pigs, to maintain group immune protection rate. Serological detection of CSFV antibody is important diagnosis methods and antibody surveillance tools. But the problem of CSF antibody detection method lacks of sensitive, specific antigen. It is necessary to study the molecular construction and character of antigen, identify the epitope with diagnosis fuction, studies on the new epitopes about CSFV.The prokaryotic expression system has many advantages of operating easily, the cost is low and the expression quantity is high, so it is an important system for recombinant protein expression, RT-PCR and PCR were used in amplifying E2 gene exclude signal peptide and transmembrane domain and 4 repeats of epitope (amino acid sequence,QTAVSPTTLRTEWK)from E2 protein, target genes were cloned into prokaryotic expression vector pMAL-P2X and pGEX-6p-l respectively, successfully constructed recombinat expression plasmids. After transformation, target protein were expressed in E.coli TB1 and BL21(DE3),expressed proteins from pMAL-4P and pGEX-4p located in the supernatant of cell disruption induced by IPTG at 37℃, expressed protein from pMAL-E2 was detected in the inclusion body and supernatant of cell disruption induced by IPTG at 20℃, expressed protein from pGEX-E2 was detected in the inclusion body of cell disruption induced by IPTG at 37℃. Target protein were purified with MBP or GST affinity chromatograph. Western-blotting confirmed target protein in E. coli were antigenic. ELISA results show that fusion protein GST-4P or MBP-4P can not react with BVDV E2 anti-rabbit serum, others not.This shows that it is important to identify CSFV as a potential antigen.Using purified expressed protein MBP-E2, MBP-4P in E.coli and E2 protein from Baculovirus in sf9 cells to immunize rabbits for generating high titer ployclonal serum. Western-blotting and IFA showed that the polyclonal serum can react with post-vaccined rabbit serum, it means that the polyclonal serum have good reactivity and specificity.These results will help to discriminate infected pig from immune pig and build up CSFV combination antigens for the antibody examination method and facilitate the structural and functional study of CSFV E2 protein.