Research On Expression Of Structure And Nonstructure Proteins Of Classical Swine Fever And Their Reactivities

Classical swine fever (CSF), a highly-infectious disease, brings severe economic losses to the pig farming worldwide. Due to the change of epidemic model of CSF, antibody titration of immune pigs too low to resist virulent strain infection and lead to CSF sporadic or epidemic. In order to effective control of CSF, 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 is the lack of sensitive, specific antigen. The prokaryotic expression system has advantages of operating easily, the cost is low and the expression quantity is high, so it is an important system for recombinant protein expression, we had expressed the gene of CSFV E2 efficiently in E. coli and developed the indirect ELISA to survey pig serum antibody and assembled the antibody monitor kit. In order to further exaltate sensitivity and particularity of the antibody dicetion method, research to manufacture the new generation detective antigen, so we expressed the structure and non-structure proteins of CSFV in E. coli and in baculovirus expression system , the recombinant proteins were analyzed by immunological methods to make sure which viral protein can induce antibody in pig. In this research, nine gene ( Npro, C, E1, NS2, NS3, NS4A, NS4B and NS5A) of CSFV were cloned and nine recombinant expression plasmids ( pGEX-Npro, pGEX-C, pGEX-E1, pGEX-NS2, pGEX-NS3, pGEX-NS4A, pGEX-NS4B, pGEX-NS5A, pGEX-NS5B) were constructed, then these recombinant expression plasmids transformed into E. coli DH5a ,after induced by IPTG, we find that only three genes (Npro, C and NS4A) were successful expressed through SDS-PAGE detection. Recently,recombinant baculovirus have become widely used as vectors to express heterologous genes in cultured insect cells and insect larvae, In most cases, the recombinant proteins are processed, modified, and targeted to their appreciate cellular locations, where they are functionally similar to their authentic counterparts. In this research , We also constructed eight recombinant transfer plasmids (pHT-E1, pHT-NS4B, pHT-NS2, pHT-NS3, pFast-E2, pFast-Erns, pFast-NS5A and pFast-NS5B), all the recombinant transfer plasmids were introduced into E. coli DH10Bac cells which included a shuttle vector , Bacmid. By site-specific transposition, different target genes were integrated into Bacmid, and the recombinant shuttle vector were constructured, named Bac-E1,Bac-4B,Bac-NS2,Bac-NS3,Bac-E2,Bac-Erns,Bac-NS5A and Bac-NS5B.The cultured Sf9 cell were directly transfected with recombinant bacmid DNA, and the pure recombinant baculovirus was obtained, named rAcV-E1, rAcV-E4B, rAcV-NS2, rAcV-NS3, rAcV-E2 rAcV-Erns, rAcV-NS5A and rAcV-NS5B. We get recombinant baculovirus were identified through electron microscope asssy, PCR analysis revealed that target genes were correctly inserted into baculovirus genome under the control of polyhedron promoter. Apply anti-6His antibody to examine recombinant proteins by...