Establishment Of Sera Antibody Detecting Kit For Classical Swine Fever And Development Of Recombinant Vaccine Against Classical Swine Fever Virus

Full E2 gene of classical swine fever virus (CSFV) shimen strain was isolated from CSFV genome by RT-PCR. Then, another 2 pairs of primers were used to amplify the A/D domains of E2 gene with the full E2 gene as template. The two target sequences were cloned into prokaryotic expression vector pET-32a one by one and the recombinant plasmids named pET-E2 and pET-2E2 were constructed. Then pET-E2 and pET-2E2 were transformed into E.coli BL21 (DE3) and induced with IPTG The target proteins can be expressed in a very high level in the form of inclusion bodies.The recombinant envelope protein 2E2 of CSFV was obtained under the optimized condition of host cell cultivation and IPTG induction. Consequently, the expression products were purified by the means of His-bind resin protein purification procedure. Following SDS-PAGE and Western-blot were used to detect the purification effect and the specificity of the purified recombinant envelope protein of CSFV. Then the purified envelope protein 2E2 was used to be coated on the well of 96-well plate, each following step such as coating concentration of recombinant glycoprotein, sample dilution and the coating time .was optimized. As a result, an indirect ELISA (i-ELISA) was set up to detect sera antibody levels against CSFV. About 120 sera samples were detected by the i-ELISA established here and the Indirect-haemagglutination detective kit, respectively. The result showed the better sensitivity of the i-ELISA established here.In this study, one gene fragment encoding swine immunoglobulin G heavy chain constant region (scIgG) was obtained with PCR method. Then, the fragment was cloned into pET-2E2 and the recombinant plasmid pET-2E2-sdgG was constructed. Mice showed both neutralizing antibodies and T cell proliferation response after having been immunized with the purified proteins of E2, 2E2 and 2E2-scIgG, respectively. The present studydemonstrated the existence of T cell epitope(s) in the antigenic domain A/D of the envelope glycoprotein E2 of CSFV and the promising potential of self-IgG as antigen carrier.