Prokaryotic Expression Of Classical Swine Fever Virus E2 Protein And Development Of Its Monoclonal Antibody

Classic swine fever,commonly known as "rotten intestinal fever",is one of the important infectious diseases that hinder the development of the pig industry.The disease,a severe swine infectious disease,is caused by classical swine fever virus(CSFV).It is characterized by hemorrhagic fever syndrome and immunosuppression,and causes economic losses worldwide.E2 is one of the most immunogenic CSFV glycoproteins,which can induce neutralizing antibodies to resist attack of the deadly viruses.The E2 protein subunit vaccine has also been applied to the immunization of CSFV,but the method for evaluating the quality of the E2 subunit vaccine is not yet perfect.In this study,the E2 envelope glycoprotein of CSFV was expressed in prokaryotic cells,and specific monoclonal antibodies were obtained by immunizing mice,which will support a tool for the evaluation of CSFV E2 vaccine and the prevention and control of CSFV1.Clone and expression of CSFV E2 proteinE2 is one of the main structural glycoproteins of CSFV.It is also the main protective antigen that induces produce of the specific neutralizing antibodies.This protein is also the key determinant of virus entry and immunity.In this study,E2 protein(removing the transmembrane region)of the swine fever virus(CSFV)was amplified by RT-PCR from the rabbit attenuated vaccine strain(strain C),and then cloned it into the prokaryotic expression vector pET-32a to construct pET-32a-CSFV-E2 recombinant plasmid.Then it was transformed into E.coli BL21(DE3).The recombinant E2 protein was expressed in E.coli cells induced by 0.1mM IPTG at 37? and 225 rpm for 5 hours.The supernatant and precipitation of the E.coli cells were analyzed by SDS-PAGE respectively.The results showed a band about 58kDa at the precipitation,which was consistent with the size of the fusion protein.Western-blot results showed that the recombinant protein could react with mouse anti-His monoclonal antibody and classical swine fever serum,indicating that the immunogenicity of the recombinant E2 protein is good.It will provide biological materials for the further development of monoclonal antibodies against E2 protein.2.Development of monoclonal antibody against classical swine fever virus E2 proteinIn order to prepare monoclonal antibodies against E2 protein,BALB/c female mice at age of 6-8 weeks old were immunized every 10 days with the recombinant HIS-E2.After three times immunization the mice sera were detected and bosted with the recombinant HIS-E2.Cell fusion is performed after 72-96h bost immunization.PK15 cells infected with CSFV were used for screening the monoclonal antibodies in supernatant of the hybridoma cells by IFA and Western-blot.As a result,four hybridoma cell lines that can stably secrete monoclonal antibodies against E2 protein were obtained,which were named as CSFV-E2-1C2,CSFV-E2-2B2,CSFV-E2-3A8 and CSFV-E2-3H11.After two subclones,the hybridoma cells reached 100%secretion of anti-E2 protein specific antibodies.The subclass identification results showed that the subclasses of CSFV-E2-1C2 and CSFV-E2-3A8 were IgGl,CSFV-E2-2B2 and CSFV-E2-3H11 were IgM,and the light chain types were Kappa.The titer of CSFV-E2-1C2 ascites was 1:6400 and that of CSFV-E2-3A8 was 1:12800 in IFA.The epitopes of two monoclonal antibodies were analyze by IFA with the expression of six E2 truancted genes transfered into 293T cells.The results showed that CSFV-E2-1C2,CSFV-E2-3A8 is located at 50-100aa and 1-50aa of E2 respectively.In this study,monoclonal antibodies against CSFV E2 protein were successfully prepared,which laid the foundation for the biological research of CSFV,the establishment of detection methods and evaluation of vaccine quality.