Expression Of Csfv E^rns Protein In Pichia Pastoris And Preparation Of Monoclonal Antibodies Against E_rns

Classical swine fever(CSFV) is one kind of high contact infective disease caused by classical swine fever virus(CSFV), which subordinate to the Pestivirus and the Flaviviridae. The characteristics of this disease are high fever,hemorrhage and high mortality. It has brought severe economic losses to the pig farming world wide since it happened.In this study, E^rns gene squence was synthesized according to the sequence of the popular Classical swine fever virus C- strain(CSFV-C), then purified products were cloned into pPIC9K expression vector. After identification with enzyme digestion and sequence analysis, the recombinant plasmid was obtained and then transformed into the host P. pastoris GS115 for expression. Induced by 0.5% Methanol at 30℃, the expression product of this gene was identified by SDS-PAGE and Western-blot. The results revealed the expression peak of E^rns protein is 240mg/L at72h and a specific reaction occured between the expression product and positive serum of classical swine fever virus,which indicates that E^rns has good immunogenicity. By using E^rns protein as the coated antigen, Indirect ELISA ascertained that the optimum concentration of coated antigen was 4μg/ml and the optimum dilution of serum samples was 1︰400.Then, immunizing 8 weeds female mouse with the purified E^rns , prepared for E^rns monoclonal antibodies(McAbs)of classical swine fever virus. Through indirect ELISA,subclone and series of basic identifications, 3 sorts of monoclonal hybridoma were confirmed, named A6H, A3E and C3G. Then McAbs(A6H,A3E,C3G)were identified at characterizations, et al. The McAbs(A6H,A3E,C3G)titers in cell supernatant and ascites were 1︰1600,1︰1600,1︰3200 and 1︰1×106,1︰4×106,1︰2×105 by the indirect ELISA, respectively.By using positive serum of classical swine fever virus, spleen and lymph node of normal pig and Rabbit as negative, Sandwich ELISA showed that 3 sorts of monoclonal hybridoma all had specific reaction with classical swine fever virus(CSFV), but not with others, which means that they were great valuable.In conclusion, we obtained recombinant Pichia pastoris of E^rns-pPIC9K-GS115, established a indirect ELISA which would be valuable to the detection of CSFV. Then we obtained the specific McAbs against E^rns protein, which would be useful for the future studies of CSFV.