High-level Expression Of E^rns Gene Of Classical Swine Fever Virus (CSFV) In Pichia Pastoris And Initial Establishment On Indirect ELISA

Classical swine fever is highly contacted and mortal contagious disease, and caused large economic losses in nation. Vaccining classical swine fever attenuated vaccine can control classical swine fever in large-scale epidemic, but it will increase the difficulty to differentiate virulent infected pigs from immunized pigs. Expression of Classical Swine Fever Virus protective antigen E^rns protein was used to establish classical swine fever antibody diagnostic method, which has a important serological identification role in development and application of marker vaccines (Marker vaccine),and in monitoring hog cholera virus prevalence situation and vaccine immuning situation ,and developping immunity procedure.E^rns can distinguish antibodies which were produced from vaccine immuning and wild virulent infection ,using marker vaccines and providing supporting diagnostic methods can improve the accuracy of clinical diagnosis and efficiency.The purpose of this study is to express the biological active E^rns protein using Pichia pastoris expression system,and to provide test information for establishing the serological ELISA diagnostic method of large-scale screening for specific antibodies against CSFV and to make the use of maker vaccine E2 widely.The total RNA was extracted from the CSFV weaken live vaccine and used as the template for the cloning of the gene E^rns by RT-PCR with specific primers designed according to the reported sequence in Genbank(Z46258). The E^rns was linked with clone vector pGM-T, and sequenced , the result showed the Similarity index was 99.0%, The expression plasmids pPIC9K-E^rns of Pichia pastoris were constructed and transformed through electroporation into Pichia pastoris GS115. Multicopy strains were screened by G418, and Mut phenotype was selected by MM plate and coenobium PCR.The selected strain was cultured in 3⁵ L flasks by level shaking and the supernatant was analyzed by SDS-PAGE and western-blotting.The results indicated that the E^rns protein was successfully expressed and secreted with a molecular weight of approximately 48 kDa. The results showed that the E^rns protein was successfully expressed, and could also be recognized by specific antibodies and had biological activity.We purified the yeast supernatants with the anion-exchange chromatography, using the pruified E^rns protein as coated antigen ,an indirect ELISA was developed for detecting the anti-E^rns antibody in the CSFV serum by exploring the concentration of coated antigen and dilution degree of serum,which would provide a good fundament for development of CSFV antibody surveillance kit.