关键词: 猪瘟病毒, E^rns/E2, 融合蛋白, 串联表达, 包涵体, 复性, 间接ELISA

Abstract: In order to establish a method for the detection of serum antibody against classical swine fever virus(CSFV),the nucleic acid sequence encoding the major antigenic region of co-expressed CSFV E^rnsand E2 was cloned into plasmid pET-32a,the recombinant bacteria were obtained by transforming Rosetta TM2(DE3) with positive recombinant plasmid.SDS-AGE,Western blot and solubility analysis indicated that the molecular weight of expressed Erns/E2 protein was about 43 ku,could be recognized by CSFV positive serum and was mainly in the form of inclusion bodies.The purified inclusion bodies protein was refolded by rapid dilution method,and the recovery efficiency was more than 40%.The purified and refolded protein of E^rns/E2 was used as the coating antigen to develop an indirect ELISA ( E^rns/E2-ELISA)for detecting CSFV antibody after optimization of reaction conditions. Results from the specificity,sensitivity and reproducibility tests showed that the Erns / E2-ELISA were specific, sensitive and roproducible.A total of 121 serum samples were detected by the Erns/E2-ELISA and IDEXX CSFV antibody test kit,the E^rns/E2-ELISA coincidence rate,sensitivity and specificity were 86.78%,92.11% and 77.78%,respectively.Four 30-day-old healthy piglets were immunized with C strain vaccine and the fluctuation dynamics of antibody was evaluated by the E^rns/E2-ELISA and IDEXX CSFV antibody test kit.The antibody can be detected as early as 7 days and 14 days respectively,reaching the highest level on the 35th—42th days and finally stabilizing on the 49th—56th days.

Key words: Classical swine fever virus, E^rns/E2;Fusion protein;Co-expression;Inclusion bodies;Refolding;Indirect ELISA