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

摘要： 为了建立猪瘟病毒（CSFV）血清抗体检测方法，将CSFV E^rns和E2蛋白主要抗原区串联表达的编码核酸序列克隆至原核表达载体pET-32a中，转化大肠杆菌Rosetta TM2（DE3）构建重组表达菌。SDS-PAGE、Western blot鉴定及蛋白质可溶性分析结果显示，表达的E^rns/E2蛋白分子质量约为43 ku，能够被CSFV阳性血清识别，主要以包涵体形式存在。通过快速稀释法复性了纯化的包涵体蛋白，复性效率大于40%。以纯化复性的E^rns/E2蛋白为包被抗原，经优化各反应条件，建立了间接ELISA检测CSFV抗体方法(E^rns/E2-ELISA)。特异性、敏感性和重复性试验结果表明，建立的检测方法特异性强、敏感性高、重复性好。利用建立的E^rns/E2-ELISA检测方法对121份随机采取的临床血清样本进行检测，与IDEXX CSFV抗体检测试剂盒相比，E^rns/E2-ELISA检测方法符合率为86.78%，敏感性为92.11%，特异性为77.78%。利用建立的E^rns/E2-ELISA检测方法和IDEXX CSFV抗体检测试剂盒同时评价CSFV C株疫苗免疫的4只30日龄健康仔猪抗体消长规律，可分别于免疫后7 d和14 d开始检出阳性，35~42 d抗体水平达到高峰，49~56 d抗体趋于稳定。

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