猪戊型肝炎病毒液相蛋白芯片检测方法的建立

Development of a fluorescent microsphere-based immunoassay for detection of antibodies against swine hepatitis E virus

构建了ORF2蛋白原核表达体系Rosetta-pMAL-c5X-ORF2,经IPTG诱导表达后,对重组蛋白进行SDSPAGE和Western blot分析,并采用镍柱亲和层析法进行纯化,以该蛋白为抗原建立液相蛋白芯片检测方法。结果显示:本试验成功表达了可溶性ORF2蛋白,具有良好的抗原性。液相蛋白芯片检测方法对猪常见的其他疾病阳性血清无交叉反应,其批内、批间变异系数分别为5%和6.6%。对102份临床血清样本检测结果显示,该方法与商品化ELISA试剂盒符合率为93.1%,关联性卡方检验显示2个方法具有一致性(P<0.01)。本试验为临床HEV血清抗体的检测提供了一种特异、灵敏的新型快速检测技术,为建立猪病多重检测方法提供了基础。

Swine hepatitis E virus（HEV） is a zoonotic virus and pigs are considered as an impor- tant reservoir of HEV. For effective monitoring it,there is a great need of rapid and sensitive as- say for detection of antibodies against swine HEV. In this study,a fluorescent microsphere-based immunoassay（MFIA） for detection of swine HEV ORF2 proteins antibodies was developed. Re- combinant ORF2 proteins was expressed by using a prokaryotic expression system, purified and covalently coupled to Luminex fluorescent microsphere. Under the optimized reaction conditions, FMIA was specific to detect the antibody against HEV in swine serum and no cross-reaction with positiVe serum from other diseases. The coefficients of variation for intra and inter-assay were 5 % and 6.6% ,respectively. Compared with ELISA,the coincidence rate of FMIA was 93.1%. These results indicate that the FMIA is rapid, sensitive and specific for detecting antibodies against swine HEV and has a potential to develop a multiplexing the FMIA that can detect evidence Of exposure to multiple swine pathogens in one single step.