口蹄疫病毒3B基因的高效表达及其表达产物的反应活性

通过PCR技术获得口蹄疫病毒非结构蛋白3B基因,将其克隆至质粒pET-32a( )和高效可溶性表达质粒pEM中,经PCR筛选,获得阳性重组子pET-3B和pEM-3B,将它们转化大肠杆菌BL21中进行诱导表达,SDS-PAGE及Western-blot检测结果证明,表达的3B融合蛋白能与FMDV感染动物血清发生特异性反应。蛋白可溶性分析表明,EM-3B融合蛋白绝大部分以可溶形式表达。以此可溶性融合蛋白EM-3B为抗原建立了可区分FMD免疫动物和感染动物的EM-3B-DIVA-ELISA方法,经检测,其特异性为95.0%,敏感性为97.5%,与2个国产试剂盒的符合率分别为96.25%和98.75%。

High expression of foot-and-mouth disease virus 3B gene and reactivity of the expressed protein

3B gene encoding non-structural protein 3B of food-and-mouth disease virus(FMDV) was amplified by PCR and inserted into expression plasmids pET-32a( ) and pEM.The positive recombinants pET-3B and pEM-3B were screened by PCR and then transformed into Escherichia coli BL21 induced by IPTG.Analyses by SDS-PAGE and Western-blotting showed that the soluble EM-3B fusion protein expressed could react specifically with sera from animals infected with FMDV.Then,with the fusion protein EM-3B as a diagnostic antigen,an ELISA to differentiate FMDV-infected animals from vaccinated animals(EM-3B-DIVA-ELISA) was established.Results of specificity and sensitivity evaluations confirmed that the specificity and sensitivity of EM-3B-DIVA-ELISA were 95.0% and 97.5%,respectively.The coincidence rates of the EM-3B-DIVA-ELISA with the two commercial ELISA kits were 96.25% and 98.75% respectively.