口蹄疫病毒非结构蛋白基因的克隆表达及免疫活性的研究

Cloning and Expression of Nonstructural Protein Gene of Food-and-mouth disease virus and Its Antigenic Activity

从口蹄疫病毒(FMDV)细胞培养物中提取总RNA,设计简并引物通过RT-PCR获得了完整的3ABC基因片段,将3AB基因和部分3ABC基因分别克隆到原核表达载体上构建重组表达质粒pET-3AB和pET-3ABC,然后转化BL21(DE3)plysS进行诱导表达,将表达产物进行SDS-PAGE分析和Western blot鉴定。结果表明,3AB基因和3ABC基因可以在大肠埃希菌中高效表达,且表达产物能够与FMDV阳性血清产生免疫反应。进一步摸索重组蛋白的纯化条件,制备纯化蛋白,以纯化的表达产物为包被抗原进行间接ELISA检测,结果表明,重组蛋白具有特异的免疫学活性,能够用于鉴别FMDV感染动物血清和免疫动物血清。

The 3ABC gene of Food-and-mouth disease virus was obtained from the FMDV genome RNA by RT-PCR using incorporate primers.Then the special amplified fragment 3AB gene and partial 3ABC gene were cloned into prokaryotic expression vector and constructed the recombinant plasmid pET-3AB and pET-3ABC.Subsequently,these recombinant plasmids were transformed into BL21（DE3） plysS and the target proteins were induced by IPTG.Expression of nonstructural protein was examined and identified by SDS-PAGE and Western blotting.The results showed that recombinant proteins of 3AB and 3ABC were expressed in E.coli successfully and they could react with FMDV infected animal serum.At last,the recombined proteins were further purified and an indirect ELISA method based on the purified recombinant proteins indicated that recombinant proteins of 3AB and 3ABC could differentiate sera of infected animals from vaccinated animals.