口蹄疫病毒非结构蛋白3AB基因的可溶性表达与生物活性分析

通过设计特异性引物扩增得到完整的口蹄疫病毒（FMDV）非结构蛋白（NSP）3AB基因，定向克隆到表达载体pET-30a中，获得了重组质粒pET-3AB，并转化大肠埃希氏菌BL21(DE3)，重组菌分别在37℃和28℃条件下诱导表达，表达产物经SDS-PAGE电泳鉴定，观察到37℃培养条件下目的蛋白形成包涵体，在28℃条件下培养目的蛋白以可溶性的形式表达。Western blotting分析表明，表达的目的蛋白能与FMDV感染牛血清发生特异性反应。以纯化的3AB蛋白作为抗原，采用间接ELISA模式检测了部分FMDV感染动物血清和健康动物血清，证明表达蛋白与FMDV感染血清有很好的反应而与健康动物血清无反应。该项研究为建立以3AB为抗原的FMDV NSP抗体检测ELISA方法奠定了基础。

Expression of Nonstructural Protein Gene 3AB of Foot-and-Mouth Disease virus and Analysis of its Bioactivity

The full length of 3AB gene of foot-and-mouth disease virus (FMDV) was amplified and subcloned into pET-30a vector by two unique restriction sites. The recombinant plasmid pET-3AB was transformed into a BL21 (DE3) strain of E.coli. The recombinants were induced with IPTG at 37℃and 28℃ for expression of target protein. The product was analyzed by SDS-PAGE. The results showed that the expressed protein was solubly expressed when induced at 28℃ and formed inclusion body when induced at 37℃. Both products could reacted well with sera derived from FMDV infected cattle by western blotting. The expressed product was further purified and used as the antigen in an indirect ELISA assay for detection of different animal sera. The result showed that the partially purified 3AB protein could only react with sera derived from FMDV infected animals. This preliminary result indicated that the expressed 3AB could be used as antigen in ELISA for discrimination FMDV naturally infected animals from vaccinated animals.