O型口蹄疫病毒结构蛋白VP1基因的克隆与原核表达

Cloning and Prokaryotic Expression of VP1 Gene of Foot-and-mouth disease virus Serotype O in E.coli

根据已经公布的O型口蹄疫病毒VP1基因序列,设计合成了1对VP1基因特异性引物,应用RT-PCR技术从O型口蹄疫病毒标准毒株扩增得到VP1基因,并将其克隆到原核表达载体pET-28a中,构建了重组原核表达质粒pET-28a-VP1,对重组表达质粒鉴定正确后,转化大肠埃希菌BL21进行诱导表达,SDS-PAGE和Western blotting检测结果表明,O型口蹄疫病毒VP1基因在大肠埃希菌BL21中得到了正常表达,所表达的融和蛋白与标准O型口蹄疫病毒阳性血清具有特异性抗原/抗体反应,说明该融和蛋白具有免疫学活性。

According to the published sequence of VP1 gene of foot-and-mouth disease virus serotype O, a pair of primers were designed and synthesized. The VP1 gene was amplified by RT-PCR method from FM- DV serotype O, and cloned into pET-28a vector. The prokaryotic expression plasmid containing VP1 gene was successfully constructed. The VP1 gene was sequenced and compared with the published sequence of VP1 gene of FMDV serotype O in the GenBank. The expression of recombinant plasmid pET-28a-VP1 in E. coli BL21 was induced and detected by SDS-PAGE and Western-blot analysis. The results showed that the structural protein VP1 gene of FMDV can express successfully in E. coli BL21. The fusion protein which was expressed in E. coli BL21 can be recognized by the positive serum of cattle that was infected by FMDV.