口蹄疫病毒OH株结构蛋白基因VP1的克隆与表达

采用RT-PCR方法扩增获得了O型口蹄疫病毒的主要免疫原VP1基因,将其插入pMD18-T载体进行序列分析,结果表明,所获得的基因片段含有完整的FMDV结构蛋白VP1编码区。根据表达载体pQE-Trisystem的克隆位点序列和该VPl基因片段的末端序列设计了1对表达引物,以重组pMD-T-VP1阳性质粒为模板,扩增获得了VP1基因,通过酶切将其克隆至表达载体pQE-Trisystem上。经测序证实,重组表达质粒所含的外源基因VP1编码框正确无误。将重组表达质粒pQE-VP1转化至大肠埃希氏菌M15,通过IPTG诱导促使VP1基因高效表达,SDS- PAGE和Western-blot分析表明,表达产物大小与预期的结果(26 ku)一致,且具有良好的反应原性。以2 mmol／L IPTG诱导表达5 h时表达量最高,其中70％-80％的目的蛋白存在于菌体裂解后的上清中,表明外源基因VP1主要以可溶性方式表达。

Cloning and expression of VP1 gene of foot-and-mouth disease virus

The FMDV VP1 gene was amplified by RT-PCR, and then cloned into the vector pMD18-T. The sequencing result showed that the amplified fragment was the whole encoding region of the VP1 gene of FMDV. According to the characteristics of pQE-Trisystem and its restriction sites the appropriate expression primers were designed and the VP1 gene was ligated to the expression vector pQE-Trisystem. The sequence analysis showed that the VP1 gene was cloned to the expression vector successfully. The VP1 gene was expressed in E. coli M15 and induced by IPTG. SDS-PAGE and Western-blot analyses showed that the expressed VP1 protein had good antigenicity. When it was induced for 5 h with 2 mmol/L of IPTG, the VP1 gene was expressed efficiently and 70%- 80% of the protein was in suspension, indicating that the expressed VP1 protein was soluble.