创伤弧菌溶血素基因的高效表达与鉴定

Efficient expression and identification of Vibrio vulnificus cytolysin genes

目的:构建创伤弧菌溶血素基因(vvc)的融合表达载体,并实现创伤弧菌溶血素在大肠杆菌中的高效表达.方法:用一对创伤弧菌溶血素基因特异性引物从创伤弧菌基因组DNA中钓取vvc基因,T-A克隆后测定核苷酸序列,构建pET-32 a(+)-vvc融合表达载体,转化大肠杆菌BL21(DE3),IPTG诱导表达,表达产物行SDS-PAGE并进行W est-ern B lot鉴定.结果:构建了pET-32 a(+)-vvc融合表达载体,DNA测序证明,获得的vvc基因长度为1311 bp,与Gen-Bank中报道的创伤弧菌溶血素基因序列完全一致.SDS-PAGE分析表明,vvc融合蛋白Mr为71×103,其表达量约占菌体总蛋白的32%.Western blot结果显示,目的蛋白可与创伤弧菌免疫后的小鼠血清特异性结合.结论:成功地实现了VVC基因在大肠杆菌中的高效表达,为进一步研究vvc蛋白的生物学功能奠定基础.

AIM: To construct the expression vector of Vibrio vulnificus cytolysin(vvc) genes,and to study its efficient expression in the E.coli.METHODS: The vvc genes from Vibrio vulnificus genome were obtained by PCR,cloned and then sequenced.PET-32a(+)vvc prokaryotic expression vector was constructed and then transformed into E.coli BL21(DE3).After induction by 1.0 mmol/L IPTG,its expression was analyzed by SDS-PAGE and Western-Blot.RESULTS: The nucleotide sequences of the cloned vvc gene matched with the NCBI Genebank report.SDS-PAGE analysis suggested that the VVC protein was efficiently expressed,the molecular mass of fusion protein was 71×103,accounting for about 32% of total protein in VVC.The Western-Blot results suggested that the prokaryotic expression of VVC fusion protein particularly combined with its anti-serum.CONCLUSION: vvc genes are efficiently expressed and identified in E.coli,which lays foundation for the further study on biological function of VVC protein.