艰难梭菌毒素A羧基端基因的克隆与表达

目的:克隆并表达艰难梭菌毒素A羧基末端受体结合区 (CDTAR)基因.方法:PCR扩增CDTAR基因并将其克隆到表达载体pET-22b( ),重组质粒转化到E.coli BL21(DE3),经IPTG诱导表达,聚丙烯酰胺凝胶电泳(SDS-PAGE)对表达产物进行分析.结果:构建了含CDTAR基因的重组质粒pET-CDTAR,IPTG诱导后SDS-PAGE显示表达出Mr约为35.7 ku的重组蛋白,占菌体总蛋白的36.1%,可溶性表达占上清的22.2%,包涵体中约占24.9%.结论:成功克隆了CDTAR基因,并构建表达了CDTAR重组蛋白,为进一步研究CDTAR功能及研制艰难梭菌疫苗奠定了基础.

Gene cloning and high expression of clostridium difficile toxin A C-terminal repeated unit

AIM: To obtain the high expression of the gene coding for clostridium difficile toxin A receptor binding zone(CDTAR). METHODS: The clostridium difficile toxin A C-terminal repeated gene was amplified by PCR and cloned into the prokaryotic expression vector pET-22b(+),and the recombined plasmid pET-CDTAR was transformed into E.coli strain BL21(DE3).The recombined vector was confirmed by digestion with EcoRI/XhoI and sequencing.The E.coli strain BL21(DE3) containing pET-CDTAR was induced with IPTG and analyzed with SDS-PAGE.RESULTS: A 35.7 ku protein was acquired after inducing with IPTG and thin layer scanning suggested that CDTAR occupied 36.1% of the total bacterial protein,22.2% of the supernatant and 24.9% of the inclusion body.CONCLUSION: The cloning and high expression of clostridium difficile toxin Areceptor gene lay a foundation for the further study on CDTAR function and clostridium difficile vaccine.