艰难梭菌毒素a羧基端基因的克隆与表达.docVIP

艰难梭菌毒素A羧基端基因的克隆与表达 作者：杨晓强，王亚东，孙勇，陈学清，姜泊 【关键词】 艰难梭菌；毒素A；受体结合区；克隆,分子；基因表达 　　Gene cloning and high expression of clostridium difficile toxin A Cterminal repeated unit 　　　【Abstract】 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 Cterminal repeated gene was amplified by PCR and cloned into the prokaryotic expression vector pET22b(+), and the recombined plasmid pETCDTAR 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 pETCDTAR was induced with IPTG and analyzed with SDSPAGE. 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 A receptor gene lay a foundation for the further study on CDTAR function and clostridium difficile vaccine. 　 　　【Keywords】 clostridium difficile; toxin A; receptor binding zone; cloning, molecular; gene expression 　　【摘要】 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pET22b(+)，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(SDSPAGE)对表达产物进行分析. 结果： 构建了含CDTAR基因的重组质粒pETCDTAR, IPTG诱导后SDSPAGE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. 　　【关键词】 艰难梭菌；毒素A；受体结合区；克隆，分子；基因表达 　　0引言 　　艰难梭菌（clostridium difficile, CD）是一种G+厌氧芽孢杆菌，是医院内肠道感染的主要致病菌之一，临床上约50%的抗生素相关性腹泻和近100%的伪膜性肠炎与此菌有关［1-2］. 万古霉素及甲硝唑是治疗该菌的主要药物. 但近年来国内外均已发现了多重耐药的菌株［3-4］；疫苗研究已经成为防治CD相关性疾病的关键. CD产生的毒素A与毒素B是其主要致病因素,毒素B通常是在毒素A作用后引起肠壁细胞损伤，而艰难梭菌毒素A羧基末端受体结合区 (clostridium difficile toxin A receptor binding zone, CDTAR)则是毒素A与肠壁细胞结合的关键蛋白区域［5］. 研究表明，针对毒素A和B的抗体可以对CD的感染有保护作用. 因此，我们拟克隆CDTAR基因，并将其在原核菌中进行表达验证，为进一步研制CD疫苗奠定