艰难梭菌毒素基因3’末端重复区域基因片段的PCR克隆  

PCR cloning of 3'-terminal repeat fragments of Clostridium difficile toxin A and toxin B gene

作　　者：杨晓强[1] 姜泊[1] 孙勇[1] 王继德[1] 

机构地区：[1]第一军医大学南方医院全军消化内科研究所,广东广州510515

出　　处：《第一军医大学学报》2002年第9期791-793,共3页Journal of First Military Medical University

基　　金：广州市科技计划项目(026G2434)

摘　　要：目的探寻一种简单、快速、灵敏的检测艰难梭菌的方法。方法 以艰难梭菌毒素A和毒素B基因3’末端高度保守重复区分别设计一对引物,进行PCR反应,同时在同一体系相同反应条件下使用相同引物分别以大肠杆菌和乳杆菌DNA为模板进行扩增,比较二者结果。结果从艰难梭菌成功克隆了毒素A基因3’端重复区域960bp的基因片段及毒素B基因3’端重复区域1851bp的基因片段,不同来源的菌株出现了相同的2条电泳带,并通过测序鉴定;而对照的大肠杆菌和乳杆菌株无特异电泳带出现。结论从艰难梭菌毒素人毒素B基因3’末端重复区域克隆的基因片段具有保守性,可以作为基因探针在临床上进行艰难梭菌检测,该方法简便、灵敏,可直接用粪便标本进行检测。此外,这些基因编码的多肽具有较高的疏水性井存在着膜受体结合区域,可以此为基础进行基因工程蛋白质疫苗的研究。Objective To explore a method to rapidly detect the presence of Clostridium difficile. Methods PCR was used to amplify DNA fragments from the highly conserved and repeated domains of the 3'-terminal of Clostridium difficile toxin A gene and toxin B genes. Results The fragments 960 bp and 1 851 bp in length were respectively amplified from toxin A and toxin B, while the control bacteria presented no distinct results. Conclusion The 960 bp and 1 851 bp fragments are specific for Clostridium difficile and PCR can be used to detect Clostridium difficile from the stool. As the peptides encoded by these conserved domains have high hydrophobicity and strong antigenicity, manufacture of the protein vaccine by gene engineering is possible on the basis of these conserved domains.

关 键 词：克隆 艰难梭菌 细菌毒素 基因克隆 聚酶链反应 厌氧芽孢杆菌 

分 类 号：R346[医药卫生—基础医学]