产毒性大肠杆菌不耐热肠毒素B亚单位基因克隆及表达  

CLONGING AND EXPRESSION OF LT B SUBUNIT OF EXTEROTOXINGENIC E COLI

作　　者：王万胜[1,2] 方利君[1,2] 王顺林[1,2] 蒋惠秋[1,2] 李宏年[1,2] 顾健人[1,2] 

机构地区：[1]上海医科大学儿科医院分子生物学实验室 [2]上海市肿瘤研究所癌基因及相关基因国家重点实验室

出　　处：《上海医科大学学报》1998年第1期31-34,共4页Journal of Fudan University(Medical Science)

基　　金：国家自然科学基金

摘　　要：目的研究克隆并表达产毒性大肠杆菌不耐热肠毒素Ｂ亚单位。方法合成２条引物用ＰＣＲ法扩增包括产毒性大肠杆菌不耐热肠毒素（ＥＴＥＣ，ＬＴ）Ｂ亚单位基因编码区及起始密码子上游７９３ｂｐ的ＤＮＡ片段。ＰＣＲ产物克隆入ｐＢｌｕｅＳｃｒｉｐｔＳＫ（－）并转化进入ＸＬ－Ｂｌｕｅ工程菌中。结果经ＥＬＩＳＡ，Ｗｅｓｔｅｒｎ－ｂｌｏｔ测定共获得２２株菌表达ＬＴ－Ｂ，表达水平比Ｈ１０４０７高２～３倍。克隆的基因经测序证实有一个碱基置换，但无氨基酸序列改变。结论本工作成功表达了产毒性大肠杆菌不耐热肠毒素Ｂ亚单位。URPOSE To clone and express the LT B subunit of enterotoxingenic E coli.METHODS A pair of synthetic primers, flanking the 793bp upstream of the LT-B coding area and the stop site of the transcription, were used to amplify LT B gene of enterotoxingenic E coli by PCR. The PCR products were cloned into pBluescript SK(一) and transformed into XL-blue.RESULTS By means of ELISA, Western-blotting, 22 strains were identified to produce LT B subunit, 2～3 times higher than H10407. The sequence of cloned genes showed single base substitution without changes of amino acid sequence.CONCLUSIONS We succeeded in the expression of B subunit of heat-labile enterotoxin of enterotoxingenic escherichia coli.

关 键 词：大肠杆菌 肠毒素 聚合酶链反应 基因工程 ETEC 

分 类 号：R378.21[医药卫生—病原生物学] R394.8[医药卫生—基础医学]