肠出血性大肠杆菌O157∶H7 TccP基因克隆、表达及其抗原性鉴定  

Prokaryotic expression of Tir-cytoskeleton coupling proteinfrom EHEC O157∶H7 and identification of its antigenicity

作　　者：杨琴[1,2] 顾江[2] 毛旭虎[2] 邹全明[2] 宋方洲[1] 丁红雷[2] 王庆旭[2] 

机构地区：[1]重庆医科大学基础医学院生物化学与分子生物学教研室,重庆400016 [2]第三军医大学医学检验系临床微生物学及免疫教研室

出　　处：《中国人兽共患病学报》2007年第4期319-322,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助项目(30670107);军队杰出人才基金资助项目(04J009);重庆市自然科学基金资助项目(CSTC2006BB5115)

摘　　要：目的克隆表达肠出血性大肠杆菌(EHEC)O157∶H7 Tir细胞骨架偶联蛋白(TccP)基因,并研究其抗原性。方法采用PCR法自EHEC O157∶H7 Sakai菌株基因组扩增TccP基因,构建TccP原核表达载体并在大肠杆菌中诱导表达。表达产物经初步纯化后,免疫新西兰白兔,制备兔抗TccP多克隆抗体。抗体效价及特异性用ELISA法和Western blotting法进行测定和分析。结果从EHEC O157∶H7 Sakai菌株中克隆出了1014bp的TccP基因。构建的重组质粒pET28a-TccP在大肠杆菌中获得了高效表达;以TccP免疫家兔获得了高效价多克隆抗体,Western blotting分析此抗体能与TccP发生特异性抗原抗体反应。结论在大肠杆菌中成功克隆表达了TccP基因,所获TccP具有良好的抗原性,为进一步研究TccP在EHEC O157∶H7致病机制中的作用奠定基础。To express the gene of TccP of EHEC O157 ： H7 in E. coli and prepare the rabbit antibody against TccP. the gene of Z3072 DNA was amplified from EHEC O157 ： H7 by PCR. After confirmed by DNA sequencing,the Z3072-DNA was cloned into expression vector pET-28a（＋） and then the Z3072 gene was expressd in E. coli under the IPTG induction. The protein was prmarily purified with the purity of about 80%. The rabbit antibody against TccP was prepared by immunization of two New Zealand white rabbits using the purified TccP as immunogen. The titer and specificity of the antisera were determined by ELISA and Western blot assay. It was demonstrated that the 1 014bp Z3072 gene was amplified,cloned, and expressed in E. coll. The expressed product existed in the bacterial inclusion body. The rabbit antibody against TccP was prepared and its titer was about 1 ： 640 000. Western blot analysis showed that the antibody cold bind to the expressed TccP protein specifically. In this way,the TccP protein was expressed in E. coli and rabbit against TccP was prepared successfully,thus providing the necessary materials for further study.

关 键 词：肠出血性大肠杆菌O157:H7 Tir-细胞骨架偶联蛋白 基因表达 多克隆抗体 

分 类 号：R378.21[医药卫生—病原生物学]