幽门螺杆菌GGT基因的克隆及其在大肠杆菌中的表达  

Cloning of Helicobacter pylori γ-glutamyl transpeptidase gene and its expression in E.coli

作　　者：张尤历[1] 唐炜[1] 王文兵[2] 陈慧娟[1] 周武松[3] 

机构地区：[1]江苏大学附属医院,江苏省镇江市212001 [2]江苏大学生命科学研究院,江苏省镇江市212013 [3]安徽农业大学生命科学研究院,安徽省合肥市230036

出　　处：《世界华人消化杂志》2009年第17期1768-1771,共4页World Chinese Journal of Digestology

基　　金：江苏省社会发展基金资助项目;No.BS2006041~~

摘　　要：目的:克隆幽门螺杆菌(Hpylori)γ-谷氨酰转肽酶(γ-glutamyl transpeptidase,GGT)基因,实现GGT基因在大肠杆菌中的表达.方法:从胃癌患者胃黏膜组织中分离培养获得Hpylori,提取其基因组DNA,对GGT基因进行PCR扩增,克隆进pMD18-T载体,酶切和测序验证,构建原核表达载体pET-28a(+)-GGT,转化大肠杆菌BL21,经IPTG诱导表达重组融合蛋白,SDS-PAGE及Western blot分析检测表达产物.结果:成功克隆了GGT基因,经酶切和测序验证正确,成功构建了pET-28a(+)-GGT质粒,高效表达出了68kDa的融合蛋白.结论:在大肠杆菌中成功表达了GGT重组融合蛋白,为进一步研究GGT与线粒体介导的细胞凋亡之间的关系奠定了基础.AIM： To clone and express H pylori γ-glutamyl transpeptidase in Escherchia coil METHODS： The strains of H pylori were isolated from human gastric mucosa with gastric cancer. The GGT amplified by PCR from H pylori DNA was cloned into apMD18-T vectors. The recombinant plasmids were confirmed by enzyme digestion and were sequenced. The prokaryotic expression vector pET-28a （＋）-GGT was constructed and GGT gene was expressed in E.coli BL21 strain. The fusion protein was produced by IPTG induction and analyzed by SDS-PAGE and Western blot. RESULTS： The GGT gene was obtained and its sequence was proved to be correct. The prokaryotic expression vector pET-28a （＋）-GGT was constructed. The fusion protein with relative molecule mass of 68 kDa was highly expressed. CONCLUSION： The GGT is successfully expressed in E.coli, which is of importance to research of the relationship between the GGT and mitochondria-mediated apoptosis.

关 键 词：幽门螺杆菌 Γ-谷氨酰转肽酶 克隆 原核表达 

分 类 号：R346[医药卫生—基础医学]