幽门螺杆菌毒素相关基因的克隆、测序及表达  

Cloning, sequencing and expression of the gene of Helicobacter pylori cagA

作　　者：李姁君[1] 韩锋产[1] 闫小君[1] 苏成芝[1] 

机构地区：[1]第四军医大学全军基因诊断技术研究所,陕西西安710033

出　　处：《第四军医大学学报》2002年第5期429-432,共4页Journal of the Fourth Military Medical University

摘　　要：目的　分段扩增、克隆幽门螺杆菌毒素相关基因cag A中间区 cag1,cag2 ,并利用大肠杆菌系统表达 .方法　PCR法扩增 cag A基因 ,双脱氧中止法测序正确后 ,克隆入大肠杆菌表达载体 p BV2 2 0 ,受控于 PRPL 启动子 ,转化大肠杆菌 DH5α,4 2℃进行温度诱导 .结果　 PCR分段扩增出 cag A中间区基因 cag1,cag2 ,分别为 975 ,75 5 bp,克隆入 p UC19质粒 ,测序正确 ;在 p BV中构建 cag1,cag2的重组表达载体p BVcag A1,p BVcag A2 ,工程菌诱导后 SDS- PAGE显示新生表达蛋白带 ,Mr:Cag136× 10 3,Cag2 2 7.9× 10 3,均与预期的 cag A蛋白 Mr一致 ,约占菌体总蛋白的 36 %和 2 4 % .结论　成功克隆分段扩增的 cag A中间区基因 ,并可在大肠杆菌DH5α中高效表达 .AIM To amplify and clone the middle region of Helicobacter pylori cag A gene, and express the proteins in E.coli . METHODS After the cag 1 and cag 2 were amplified by PCR and sequencd by dideoxy methods, they were inserted into expression vector pBV220 in which exogenous genewas controlled by P RP L promoters. The recombinant plasmids pBV cag 1 and pBV cag 2 were transformed into E.coli DH5α and induced at 42℃ respectively to express the encoded proteins. RESULTS The middle region of cag A was amplified and 975, 755 bp products were got as cag 1, cag 2, which were cloned into pUC19 and sequenced correctly. Then, their recombination expression clones pBV cag A1 and pBV cag A2 were constructed. When their engineered bactera were induced, the anticipated M r 36×10 3 and 27.9×10 3 proteins band appeared on SDS PAGE gel and amounted to 36% and 24% of total bacterial protein respectively. CONCLUSION The H.pylori cag A middle region might be successfully cloned and efficiently expressed fractionally in E.coli .

关 键 词：毒素相关基因 基因克隆 基因表达 温度诱导 幽门螺杆菌毒素 

分 类 号：R378.99[医药卫生—病原生物学] Q78[医药卫生—基础医学]