幽门螺杆菌Mr26000外膜蛋白真核载体的构建及表达蛋白的鉴定  

Construction of the eukaryotic expression vector for the gene encoding the 26 kDa outer membrane protein of Helicobacter pylori and identification of the expressed proteins

作　　者：姜政[1] 汪志群[1] 王新渝[1] 黄爱龙[1] 王丕龙[1] 

机构地区：[1]重庆医科大学第一附属医院,重庆400016

出　　处：《中国人兽共患病杂志》2004年第7期596-600,共5页Chinese Journal of Zoonoses

基　　金：渝科发〔2 0 0 2〕18号 86;渝教科〔2 0 0 2〕18号文 6;国家自然科学基金〔NO 3 0 3 713 18〕资助

摘　　要：目的　构建含人幽门螺杆菌 (Helicobacterpylori,H pylori) 2 6 0 0 0外膜蛋白编码基因的真核重组载体 ,并在COS- 7细胞中表达 ,为核酸疫苗的开发奠定基础。方法　从原核表达质粒 pET32a(+) / 5 94中 ,酶切H pylori 2 6 0 0 0外膜蛋白编码基因片段 ,将目的基因与同样进行酶切、纯化的载体pcDNA3 1进行连接 ,而后转化并筛选含有目的基因的重组载体pcDNA3 1/ 5 94 ,并在COS - 7细胞中表达 ,以RT -PCR ,Dotblot法检测其表达产物。 结果　经酶切证实插入的基因片段为H pylori 2 6 0 0 0外膜蛋白编码基因 ;采用RT -PCR方法 ,能够从转染COS - 7细胞中扩增出一条与目的基因大小一致的DNA片段 ;同时Dotblot法等检测显示 ,该重组质粒能够在COS - 7细胞中表达目的蛋白。结论　成功地构建了真核重组载体 pcDNA3 1/ 5 94 ,并在COS - 7细胞中表达 ,为H pylori核酸疫苗的研制奠定了良好的基础。To construct a prokaryotic expression vector for the gene encoding the 26 kDa outer membrane protein of Helicobacter pylori to express in COS-7 cells so as to lay the foundation for the development of the DNA vaccine for H.pylori,the target gene encoding the 26 kDa outer membrane protein was obtained from the prokaryotic expression vector pET32a(+)/594 by digestion with Xho Ⅰ and Kpn Ⅰ simultaneously,and in the same way,the pcDNA3.1 was digested by Xho Ⅰ and Kpn Ⅰ simultaneously.Then,the objective genes and pcDNA3.1 were extracted by agarose electrophoresis with gel kid,and connected with T4 ligase at 4∶1 molar rate.The recombinant vector pcDNA3.1/594 containing the objective genes was transformed and screened to COS-7 cells,and the expression of the recombinant eukaryotic vector in COS-7 cells was investigated by using RT-PCR and dot blotting.It was found that the inserted gene fragment was the gene encoding the 26 kDa outer membrane protein of H.pylori as demonstrated by the enzyme digestion analysis,and a DNA fragment with the same size of the objective gene could be amplified from the transfected COS-7 cells by RT-PCR.This recombinant plasmid could express objective proteins in COS-7 cells as demonstrated by the dot blotting.It concludes that the recombinant eukaryotic vector pcDNA3.1/594 has been constructed and expressed in COS-7 cells successfully,and these results obtained may lay the foundation for researches on the development of DNA vaccine for H.pylori.

关 键 词：幽门螺杆菌 外膜蛋白 斑点印迹 真核表达载体 

分 类 号：R387.2[医药卫生—医学寄生虫学]