人幽门螺杆菌外膜蛋白18000DNA疫苗的构建及表达  

DNA vaccine construction and expression of encoding gene of 18 000 outer membrane protein of human Helicobacter pylori

作　　者：姜政[1] 余剑平[2] 王新渝[3] 黄爱龙[4] 王丕龙[1] 

机构地区：[1]重庆医科大学附属第一医院,重庆400016 [2]自贡市第三人民医院,自贡643000 [3]重庆医科大学应用技术学院,重庆400016 [4]重庆医科大学肝炎研究所,重庆400016

出　　处：《中国免疫学杂志》2005年第2期134-138,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金No 3 0 3 713 18;重庆市科委 [2 0 0 2 ] 18 86项目资助

摘　　要：目的 :构建含人幽门螺杆菌 (Helicobacterpylori,H .pylori) 180 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 2 8中 ,酶切H .pylori180 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 2 8,并在COS-7细胞中表达 ,以RT-PCR ,Western法检测其表达产物。结果 :经单、双酶切证实插入的基因片段为H .pylori 180 0 0外膜蛋白编码基因 ;采用RT PCR方法 ,能够从转染的COS-7细胞中扩增出一条与目的基因大小一致的DNA片段 ,Western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 ,而且具有生物活性。结论 :成功地构建了核酸疫苗pcDNA3 1 5 2 8,并在COS-7细胞中表达 ,为H .Objective:To construct a recombinant eukaryotic expression vector containing gene encoding 18 000 outer membrane protein from human Helicobacter pylori(H.pylori) and be expressed in COS-7 cell,and lay the foundation for the exploiting DNA vaccine of Hp.Methods:The target genes encoding 18 000 outer membrane protein were acquired from the prokaryotic expression vector pET32a(+)/528 by XholⅠ,KpnⅠ digestion simultaneously,in the same way,the pcDNA3.1 was digested by XhoⅠ,KpnⅠ digestion simultaneously,and the objective genes and pcDNA3.1 were extracted out of agarose electrophoresis with gel kit,and connected by T4 ligase.The recombinant vector pcDNA3.1/528 was used to select and transform,meanwhile express in COS-7 cell.The expressions of recombinant eukaryotic vector in COS-7 cell were investigated by reverse transcriptive-polymerase chain reaction and Western.Results:The gene had inserted into eukaryotic vector was the target gene encoding 18 000 outer membrane protein by enzyme digestion analysis,and objective gene was amplified from COS-7 cell transfected with pcDNA3.1/528 by RT-PCR,the Western showed that recombinant eukaryotic vector could be expressed in COS-7 cell.Conclusion:The recombinant eukaryotic vector pcDNA3.1/528 was constructed and expressed in COS-7 cell successfully.The results obtained lay the foundation for research on development of H.pylori DNA vaccine.

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：R392.7[医药卫生—免疫学]