幽门螺杆菌glmM DNA重组质粒的构建及体外表达  

Construction, expression in vitro of DNA recombinant plasmid of pVAX1-glmM of Helicobacter pylori

作　　者：吴小茜[1] 佘菲菲[1] 丁惠[1] 张静[1] 陈月秀[1] 

机构地区：[1]福建医科大学分子医学微生物学实验室,福州350004

出　　处：《中国人兽共患病学报》2006年第7期617-620,共4页Chinese Journal of Zoonoses

基　　金：福建省自然科学基金重大项目(2001F003)

摘　　要：目的构建幽门螺杆菌(Hp)pVAX1-glmMDNA重组质粒,体外转染SGC-7901细胞并鉴定其表达蛋白的抗原性。方法RT-PCR方法从国际标准株NCTC11637中获取glmM全长基因,克隆插入T载体。测序后经酶切、连接反应将glmM的开放读码框架定向克隆入真核表达载体pVAX1,酶切初步鉴定后测序确认。通过脂质体法将pVAX1-glmMDNA重组质粒转染SGC-7901细胞,检测其转染效率,确定转染成功后RT-PCR法检测glmM在转录水平的表达,ELISA法鉴定表达蛋白的抗原性。结果DNA测序结果显示扩增出的glmM全长基因与GenBank公布的HpglmM序列有96%的同源性。PCR、酶切和测序结果证实glmM目的基因成功克隆入真核表达载体pVAX1。重组质粒转染的细胞经RT-PCR扩增获得1.4kb片段,与目的基因大小相符。ELISA结果显示重组质粒转染细胞的超声破碎物及培养上清液中抗原的滴度比对照组高2倍以上。结论成功构建了pVAX1-glmMDNA重组质粒,其表达蛋白具有良好的抗原性,为进一步研究其抗Hp感染的效果及制备相应有效DNA疫苗奠定基础。To construct Helicobacter pylorio （Hp）DNA recombinant plasmid pVAX1-glmM and to identify the antigenicity of the proteins expressed, total length gene of glmM was amplified with RT-PCR from the RNA of standard Hp strain NCTCl1637 and was cloned into T vector. After sequencing, the ORFs of Hp glmM was cloned into the eukaryotic expression vector pVAX1 through enzyme digestion and ligation reactions. The recombinant plasmid was firstly identified by enzyme digestion and then subjected to sequencing. Recombinant was transfected into SGC-7901 using Lipofectin 2000, and the effect of transfection was detected to determine the success of transfection. At the level of transcription in cells, the expression of glmM had been detected by RT-PCR. ELISA reaction was used to identify the antigenicity of the proteins expressed. The DNA se- quence analysis showed that total length gene of glmM （amplified with RT-PCR） had 960% homology compared with the fragment published by GenBank. The results of enzyme digestion and sequencing displayed that glmM gene was＇inserted into the eukaryotic expression vector pVAX1. A fragment （1.4kb） amplified with RT-PCR from the cell which transfected by pVAX1- glmM was agreed with the size of destine gene. To either ultrasound quassation or cultivated supernatants of cell which transleered by recombinant plasmid, the result of ELISA showed that the titre of antigen was two times higher than control group. It is concluded the recombinant plasmid pVAX1-glmM had been constructed successfully, and the proteins expressed had good antigeniclty. It may be helpful for further investigantion about its protection against the infection of Hp ,and for the preparation of DNA vaccine.

关 键 词：幽门螺杆菌 glmM重组质粒 表达 

分 类 号：R378[医药卫生—病原生物学]