鳗弧菌W-1金属蛋白酶基因真核表达载体的构建及其在动物细胞中的表达  

Construction of Eukaryotic Expression Vector for the Mutated empA(m-empA7) Gene from Vibrio anguillarum W-1 and Its Expression in Animal Cells

作　　者：杨慧[1] 陈吉祥[2] 张晓华[2] 李筠[2] 薛小平[1] 

机构地区：[1]西北工业大学生命科学院,陕西西安710072 [2]中国海洋大学海洋生命学院,山东青岛266003

出　　处：《化学与生物工程》2010年第2期42-45,66,共5页Chemistry & Bioengineering

基　　金：国家自然科学基金资助项目(30371108;30972275);国家863计划资助项目(2007AA09Z416);国家973计划资助项目(2006CB101803)

摘　　要：采用基因克隆方法,以带有突变的鳗弧菌W-1金属蛋白酶基因的原核表达质粒pET24d(+)/m-empA7为模板,通过PCR扩增得到m-empA7基因,将其插入pCDNA3.1(+)构建重组真核表达质粒pCDNA3.1(+)/m-empA7;然后用磷酸钙法将其转染入动物细胞CHO和HEK293T中并进行瞬时表达,分别以RT-PCR和Western-blot检测重组质粒的基因转录和蛋白表达情况。结果表明,成功将突变的鳗弧菌W-1金属蛋白酶基因转染至真核细胞中,并能够在动物细胞中正确地转录和表达,为鳗弧菌基因工程疫苗的研制奠定了基础。The mutated empA （m-empA7） gene was amplified by PCR with the prokaryotic expression plasmid pET24d （＋）/m-empA7 as a template, and cloned into the eukaryotic expression plasmid pCDNA3.1（＋） by gene cloning methods, resulting the recombinant plasmid pCDNA3, 1（＋）/m-empAT. It was then transfected into CHO and HEK293T cells by calcium-phosphate transfection method. The transcription of pCDNA3.1 （＋）/m-empA7 was assayed by reverse transcription polymerase chain reaction （RT-PCR）. The expression product of pCDNA3.1 （＋）/m-empA7 was analyzed by Western-blot. It proved that the eukaryotic expression plasmid pCDNA3, l（＋）/m-empA7 was successfully constructed and it could be transcribed and expressed in mammalian cells. The results indicated that the expression product of the recombinant plasmid had high antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidate in the development of anti-vibriosis and paved the way for further study.

关 键 词：鳗弧菌W-1 金属蛋白酶 真核表达 基因疫苗 

分 类 号：Q78[生物学—分子生物学]