布鲁氏菌磷酸葡萄糖变位酶基因的原核表达与鉴定  

Expression and Identification of Phosphoglucomutase Gene of Brucella

作　　者：张豫[1] 张辉[1] 张艳[1] 陈瑞花[1] 王震[1] 桂丹[1] 孙志华[1] 陈创夫[1] 

机构地区：[1]石河子大学动物科技学院/新疆地方病与民族高发病教育部重点实验室,石河子832003

出　　处：《石河子大学学报（自然科学版）》2013年第1期35-38,共4页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(31001046;30960288)

摘　　要：对布鲁氏菌磷酸葡萄糖变位酶(pgm)蛋白进行了表达与纯化,为研究该基因的功能提供了物质基础。首先利用PCR方法从羊种布鲁氏菌基因组DNA中PCR法扩增pgm基因,并将该基因亚克隆至pET-28a(+)载体中,转化入大肠杆菌(DE3),诱导表达融合蛋白;表达产物经Ni-NTA Agarose亲和层析纯化后,进行SDS-PAGE和Western blot分析。结果显示:扩增了pgm的全基因,成功构建了布鲁氏菌pgm基因的原核表达载体pET-28a(+)-pgm,并且在大肠杆菌中进行了表达,经Western blot鉴定,该蛋白可以与布鲁氏菌免疫血清产生特异性结合反应,且具有良好的免疫反应性。本研究为进一步研究布鲁氏菌pgm蛋白的生物学功能提供基础物质,也为进一步开发基因工程疫苗提供理论基础。To express pgm fusion protein in E.coli BL21 and purify and identify the expressed product,so as to provided the material basis for the study of the function of the gene.The full-length pgm gene was synthesized by PCR and cloned into vector pET-28a（＋）.The constructed recombinant plasmid pET-pgm was transformed to competent E.coli BL21 expression under induction of IPTG.The expressed protein was purified with Ni-NTA Agarose,identified by Western blot,and the full-length pgm gene was sequenced.Both restriction analysis and sequencing proved that recombinant plasmid pET-pgm was constructed correctly.The SDS-PAGE and western blot analysis indicated that the recombinant protein induced by IPTG was about 64 ku.The protein can produce specific binding reaction,and has a good immune response with Brucella sera.

关 键 词：布鲁氏菌 pgm基因 原核表达 

分 类 号：S852.5[农业科学—基础兽医学]