尤氏泰勒虫OPRT基因的优化表达  

Optimized expression of OPRT gene from Theileria uilenbergi

作　　者：李倩[1] 李有全[1] 李亚琼[1] 陈泽[1] 杨吉飞[1] 刘军龙[1] 关贵全[1] 罗建勋[1] 殷宏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2015年第4期419-422,共4页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31272556);PiroVAC(KBBE-3-245145);国家农业公益性行业科研专项(201303035)

摘　　要：为克隆和表达尤氏泰勒虫OPRT基因的功能区片段,并对重组OPRT蛋白的反应原性进行分析,以反转录得到的尤氏泰勒虫cDNA链为模板,PCR扩增出OPRT基因片段,然后将其克隆至pET-30a(+)载体;把构建成功的重组质粒转化至感受态大肠杆菌BL21进行优化表达,对诱导表达的蛋白纯化后用Western-blot分析它与吕氏泰勒虫和绵羊泰勒虫阳性血清是否发生交叉反应。结果显示,OPRT基因获得了不可溶性表达,最佳表达时间为6h,最佳表达温度为28℃,且表达的重组OPRT蛋白分别与上述2种泰勒虫阳性血清发生交叉反应。结果表明,OPRT同源基因也存在于吕氏泰勒虫和绵羊泰勒虫之中。To clone and express the functional domain fragment of orotate phosphoribosyl transferase(OPRT)gene of Theileria uilenbergi and to analyze the reactionogenicity of the recombinant protein,the functional domain fragment of OPRT gene was amplified and cloned into pET-30a(+)vector,then transformed into Escherichia coli BL21.To detect whether there were cross reactions with positive sera against Theileria luwenshuni and Theileria ovis,the expressed protein was purified and analyzed by Western-blot.In result,the purified OPRT protein was insoluble,the optimized expression time was 6h,the optimized expression temperature was at 28℃,and there was cross reaction with the positive sera against T.luwenshuni or T.ovis,respectively.The result showed that the homologous OPRT gene also exists in the two parasites.

关 键 词：尤氏泰勒虫 乳清酸磷酸核糖转移酶基因 优化表达 免疫印迹 

分 类 号：S852.7[农业科学—基础兽医学]