小反刍兽疫病毒N基因的原核表达  

The Prokaryotic Expression of N Gene from Peste Des Petits Ruminants Virus

作　　者：阮洋[1,2] 秦峻岭[2,3] 高玉伟[2] 孙玮[2,3] 张涛[2,1] 杨玉姣[2,3] 杨松涛[2] 王承宇[2] 王铁成[2] 黄耕[2] 夏咸柱[1,2,3] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130062 [3]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《动物医学进展》2011年第3期21-25,共5页Progress In Veterinary Medicine

基　　金：公益性行业科研专项项目(200903037)

摘　　要：目的是表达出小反刍兽疫病毒的核蛋白,并鉴定其活性。根据GenBank发表的小反刍兽疫病毒(PPRV)Nigeria 75/1株N基因序列,对其进行基因优化并合成。设计引物,利用PCR的方法扩增PPRV-N基因,将该基因片段定向克隆到原核表达载体pET-28a(+)中,构建原核表达载体pET-28a-N。阳性质粒转化原核表达宿主菌Transetta(DE3),用IPTG诱导表达并确定表达N基因的最佳诱导时间、温度。通过SDS-PAGE电泳和Western blot分析确定表达蛋白的表达量和活性。扩增出PPRV-N基因,构建的原核表达载体pET-28a-N和原核表达菌,表达了N蛋白,表达蛋白能与PPRV阳性血清发生特异性反应。To acquire the N protein of peste des petits ruminants virus（PPRV） by expressing N gene in prokaryotic system,the complete length of N gene from peste des petits ruminants virus was amplified by PCR using a pair of specific primers designed according to the sequences in GenBank,and the PCR products were cloned into prokaryotic expression vector pET28a（＋） to obtain the prokaryotically expressed plasmid pET-28a-N.The target gene was then expressed in E.coli Transetta（DE3） cells inducted with IPTG,and the expression was optimized with a proper induction time and temperature.the good immunological activity to PPRV antibodies was proved by SDS-PAGE and Western blot analysis.The protein was proved to be soluble,and the target protein was proved to be immunological activity

关 键 词：小反刍兽疫病毒N基因 原核表达 SDS-PAGE电泳 WESTERN-BLOT 

分 类 号：Q386[生物学—遗传学]