猪圆环病毒2型Cap基因的克隆与原核表达  

Cloning and Prokaryotic Expression of Gene Cap in Porcine Circovirus Type 2

作　　者：杨克礼[1] 时代 段正赢[1] 田永祥[1] 周丹娜[1] 郭锐[1] 刘泽文[1] 袁芳艳[1] 刘威[1] 孟丽[1] 

机构地区：[1]湖北省农业科学院畜牧兽医研究所动物胚胎工程与分子育种湖北省重点实验室,湖北武汉430064 [2]河南省信阳市浉河区畜牧局,河南信阳464000

出　　处：《江西农业学报》2017年第1期96-101,共6页Acta Agriculturae Jiangxi

基　　金：科技部重点专项课题(2016YFD0500703);湖北省农业科技创新中心项目(2016-620-000-001-026);湖北省科技支撑计划(公益性科技研究类)项目(2014BKB063);动物胚胎及分子育种湖北省重点实验室开放课题(2016ZD156)

摘　　要：利用PCV2鄂州株的基因序列设计特异性引物,分别在上、下游引物中加入Sac I和Hind III酶切位点,扩增出PCV2鄂州株的ORF2基因;将该片段克隆至p ET-28a原核表达载体,经PCR、酶切鉴定,成功构建了阳性重组表达质粒p ET-ORF2。将其转入大肠杆菌Rosetta中,经IPTG诱导,SDS-PAGE电泳分析确定重组蛋白大小为34.5 k D,主要以包涵体的形式表达。优化后确定最佳诱导条件为:37℃,至OD值0.4~0.6时加入终浓度为1 mmol/L的IPTG,诱导5 h。表达产物经His-tag镍柱纯化后进行Western-blot分析,结果表明表达的重组蛋白能够与PCV2标准阳性血清发生特异性反应,具有良好的反应原性。According to the complete genome of Porcine Circovirus Type 2 （PCV2） strain HBEZ, a pair of specific primers were designed to amplify ORF2 gene in HBEZ, and Sac I and Hind III were used in the primers. ORF2 gene fragment was cloned to the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-ORF2. PCR and restrict enzyme identification confirmed that the recombinant plasmid was constructed successfully. Then pET-ORF2 was transformed into E. coli strain Rosetta. After being induced by IPTG, the recombinant protein with the molecular weight of 34.5 kD could express mainly in the form of inclusion body in SDS-PAGE eleetrophoresis. The best inducement conditions were acquired as follows ： bacterial culture at 37 ℃, adding 1 mmol/L IPTG when the OD-value was between 0.4 and 0.6, and inducement by IPTG for 5 h. The expression product Cap protein was purified by His-tag Ni-eolumn and then was examined by Western-blot. The results showed that the expressed recombinant protein could react specifically with the polyclonal antibody against PCV2, and it possessed a good reactoge- nicity.

关 键 词：猪圆环病毒2型 Cap基因 克隆 原核表达 

分 类 号：S852.659.5[农业科学—基础兽医学]