江西农业学报2017，Vol.29Issue(1)：96-101,6.DOI:10.19386/j.cnki.jxnyxb.2017.01.19
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
摘要
Abstract
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 Iand 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 identifi-cation 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 electrophoresis.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-column and then was examined by Western-blot.The results showed that the ex-pressed recombinant protein could react specifically with the polyclonal antibody against PCV 2, 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Key words
Porcine circovirus type 2/Gene Cap/Cloning/Prokaryotic expression分类
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杨克礼,孟丽,时代,段正赢,田永祥,周丹娜,郭锐,刘泽文,袁芳艳,刘威..猪圆环病毒2型 Cap 基因的克隆与原核表达[J].江西农业学报,2017,29(1):96-101,6.基金项目
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（2014BKB 063）；动物胚胎及分子育种湖北省重点实验室开放课题（2016ZD156）。 （）
