猪圆环病毒2型Cap基因原核表达载体的构建及表达

Construction and expression of prokaryotic expression vector of porcine circovirus type 2 Cap gene

根据GenBank上已发表的PCV2的衣壳蛋白(Cap蛋白)基因设计1对引物,利用PCR方法从已知PCV2病毒中扩增到l条DNA片段,将PCR产物克隆至pMD18-T载体上获得稳定质粒,酶切及测序鉴定后,获得大小为578 bp的Cap基因序列,将该基因插入表达载体pET-30a质粒中,获得重组质粒pET-30a-PCV2-Cap,并转化到大肠杆菌BL21(DE3)中。重组质粒pET-30a-PCV2-Cap经IPTG诱导后表达的重组蛋白分子量为28.0kD,经破碎后SDS-PAGE电泳分析表明该蛋白具有可溶性。Western blot分析表明,该重组蛋白与PCV2阳性血清发生特异性反应,表明该重组蛋白具有良好的反应原性。

A pair of primers were designed according to the capsid protein gene of porcine circovirus type 2（PCV2） published in the Genbank,and a DNA fragment from the known PCV2 was amplified by PCR. The PCR product was cloned onto pMD-18T vector to obtain a stable plasmid. The restriction analysis and,sequencing indicated that a 578 bp Cap gene sequence was obtained. The gene was inserted into the expression vector pET-30a,and the gained recombinant plasmid pET-30a-PCV2- Cap was transformed into E. coli BL21 （DE3）. The molecular weight of the recombinant protein expressed by pET-3Oa-PCV2-Cap was 28.0 kD after IPTG induction. The SDS-PAGE showed that the expressed protein was soluble after fragmentation. The Western blot analysis showed that the recombinant protein reacted specifically with PCV2 positive serum, implying that the recombinant protein possessed good reactionogenicity.