猪圆环病毒2型ORF3基因的克隆及原核表达

以细胞总基因组提取试剂盒抽提PCV2细胞毒株总DNA为模板,用特异性引物扩增PCV2 ORF3基因。将ORF3和表达载体pGEX-6p-1用BamH I和Sal I双酶切回收后连接,将测序验证为阳性的重组质粒命名为pGEX-6p-1-ORF3。重组质粒pGEX-6p-1-ORF3转化BL21(DE3)后用IPTG诱导表达并纯化,对获得的表达产物进行SDS-PAGE电泳、Western blot检测。结果表明:PCV2 ORF3在pGEX-6p-1中获得了高效表达,其表达蛋白主要以包涵体形式存在,其分子质量约为37 kD,且能够和PCV2阳性血清发生特异性相互作用,具有免疫学活性。

Clone and Prokaryotic Expression of ORF3 Gene in Porcine Circovirus Type 2

Total DNA of porcine circovirus type 2(PCV2) was extracted by the total cellular genome extraction kit,and a special primer was designed to amplify PCV2 ORF3 gene.Then,ORF3 and expression vector pGEX-6p-1 were digested by BamH I and Sal I,and then were ligated together.The recombinant plasmid which sequencing test was positive was named as pGEX-6p-1-ORF3,and it was transformed into BL21(DE3),then the fusion proteins were expressed when it was induced by 1 mmol/ L of IPTG and purified.The obtained expression products were tested by SDS-PAGE and Western blot.The results showed that the PCV2 ORF3 was expressed in pGEX-6p-1,which mainly existed with the form of inclusion body;the ORF3 protein was 37 kD in molecular mass and it can generate specificity interaction with the positive serum of PCV2.Therefore,the ORF3 protein have a good immunology activity.