猪圆环病毒2型ORF2基因的原核表达及反应原性分析

摘要

研究猪圆环病毒2型(PCV2)Cap蛋白的抗原性,为开发PCV2的检测方法奠定基础.以PCV2 CAU0673毒株的DNA为模板,扩增获得702 bp的目的片段,扩增产物克隆入pET30a(+)原核表达载体,构建pET30a-PCV2-ORF2重组质粒,转入大肠埃希菌BL21(DE3);在37 ℃以1 mmol/L IPTG诱导表达6 h;采用Ni-NTA树脂亲和层析纯化重组蛋白,并用不同浓度的尿素对纯化蛋白进行复性.SDS-PAGE分析表明,该ORF2编码基因在大肠埃希菌中得到表达,蛋白大小约为34 ku;Western blot检测结果表明,该重组Cap蛋白与PCV2阳性血清发生特异性反应,与NA-PRRSV和PPV1血清不发生交叉反应.成功构建了PCV2-ORF2原核表达载体,实现了在大肠埃希菌中的表达,纯化后的复性蛋白具有较好的反应原性,为猪圆环病毒2型检测方法建立或试剂盒的开发奠定了基础.%This experiment was aimed to study the antigenicity of Cap protein of porcine circovirus type 2(PCV2).The gene was amplified from the DNA of PCV2-CAU0673 strain by PCR,whose product was approximately 702 bp.The product was cloned into pET30a(+)vector,PCR,digestion and sequencing were used to identify positive plasmid.The positive recombined plasmid was expressed by E.coli BL21(DE3)and was induced by IPTG.The recombinant protein was purified by Ni-NTA and refolding.The results of SDS-PAGE showed that the recombined protein was successfully expressed in E.coli BL21(DE3)with a relative molecular weight of 34 ku.The results of Western blot showed that this recombined protein was specifically reacted with PCV2 positive serum;no cross reacted with NA-PRRSV and PPV1 positive serum.The recombinant vector pET30a-PCV2-ORF2 was successfully constructed,and the recombined Cap protein with excellent reactinogenicity was successfully expressed in E.coli which laid a foundation for the diagnosis of PCV2.