猪圆环病毒2型ORF2基因的原核表达及反应原性分析  

Prokaryotic Expression of ORF2 Gene Fragment of Porcine Circovirus Type 2 and the Reactinogenicity Analysis of the Expressed Protein

作　　者：欧云文 马小元[2] 王俊[2] 丁耀忠[2] 张永光[2] 张杰[2] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室,甘肃兰州730046

出　　处：《动物医学进展》2017年第4期1-6,共6页Progress In Veterinary Medicine

基　　金：国家国际合作项目(2012DFG31890);国家自然科学基金项目(31072143)

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PCV2CAU0673毒株的DNA为模板,扩增获得702bp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1mmol/L IPTG诱导表达6h;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。SDSPAGE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34ku;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.

关 键 词：猪圆环病毒2型 ORF2基因 原核表达 反应原性 

分 类 号：S852.659.2[农业科学—基础兽医学]