| 猪圆环病毒2型ORF2基因的原核表达及反应原性分析 |
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| 引用本文： | 欧云文,马小元,王俊,丁耀忠,张永光,张杰.猪圆环病毒2型ORF2基因的原核表达及反应原性分析[J].动物医学进展,2017,38(4). |
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| 作者姓名： | 欧云文 马小元 王俊 丁耀忠 张永光 张杰 |
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| 作者单位： | 1. 甘肃农业大学动物医学院,甘肃兰州 730070;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;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 |
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| 基金项目： | 国家国际合作项目，国家自然科学基金项目 |
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| 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。
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| 关 键 词： | 猪圆环病毒2型 ORF2基因 原核表达 反应原性 |
Prokaryotic Expression of ORF2 Gene Fragment of Porcine Circovirus Type 2 and the Reactinogenicity Analysis of the Expressed Protein |
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| OU Yun-wen,MA Xiao-yuan,WANG Jun,DING Yao-zhong,ZHANG Yong-guang,ZHANG Jie.Prokaryotic Expression of ORF2 Gene Fragment of Porcine Circovirus Type 2 and the Reactinogenicity Analysis of the Expressed Protein[J].Progress In Veterinary Medicine,2017,38(4). |
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| Authors: | OU Yun-wen MA Xiao-yuan WANG Jun DING Yao-zhong ZHANG Yong-guang ZHANG Jie |
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| Abstract: | 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. |
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| Keywords: | Porcine circovirus type 2 ORF2 gene prokaryotic expression reactinogenicity |
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