| 猪伪狂犬病病毒流行株gE蛋白表达及其多克隆抗体制备 |
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| 引用本文： | 曲哲会,赵瑜,张喜文,连慧香,赵云焕,陈斌,郭晓秋.猪伪狂犬病病毒流行株gE蛋白表达及其多克隆抗体制备[J].中国畜牧兽医,2021,48(2):631-639. |
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| 作者姓名： | 曲哲会 赵瑜 张喜文 连慧香 赵云焕 陈斌 郭晓秋 |
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| 作者单位： | 1. 信阳农林学院牧医工程学院, 信阳 464000;2. 河南省大别山区生态畜禽健康生产工程研究中心, 信阳 464000;3. 信阳市畜禽养殖与环境控制重点实验室, 信阳 464000 |
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| 基金项目： | 河南省科技攻关项目(172102110184);信阳农林学院校级科技创新团队(CXTD-201801)。 |
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| 摘 要： | 本研究旨在获得重组伪狂犬病病毒（PRV）gE蛋白及其多克隆抗体。利用PCR方法从PRV感染猪的肺脏、脑和扁桃体混合组织中扩增PRV gE基因，连接至克隆载体pMD18-T（pMD-gE）后进行测序与进化树分析。以pMD-gE为模板，利用PCR方法扩增其膜外结构域部分基因（gE-outside），将其连接至原核表达载体pET-30a（+），转化大肠杆菌DH5α感受态细胞，构建重组质粒pET-gE-outside。将重组质粒pET-gE-outside转化大肠杆菌RosettaTM（DE3）pLysS感受态细胞，经IPTG诱导后，通过SDS-PAGE和Western blotting进行表达产物的分析与鉴定。经亲和层析技术纯化重组PRV gE蛋白并免疫小鼠，通过间接ELISA和Western blotting分别进行三免3周血清中鼠抗PRV gE多克隆抗体效价的测定和鉴定。PCR和测序结果表明，本研究成功克隆了PRV gE基因，与国内2011年以后流行毒株属于相同分支。SDS-PAGE和Western blotting结果证实，PRV gE-outside基因在原核表达系统获得正确表达，分子质量约为55 ku，且可与猪抗PRV多克隆抗体发生免疫反应。经亲和层析纯化的gE-outside蛋白浓度为1.23 mg/mL。将其免疫小鼠，三免3周的小鼠血清中鼠抗PRV gE-outside多克隆抗体效价为1：204 800，并可与gE-outside蛋白发生免疫反应。综上，本研究制备了重组PRV gE蛋白和鼠抗PRV gE蛋白多克隆抗体，可为PRV感染机制研究及建立快速、高效免疫学检测技术提供技术指导和材料。
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| 关 键 词： | 伪狂犬病病毒(PRV) gE蛋白 原核表达 多克隆抗体 |
| 收稿时间： | 2020-08-10 |
Expression of Pseudorabies Virus gE Protein and Preparation of Its Polyclonal Antibody |
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| QU Zhehui,ZHAO Yu,ZHANG Xiwen,LIAN Huixiang,ZHAO Yunhuan,CHEN Bin,GUO Xiaoqiu.Expression of Pseudorabies Virus gE Protein and Preparation of Its Polyclonal Antibody[J].China Animal Husbandry & Veterinary Medicine,2021,48(2):631-639. |
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| Authors: | QU Zhehui ZHAO Yu ZHANG Xiwen LIAN Huixiang ZHAO Yunhuan CHEN Bin GUO Xiaoqiu |
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| Institution: | 1. College of Animal Science and Veterinary Medicine, Xinyang Agriculture and Forestry University, Xinyang 464000, China;2. Engineering Research Center for Health Production of Ecological Livestock and Poultry of Henan Dabieshan District, Xinyang 464000, China;3. Xinyang Key Laboratory of Animal Husbandry and Environmental Control, Xinyang 464000, China |
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| Abstract: | The purpose of this study was to obtain recombinant PRV gE protein and polyclonal antibody against PRV gE protein.The PRV gE gene was amplified from PRV-infected pig tissues(lung,brain and tonsil)by PCR,and then ligated to the cloning vector pMD18-T(pMD-gE)for sequencing and phylogenetic tree analysis.Using pMD-gE as a template,part of the gene encoding gE protein extramembrane domain(gE-outside)was amplified by PCR,then connected to the prokaryotic expression vector pET-30a(+),transformed into E.coli DH5αcompetent cell to construct recombinant plasmid pET-gE-outside.The recombinant plasmid pET-gE-outside was transformed into the E.coli Rosetta TM(DE3)pLysS competent cell.After induction by IPTG,the expressed product was analyzed and identified by SDS-PAGE and Western blotting,respectively.The recombinant PRV gE protein was purified by affinity chromatography and used to immunize mice.The titers measure and identification of the mouse anti-PRV gE polyclonal antibody in the serum from the immunized mice after the third immunization for 2 weeks were detected by indirect ELISA and Western blotting,respectively.The PCR and sequencing results showed that the PRV gE gene was successfully cloned in this study,which belonged to the same branch as the domestic virus strain after 2011.The SDS-PAGE and Western blotting results confirmed that the PRV gE-outside protein was correctly expressed by the prokaryotic expression system,with a molecular weight of about 55 ku,and could react with porcine anti-PRV polyclonal antibody.The concentration of gE-outside protein purified by affinity chromatography was 1.23 mg/mL.3 weeks after immunizing mice with gE-outside protein,the mouse anti-PRV gE-outside polyclonal antibody was prepared with a titer of 1∶204800,and had an immune reaction with gE-outside protein.In this study,recombinant PRV gE protein and mouse anti-PRV gE polyclonal antibody were prepared,which could provide technical guidance and material for the study on the mechanism of PRV infection and the establishment of rapid and efficient immunological detection technology. |
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| Keywords: | Pseudorabies virus(PRV) gE protein prokaryotic expression polyclonal antibody |
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