鸭坦布苏病毒NS1蛋白的原核表达与多克隆抗体制备  

Prokaryotic expression of duck Tembusu virus NS1 protein and preparation of its polyclonal antibody

作　　者：张喜文 鲁绍芳 李盼盼 冉梦媛 李卓燕 焦凤超[1,3] 董建国 黄立[1] 赵聘[1,3] 曲哲会[1,2,3] 时代 ZHANG Xiwen;LU Shaofang;LI Panpan;RAN Mengyuan;LI Zhuoyan;JIAO Fengchao;DONG Jianguo;HUANG Li;ZHAO Pin;QU Zhehui;SHI Dai(College of Animal Science and Veterinary Medicine,Xinyang Agriculture and Forestry University,Xinyang 464000,China;Xinyang Key Laboratory of Integrated Technology for Prevention and Control of Major Livestock and Poultry Diseases,Xinyang 464000,China;Engineering and Technology Research Center for Waterfowl Resources Development and Utilization and Epidemic Disease Prevention and Control of Henan Province,Xinyang Agriculture and Forestry University,Xinyang 464000,China;Bureau of Agriculture and Rural Affairs of Shihe District,Xinyang 464000,China)

机构地区：[1]信阳农林学院动物科技学院,河南信阳464000 [2]信阳市畜禽重大疫病防控综合技术研究重点实验室,河南信阳464000 [3]河南省水禽资源开发利用与疫病防控工程技术研究中心,河南信阳464000 [4]河南省信阳市浉河区农业农村局,河南信阳464000

出　　处：《养殖与饲料》2023年第4期19-25,共7页Animals Breeding and Feed

基　　金：河南省科技攻关项目(222102110188);信阳农林学院高水平科研孵化器建设项目(FCL202004),信阳农林学院科技创新团队建设项目(XNKJTD-014),信阳农林学院青年科研基金项目(20200113),信阳农林学院青年科研基金项目(QN2021012);信阳市创新应用专项(20200016)。

摘　　要：[目的]利用原核表达系统制备鸭坦布苏病毒(duck Tembusu virus,DTMUV)NS1蛋白及其多克隆抗体。[方法]利用PCR与核苷酸序列测定方法克隆DTMUV NS1基因,连接至重组表达载体p ET-30a(+),构建重组质粒p ET-NS1,转化至大肠杆菌Rosetta(DE3)感受态细胞,SDS-PAGE和Western blot进行IPTG诱导表达产物分析与鉴定,从IPTG的浓度和诱导时间进行表达条件优化,并进行表达产物可溶性分析,重组DTMUV NS1蛋白经亲和层析纯化后免疫小鼠,制备鼠抗DTMUV NS1蛋白多克隆抗体,并通过间接免疫荧光试验(IFA)鉴定。[结果]成功克隆DTMUV NS1基因,约1065 bp,获得重组质粒p ET-NS1,转化至大肠杆菌Rosetta(DE3)后,可见分子质量约43 ku的特异蛋白,可以与鸡抗DTMUV多克隆抗体发生免疫反应,终浓度为0.8 mmol/L的IPTG诱导2~5 h为DTMUV NS1蛋白最佳表达条件,呈包涵体形式表达,经亲和层析纯化后,成功获得分子质量约43ku的NS1蛋白,蛋白质量浓度为329μg/m L,鼠抗DTMUV NS1蛋白多克隆抗体可以与DTMUV发生特异性免疫反应。[结论]本研究利用原核表达系统表达和纯化DTMUV NS1蛋白,并制备鼠抗DTMUV NS1蛋白多克隆抗体,可为DTMUV致病机制研究和免疫学检测方法建立提供物质材料与技术指导。[Objectives]This study aimed to prepare duck Tembusu virus(DTMUV)NS1 protein and its polyclonal antibody by prokaryotic expression system.[Methods]The DTMUV gene was cloned by PCR and nucleotide sequencing,and linked to the recombinant expression vector pET-30a(+),recombinant plasmid pET-NS1 was constructed,and then transformed into Rosetta(DE3)competent cells.The induced expression products by IPTG were analyzed and identified by SDS-PAGE and Western blotting,respectively.Expression conditions were optimized by IPTG concentration and induction time,and solubility of expression products was conducted.Mice were immunized with recombinant DTMUV NS1 protein,which was purified by affinity chromatography,to prepare polyclonal antibodies against DTMUV NS1 protein and identified by indirect immunofluorescence assay.[Results]The results showed that DTMUV NS1 gene was successfully cloned with 1065 bp and the recombinant plasmid pET-NS1 was obtained.After bing transformed into Rosetta(DE3)competent cells,the specific protein with molecular weight of about 43 ku was observed,which could react with chicken anti-DTUMV polyclonal antibody.The optimal expression condition of DTMUV NS1 protein was induced by IPTG with the final concentration of 0.8 mmol/L for 2-5 h,and expressed in the form of inclusion body.After affinity chromatography purification,the purified NS1 protein with a molecular weight of about 43 ku was obtained with a protein concentration of 329μg/mL.The mouse anti-DTMUV NS1 protein polyclonal antibody was obtained,which could specifically react with DTMUV.[Conclusions]In summary,the DTMUV NS1 protein was expressed and purified by the prokaryotic expression system,and the mouse anti-DTMUV NS1 protein polyclonal antibody was prepared,which could provide technical guidance and material basis for the study on the pathogenic mechanism of DTMUV infection and the establishment of immunological detection technology.

关 键 词：鸭坦布苏病毒 NS1蛋白 原核表达系统 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]