猪MAVS基因原核表达载体的构建及其多克隆抗体的制备  

Antigenic specificity ofswine MAVS by prokaryotic expression and its preparation of polyclonal antibody

作　　者：赵倩楠[1,2] 应雪[1,2] 李晓泉[1,2] 张珂[1,2] 李晓宁[1,2] 梁晶晶[1,2] 罗廷荣[1,2] ZHAO Qian-nan YING Xue LI Xiao-quan ZHANG Ke LI Xiao-ning LIANG Jing-jing LUO Ting-rong(Laboratory of Animal Infectious Diseases, College of Animal Science and Technology, Guangxi University, Nanning 530004, China State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, China)

机构地区：[1]广西大学动物科学技术学院动物传染病研究室,广西南宁530004 [2]广西大学亚热带农业生物资源保护利用国家重点实验室,广西南宁530004

出　　处：《中国兽医杂志》2017年第4期39-42,共4页Chinese Journal of Veterinary Medicine

基　　金：广西自然科学基金重点项目(2012GXNSFDA053009);国家自然科学基金项目(31460653)

摘　　要：通过RT-PCR从PK-15细胞系中扩增克隆MAVS(mitochondrial antiviral signaling protein)基因,构建原核表达载体p ET-MAVS220,转化感受态细胞Rosetta(DE3),利用IPTG诱导表达,重组MAVS经纯化后免疫4周龄昆明系小鼠制备抗线粒体抗病病毒信号蛋白(MAVS)多克隆抗体。诱导表达的最佳条件为IPTG 0.05 mmol/L,37℃诱导6 h,重组MAVS以可溶性蛋白和包涵体两种形式表达。应用该重组蛋白免疫小鼠获得的抗MAVS多克隆抗体与纯化的重组MAVS蛋白反应效价可达1∶16 000;该抗体与Poly(I∶C)刺激PK-15细胞产生的MAVS及与转染了重组MAVS基因真核表达载体pcDNA3.0-MAVS的BHK-21细胞表达的MAVS蛋白发生特异性反应,效价可达1∶1 000,特异性良好。The MAVS gene was amplified from PK-15 cells by RT-PCR and was then subcloned into the prokaryotic expression vector to construct pET-MAVS220. The pET-MAVS220 was transfected into an E. coli Rosetta（DE3） to express the recombinant MAVS via IPTG induction. The recombinant MAVS was purified and used to immunize the four-week Kunming mice to prepare anti-MAVS polyclonal antibody. The constructed recombinant vector pET-MAVS220 was transformed into E. coli Rosetta（DE3） cells and the MAVS was expressed by induction of 0.05 mM IPTG at 37 ℃ for 6 h. The recombinant MAVS was identified in two forms of soluble protein and/or inclusion body. The purified MAVS was used to immunize the four-week Kunming mice to prepare a polyclonal anti- body. The reactivity of the anti-MAVS polyclonal antibody with prokaryotic expressed MAVS was identified to reach a high dilution of 1 ： 16000, and reach a dilution of 1：1000 with MAVS expressed in eukaryotic PK-15 cells by poly（I：C） and in BHK-21 cells transfected with the eukaryotic vector pcDNA3.0-MAVS. The anti-swine MAVS polyclonal antibody showed a high reactivity and specificity.

关 键 词：猪MAVS 原核表达 多克隆抗体 

分 类 号：S858.28[农业科学—临床兽医学]