牛结节性皮肤病病毒p32蛋白膜外区可溶性表达及多克隆抗体制备  

Soluble Expression of Extracellular Domain of Lumpy Skin Disease Virus p32 Protein and Preparation of Polyclonal Antibodies

作　　者：黄敏霞 翟颀 吕殿红 温肖会 翟少伦 罗胜军 周秀蓉 贾春玲 魏文康 HUANG Min-xia;ZHAI Qi;Lü Dian-hong;WEN Xiao-hui;ZHAI Shao-lun;LUO Sheng-jun;ZHOU Xiu-rong;JIA Chun-ling;WEI Wen-kang(College of Animal Science&Technology,Zhongkai University of Agriculture and Engineering,Guangzhou,Guangdong,510225,China;Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Key Laboratory of Livestock Disease Prevention of Guangdong Province,Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province,Ministry of Agriculture and Rural Affairs,Guangzhou,Guangdong,510640,China;Agro-biological Gene Research Center,Guangdong Academy of Agricultural Sciences,Guangzhou,Guangdong,510640,China)

机构地区：[1]仲恺农业工程学院动物科技学院,广东广州510225 [2]广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站,广东广州510640 [3]广东省农业科学院农业生物基因研究中心,广东广州510640

出　　处：《动物医学进展》2022年第12期20-25,共6页Progress In Veterinary Medicine

基　　金：广州市基础与应用基础研究项目(202201011182);广东省现代农业产业技术体系创新团队建设专项资金项目(2022KJ119,2022KJ114);广东省农业科学院“十四五”学科团队项目(202122TD)。

摘　　要：为获得牛结节性皮肤病病毒(LSDV)p32可溶性蛋白并制备其多克隆抗体,截取了LSDV的P 32基因膜外区序列,并构建了重组表达质粒pColdⅠ-P32,在大肠埃希氏菌BL21(DE3)pLysS细胞中经低温诱导表达,然后用钴离子亲和层析纯化以可溶性形式表达的重组蛋白,使用纯化的p32截短蛋白免疫昆明小鼠制备多克隆抗体,并通过Western blot鉴定。结果表明,成功使用钴离子亲和层析纯化获得LSDV p32可溶性膜外区蛋白,制备的多克隆抗体可以特异性识别LSDV感染的牛组织蛋白。获得的纯度较高的LSDV p32截短蛋白和特异性良好的多克隆抗体,为建立LSDV抗原检测方法及亚单位疫苗的研制奠定了基础。To obtain the soluble protein of lumpy skin disease virus p32 and prepare polyclonal antibodies,this study intercepted the extracellular domain sequence of LSDV P 32 gene and constructed the recombinant expression plasmid pCold I-P32.Then the recombinant plasmid was induced to express in E.coli BL21(DE3)pLysS at low temperature condition.The recombinant protein expressed in soluble form was purified by cobalt ion affinity chromatography,and the polyclonal antibodies were prepared by immunizing Kunming mice with the p32 purified truncated protein,then the specificity was identified by Western blot.The results showed that the LSDV p32 soluble outer membrane region protein was successfully purified by cobalt affinity chromatography.Western blot showed that the polyclonal antibodies could recognize LSDV-infected bovine tissue proteins specifically.The obtained truncated protein of LSDV p32 with high purity and polyclonal antibodies with good specificity laid the foundation for the establishment of LSDV antibody/antigen detection methods and the development of subunit vaccine.

关 键 词：牛结节性皮肤病病毒 p32蛋白 可溶性表达 多克隆抗体 

分 类 号：S852.659.1[农业科学—基础兽医学] S858.23[农业科学—兽医学]