施马伦贝格病毒N蛋白的原核表达及其单克隆抗体的制备  

Prokaryotic expression of Schmallenberg virus N protein and preparation of its monoclonal antibody

作　　者：孔玉方 陈冬杰 魏方 王慧煜[1] 吴绍强[1] KONG Yu-fang;CHEN Dong-jie;WEI Fang;WANG Hui-yu;WU Shao-qiang(Institute of Animal Inspection and Quarantine,Chinese Academy of Inspection and Quarantine,Beijing 100176,China)

机构地区：[1]中国检验检疫科学研究院动物检验与检疫研究所,北京100176

出　　处：《中国兽医科学》2021年第8期1029-1034,共6页Chinese Veterinary Science

基　　金：“十三五”国家重点研发计划项目(2017YFD0501803);中国检验检疫科学研究院科研项目(2018JK013)。

摘　　要：为了制备针对施马伦贝格病毒(SBV)N蛋白的单克隆抗体,将SBV N基因克隆至原核表达载体pET-28a,构建重组质粒pET28a-SBV-N,转化至BL21(DE3)后进行原核表达并纯化。以纯化的重组SBV-N蛋白免疫BALB/c小鼠制备单克隆抗体(McAbs),最终获得了3株McAbs,依次命名为2D6、4F10和4G8。Ig亚类鉴定结果显示:2D6为Ig G2b亚型,轻链为κ型;4F10为Ig M亚型,轻链为κ型;4G8为Ig M亚型,轻链为λ型。Western-blot结果显示,这3株McAbs均可与重组SBV-N和赤羽病病毒(AKAV)N蛋白产生特异性反应,进一步表明这些McAbs具有通用性。IFA结果表明,McAb 2D6有望用于SBV与AKAV的血清学鉴别诊断。SBV N蛋白和单克隆抗体的成功制备,为施马伦贝格病血清学诊断提供了生物原料。To prepare the monoclonal antibodies against N protein of Schmallenberg virus(SBV),we cloned the SBV N gene into the prokaryotic expression vector p ET-28 a and constructed the recombinant plasmid p ET28 a-SBV-N.Then the plasmid was transformed into Escherichia coli BL21(DE3) for further prokaryotic expression and purification.The purified recombinant SBV-N protein was used to immunize BALB/c mice to prepare the monoclonal antibodies(Mc Abs).After screening,three Mc Abs were obtained and named as 2 D6,4 F10 and 4 G8,respectively.The antibody subtype assay indicated that 2 D6,4 F10 and 4 G8 was Ig G2 b,Ig M and Ig M subtype,respectively,and the light chain was κ type,κ type,and λ type,respectively.Western-blot results showed that the three Mc Abs could react with both recombinant SBV-N and Akabane virus(AKAV) N protein,which further indicates that the three Mc Abs are versatile.IFA results indicate that Mc Ab 2 D6 is expected to be used in the serological differential diagnosis of SBV and AKAV.The successful preparation of SBV N protein and monoclonal antibodies provides biological materials for the serological diagnosis of Schmallenberg disease.

关 键 词：施马伦贝格病毒 N蛋白 表达 单克隆抗体 

分 类 号：S852.659.5[农业科学—基础兽医学]