伪狂犬病病毒gE基因的表达及gE蛋白单克隆抗体的制备  

Expression of gE gene of pseudorabies virus and preparation of monoclonal antibody against gE protein

作　　者：王淑杰[1] 蔡雪辉[1] 刘永刚[1] 吴国军[1] 刘狄萩 张琦[1] 马平[1] 李成君[1] 石文达[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点猪病实验室,黑龙江哈尔滨150001

出　　处：《中国兽医科学》2007年第8期679-683,共5页Chinese Veterinary Science

基　　金：国家科技攻关计划项目(2005BA711A10);"十一五"国家科技支撑项目(2006BAD06A18)

摘　　要：以猪伪狂犬病病毒(PRV)Min-A株pMD-gE质粒为模板,利用所合成的特异性引物进行PCR扩增,获得了1 000 bp的DNA片段,并将其克隆至表达载体pET-30a中,转化到大肠杆菌中进行了原核表达,对表达产物进行了抗原性分析;在此基础上,用纯化的gE蛋白免疫BALB/c小鼠,筛选和鉴定了分泌抗gE蛋白单抗的杂交瘤细胞株。结果显示,原核表达的gE蛋白具有良好的抗原性,其分子质量为46 ku。筛选获得3株分泌抗gE蛋白单抗的杂交瘤细胞株,Western-blot和间接免疫荧光试验证实,所获得的3株单抗具有良好的特异免疫反应原性。3株单抗均为IgG1亚类,且轻链均为κ链。The gE gene of 1 000 bp was amplified with pMD-gE of pseudorabies virus（PRV） strain Min-A as template by a pair of specific primers,and the amplicon was cloned into prokaryotic expression vector pET-30a and expressed in Escherichia coli. Antigenicity of the expressed protein was analysed. BALB/c mice were immunized with the purified gE protein. Murine myeloma cells of excreting specific anti-gE antibody were screened and characterized. The results showed that prokaryotically expressed gE protein possessed good immunogenicity,and molecular mass was 46 ku. Three strains of murine myeloma cells of excreting specific anti-gE antibody were obtained by screening,and the sensitivity and specific reactionogenicity of these McAbs were confirmed by Western-blotting and immunofluorescence assay. All the McAbs possessed to IgG1 subclass,and their light chains belonged to κ chain.

关 键 词：伪狂犬病病毒 gE蛋白 原核表达 单克隆抗体 

分 类 号：S852.659[农业科学—基础兽医学] Q813.2[农业科学—兽医学]