抗狂犬病病毒核蛋白单链抗体的表达及活性鉴定  

Expression and activity identification of single chain variable fragments against nucleoprotein of rabies virus

作　　者：程朝飞[1] 宫苗苗[1] 李刚[1] 赵占中[1] 朱鸿飞[1] 袁维峰[1] 史利军[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国兽医科学》2013年第9期963-968,共6页Chinese Veterinary Science

基　　金：中央级公益性科研院所基本科研业务费专项资金项目(2010js-2;2011js-3)

摘　　要：为了获得抗狂犬病病毒(RV)核蛋白(N)的单链抗体,从分泌抗狂犬病病毒核蛋白单克隆抗体的杂交瘤细胞株1D9中提取总RNA,经RT-PCR扩增得到抗体的轻链可变区基因和重链可变区基因,利用重叠引物延伸法将轻链可变区基因和重链可变区基因连接,同时引入连接肽编码序列,成功扩增得到单链抗体(ScFv)基因,将ScFv基因克隆至原核表达载体pET-32a(+),转化至E.coli BL21(DE3)感受态细胞,经IPTG诱导表达后得到融合蛋白。SDS-PAGE和Western-blot分析结果表明,融合蛋白大小约为32ku,主要以包涵体的形式存在,能被抗His标签的单克隆抗体特异性识别。间接竞争ELISA检测结果表明,该单链抗体能够与RV-His-N重组蛋白和RV结合。抗RV核蛋白单链抗体的成功表达,为单链抗体在狂犬病的诊断和治疗中的进一步研究奠定了基础。To obtain a single chain variable fragment（ScFv） against nucleoprotein of rabies virus（RV）, the genes coding for the light and heavy chain variable regions were cloned by RT-PCR from a murine mo- noclonal hybridoma cell line 1D9,which could be used to produce monoclonal antibody（McAb） to recognize the nucleoprotein. Then the light and heavy chain variable regions were fused together by a short peptide linker containing 15 amino acid（Gly4 Set）3 using splice-overlap extensive PCR. The recombinant single chain variable fragments were subcloned into the expression vector pET-32a（＋） and transformed into Escherichia coli BL21（DE3）. The expression of ScFv which was in the form of inclusion bodies induced by IPTG showed to be 32 ku in a molecular weight and anti-His-tag McAb recognized-activity by SDS-PAGE and Western-blotting, respectively. The results of indirect competitive ELISA indicated that the ScFv bound to the recombinant RV-His-N and RV. This study laid the foundation for the use of SeFv in the treatment and diagnosis of rabies.

关 键 词：狂犬病病毒核蛋白 单链抗体 原核表达 活性鉴定 

分 类 号：S855.3[农业科学—临床兽医学] S852.659.5[农业科学—兽医学]