抗犬细小病毒单链抗体的制备  

Preparation of anti-canine parvovirus single chain antibody

作　　者：刘欢[1,2] 金红岩[1] 侯强[1] 史秋梅[2] 董瑞凯 张通明 沈萍[2] 侯绍华[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 [2]河北科技师范学院预防兽医实验室,河北秦皇岛066003

出　　处：《中国兽医科学》2016年第8期959-964,共6页Chinese Veterinary Science

基　　金：公益性行业(农业)项目(201303024)

摘　　要：提取分泌犬细小病毒的杂交瘤细胞株3H9的总RNA,反转录获得c DNA链,并以此为模板扩增重链可变区(VH)基因和轻链可变区(VL)基因,利用SOE PCR方法连接VH基因和VL基因,同时引入(Gly4Ser)3连接肽序列,扩增得到大小为765 bp的单链抗体(Sc Fv)基因。将Sc Fv基因连接至原核表达载体p ET-32a(+),构建成重组质粒p ET-32a-Sc Fv,转化至BL21(DE3)感受态细胞,并在37℃条件下进行IPTG诱导表达,得到融合蛋白。经SDS-PAGE分析大小约为46 ku,与预期结果相符。经Western-blotting鉴定,该蛋白能被抗His标签的单克隆抗体特异性识别。间接ELISA试验以及中和试验结果表明,Sc Fv能够与犬细小病毒结合,具有中和犬细小病毒的活性。本试验成功构建了抗犬细小病毒单链抗体,并获得了高效表达。Total RNAwas extracted from a canine parvovirus hybridoma cell line 3H9,and the regions of genes were cloned by RT-PCR. Then the light and heavy chain variable regions were fused together by a short peptide linker containing 15 amino acid（Gly4Ser）3through splice-overlap extensive PCR. The recombinant single chain variable fragments containing 765 bp bases. ScFv genes were cloned into pET- 32a（＋） expression vector and transformed into BL21（DE3）.The positive clones were induced by IPTGfor 6 hours at 37℃,and the recombinant protein was detected by SDS-PAGE and Western-blotting. The ScFv antibody was expressed by pET-32a（＋） fused with His tag and the relative molecular mass of fusion protein was about 46 ku. The results of indirect ELISA and the neutralization test indicated that the ScFv could combine with canine parvovirus and had the activity to neutralize canine parvovirus. The pEr32a- ScFv fusion protein was constructed successfully,and obtained high performance expression.

关 键 词：犬细小病毒 单链抗体 原核表达 活性鉴定 

分 类 号：S852.165[农业科学—基础兽医学]