抗犬细小病毒单链抗体的制备

发布时间：2018-03-24 08:42

  本文选题：犬细小病毒　切入点：单链抗体　出处：《中国兽医科学》2016年08期

【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。
[Abstract]:The total RNAs of hybridoma cell line 3H9 secreting canine parvovirus were extracted, and c DNA strands were obtained by reverse transcription. The heavy chain variable region (VH) gene and light chain variable region (LV) gene were amplified by reverse transcription. VH gene and VL gene were ligated by SOE PCR method. At the same time, the gly4Serf3 ligated peptide sequence was introduced to amplify the Sc Fv gene, which was 765 BP in size. The Sc Fv gene was ligated to the prokaryotic expression vector pET-32a (pET-32a), and the recombinant plasmid p ET-32a-Sc FV was constructed and transformed into BL21 DDE3. The fusion protein was induced by IPTG at 37 鈩，

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