本文選題：犬細(xì)小病毒　切入點(diǎn)：單鏈抗體　出處：《中國獸醫(yī)科學(xué)》2016年08期

【摘要】：提取分泌犬細(xì)小病毒的雜交瘤細(xì)胞株3H9的總RNA,反轉(zhuǎn)錄獲得c DNA鏈,并以此為模板擴(kuò)增重鏈可變區(qū)(VH)基因和輕鏈可變區(qū)(VL)基因,利用SOE PCR方法連接VH基因和VL基因,同時(shí)引入(Gly4Ser)3連接肽序列,擴(kuò)增得到大小為765 bp的單鏈抗體(Sc Fv)基因。將Sc Fv基因連接至原核表達(dá)載體p ET-32a(+),構(gòu)建成重組質(zhì)粒p ET-32a-Sc Fv,轉(zhuǎn)化至BL21(DE3)感受態(tài)細(xì)胞,并在37℃條件下進(jìn)行IPTG誘導(dǎo)表達(dá),得到融合蛋白。經(jīng)SDS-PAGE分析大小約為46 ku,與預(yù)期結(jié)果相符。經(jīng)Western-blotting鑒定,該蛋白能被抗His標(biāo)簽的單克隆抗體特異性識(shí)別。間接ELISA試驗(yàn)以及中和試驗(yàn)結(jié)果表明,Sc Fv能夠與犬細(xì)小病毒結(jié)合,具有中和犬細(xì)小病毒的活性。本試驗(yàn)成功構(gòu)建了抗犬細(xì)小病毒單鏈抗體,并獲得了高效表達(dá)。
[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，

本文編號(hào)：1657549