抗犬细小病毒单链抗体库构建及其亲和力检测

为获得高亲和力抗犬细小病毒单链抗体,构建抗犬细小病毒(CPV)细菌展示单链抗体文库。研究提取犬脾脏总RNA,反转录c DNA,以此为模板,分别扩增犬抗体VH和VL编码序列,插入克隆载体p Tlinker。利用SfiⅠ酶切位点将sc Fv基因构建至细菌展示载体p BSD中,将重组质粒电转化入E.coli DH5α构建p BFD-sc Fv细菌展示库。通过标记FITC的VP2蛋白,利用流式细胞术筛选12株阳性sc Fv。将12株sc Fv基因分别插入p ET-28a,构建重组表达质粒p ET28a-sc Fv。转化到E.coli Rosetta中诱导表达,获得约28 ku目的蛋白。经ELISA检测,纯化sc Fv对VP2蛋白P/N值均大于2.1,其中sc Fv-23、sc Fv-33、sc Fv-34对VP2蛋白亲和力较强。研究通过免疫本动物源动物,在获得高价抗体同时,避免传统单克隆抗体的免疫排斥现象;结合流式细胞术和细菌展示技术可对单链抗体文库高效、快速筛选。抗犬细小病毒同源单链抗体研究在填补市场同源基因工程抗体空白的同时,可为研制具有中和活性全长抗体奠定基础。

Construction and affinity detection of an anti-CPV scFv library

In order to obtain high affinity anti-canine parvovirus single chain antibody,bacteriadisplayed recombinant scFv libraries of Canine parvovirus were constructed.Total RNA was extracted from the spleen of an immunized canine and reverse transcribed into cDNA as template.VH and VL coding sequences of the canine antibody were inserted into the cloning vector pTlinker,respectively.The scfv gene was constructed into the bacterial display vector pBSD by Sfi I digestion site,and the recombinant plasmid was electroconductive into E.coii DH5α to construct pBFD-scFv bacteria display library.Twelve VP2-binding scFv clones with unique sequences were obtained after screening scFv library by FCM with FITC-labeled VP2.The 12 recombinant scFv genes were inserted into pET-28a to construct the recombinant expression plasmid pET28a-scFv.They were transformed into E.coli Rosetta for induction and expression,in order to obtain about 28 ku of the target protein.ELISA results showed that all scFvs exhibited binding abilities and specifities to VP2,among them scFv-23,scFv-33 and scFv-34 were stronger than others.In this study,canine was immunized in order to acquire high-priced antibodies,which avoiding the traditional immune rejection of monoclonal antibodies.Flow cytometry and bacterial display technology was combined for screening in more efficient and rapid.Anti-canine parvovirus homologous single-chain antibodies fill the gaps in homologous antibodies in the market and bulid the foundation for the development of full-length antibodies with neutralization activity.