抗犬细小病毒单链抗体库构建及其亲和力检测

发布时间：2018-04-16 00:36

本文选题：CPV + 单链抗体　；参考：《东北农业大学学报》2017年11期

【摘要】：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。
[Abstract]:In order to obtain the high affinity scFv antibody against canine parvovirus, construct anti canine parvovirus (CPV) bacteria displayed antibody library. Extraction of canine spleen total RNA, reverse transcription C DNA as template, VH and VL were amplified in canine antibody encoding sequence is inserted into the cut site of Fv gene to construct SC bacteria display vector P BSD P Tlinker. by Sfi cloning vector of enzyme, the recombinant plasmid was transformed into P BFD-sc Fv electrical construction of bacterial display library E.coli DH5 alpha. Through labeled FITC VP2 protein by screening 12 strains of positive SC Fv. SC Fv gene of 12 strains were inserted into the P ET-28a flow cytometry, the recombinant plasmid P ET28a-sc Fv. transformation to induce expression of E.coli expression in Rosetta, about 28 Ku. The target protein detected by ELISA SC Fv of VP2 purified protein P/N values are greater than 2.1, including SC Fv-23, SC Fv-33, SC Fv-34 of VP2 protein through the strong affinity. Research on animal immunity The source of animal, in obtaining high antibody at the same time, to avoid the traditional immune rejection with monoclonal antibody; flow cytometry and bacteria of single chain antibody library display technology can be efficient, rapid screening of scFv. Research on homologous gene engineering antibody to fill the market blank at the same time against canine parvovirus homologous, can be developed with neutralizing activity in length antibody to lay the foundation.

【作者单位】：东北农业大学生命科学学院;
【基金】：国家重点研发计划项目(2016YFD0501003)
【分类号】：S858.292

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