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cDNA，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pTlinker。利用SfiⅠ酶切位点将scFv基因构建至细菌展示载体pBSD中，将重组质粒电转化入E.coliDH5α构建pBFD-scFv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scFv。将12株scFv基因分别插入pET-28a，构建重组表达质粒pET28a-scFv。转化到E.coliRosetta中诱导表达，获得约28ku目的蛋白。经ELISA检测，纯化scFv对VP2蛋白P...

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Bibliographic Details
Published in东北农业大学学报 Vol. 48; no. 11; pp. 26 - 34
Main Author 李德山;车瑞香;郭瑞;王宇阳;黄涛;郭笑辰;任桂萍
Format Journal Article
LanguageChinese
Published 东北农业大学生命科学学院,哈尔滨,150030 2017
Subjects
CPV;单链抗体;流式细胞术;亲和力
亲和力
Canine parvovirus
bacterial display
CPV
single chain antibody
单链抗体
flow cytometry
流式细胞术
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ISSN1005-9369

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Summary: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cDNA，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pTlinker。利用SfiⅠ酶切位点将scFv基因构建至细菌展示载体pBSD中，将重组质粒电转化入E.coliDH5α构建pBFD-scFv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scFv。将12株scFv基因分别插入pET-28a，构建重组表达质粒pET28a-scFv。转化到E.coliRosetta中诱导表达，获得约28ku目的蛋白。经ELISA检测，纯化scFv对VP2蛋白P/N值均大于2.1，其中scFv-23、scFv-33、scFv-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。
Bibliography:Canine parvovirus; single chain antibody; flow cytometry; bacterial display
23-1391/S
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.coli DH5αto 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.The12recombinant 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 about28ku of the target
ISSN:1005-9369