抗人B7-2单链抗体基因的构建及其在大肠杆菌中的表达  

Construction of Anti-B7-2 Single Chain Antibody Genes and Expression of the ScFv in Escherichia coli

作　　者：董海辉[1] 邱玉华[1,2] 陈永井[1,2] 胡静平[1] 黄光镁[1] 朱江[1] 

机构地区：[1]苏州大学医学部基础医学与生物科学学院,苏州215123 [2]苏州大学医学生物技术研究所,苏州215007

出　　处：《科技通报》2010年第3期367-373,共7页Bulletin of Science and Technology

基　　金：国家科技重大专项课题(2009ZX09103-705);江苏省自然科学基金(BK2004203);江苏省高校高新技术发展基金(JHB05-45)

摘　　要：在成功建立稳定分泌鼠抗人B7-2杂交瘤细胞株基础上,扩增并克隆出该单克隆抗体的重链(VH)和轻链(VL)可变区基因。通过重叠延伸PCR(SOE-PCR)方法,在VH和VL可变区基因之间引入连接肽(Gly4Ser)3,体外构建抗人B7-2单链抗体(B7-2 ScFv)基因。为便于表达产物的纯化,在抗人B7-2 ScFv的C端增加了6×His tag序列。将其克隆至表达载体pET32a并在大肠杆菌中进行原核表达。SDS-PAGE和Western-blot分析结果表明,抗人B7-2 ScFv在大肠杆菌BL21(DE3)菌中获得表达,重组融合蛋白的相对分子量约为43 kD,表达产物以不溶性包涵体形式存在,经溶解包涵体,体外复性和Ni-NTA亲和柱纯化,获得了高纯度的抗人B7-2 ScFv,纯化蛋白得率约16.2%。成果为抗人B7-2单链抗体的生物学活性研究和抗肿瘤药物开发奠定了基础。The variable domain genes encoding heavy（VH） and light（VL） chain were cloned from murine anti-human B7-2 hybridoma cell line,which can steadily secrete high titers of mouse anti-human B7-2 mAb.Using SOE-PCR method,short polypeptide linker（Gly4Ser）3 was inserted between the VH and VL gene fragments of anti-B7-2 mAb to form anti-B7-2 singlechain Fv antibody（ScFv） gene.In addition,6 ×His tag was added due to convenience of purification.Then,the ScFv gene fragment was inserted into expression vector pET32a and prokaryotic expression experiment was carried out in E.coli BL21.SDS-PAGE and Western Blotting analysis confirmed the expression of the ScFv with the molecular weight of about 43 kD.The ScFv expression was in the form of an inclusion bodies and the purified protein was obtained after a series purifications,including inclusion body solubilization,protein refolding and NiNTA affinity chromatography.The yield of the ScFv achieved approximately 16.2%.

关 键 词：单克隆抗体 单链抗体 B7-2 原核表达 

分 类 号：Q789[生物学—分子生物学]