抗CD47单链抗体制备及抗原结合活性分析  

Preparation single-chain Fv fragment against CD47 and antigen binding activity analysis

作　　者：席欧彦 贾二坷 赵婷[1] 秦瑞坪 马晓玲[1] 李江伟[1] 

机构地区：[1]新疆大学生命科学与技术学院生物资源基因工程重点实验室,新疆乌鲁木齐830046 [2]新疆天康畜牧生物技术股份有限公司,新疆乌鲁木齐830011

出　　处：《生物技术》2017年第4期370-377,共8页Biotechnology

基　　金：国家自然科学基金项目("骆驼单域抗体框架区作为小分子抗体通用移植骨架的研究";No.31370933;"基于高通量测序对骆驼抗体库的多样性分析和数据挖掘及新纳米抗体的发现";No.31570935)

摘　　要：[目的]研究旨在制备抗CD47小分子单链抗体,并分析其与抗原的结合能力和阻断作用的活性。[方法]采用DNA合成方法,将抗CD47的单克隆抗体B6H12的重链可变区(VH)和轻链可变区(VL),通过短肽(Gly4Ser)3连接形成B6H12单链抗体(B6H12-scFv),在大肠杆菌中可溶性表达并纯化B6H12-scFv。ELISA和Western Blot方法检测与人重组CD47的结合。检测EC9706和KYSE150两种食管癌细胞表面CD47的表达,细胞ELISA和流式细胞术分析B6H12-scFv与细胞表面CD47的结合。最后采用竞争ELISA分析B6H12-scFv对CD47与髓样细胞表面的信号调节蛋白α(SIRPα)结合的阻断能力。[结果]成功获得纯度达到90%以上的可溶性单链抗体。EC9706和KYSE150细胞均高表达CD47。细胞ELISA和流式检测B6H12-scFv浓度为50μg/m L可与EC9706表面CD47有较好结合。B6H12-scFv浓度为40μg/m L时也可以竞争性阻断CD47与SIRPα的结合。[结论]成功地构建了抗CD47单链抗体,浓度在20μg/m L具有EC9706的结合活性,也具有阻断作用。[Objective]The purpose of this research is to generate anti-CD47 single chain antibody and evaluating its antigen binding and blocking activities. [Methods]By means of DNA synthesis technique,the variable region antibody B6H12 were ligated with a 15 peptide linker（ Gly4Ser） 3 to form B6H12 single chain antibody（ B6H12-scFv）. The scFv was expressed soluble in the cytoplasm of in E. coli BL21. B6H12-scFv was purified. B6H12-scFv was strongly binding with human CD47 by ELISA and Western Blotting assay. It confirmed CD47 was over expressed on esophageal cancer cells. Furthermore,the binding ability of B6H12-scFv with cell surface CD47 was revealed by flow cytometry and cell ELISA. Finally,The blocking capacity of B6H12-scFv against CD47 bind with Signal regulatory protein alpha（ SIRPα） was analyzed by competitive ELISA. [Results]Successful access to more than 90% purity of soluble single chain antibody. EC9706 and KYSE150 were highly expressed CD47. Cell ELISA and flow cytometry showed that B6H12-scFv concentration of 50μg/m L could bind to CD47 on EC9706 surface. The results showed that B6H12-scFv concentration of 40μg/m L competitively blocks the binding of CD47 with SIRPα. [Conclusion]This study suggested that E. coli expressed anti-CD47 single-chain antibody has a functional activities and the concentration of 20μg/m L with EC9706 binding activity,and ligand blocking.

关 键 词：CD47 SIRPα 单链抗体 抗原结合 阻断作用 

分 类 号：Q784[生物学—分子生物学]