抗CD47单链抗体制备及抗原结合活性分析

发布时间：2018-06-30 17:45

本文选题：CD + SIRPα　；参考：《生物技术》2017年04期

【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。
[Abstract]:[aim] to prepare anti CD 47 small molecule scFv and to analyze its binding ability to antigen and its blocking activity. [methods] DNA synthesis, The heavy chain variable region (VH) and light chain variable region (VL) of monoclonal antibody B6H12 against CD47 were ligated to form B6H12 single chain antibody (B6H12-scFv) by means of short peptide (Gly4Ser) 3. B6H12-scFv.ELISA and Western Blot were used to detect the binding of B6H12-scFv.ELISA and Western Blot to human recombinant CD47. The expression of CD47 on EC9706 and KYSE150 esophageal carcinoma cells was detected. The binding of B6H12-scFv to CD47 was analyzed by cell Elisa and flow cytometry. Finally, the ability of B6H12-scFv to block the binding of CD47 to signal regulatory protein 伪 (SIRP 伪) on the surface of myeloid cells was analyzed by competitive Elisa. [results] soluble scFv. EC9706 and KYSE150 cells with purity of more than 90% were highly expressed CD47. When the concentration of B6H12-scFv was 50 渭 g / mL and the concentration of B6H12-scFv was 40 渭 g / mL, the binding of CD47 to SIRP 伪 could be blocked competitively. [conclusion] the anti-CD47 scFv was successfully constructed. The EC9706 binding activity and the blocking effect were observed at the concentration of 20 渭 g / mL.
【作者单位】：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;新疆天康畜牧生物技术股份有限公司;
【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)
【分类号】：R392

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