论文部分内容阅读
[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 research aimed to study the preparation of anti-CD47 small molecule single chain antibody and analyze its activity of binding and blocking with antigen. [Method] The heavy chain variable region (VH) and light chain variable region (VL) of anti-CD47 monoclonal antibody B6H12 were linked by DNA (Gly4Ser) 3 to form B6H12 single chain antibody -scFv), B6H12-scFv was expressed and purified soluble in E. coli. ELISA and Western Blot method to detect binding to human recombinant CD47. The expression of CD47 on the surface of EC9706 and KYSE150 esophageal cancer cells was detected. The binding of B6H12-scFv to cell surface CD47 was analyzed by cell ELISA and flow cytometry. Finally, competitive ELISA was used to analyze the ability of B6H12-scFv to block the binding of CD47 to signal regulatory protein alpha (SIRPα) on the surface of myeloid cells. [Result] Soluble single chain antibody with the purity above 90% was successfully obtained. Both EC9706 and KYSE150 cells highly express CD47. Cell ELISA and flow cytometry B6H12-scFv at a concentration of 50 μg / mL showed good binding to EC4770 surface CD47. The binding of CD47 to SIRPα could also be competitively blocked by B6H12-scFv at a concentration of 40 μg / mL. [Conclusion] The anti-CD47 single-chain antibody was successfully constructed, and its binding activity with EC9706 at a concentration of 20 μg / mL was also blocked.