抗CD3×CD87单链双特异抗体介导的单个核细胞对前列腺癌细胞杀伤作用的体外研究  

Bispecific Anti-CD3×Anti-CD87 Single-Chain Antibody Mediates Cytolytic Activity of Mononuclear Cells Against CD87-Positive Prostate Cancer in vitro

作　　者：高婉莉 杨黎[1] 刘姗[1] 赵智辉[1] 

机构地区：[1]南京师范大学生命科学学院生物化学与生物制品研究所江苏省分子与医学生物技术重点实验室,江苏南京210023

出　　处：《南京师大学报（自然科学版）》2017年第2期51-56,64,共7页Journal of Nanjing Normal University(Natural Science Edition)

基　　金：国家自然科学基金重大研究计划集成项目子课题(2013104GZ90073)

摘　　要：本研究尝试探讨抗CD3×CD87单链双特异抗体介导的单核细胞对CD87+前列腺癌细胞的杀伤作用.首先,利用基因工程手段,构建了pET24a/抗CD3×CD87单链双特异抗体原核表达载体;将重组载体转化BL-21(DE3)感受态细胞,用IPTG诱导蛋白表达;获得的工程菌经超声破碎,先在变性条件下亲和纯化目的蛋白,再采用稀释复性方法对scBsAb进行复性.其次,利用流式细胞术检测表达纯化的scBsAb与两种相应抗原的结合情况.最后,通过CCK-8法检测外周血单个核细胞(PBMCs)对CD87阳性前列腺癌细胞PC-3的杀伤效应.结果表明,利用基因工程手段获得了较高纯度的scBsAb;scBsAb能与PC-3细胞表面CD87特异性结合,并且能与CD3特异性结合;scBsAb的浓度及效靶比影响scBsAb介导的杀伤作用.在效靶比为10∶1,scBsAb浓度为100μg/mL时最大杀伤率达到40.86%;在scBsAb浓度为100μg/mL,效靶比为40∶1时达到最大杀伤率60.9%.此外,ELISA检测表明,在scBsAb介导的PBMCs靶向CD87阳性肿瘤细胞杀伤过程中IFN-γ的水平显著增高.结果证明,scBsAb能够在体外有效介导效应细胞杀伤CD87阳性肿瘤细胞.In this study,the aim was to investigate whether anti-CD3xanti-CD87 single-chain bi-functional specific anti-body （ scBsAb） could mediate the cytotoxicity of peripheral blood mononuclear cells（ PBMCs） against CD87-positive pros-tatic cancer cells. First,the prokaryotic expression vector of pET24a/scBsAb was constructed and transformed into com-petent cells of E. coil BL-21 （DE3） by genetic engineering technique. After induction with IPTG,the bacteria were ultra- sonicated, and scBsAb in the form of inclusion bodies were harvested by centrifugation. The scBsAb was further purified by affinity chromatography under denaturing conditions and refolded by dilution. Second,the antigens binding activity of scBsAb was examined by flow cytometry. Lastly,the scBsAb-mediated cytotoxicity of PBMCs against CD87-positive pros-tatic cancer cell line PC-3 was determined by using CCK-8 assay. Our results showed that the scBsAb with high purity was obtained. scBsAb could both specifically bind to CD87 and CD3. The scBsAb-mediated killing of PBMCs against CD87＋ PC-3 cells was related to the concentration of used scBsAb and the ratio of effector to target（ E -T） . When E -T was 10-1 and the scBsAb concentration was 100 jjig/mL,the killing rate was 40.86% ; whereas when the E -T ratio was 40-1 and scBsAb concentration was 100 jjig/mL,the killing rate was 60.9%. In addition,ELISA assay showed that IFN-7 level in the co-culture system was significantly increased during the killing process. Our data indicate that anti-CD3xanti- CD87 scBsAb could mediate PBMCs to kill CD87-positive tumor cells in vitro.

关 键 词：单链双特异抗体 胞毒作用 前列腺癌 原核表达 

分 类 号：Q819[生物学—生物工程]