[1]高婉莉,杨 黎,刘 姗,等.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[J].南京师范大学学报(自然科学版),2017,40(02):51.[doi:10.3969/j.issn.1001-4616.2017.02.009]
　Gao Wanli,Yang Li,Liu Shan,et al.Bispecific Anti-CD3×Anti-CD87 Single-Chain Antibody Mediates CytolyticActivity of Mononuclear Cells Against CD87-Positive Prostate Cancer in vitro[J].Journal of Nanjing Normal University(Natural Science Edition),2017,40(02):51.[doi:10.3969/j.issn.1001-4616.2017.02.009]
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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()
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《南京师范大学学报》（自然科学版）[ISSN:1001-4616/CN:32-1239/N]

卷:
第40卷
期数:
2017年02期
页码:
51
栏目:
·生命科学·
出版日期:
2017-06-29

文章信息/Info

Title:
Bispecific Anti-CD3×Anti-CD87 Single-Chain Antibody Mediates CytolyticActivity of Mononuclear Cells Against CD87-Positive Prostate Cancer in vitro
文章编号:
1001-4616(2017)02-0051-06
作者:
高婉莉; 杨 黎; 刘 姗; 赵智辉
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
Author(s):
Gao Wanli; Yang Li; Liu Shan; Zhao Zhihui
School of Life Sciences,Nanjing Normal University,Institute of Biochemistry and Biological Products,Jiangsu Key Laboratory for Molecular and Medical Biotechnology,Nanjing 210023,China
关键词:
单链双特异抗体; 胞毒作用; 前列腺癌; 原核表达
Keywords:
single chain bispecific antibody; cytotoxity; prostate cancer; prokaryotic expression
分类号:
Q819
DOI:
10.3969/j.issn.1001-4616.2017.02.009
文献标志码:
A
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100 μg/mL时最大杀伤率达到40.86%; 在scBsAb浓度为100 μg/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.
Abstract:
In this study,the aim was to investigate whether anti-CD3×anti-CD87 single-chain bi-functional specific antibody(scBsAb)could mediate the cytotoxicity of peripheral blood mononuclear cells(PBMCs)against CD87-positive prostatic cancer cells. First,the prokaryotic expression vector of pET24a/scBsAb was constructed and transformed into competent cells of E.coil BL-21(DE3)by genetic engineering technique. After induction with IPTG,the bacteria were ultrasonicated,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 prostatic 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 μg/mL,the killing rate was 40.86%; whereas when the E:T ratio was 40:1 and scBsAb concentration was 100 μg/mL,the killing rate was 60.9%. In addition,ELISA assay showed that IFN-γ level in the co-culture system was significantly increased during the killing process. Our data indicate that anti-CD3×anti-CD87 scBsAb could mediate PBMCs to kill CD87-positive tumor cells in vitro.

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备注/Memo

备注/Memo:
收稿日期:2017-01-08.
基金项目:国家自然科学基金重大研究计划集成项目子课题(2013104GZ90073).
通讯联系人:赵智辉,博士,教授,研究方向:抗肿瘤新药研究. E-mail:zhaozhihui_1964@aliyun.com
更新日期/Last Update: 2017-06-30