Construction of humanized carcinoembryonic antigen specific single chain variable fragment and mitomycin conjugate

作     者：De-Jie Chen Zui Tan Feng Chen Tian Du 

作者机构：Department ofGeneral Surgery Zhongnan Hospital of Wuhan University Wuhan430071 Hubei Province China 

出 版 物：《World Journal of Gastroenterology》 (世界胃肠病学杂志（英文版）)

年 卷 期：2007年第13卷第43期

页      面：5765-5770页

核心收录：

学科分类：1002[医学-临床医学] 100214[医学-肿瘤学] 10[医学] 

主　　题：Carcinoembryonic antigen Single chainvariable fragment Mitomycin Immunoconjugates Colorectal cancer 

摘      要：AIM： To construct a new target-oriented conjugate of humanized carcinoembryonic antigen （CEA） specific single chain variable fragment （scFv） and mitomycin （MMC） against colorectal cancer, and to investigate its influence on the growth and apoptosis of colorectal cancer cells. METHODS： The primer was designed according to the gene sequence described in reference 16, which respectively contains restriction enzyme cleavage sites BamH I and EcoR I in its upstream and downstream. PCR was performed with the plasmid as template containing genes of humanized anti-CEA scFv. The product was digested by BamH I and EcoR I, and connected to an expression vector which also has the restriction enzyme cleavage sites BamH I and EcoR. Expression of the reaction was induced by isopropy-β -D-thiogalactoside （IPTG）. Then the expression product was covalently coupled with MMC by dextran T-40. The immunoreactivity of the conjugate against colorectal cancer cells as well as CEA was measured by enzyme linked immunosorbent assay （ELISA）. The inhibiting ratio of conjugate on the growth of colorectal cancer cells was also measured by ELISA. The effect of conjugate on the apoptosis of colorectal cancer cells was determined by flow cytometry （FCM）. RESULTS： Restriction endonuclease cleavage and gene sequencing confirmed that the expression vector was successfully constructed. Sodium dodecyl sulfate polyacrylamide gel electropheresis （SDS-PAGE） confirmed that this vector correctly expressed the fusion protein. ELISA confirmed that the conjugate had quite a strong immunoreactivity against colorectal cancer cells and CEA. The conjugate had inhibitory effects on colorectal cancer cells in a concentration-dependent manner and could induce apoptosis of colorectal cancer cells in a concentration-dependent ***： The CEA-scFv-MMC conjugate can be successfully constructed and is able to inhibit the growth and induce apoptosis of colorectal cancer cells.