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World J Gastroenterol. Nov 21, 2007; 13(43): 5765-5770
Published online Nov 21, 2007. doi: 10.3748/wjg.v13.i43.5765
Construction of humanized carcinoembryonic antigen specific single chain variable fragment and mitomycin conjugate
De-Jie Chen, Zui Tan, Feng Chen, Tian Du
De-Jie Chen, Zui Tan, Feng Chen, Tian Du, Department of General Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei Province, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Professor Zui Tan, Department of General Surgery, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071, Hubei Province, China. docdj_1130@yahoo.com.cn
Telephone: +86-27-63436739
Received: April 26, 2007
Revised: August 23, 2007
Accepted: October 18, 2007
Published online: November 21, 2007
Abstract

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 BamHI and EcoRI 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 BamHI and EcoRI, and connected to an expression vector which also has the restriction enzyme cleavage sites BamHI 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 manner.

CONCLUSION: The CEA-scFv-MMC conjugate can be successfully constructed and is able to inhibit the growth and induce apoptosis of colorectal cancer cells.

Keywords: Carcinoembryonic antigen; Single chain variable fragment; Mitomycin; Immunoconjugates; Colorectal cancer