Construction of humanized carcinoembryonic antigen specific single chain variable fragment and mitomycin conjugate.
OBJECTIVE
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-beta-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.


CONCLUSIONS
The CEA-scFv-MMC conjugate can be successfully constructed and is able to inhibit the growth and induce apoptosis of colorectal cancer cells.
Chen
De-Jie
DJ
Department of General Surgery, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071, Hubei Province, China.
Tan
Zui
Z
Chen
Feng
F
Du
Tian
T
D000903:Antibiotics, Antineoplastic / Q000276:immunology* / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002272:Carcinoembryonic Antigen / Q000235:genetics / Q000276:immunology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000473:pathology; D015245:Deoxyribonuclease BamHI; D015252:Deoxyribonucleases, Type II Site-Specific; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D018796:Immunoconjugates / Q000276:immunology* / Q000494:pharmacology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D016685:Mitomycin / Q000276:immunology* / Q000494:pharmacology; D010957:Plasmids
D000903:Antibiotics, Antineoplastic; D002272:Carcinoembryonic Antigen; D018796:Immunoconjugates; D007135:Immunoglobulin Variable Region; D016685:Mitomycin; D015245:Deoxyribonuclease BamHI; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific