Published online Aug 15, 2001. doi: 10.3748/wjg.v7.i4.510
Revised: March 28, 2001
Accepted: April 6, 2001
Published online: August 15, 2001
AIM: To develop the single chain variable fragment of MG7 murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator.
METHODS: mRNA was isolated from MG7 producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG7 recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATOIII of highly expressing MG7-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E.coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG7 ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG7 ScFv. Finally, the relative molecular mass of soluble MG7 ScFv was measured by SDS-PAGE.
RESULTS: The V-H, V-L and ScFv DNAs were about 340 bp, 320 bp and 750 bp, respectively. The volume of the library was up to 2 × 106 and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG7 antibody for binding to the antigen expressed on KATOIII cells. Within 2 strong positive phage clones, the soluble MG7 ScFv from one clone was found to have the binding activity with KATOIII cells. SDS-PAGE showed that the relative molecular weight of soluble MG7 ScFv was 32.
CONCLUSION: The MG7 ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.