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AIM To develop the single chain variable fragment ofMG_7 murine anti-human gastric cancer monoclonalantibody using the phage display technology for obtaininga tumor-targeting mediator.METHODS mRNA was isolated from MG_7-producingmurine hybridoma cell line and converted into cDNA.Thevariable fragments of heavy and light chain were amplifiedseparately and assembled into ScFv with a speciallyconstructed DNA linker by PCR.The ScFvs DNA wasligated into the phagmid vector pCANTAB5E and theligated sample was transformed into competent E.ColiTG1,The transformed cells were infected with M13K07helper phage to form MG_7 recombinant phage antibodylibrary.The volume and recombinant rate of the librarywere evaluated by means of bacterial colony count andrestriction analysis.After two rounds of panning withgastric cancer cell line KATOⅢ of highly expressing MG_7-binding antigen,the phage clones displaying ScFv of theantibody were selected by ELISA from the enriched phageclones.The antigen-binding affinity of the positive clonewas detected by competition ELISA,HB2151 E.coil wastransfected with the positive phage clone demonstrated bycompetition ELISA for production of a soluble form of theMG_7 ScFv.ELISA assay was used to detect the antigen-binding affinity of the soluble MG_7 ScFv.Finally,therelative molecular mass of soluble MG_7 ScFv wasmeasured by SDS-PAGE.RESULTS The V_H,V_L and ScFv DNAs were about 340bp,320bp and 750bp,respectively.The volume of the librarywas up to 2 × 10~6 and 8 of 11 random clones wererecombinants.Two phage clones could strongly competewith the original MF_7 antibody for binding to the antigenexpressed on KATO Ⅲ cells.Within 2 strong positivephage clones,the soluble MG_7 ScFv from one clone wasfound to have the binding activity with KATO Ⅲ cells,SDS-PAGE showed that the relative molecular weight ofsoluble MG_7 ScFv was 32.CONCLUSION The MG_7 ScFv was successfully producedby phage antibody technology,which may be useful forbroadening the scope of application of the antibody.
AIM To develop the single chain variable fragment ofMG_7 murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaininga tumor-targeting mediator.METHODS mRNA was isolated from MG_7-producingmurine hybridoma cell line and converted into cDNA.The variable fragments of heavy and light chain were amplifiedseparately and assembled into ScFv with a speciallyconstructed DNA linker by PCR.The ScFvs DNA wasligated into the phagmid vector pCANTAB5E and theligated sample was transformed into competent E.ColiTG1, The transformed cells were infected with M13K07helper phage to form MG_7 recombinant phage antibodylibrary. The volume and recombinant rate of the librarywere evaluated by means of bacterial colony count and restriction analysis.After two rounds of panning withgastric cancer cell line KATOIII of highly expressing MG_7-binding antigen,the phage clones displaying ScFv of theantibody were selected by ELISA from the enriched phageclones.The antigen-binding a ffinity of the positive clonewas detected by competition ELISA, HB2151 E.coil wastransfected with the positive phage clone demonstrated bycompetition ELISA for production of a soluble form of theMG_7 ScFv.ELISA assay was used to detect the antigen-binding affinity of the soluble MG_7 ScFv. Finally, there are molecular mass of soluble MG_7 ScFv wasmeasured by SDS-PAGE.RESULTS The V_H,V_L and ScFv DNAs were about 340bp,320bp and 750bp,respectively.The volume of the library was up to 2 × 10~6 and 8 of 11 random clones wererecombinants.Two phage clones could strongly competewith the original MF_7 antibody for binding to the antigenexpressed on KATO ⅲ cells.Within 2 strong positivephage clones, the soluble MG_7 ScFv from one clone wasfound to have the binding activity with KATO ⅲ cells, SDS-PAGE Showed that the relative molecular weight ofsoluble MG_7 ScFv was 32.CONCLUSION The MG_7 ScFv was successfully producedby phage antibody technology,which may be useful forbroadening the scope of ap PlicatIon of the antibody.