To construct ScFv and Fab from murine anti gastric cancer monoclonal antibody(mAb)3H11. Methods.At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1.The bacterial expres...To construct ScFv and Fab from murine anti gastric cancer monoclonal antibody(mAb)3H11. Methods.At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1.The bacterial expressed 3H11 Ab fragments showed no antigen binding activity.Then,phage antibody library and random mutated library were constructed from 3H11 hybridoma cells and panning selection was performed.Again the identification of positive clone was failed.Finally the N terminal sequences of V regions were resumed to 3H11 original sequences by site directed mutagenesis via PCR. Results.Binding activity to gastric cancer cells was detected only from N terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fragments was not affected.Correction of either VL or VH N terminal sequences could partially resume the antigen binding activity. Conclusion.Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody.展开更多
The object of this study is to investigate the properties of gastric cancer assoiciated antigen by McAb3H11 against gastric cancer cell line. Antigen purified by affinity chromatography and further charac- terized by ...The object of this study is to investigate the properties of gastric cancer assoiciated antigen by McAb3H11 against gastric cancer cell line. Antigen purified by affinity chromatography and further charac- terized by biochemical and immunological techiques. Results showed that assoiciated antigen of McAb 3H11 was mainly located on the membrane of target cells. It was sensitive to heat and digestion of proteases,but resistant to treatment with sodium periodate. Schiff’s reagent staining was negative. SDS-PAGE and IEF- analysis showed that the antigen was a 210 kD molecular weight with pl of 8. 5 protein. The molecular weight of antigen extracted from target cell with tunicamycin was not changed. We concluded that the cor- responding antigen of McAb 3H11 might be an alkaline protein.展开更多
文摘To construct ScFv and Fab from murine anti gastric cancer monoclonal antibody(mAb)3H11. Methods.At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FR1.The bacterial expressed 3H11 Ab fragments showed no antigen binding activity.Then,phage antibody library and random mutated library were constructed from 3H11 hybridoma cells and panning selection was performed.Again the identification of positive clone was failed.Finally the N terminal sequences of V regions were resumed to 3H11 original sequences by site directed mutagenesis via PCR. Results.Binding activity to gastric cancer cells was detected only from N terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fragments was not affected.Correction of either VL or VH N terminal sequences could partially resume the antigen binding activity. Conclusion.Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody.
文摘The object of this study is to investigate the properties of gastric cancer assoiciated antigen by McAb3H11 against gastric cancer cell line. Antigen purified by affinity chromatography and further charac- terized by biochemical and immunological techiques. Results showed that assoiciated antigen of McAb 3H11 was mainly located on the membrane of target cells. It was sensitive to heat and digestion of proteases,but resistant to treatment with sodium periodate. Schiff’s reagent staining was negative. SDS-PAGE and IEF- analysis showed that the antigen was a 210 kD molecular weight with pl of 8. 5 protein. The molecular weight of antigen extracted from target cell with tunicamycin was not changed. We concluded that the cor- responding antigen of McAb 3H11 might be an alkaline protein.
