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
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.
