Cloning And Expression Of Fab Genes Of Anti-p185 Monoclonal Antibody 5E12

Objective: To clone Fab genes of anti-p185 monoclonal antibody 5E12 ,construct its expression vector and express it in E .coli. Methods: Fd and κ genes of anti- p185 monoclonal antibody 5E12 were cloned by RT-PCR, using general primers for framework region 1(FR1), then were inserted into Fab expression vector and expressed in E.coli. The N-terminal sequences of V regions was resumed by PCR mediated mutagenesis .The antigen-binding activity of the Fab were tested by ELISA and immunohistochemistry. Results: At firt, Fd and ( genes were cloned and expressed in E.coli. But the bacterially expressed Fab fragments showed no antigen binding activity. Then, a phage antibody library were constructed and panning selection was performed. Again the identification of positive clone was failed. Finally, the N-terminal sequences of V regions was corrected to the original sequences by PCR mediated mutagenesis, and the Fab expressed in bacterial culture supernatant was able to target HER2/neu-expressing cells (3T3/erbB-2 cells). Comparing the expression of unmutated and mutated anti-p185 Fab, we found that the changes of N-terminal sequences of V regions introduced by PCR may seriously affect antigen binding activity but not the expression of antibody. Correction of N-terminal sequences of Fd could resume the antigen binding activity. But Correction of N terminal sequences of κ chain was shown no expected result. Conclusion: We were successful in constructing and expressing anti-p185 Fab, which will benefit the construction of other engineering antibody and humanization of murine anti-p185 McAb. We also found that the N terminal changes of V regions introduced by PCR primers may affect antigen binding activity seriously, to which more attention should be paid during antibody engineering.