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Cloning of Fab Gene of an Anti-Human Bladder Cancer Monoclonal Antibody and Its Expression in E.coli
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Abstract:
Objective:To clone the Fab gene of a monoclonal antibody (mAb) BDI against human bladder cancer and its expression in E.coli. Methods: Fd and κ genes of mAb BDI were cloned by RT-PCRand inserted into an Fab expression vector. Phage displaying Fab and soluble Fab were expressed in E.coli. The N-terminal sequence of VH region was corrected by PCR mediated mutagenesis. The antigen-binding characteristics of the Fab were tested by ELISA and immunohistochemistry. Results: Fd and κ genes were cloned into the expressing vector p3MH and the phage displaying antibody and soluble Fab were expressed in E.coli, which showed weak binding activity to bladder cancer cells. Correction of the N-terminal sequence of the VHimproved the biding activity dramatically. The feasibility of the application of the Fab in phage antibody library screening was confirmed by a simulated panning procedure. Conclusion:The Fab gene of an anti-human bladder cancer mAb was expressed in E.coli. The importance of the N-terminal sequence on antibody binding activity was suggested.
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