Construction And Detection Of Anti-human CD40Single Chain Frgment Variable Phage Display Library

Objective:Construct a phage display library of anti-human CD40single chain fragment variable (ScFv) and lay a foundation for get a high affinity CD40ScFv from it.Methods:①The BALB/c mice were immuned by injecting human recombination CD40protein to neck subcutaneously.②The gene of anti-CD40antiboby’Heavy Variable and Light Variable were amplified by RT-PCR from total RNA isolated from spleen of the immunized BALB/c mice.③ScFv were constructed by overlap-PCR.④The ScFv were cloned into phage plasmid pCANTAB5E by double restriction enzyme to construct recombinant plasmid pCANTAB5E-ScFv.⑤The recombinant plasmid pCANTAB5E-ScFv was transformed into TGI E.coli, thus the prokaryotic expression library was created.⑥, Then, the phage library of CD40-ScFv was built by reinfection of help phage M13KO7.Results:①The VH、VL gene were successfully gained from the immuned BALB/c mice.②Total ScFv gene were gained by over-lap PCR, and gene length was750bp.③The recombinant plasmid pCANTAB5E-ScFv was cut by double restriction enzyme, the result indicated that there were two stripes in750bp and4200bp.④The anti-CD40ScFv phage display library containing1.75×106were successfully constructed after the TG1transformed by recombination plasmid pCANTAB5E-ScFv was rescued by help phage M13KO7.⑤8positive clones from the library was selected to sequencing randomly. The results indicated that the alignment and length of insertion sequence were right, which were matched on the basis structure of mice antibody.Conclusion:The phage display library of anti-CD40ScFv was constructed successfully, and it’s capacity and diversity can afford the penning for receiving the high binding affinity anti-CD40ScFv in the next procedure.