Construction Of Human Phage Antibody Library And Screening Of Phage Antibodies Against BFGF

Objective: To construct human phage antibody library from peripheral blood mononuclear cells (PBMC) of autoimmunity patients, which contains high titer of anti-bFGF antibody, select the anti-bFGF phage antibody from the library, and obtain the gene of high titer anti-bFGF antibody.Methods: The Fd fragment and κ chain of human IgG were separately cloned by RT-PCR from PBMC of the autoimmunity patients (SLE, RA, DM, et al). The κ chain was inserted into the phagemid pComb3 after digested with restriction enzymes of Sac â…  /Xba â… . The recombinant vector phagemid was electro-transformed into competent E.coli XL1-Blue cells to construct the κ chain library. The Fd fragment was inserted into the phagemid collection containing the κ chain repertoires after digested with Spe â…  /Xho â…  . The recombinant phagemid then was electro-transformed into XL1-Blue cells to construct the Fab library. The recombinant phage was rescued by coculturing with helper phage VCSM13. After three rounds of panning, the specific phage antibodies against bFGF were highly enriched. The high affinity phage antibodies against bFGF were identified by phage-ELISA.Results: The results showed that the single κ chain library contained 1.2×10⁸ clones, and the human Fab phage antibody library contained 2.5×10⁷ clones. Under the super-infection of helper phage VCSM13, the phage antibody titer was about 4×10¹¹ pfu/mL. After three rounds of "adsorption-elution-amplification", phage antibodies against bFGF have been enriched effectively. 26 phage antibody clones were rescued and 4 Fab phage antibody clones with high affinity and specificity to bFGF were obtained by phage-ELISA.Conclusion: The constructed the human phage antibody library provides an effective method to select the high specific and high affinity human anti-bFGF antibody, which provides a basis for the research of anti-bFGF drugs.