Construction of a large single-chain phage antibody library
Objective To construct a large single chain phage antibody library for human single chain antibody cloning. Methods Diverse V L and V H genes were amplified by RT PCR from lymphocytes collected from adult peripheral blood and new born cord blood. The V genes were spliced to form ScFv by overlap PCR and cloned into vector PDF, obtaining a primary library in which the V H genes were flanked by two non homologous loxp sites. The V H and V L genes in this primary library were shuffled by infecting the phagemid into cre expressing bacteria at high multiplicity of infection(MOI) to create a very large phage antibody library. The library was tested by selecting human antibodies against various antigens through bio panning. Results A large phage antibody of 6×10 10 in size was constructed. Diverse specific human ScFvs against all the 6 tested antigens were obtained from the library by biopanning. Conclusion The loxp cre mediated recombination in single bacteria cell is efficient in constructing very large phage antibody library. The success in cloning various human antibodies suggests that the constructed phage antibody library can be used in human antibody preparation.