Construction of phage antibody library and screening of anti-FasFab antibody

Aim The phage display antibody library was constructed to obtain functional anti Fas Fab. Methods Balb/c mice were immunized with recombinant Fas protein. mRNA was isolated from splenocytes and antibody heavy chain genes Fd and κ chain genes were amplified by RT PCR, and combinatorial Fab antibody library. This iuelicated that the screening of antibody library was done with recombinont Fas antigen and the immunifacient function of the antibody was determined by ELISA. Results The amplified products were the desired fragments and contained 700 bp. The amplified antibody heavy chain Fd genes were cloned into pcomb3 vector at first, and transformed into E.coli XL1 blue to construct an antibody heavy chain library with a size of 3.5×106 members. The κ chain genes were then randomly combined with heavy chain genes to generate a combinatorial vector encoding both chains and capable of generating Fab fragments. These combinatorial vectors were transformed to E.coli XL1 Blue,and the combinatorial Fab antibody library was 3.7×106. The phage antibody library was further prepared by infection of helper phage VCSM13 to display the antibody Fab fragments on the surface of phage. The titer of phage in the library was 8.9×1015 cfu/L. The phage displaying antibody fragments were subjected to four rounds of panning with recombinant Fas antigen at solid phase. The eluted phage was enriched for nearly 100 fold and the percent of recombinant clones increased from 27% to 100% after four rounds of panning. Anti Fas Fab phage antibody displaying on phage surface and soluble anti Fas Fab showed specific binding activity to Fas by ELISA. Conclusion The successful preparation of anti Fas Fab antibody paved a way for further study and application.