Construction Of Fab Phage Antibody Libraries From Chickens Against Avian Influenza Virus

Avian influenza (AI) is a disease infected by influenzavirus A. Avian influenza virus is the major etiologic agent of orthomyxoviridae. Influenzavirus A can infect swine, horse, mammal in ocean, poultry and human. Poultry and human which infected by this disease will also die.Phage display of antibody libraries has provided a powerful tool for the isolation of important viral pathogens .Large repertoires of antibodies can be displayed on the surface of filamentous phage particles, and antibodies with desired specificity can be isolated by panning on the antigen of interest.In this article ,we report the construction of chicken combinatorial immunoglobulin library to Avian Influenza Virus.The mRNA isolated from peripheral blood lymphocytes of chickens with AIV antibodies were reverse transcribed to the first strand cDNA using Oligo-dT as the primer.The genes of heavy Fd fragment and light chain of antibodies were amplified by a group of specific primers for chicken IgG Fab fragment. The gel purified light chain genes were firstly cloned into phagmid vector pComb3 to construct a chicken recombinatorial immunoglobulin library. The constructed library size was 1 x 10~5,and the cloning efficiencies of light chain genes were 50%.The heavy chain genes were subsequently combined with light chain genes randomly to constructed a combinatorial vectors.then transformed to E.Coli.XLI-Blue. the combinatorial Fabs libraries were constructed successfully. The chicken combinatorial Fab immunoglobulin library quantities were about 2.5x10~5 and the cloning efficiencies of Fab genes were 40%. Finally, the phage antibody library was further prepared by infecting chicken Fab combinatorial immunoglobulin library with help phage for displaying the titer of Fab fragment on the surface of phage .The titer of phages in library was lx10~13CFU/ml .The phage displaying antibody fragments were subjected to five rounds of paning with AIV antigen coated in solid phage. The eluted phages were enriched nearly 100-folder,and the percentase of recombinant clones increased from 15% to 65% after the five rounds of panning, choosed severy positive clones were sequenced and the data was analyzed by aligment with GeneBank, chicken immunoglobulin sequence was identified and confirmed. The light chains were identified as k type.We constructed chicken Fab recombinant library to AIV successfully. This specific Fab antibody combinatorial library laid a basis on panning and secreening specific Fab antibody to avian influenza virus. The antibodies panned and screened with puried AIV will be used as a tool to diagnose and study AIV. The antibodies will also be used as potential therapeutic medicine for the therapy of avian influenza after exposure to the virus.