Generation Of HPAIV Single-Chain Variable Fragment Antibody By Phage Surface Display Technique

H5N1 avian influenza (AI) poses big threat to public health, and over 200 people around the world have been confirmed with the H5N1 infection and more than half of them died, the development of effective drugs to this virus is one of the top-priority for the H5N1 pandemic plan. Our goal of the present study is to contract a single-chain variable fragment (ScFv) antibody against H5N1 avian influenza for potential human H5N1 influenza treatment. We previously developed a series of mouse hybridoma cells the secret the monoclonal antibody against H5N1 virus. In the present study, we amplified the variable heavy chain (VH) and variable light chain (VL) antibody genes from the total RNA of a hybridoma cell line that secrets the monoclonal antibody against H5N1 virus using a series of 4 pair specific primers and overlapping extention PCR technique. The ScFv gene was ligated into the phagemid vector pCANTAB5E and transformed into the competent E.coli TGl. After three rounds of panning using H5N1 avian influenza virus (AIV) as subtractive selection antigen, one phage-ScFv with high affinity to the H5N1 AIV was selected from the enriched phages. Sequence blast results revealed that our phage-ScFv antibody is noval and has not been previously reported. We expressed the ScFv antibody in E. Coli TG1 strain, and the expressed antibody retains all of the characters of the original monoclonal antibody, which was confirmed by western blot, ELISA and HI assay. The ScFv antibody developed in this study could be used for further treatment for H5N1 avian influenza infection in animal models.