Development Of Monoclonal Antibodies And Epitope Identification On Hemagglutinin In H5N2Avian Influenza Virus

Avian influenza (AI) was first outbreak in Italy in1878which caused by type A influenza virus (AIV). The disease can caused head edema, cyanosis in comb and legs, diarrhea, drop in egg production as well as severe neurological symptoms in poultry. AI can also cause mammalian disease which has aroused worldwide attention. The purpose of this study is to produce monoclonal antibodies (McAb) and select epitope on Hemagglutinin (HA) of H5N2AIV which plays a fundamental role in further study on antigenic structure of HA proteins and building new methods for AIV detection.In this study, AIV were amplified by embryonated eggs and the purified virus was collected after ultracentrifugation, sucrose density gradient centrifugation and off sugar. SPF BALB/c mice were immunized with purified virus. Spleen cells from the immunized mice were fused with SP2/0myeloma cells. Positive clones were screened by indirect ELISA. A total of four monoclonal antibodies were obtained:GE5, DD4, FF4, DE1. After Western-blot and laser confocal experiment, they showed that GE5McAb was identified to secret HA antibody, and its hybridoma supernatant and ascites titers were1:2000and1:64000. The McAb is belong to IgG1subclass and its light chain is κ chain. Ascites was purified by Protein G which lay the foundation for subsequent epitope screening experiments.Screening epitope with PH.D.-12phage peptide library. After three panning, the phage was specific enrichment. Identify positive clones with indirect ELISA and amplify plaque for sequencing. The result showed that32phage samples have the same amino acids sequence:N I H H G A L V R H Q V. Analysis the sequence of H5N2AIV and12peptides with DNAStar software, the result indicates that193aa-204aa in HA sequence of virus shows33.3%similarity with12peptides. Using synthetic peptide as coating antigen, the result of ELISA is positive which proves that GE5McAb can bind with synthetic peptide specifically.Consensus amino acids (194aa、195aa、196aa、203aa) were selected to actualize site-directed mutation, testing the binding of McAb to the HA proteins expressed by mutant HA genes. Complete HA gene was amplified by HA specific primers. Mutant HA genes were amplified by mutant specific primers. Cloned the gene into PMD18-T Vector. After digested with specific enzymes (Smar Ⅰ and Xhol Ⅰ), the mutant HA gene was correctly subcloned into a eukaryotic expression vector pCAGGs and transfected it into SF9cells. Taking confocal laser experiment with GE5McAb and SF9cells expressed HA protein. According to the result, the intensity of fluorescence signal of SF9cells which express normal HA proteins is stronger than SF9cells which express mutant HA proteins. It suggested that the epitope against McAb maybe at193aa-204aa in HA of virus. The research plays a fundamental role in further study on antigenic structure of HA proteins and building new methods for AIV detection.