Preparation Of Monoclonal Antibodies Against Non-structural Protein Of H5N1Subtype Avian Influenza Virus And Identification Of Its Antigenic Epitope

Avian influenza(AI) is an infectious disease of birds and poultry caused by RNA viruses of thefamily Orthomyxoviridae, the avian influenza viruses(AIV). In recent years, the epidemic of AIrepresented by the H5N1subtype continues to cause a severe loss for poultry industry, and threaten bothhuman health and public health security of the world.Vaccination against AIV, a powerful tool forcontrol of the disease, may result in issues related to surveillance programs that cannot identify infectedand vaccinated animals through serological tests. To overcome this problem, several researches havefocused on a differentiation of infected and vaccinated animals (DIVA) program of AI, which is mostlycentralizing on the non-structural（NS1）protein based on antibody reponse. One of the most essentialbasis is monoclonal antibody (MAb) against NS1protein, and that is the reason why our study started.To prepare MAb against NS1of AIV, BALB/c mice were immunized with purified recombinantNS1expressed in E.coli. The immunized mice spleen cells were fused with myeloma cells SP2/0, andthen2MAbs (D9and D7) against NS1were screened by indirect ELISA. Western blot assay showedthat the MAbs were reacted with recombinant NS1. Indirect immunofluorescence assay (IFA) indicatedthat the MAbs were reacted with NS1expressed in SF9cells transfected with eukaryocyte recombinantplasmids.Screening a phage display random7-mer peptide library with the2MAbs. The MAb D9recognized phages displaying peptides with the consensus peptide WNLNT-VS, which was substantiallymatched with182WNDNTVRVS190of AIV NS1. The result suggested that the peptide is a linear epitopeof NS1.The synthetic peptide WNDNTVRVS was verified the specific binding with D9by ELISA.Similarly, the MAb D7recognized phages displaying peptides with the consensus peptide NFFTQAL,which was no matched with AIV NS1. Then, using peptide microarray identified D7epitope, and29DAPF32was finally identified. Dot-blot immunoassay indicated D7can bind with synthetic peptideDAPF specifically. IFA showed that D7was reacted with deletion DAPF NS1protein expressed in293Tcells significantly weaker than with NS1protein expressed in293T cells, further validate the binding ofD7with DAPF.In present study, two MAbs were obtained, named D9and D7. The identified sequence of theantigenic epitope against D9is WNDNTVRVS which located in AA182to AA190and the other oneagainst D7is DAPF, located in AA29to AA32.The result facilitate for further study on the function ofNS1and diagnostic method development for AIV detection.