Screening HA Epitopes Polypeptide Of H5 Subtype Avian Influenza Virus And Establish Polypeptide ELISA For Detecting Antibody

Avian influenza is an infectious disease caused by avian influenza virus A in poultry and wildfowl syndrome. AIV mainly infected poultry, causing respiratory disease and lead to systemic disease, resulting in decreased egg production, severe mortality can be caused by 100%. In recent years, AIV mutation frequent, which makes some mutant strains can directly infect humans and cause death, seriously endanger public health, and cause huge economic losses, has been closely watched. Inspection of local mainly testing AIV antibody in chicken serum by enzyme linked immunosorbent assay (ELISA) and judging the immunity of chickens and whether there is wild virus infection, at present the envelope antigen of ELISA kit which testing AIV antibody mainly include inactivated H5 subtype virus and purified H5 subtype HA protein, these ELISA kits are mainly imported and the price is high and not conducive to the monitoring of the disease.In the study, we prepared the inactivated antigen with the H1-H15 subtypes influenza virus and Newcastle disease virus. Synthesis of polypeptides containing 15 aa with a peptide-peptide overlap of 14 aa by the HA amino acid sequence according to H5 subtype AIV(A/DuTurkey/England/N28/1973) which saved by our laboratory. These H5 subtype HA antigen peptides are immobilized on solid supports and screen out 4 antigenic polypeptide which can binding specificity with chicken serum of H5 subtype and named H5-1, H5-2, H5-3, H5-4.The screened H5-1 epitopes polypeptide is used as the coating antigen of the ELISA test, determination of the optimal amount of antigen and the dilution of serum sample by matrix test, and optimized the test conditions, such as the Coating liquid, the sample dilution, the second antibody dilution and the developing time. Detection of H5 subtype chicken serum with 4 antigen peptides under the optimal conditions, the results show that only H5-1 antigen peptide can specifically detect H5 subtype of chicken serum and not interferenced from other subtypes of chicken serum, H5-2, H5-3 and H5-4 were interfered by other subtypes of chicken serum and their specificity is not strong. Detection chicken serum of Newcastle disease virus with H5-1 epitopes polypeptide, the results show that H5-1 epitopes polypeptide can not be combined with chicken serum of Newcastle disease virus. Series of gradient titration test show that the best sample dilution of this ELISA test was 1:200, compliance test show that the positive detection rate of this method was more than 87.6% when the sample’s HI greater than 1 and the positive detection rate of this method was more than 98.3% when the sample’s HI greater than 4.H5-1 epitopes polypeptide was truncated respectively from the amino terminal and carboxyl terminal, and insert the nucleic acid fragment which corresponding the truncated polypeptides into prokaryotic expression vector pgex-6p-l.The epitope of H5-1 epitopes polypeptide is HNIHP confirmed by the Western blot test ultimately.