Expression Of NA1 And NA2 Genes From Avian Influenza Virus In The Baculovirus/insect Cell System And Development Of ELISA To Detect Antibodies Against Avian Influenza Virus Neuraminidase N1 And N2 Antibody

In order to identify the N1-antibody and N2-antibody in the serum sample,an ELISA assay,based on preliminary purified N1 and N2 neuraminidase protein produced in insect cells by recombinant baculovirus was developed.The DNA fragments of NA1 from H5N1 Avian influenza virus(AIV) and NA2 from H9N2 Avian influenza virus were amplified by RT-PCR.The genes of interest were cloned into pFastBactHTA transfer vector and named as pFastBacHTA-NA1 and pFastBacHTA-NA2.The recombinant vectors were introduced into the target bacmid in DH10Bac cells and named the recombinant bacmids as Bacmid-NA1 and Bacmid-NA2.Sf9 cells were transfected with recombinant bacmid DNA and the recombinant baculovirus which named as vBac-NA1 and vBac-NA2 were collected from the culture medium.The appropriate harvesting time of baculovirus-infected cells was 120h post-transfection,and the recombinant protein N1 and N2 respectively amounted to 2.1%and 1.7%of total cellular protein. The recombinant protein N1 and N2 were analyzed by western-blot and neuraminidase inhibition test (NIT).The results showed the recombinant NAs respectively had good reactivity with antiserum of H5N1 and H9N2 AIV and no cross-reaction.The recombinant NAs were enzymatically active.The indirect ELISA for identify the N1-antibody and N2-antibody was established.This assay optimal working dilution was ascertained,including goat anti-chiken immunoglobulin conjugated to horseradish peroxidase,antigen,testing sera,et al.Using this method detected 64 serum samples, compared to NIT,for idenfication of N1-antibody,the coincidence rate between them was 95.3%,for idenfication of N2-antibody,the coincidence rate between them was 96.8%.This method could correctly detect the N1-antibody and N2-antibody which were induced by 7 kinds of avain influenza vaccines.In conclusion,the method is simple,rapid and can be used to identify the N1-antibody and N2-antibody in the serum sample.