Expression Of Avian Influenza Virus Subtype H5N1 Neuraminidase (NA) Gene In Sf9 Insect Cells

Avian influenza is caused by avian influenza virus (AIV) which is a highly contagious infectious disease. It first occurred in Italy in 1878, and it covered almost all the world nowdays. The highly pathogenic avian influenza subtype H5N1 caused poultry not only the large-area death but also which threat the health of human, in recent years. Influenza virus has two surface glycoprotein which are hemagglutinin (HA) and neuraminidase (NA), NA gene encoding the NA protein of AIV are one of the major surface antigen, and humoral immunity are the target antigen.which could induce the production of specific antibodies, inhibit the release of virus from infected cells and then infect other cells, reduce the proliferation of virus.The research studied Influenza Virus subtype H5N1 neuraminidase gene expressed in Sf9 Insect Cells. NAs, a truncation of the neuraminidase (NA) gene of H5N1 subtype avian influenza virus (AIV), was successfully amplified by polymerase chain reaction (PCR) to truncate the signal peptide sequence of NA. The gene was inserted into plasmid pFastBacHTb to obtain recombinant transfer vector pFastBacHTb-NAs. pFastBacHTb-NAs was transformed into DH10B cells carrying both AcBacmid and helper plasmid. Recombinant bacmid rAcBacmid-NAs with the insertion of NAs gene was obtained by specific transposition between pFastBacHTb-NAs and AcMNPV bacmid. Recombinant virus vAc-NAs was obtained by transfecting rAcBacmid-NAs into Sf9 insect cells. Supernatant containing vAc-Nas was collected and Sf9 cells were infected by vAc-NAs. Cells collected 3~5days after transfection and infection were broken by the sonicator. Then the supernatant and the sediment were detected by SDS-PAGE and western blotting. That truncated NA protein NAs had been highly expressed in Sf9 insect cells with the approximate molecular weight 52kDa which was equal to the theoretical size, but cell supernatant without expression products.This study we cloned the source of Influenza Virus subtype H5N1 neuraminidase gene, the gene was inserted into plasmid pFastBacHTb to obtain recombinant transfer vector pFastBacHTb-NAs. Influenza Virus Subtype H5N1 Neuraminidase Gene (NAs) had been expressed in Sf9 insect cells. With a view to study of influenza Virus subtype H5N1 neuraminidase gene and development of new types of influenza vaccine to lay the foundation.