Expression and identification of H5 subtype hemagglutinin of avian influenza A virus in insect cells / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To clone and express avian influenza A virus [A/Hong Kong/482/97(H5N1)] H5 subtype hemagglutinin in baculovirus-insect cell expression system and investigate the antigenicity and bioactivity of the recombinant protein.</p><p><b>METHODS</b>H5 gene of influenza A virus was amplified by PCR. The recombinant bacmid was obtained by cloning the gene to the donor plasmid of pFastBacHTB and transformed into DH10Bac competent cells. The recombinant baculovirus stock was prepared by transfecting the recombinant bacmid DNA into the insect cell line for protein expression after amplification. Immunofluorescene assay (IFA) and Western blotting were performed to identify the antigenicity of the recombinant protein, and hemagglutination assay was used to identify its bioactivity.</p><p><b>RESULTS</b>The recombinant his-H5 protein was expressed in the insect cells with a relative molecular mass of 64,000, which showed erythrocyte-agglutinating activities with the red blood cells of guinea pig. Western blotting and IFA demonstrated that the recombinant his-H5 could be recognized and bound by standard anti-H5 sera.</p><p><b>CONCLUSION</b>The recombinant his-H5 with a post-translation modification is successfully obtained in insect cells, which may provide a potential source for further study of the antigen's biological function and for production of the subunit vaccine or monoclonal antibodies.</p>