Expression of H5N1 avian influenza A virus hemagglutinin in sf9 insect cells and its identification

Abstract

Objective To express hemagglutinin (HA) of H5N1 avian influenza A virus using a baculovirus-insect cell expression system and identify the expression product. Methods The HA gene of A/Goose/Guangdong/1/96 (H5N1) avian influenza A virus was optimized according to the sf9 insect cell codon preference and full-length synthesized. The recombinant donor plasmid pFastHA was constructed, and then transformed into DH10Bac competent cells after PCR and sequence identification. The recombinant shuttle plasmid BacmidHA was constructed, identified by PCR with M13 primer, and transfected into sf9 cells with liposome method to obtain recombinant baculovirus rBacHA. The expression product of rBacHA was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and indirect immunofluorescence assay. Results A band with the expected size of 1 707 bp was observed after the donor plasmid pFastHA was double-enzyme digested. No mutation in the target gene was found by gene sequencing. A band with the excepted size of 3 180 bp was obtained from shuttle plasmid BacmidHA by PCR amplification. SDS-PAGE showed that the relative molecular weight of the rBacHA expression product was about 65 000. Western blotting analysis revealed that the expression product was able to react with avian influenza A virus positive serum. The rBacHA-infected sf9 cells emitted green fluorescence under a fluorescence microscope, indicating the correct expression of HA gene. Conclusion The recombinant HA protein with good antigenicity is expressed successfully by the baculovirus-insect cell expression system. Key words: Influenza A virus, H5N1 subtype; Hemagglutinins; Spodoptera; Baculovidae; Protein biosynthesis