[Establishment of a lentivirus-mediated rapid eukaryotic expression system of novel avian influenza H7N9 virus hemagglutinin gene].

OBJECTIVE

To establish a rapid eukaryotic expression system of hemagglutinin (HA) gene of novel avian influenza H7N9 using lentiviral vector, express the recombinant protein and study its functions in human embryonic kidney HEK293T cells.

METHODS

The full-length HA gene was amplified from H7N9 genomic RNA by reverse transcription PCR (RT-PCR) and linked with pMD18-T vector to generate pMD18-T-HA plasmid. Blunt-end HA gene with Kozak sequence was amplified from pMD18-T-HA vector, and then pLenti-HA-V5 expression vector was constructed by Topo cloning for transient expression in HEK293T cells. Expression of HA-V5 recombinant protein was confirmed by immunofluorescence assay (IFA) and Western blotting. Hemagglutination test was performed to evaluate the biological activity of the recombinant protein.

RESULTS

The full-length HA gene (1 683 bp) was obtained and eukaryotic expression plasmid was constructed successfully. A recombinant protein with relative molecular mass (Mr) 70 000 was expressed and the antigenicity and binding specificity to positive serum were demonstrated by IFA and Western blotting. The hemagglutination activity was proved by hemagglutination test. IFA and Western blotting showed that the Mr 70 000 recombinant protein had an immuoreactivity to positive serum. The hemagglutination activity was confirmed by hemagglutination test.

CONCLUSION

The rapid eukaryotic expression system of HA gene was successfully constructed, which laid a solid foundation for further research on subunit vaccine development, neutralizing epitope mapping and packaging pseudovirus.