[Expression of neuraminidase gene of influenza virus H1N1 in baculovirus-expression system].

OBJECTIVE

To construct the recombinant baculovirus with NA gene of Influenza H1N1 virus.

METHODS

Full-length NA gene of Influenza virus H1N1 (A/PR/8/34) was amplified by PCR and inserted into pFastBacdual vector to construct the recombinant baculovirus transfer vector pFBD-NA. Recombinant shuttle vectors rBacmid-NA was then obtained after transforming DH10B competent cells containing bacmid plasmids. After transfecting into sf9 cells, recombinant baculovirus rBac-NA was obtained. The rBac-NA genome was extracted and identified by PCR. NA protein expressed by recombinant baculovirus-infected sf9 cells was determined by IFA, Western Bolt and ELISA.

RESULTS

PCR results proved that recombinant shuttle vectors rBacmid-NA was successfully constructed. NA protein was detected by IFA and showed strong specific green fluorescence on the surface of infected cells. NA protein was recognized by two polyclonal antibodies specific for NA in Western Blot. ELISA showed specific reaction of recombinant NA protein with mouse polyclonal antibody against influenza virus (PR8), indicating high antigenicity.

CONCLUSION

Recombinant baculovirus rBac-NA that expresse NA protein of influenza virus was successfully constructed. This work provides a basis for further study on NA protein function and novel influenza vaccine development.