Optimization of expression process of recombinant hepatitis E vaccine by insect cell-baculovirus expression vector system
Objective To optimize the culture process of insect cell-baculovirus expression vector system(BEVS)in order to further improve the protein expression of recombinant hepatitis E virus-like particles(HEV-LPs),and to scale up and verify the process.Methods Serum-free suspension culture of Sf9 insect cells was carried out and a 7 L bioreactor was established through shaking flask cascade amplification.The parameters such as viable cell density,culture temperature and recombi-nant virus inoculation ratio were optimized when the cells were inoculated with recombinant baculovirus.The cell growth status was observed and the expression level of the target protein was analyzed.The target protein was detected by SDS-PAGE,Western blot,high-performance liquid chromatography(HPLC)and transmission electron microscope(TEM).The morphology of HEV-LPs and protein yield were integrated,and the suitable expression parameters of recombinant HEV-LPs were determined to prepare three batches of 7 L recombinant hepatitis E vaccine.Results The optimal parameters for the cultivation of insect cells in a 7 L bioreactor were as follows:stirring 90 r/min,pH(6.2±0.1),optimal incubation tempera-ture 27 ℃,cell density of recombinant baculovirus inoculation 6.0 × 106 cells/mL,and dissolved oxygen 90％.Under this condition,the relative molecular mass of the expressed HEV-LPs target protein was about 58 000,which had a specific binding with HEV mouse monoclonal antibody.The final yield was 60-70 mg/L,with the purity of more than 95％.Spherical particles with a diameter of about 20 nm were observed under TEM.The HEV-LPs recombinant protein prepared by the opti-mized parameters had stable yield and uniform particle morphology.Conclusion The culture and expression process of recombinant hepatitis E vaccine was successfully optimized,and the stability of the amplification process was verified,which lays a foundation for the development of recombinant hepatitis E vaccine production process.
Hepatitis E VaccineInsect cell-baculovirus expression vector systemBioreactorCulture processOptimiza-tionVirus-like particles(VLPs)
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