Expression and Purification of H7 Subtype Avain Influenza Viruses HA Protein in Sf9

Abstract

【Objective】The experiment is aimed to express the HA protein of H7 subtype avian influenza virus (AIV) in eukaryotic cells, in order to provide bass for futher preparation of H7 subunit vaccine and evaluation of their immunogenicity.【Method】First, the preferred codons of Spodoptera frugiperda clone 9 (Sf9) insect cells were used to optimize and synthesize the HA sequence of H7 subtype AIV, which was cloned into the shuttle plasmid pFastBac1. Then the baculovirus plasmid of the recombinant HA gene was transfected into Sf9 insect cells, and whether the recombinant virus were successfully rescued was identified by indirect immunofluorescence and western blots assay. Later on, through the SYBR green dye fluorescent quantitative PCR test, a baculovirus qPCR quantitative method based on gp64 gene was established to screen out the optimal multiplicity of infection and incubation time for expressing HA recombinant protein in Sf9 cells. Finally, the HA recombinant protein was affinity purified by the His nickel strain.【Result】After the recombinant baculovirus expressing the H7 subtype AIV HA protein was passed on to Sf9 cells for 3 generations, the Sf9 cells began to exhibit cell lesions such as swelling, shedding, and death, indicating that the recombinant baculovirus has been rescued successfully. Through indirect immunofluorescence and western blot tests, it was found that a green fluorescence signal specifically bound to the HA recombinant protein is appeared in Sf9 cells, and the band size of the specifically bound to the HA recombinant protein was about 70 kD, which was consistent with expectations, and the HA recombinant protein can react with H7 subtype AIV positive serum, indicating successful expression of the HA recombinant protein. A baculovirus qPCR detection method was established based on gp64 gene using the constructed pUC19-gp64 plasmid as a standard, and it exhibited a good linear relationship between 1×103~1×108 copy/μL, while the correlation coefficient of standard curve R2=0.993. Additionally, it was found that the protein expression effect was the best when the Sf9 cells were inoculated with a ratio of 10 MOI and incubated for 72 h using western blots test, the HA recombinant protein was purified through affinity purification of His nickel strain, with a protein concentration of 0.268 mg/mL, the agglutination titer of chicken red blood cells is 4 log2.【Conclusion】This experiment successfully expressed the H7 subtype AIV HA protein in Sf9 insect cells and established the corresponding fluorescence quantitative PCR detection method for baculovirus, which can provide refference for further evaluation of the immunogenicity of H7 subunit vaccine.

Keywords

• h7 subtype avain influenza viruses
• hemagglutinin (ha)
• insect cell
• baculovirus
• gene expression
• protein purification