Abstract
BackgroundHemagglutinin (HA), as the surface immunogenic protein, is the most important component of influenza viruses. Previous studies showed that the stability of HA was significant for HA’s immunogenicity, and many efforts have been made to stabilize the expressed HA proteins.MethodsIn this study, the protein disulfide isomerases (PDIs) were investigated for the ability to improve the stability of HA protein. Two members of the PDIs family, PDI and ERp57, were over-expressed or down-expressed in 293 T cells. The expression of H3 HA and PDIs were investigated by real-time qPCR, western-blot, immunofluorescence assay, and flow cytometry. The stability of HA was investigated by western-blot under non-reducing condition. Moreover, BALB/c mice were immunized subcutaneously twice with the vaccine that contained HA proteins from the ERp57-overexpressed and conventional 293 T cells respectively to investigate the impact of ERp57 on the immunogenicity of H3N2 HA.ResultsThe percentage of the disulfide-bonded HA trimers increased significantly in the PDIs-overexpressed 293 T cells, and ERp57 was more valid to the stability of HA than PDI. The knockdown of ERp57 by small interfering RNA significantly decreased the percentage of the disulfide-bonded HA trimers. HA proteins from ERp57-overexpressed 293 T cells stimulated the mice to generate significantly higher HA-specific IgG against H1N1 and H3N2 viruses than those from the conventional cells. The mice receiving H3 HA from ERp57-overexpressed 293 T cells showed the better resistance against H1N1 viruses and the higher survival rate than the mice receiving H3 HA from the conventional cells.ConclusionERp57 could improve the stability and immunogenicity of H3N2 HA.
Highlights
Hemagglutinin (HA), as the surface immunogenic protein, is the most important component of influenza viruses
Our results showed that ERp57 could improve the stability and immunogenicity of H3N2 HA, which may help the development of vaccine against influenza against pandemics
Overexpression of Protein disulfide isomerases (PDI) did not impact the expression and characterization of HA proteins in 293 T cells To investigate the impact of the overexpression of PDIs on the expression of H3 HA proteins, the recombinant H3 HA and PDIs plasmids were co-transfected into 293 T cells
Summary
Hemagglutinin (HA), as the surface immunogenic protein, is the most important component of influenza viruses. Previous studies showed that the stability of HA was significant for HA’s immunogenicity, and many efforts have been made to stabilize the expressed HA proteins. HA plays an important role in infection by mediating virus entry into the host cell and fusion between viral and cellular target membrane and is used as a crucial component of the broad-spectrum vaccine [6,7,8]. Previous studies showed that HAs from H3N2 have two conversed cysteines, in the 540 and 544 sites of the transmembrane domain, which were significant for the stability of HA trimer [14, 15]. The protein family of protein disulfide isomerases (PDIs) which assist the formation of disulfide bonds [23] were investigated for the ability to improve the stability of HA proteins in this study
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.