Subject(s)
Adolescent , Adult , Humans , Middle Aged , Young Adult , Age Distribution , Betacoronavirus , Physiology , China , Epidemiology , Coronavirus Infections , Epidemiology , Virology , Disease Outbreaks , Health Behavior , Masks , Pandemics , Pneumonia, Viral , Epidemiology , Virology , Social Media , Surveys and QuestionnairesABSTRACT
<p><b>OBJECTIVE</b>To express the rubella virus E1-374 glycoprotein in Pichia pastoris and study the immunogenecity of the recombinant protein.</p><p><b>METHODS</b>The cDNA of protein E1-374 was cloned into the expression vector pGAPZalphaA and transformed into Pichia pastoris GS115 cells by electrotransfection. The expressed protein was confirmed by indirect immunofluorescence and demonstrated immunoreactivity by Western Blot. Rubella virus IgG antibody was assayed with ELISA after mice were inmmunized by E1-374 glycoprotein.</p><p><b>RESULTS</b>SDS-PAGE analysis and Western Blot analysis of E1-374 protein revealed this protein to be 46.89 x 10(3). Antiserum (1:100) and E1-374 (5.5 microg/ml) was chosen for ELISA optimization. The intra-assay coefficient of variation for the ELISA was 0.36%-12.45%.</p><p><b>CONCLUSION</b>Protein E1-374 was highly expressed in Pichia pastoris cells, and it was a good choice to prepare rubella virus recombinant protein vaccines.</p>
Subject(s)
Animals , Female , Humans , Mice , Enzyme-Linked Immunosorbent Assay , Gene Expression , Mice, Inbred BALB C , Pichia , Genetics , Metabolism , Recombinant Proteins , Genetics , Allergy and Immunology , Rubella virus , Genetics , Allergy and Immunology , Viral Envelope Proteins , Genetics , Allergy and ImmunologyABSTRACT
To determine the functions of N-carbohydrate chains in human parainfluenza virus type 3 hemagglutinin-neuraminidase(HN) protein, a PCR-based site-directed mutagenesis method was used to obtain N-glycan mutants. Protein electrophoresis rate, cell surface expression,receptor binding activity, neuraminidase activity and cell fusion promotion activity were determined. The HN proteins of single mutants (G1, G2, and G4) and multiple mutants (G12, G14, G24 and G124) migrated faster than the wild-type (wt) HN protein on polyacrylamide gels, while G3-mutated protein and wt HN protein migrated at the same position. There was no statistic difference in cell surface expression and neuraminidase activity between wt and each mutant HN protein (P>0.05), but receptor binding activity and cell fusion promotion activity of each mutant protein was reduced to significant extent (P<0.05). G1, G2 and G4 mutants exhibited re duced receptor binding activity, which was 83.94%, 76.45% and 55.32% of the wt level, respectively. G1, G2 and G4-mutated proteins also showed reductions in fusion promotion activity, which was 80.84%, 77.83% and 64.16%, respectively. Multiple mutants with G12-, G14-, G24- and G124- substitutions could further reduce receptor binding activities, 33.07%, 20.67%, 19.96% and 15.11% of the wt HN level, respectively. G12, G14, G24 and G124 mutants exhibited levels of fusion promotion activity that were only 46.360, 12.04%, 13.43% and 4.05% of the wt amount, respectively. As N-glycans of hPIV3 HN protein play an important role in receptor binding activity and cell fusion promotion activity of HN protein. We propose that the loss of N-glycans change the conformation or orientation of globular domain that is responsible for receptor binding and lower receptor binding activity and cell fusion promotion activi ty.