| Objective: To construct the chimeric vector encoding DCP-Ih C-ProDer f1gene through prokaryotic expression and purify this protein in plasmid p ET28a(+)-DCP-Ih C-ProDer f1 in large scale,and investigate the effects on the signaling pathway of JAK2/STAT3 in asthmatic mice undergong immunotherapy with the recombinant protein DCP-Ih C-ProDer f1 through blocking JAK2/STAT3 using inhibitor AG-490 specific to JAK 2.Methods:1)Specific DC cell binding peptides(peptides 3,FYPSYHSTPQRP),and peptide sequences of MHC II Ii molecular 1-110 and the sequence of Dermatophagoides farinae allergen ProDer f1 were initially synthesized.Molecular biology technology was used to construct chimeric gene DCP-Ih C-ProDer f1.Then the gene fragments were inserted into Escherichia coli expressed vector p ET28a(+)to develop recombinant vector p ET28a(+)-DCP-Ih C-ProDer f1 via parokaryotic expression.All recombinant vectors were verified by restriction enzyme digestion and sequencing;2)The constructed recombinant plasmid p ET28a(+)-DCP-Ih C-ProDer f1 were transferredinto the Escherichia coli expression strain E.coli BL21(DE3).Low dosage of IPTG was used to induce DCP-Ihc-ProDer f1 expression for optimizing the conditions for expression and purification of the protein in large scale;3)The asthmatic murine models were established by using the crude extracts of acaroid mites.Then the purified DCP-Ih C-ProDer f1 protein was used as a vaccine for specific immunotherapy of asthmatic mice,in which one group of mice were intraperitoneally injected with JAK2 inhibitor(AG-490)0.5 h before specific immunotherapy via aerosol inhalation.ELISA kit was used to detect the cytokines,including IFN-γ,IL-10,IL-4 and IL-17 in the bronchoalveolar lavage fluid and serum levels of Ig E and Ig G2 a.Lung tissues were taken for histological observation of the pathological changes,and Western blot was performed to determine the p-JAK2 and p-STAT3 in whole protein extracts of lung tissues.Results: 1)Verification by restriction endonuclease digestion and sequencing indicated that prokaryotic expression vector p ET28a(+)-DCP-Ih C-ProDer f1 was successfully constructed;2)The final concentration of IPTG in dose of 0.4mmol/L was capable of inducing E.coli BL21(DE3)p ET28a(+)-DCP-Ih C-ProDer f1 strain to express DCP-Ih C-ProDer f1 protein,which was subjected to SDS-PAGE analysis before expression induction and purification in large scale;3)The purified DCP-Ih C-ProDer f1 protein was used as vaccine for specific immunotherapy(SIT)of the asthmatic mice.The lung biopsies showed significantly alleviated inflammation with fewer inflammatory cells seen in mice following SIT compared to those administered with the inhibitor.However,the condition remained no significant difference between mice treated with the inhibitor and conventional protocol.ELISA results showed that the mice in the SIT group and inhibitor treatment group had significantly reduced serum antibody Ig E than those in the asthma group(P<0.05),yet had higher serum Ig G2 a level(P<0.05),and Ig G2 a level was not significantly different between inhibitor group and SIT group(P>0.05).Mice with SIT and inhibitor had lower IL-4,IL-17,IFN-γ and IL-10 levels in the BALF than those of asthma group(P<0.05),whereas the difference was not significant between SIT group and inhibitor group(P>0.05).Western blot detection of the lung tissue protein extraction showed that the expression of p-JAK2 and p-STAT3 in asthmatic group was significantly higher than that of the control group(P<0.05),yet expression of p-JAK2 and p-STAT3 was significantly downregulated in SIT group and inhibitor group(P<0.05).Conclusions: p ET28a(+)-DCP-Ih C-ProDer f1 was successfully constructed via prokaryotic expression and purified in largescale;Chimeric DCP-Ih C-ProDer f1 f peptide vaccine can lead to better results by SIT of asthmatic mice;DCP-Ih C-ProDer f1 functioning in SIT may be associated with JAK2/STAT3 signaling pathway. |
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