The Specific Immunotherapy Of House Dust Mite Sensitized Asthma By Using Der P1 T Cell Epitome Fusion Peptide And Its Underlying Mechanism

Objectives:To construct the chimeric vector encoding house dust mites T cell antigen epitope Der p 1 T gene through prokaryotic expression and purify this protein in plasmid pET28a(+)-Der p 1 T in large scale.Investigate the effects of house dust mites extract on mouse lung epithelial cells MLE12 and the underlying molecular mechanism.To explore the effects of Der p 1 T on immune cells in asthmatic mice undergoing immunotherapy with the recombinant protein Der p 1 T and evaluate the treatment of allergic asthma.Methods: The nucleotide sequence of house dust mites T cell antigen epitope Derp 1 T were initially synthesized.Molecular biology technology was used to construct chimeric gene Der p 1 T.Then the gene fragments were inserted into Escherichia coli expressed vector p ET28a(+)to develop recombinant vector pET28a(+)-Der p 1 T via prokaryotic expression.All recombinant vectors were verified by restriction enzyme digestion and sequencing.Low dosage of IPTG was used to induce Der p 1 T expression for optimizing the concentration and conditions for expression and purification of the protein in large scale.MLE12 cells were treated with house dust mites extract,the expression of cytokines and chemokine was analyzed by Real time PCR,the activation of NF-?B and MAPK was detected by Western blot.Real time PCR was used to evaluate the knock-down efficiency of Tlr4,Myd88 and Traf6.The expression of cytokines and chemokine was determined by Western blot.The asthmatic mouse models were established by using the house dust mites extract.Then the purified Der p 1 T recombinant protein was used as a vaccine for specific immunotherapy of asthmatic mice.After the treatment,the symptoms and signs of the mice were recorded.ELISA kit was used to detect the cytokines,including IL-10,IL-4,IFN-? and IL-17 A in the bronchoalveolar lavage fluid and serum levels of Ig E and Ig G2 a.Flow cytometry analysis was used to determine Th1/Th2 and Th17/Treg cell community changes.Lung tissues were taken for histological observation of the pathological changes.Results:The results of restriction enzyme digestion and sequencing indicated that prokaryotic expression vector pET28a(+)-Der p 1 T was successfully constructed.The final concentration of IPTG in dose of 1.0mmol/L was capable of inducing Der p 1 T expression,which was subjected to SDS-PAGE analysis before expression induction and purification in large scale.The results of western blot confirmed the purification of Der p1 T protein.The results of Real time PCR and Western blot showed that the expression of TNF-??IL-8?IL-25?TGF-??MCP-1and Eotaxin was induced by house dust mites extract in MLE12 cells.House dust mites extract significantly induced the phosphorylation of NF-?B and JNK.Knockdown of TLR4,MyD88 and TRAF6 significantly inhibited the expression of COX-2 and MCP-1 induced by the crude extract of house dust mite.After the specific immunotherapy,the symptom was significantly alleviated,with fewer inflammatory cells seen in mice following SIT compared to the asthma group.The results of ELISA showed that the SIT mice had significantly decreased serum antibody Ig E than those in the asthma group(P<0.01),whereas had higher serum Ig G2 a level(P<0.01).The level of IL-4 and IL-17 A in the BALF in SIT mice was lower than the asthma group(P<0.01? P<0.01),conversely,the level of IFN-? and IL-10 was higher than the asthma group(P<0.05?P<0.01).Consistently,the results of flow cytometry showed that compared with the asthma group,the number of TH1 cells in the spleen of the SIT group significantly increased(P<0.05),while the number of TH2 cells decreased(P<0.05).Meanwhile,the number of TH17 cells in the spleen of the SIT group was significantly decreased compared with that in the asthma group(P<0.05),while the Treg cells increased(P<0.05).Pathological results showed that Der p1 T recombinant proteins specific immune therapy can effectively relieve the airway inflammatory and cells infiltration,and greatly improved the lung tissue pathology.Conclusion: House dust mites extract can significantly induced the expression of TNF-??IL-8?IL-25?TGF-??MCP-1and Eotaxin in MLE12 cells.The transcription of key inflammatory factors and chemokine were activated by the activation of NF-?B and JNK signaling pathway through the Tlr4-Myd88-Traf6 axis.Der p1 T chimeric peptide vaccine specific immunotherapy for asthma mice has a efficient therapeutic effect.Our study provides a solid theoretical basis for clinical treatment of acaric allergic asthma.