The Effect Of Der F1 T Cell Epitope Vaccine Specific Immunotherapy For Asthmatic Mice And STAT4/STAT6 Signal Pathway

Objective:To investigate the Der f IT cell epitope fusion peptide vaccine for immunotherapy of allergic asthma in mice and the effect of STAT4/STAT6 signal pathway.Methods:1, Eight T-cell epitopes from Derf 1 gene were synthesized, including genes of T1 (116-136)、T2(142-162)、T3(173-192)、T4(226-246)、T5(247-255、T6(256-273)、T7(298-315)、T8(316-321), using molecular biological methods to synthesize epitope chimeric gene by the sequence of T1-T2-T3-T4-T5-T6-T7-T8 tandem nucleotide, named Der f 1T. The fusion gene was cloned into pET28a (+),to construct pET28a (+)-Der f 1 T recombinant vector,and verificated by restriction endonuclease, The vectors were transformed into the E. coli cell lines BL21 (DE3).Expression the Der f 1 T induced by different concentrations of IPTG in small doses, to determine the optimal concentration, then expression and purification Der f IT in high dose under the conditions of the optimum concentration IPTG. Analysis and identification of expressed protein by SDS-PAGE, Western bloting.2, Use the dust mite extract allergens, to construct a murine model of allergic asthma,30 mice were randomly divided into three groups, PBS group, asthma group and immune therapy group. The purified Der f 1 T cell epitope fusion peptide vaccine was specific immunotherapy for asthma mouse model. The mice were sacrificed in each group of and were collected respectively. Detected the cytokines IL-13 and IFN-gamma content by ELISA in the Spleen cells culture supernatant and BALF and serum specific IgE (sIgE) and the level of IgG2a. Extraction lung tissue protein in each group underwent Western bloting, identification expression of STAT4/STAT6 protein.Results:Double enzyme digestion and sequencing results showed that the prokaryotic expression vector pET28a (+)-Der flT was successfully constructed; SDS-PAGE showed that Der flT induced and purified successfully; Western bloting further indicated that Der flT protein was purified successfully; The lung tissue pathological examination shows that the immune inflammatory reaction in mice in treatment group was significantly better than in asthma group inflammation, reduce infiltration of inflammatory cells. The number of eosinophils in BALF is that immunotherapy group eosinophil count was significantly lower than that of asthma group (P<0.01). Immunotherapy group of mice in BALF cytokine IL-13 content is significantly decreased compared asthma group(P< 0.01), IFN-y content changes with different, immunotherapy group was significantly higher than that of asthma group (P< 0.01). Spleen cells culture supernatant were detected the content of IL-13,immunotherapy group were significantly lower than those in asthma group (P< 0.01), the change of IFN-gamma content with different, immune therapy group was significantly higher than the asthma group (P< 0.01). The content of serum specific IgE in immune therapy group was significantly lower than that of asthma group (P< 0.01), IgG2a level was different with IgE changes, the immune treatment group were significantly higher than asthma group (P< 0.01).Conclusion:Der f 1 T cell epitope recombinant protein was expressed successful, and the protein can alleviate the inflammatory reaction of asthmatic mice effectively and correct the imbalance of Thl/Th2.