Design Of A ProDer P1 T Cell Epitope Chimeric Peptide From Dermatophagoides Pteronyssinus By The MHC Class Ⅱ Pathway Of Antigen Presentation And Analysis Of Its Effectiveness For Specific Immunotherapy

Objective: To construct recombinant vector p ET28a(+)-TAT-Ihc-DPTCE for expressing TAT-Ihc-DPTCE and explore its effect for allergen specific immunotherapy.Methods: 1. Three c DNA fragments were synthesized, including the TAT c DNA, the c DNA encoding the first 1-110 amino-acids of the invariant chain(Ih C) for targeting the fusion protein via MHC class II pathway and the fragment encoding a chimeric peptide containing five T-cell epitopes from Pro Der p1 gene. The fused genes were constructed, and cloned into the prokaryotic expression vector p ET28a(+), respectively. 2. The vectors were transformed into the E. coli cell lines BL21(DE3), respectively. Recombinant proteins, DPTCE, TAT-DPTCE and TAT-Ihc-DPTCE, were expressed and purified after induction with IPTG. 3. After preparing murine model of asthma with allergen extract from Dermatophagoides pteronyssinus, the asthma models were performed for specific immunotherapy using DPTCE, TAT-DPTCE and TAT-Ihc-DPTCE, respectively. Eosinaphil in the bronchoalveolar lavage fluid(BALF) was counted under microscopy. The levels of cytokines were measured by ELISA, including IFN-γ, Interleukin(IL)-13, IL-10 and TGF-β in the BALF, as well as serumantibodies of Ig E, Ig G1 and Ig G2 a. Lung tissues were also detected by histologic examination.Results: The recombinant vectors, p ET28a(+)-DPTCE, p ET28a(+)-TAT-DPTCE and p ET28a(+)-TAT-Ihc-DPTCE were constructed successfully. The recombinant proteins, DPTCE, TAT-DPTCE and TAT-Ih C-DPTCE were expressed successfully. 3. The effect of ASIT: compared with asthma group, the count of EOS in the BALF was decreased in the Pro Der p1 group, DPTCE group, TAT-DPTCE group, and TAT-Ih C-DPTCE group, respectively(p < 0.01). The levels of IFN-γ, IL-10 and TGF-β were increased significantly, compared with asthma group(p < 0.01), but the levels of IL-13 in BALF from every group for immunotherapy were lower than asthma group(p < 0.01). The levels of antigen-specific Ig E and Ig G1 in sera from every group for immunotherapy were decreased significantly than asthma group(p < 0.01), but the levels of Ig G2 a were higher than asthma group(p < 0.01). Compared with the asthma group, infiltration of inflammatory cells was alleviated in the lung tissues from treatment group(p < 0.01). Meanwhile, the effect of TAT-Ih C-DPTCE as vaccine for SIT was superior to those of TAT-DPTCE and DPTCE(p < 0.01).Conclusion: The chimeric protein TAT-Ih C-DPTCE、DPTCE and TAT-DPTCE are effective for asthma models, in which the TAT-Ih C-DPTCE is the best.