Construction And Expression Of A Chimeric Gene With T-/B-cell Epitopes From Major Allergen Group1Dust Mites And Study On Their Effectiveness For Allergen-specific Immunotherapy

Objective To construct and express a chimeric gene with T-/B-cell epitopes of the major allergen group1from Dermatophagoides farina (Der f1) and To investigate the effect of immunotherapy of recombinant chimeric epitopes of major allergen group1from Dermatophagoides farina on asthma of mice.Methods (1)Two chimeric genes, Der f1A and Der f1B, were synthesized as B1-T1-B2-T2-B3-T3-B4-T4-B5-T5-B6and B1-B2-B3-B4-B5-B6-T1-T2-T3-T4-T5pattens. Two recombinant vectors, pET-28a(+)-Der f1A and pET-28a(+)-Der f1B, and identified via digestion by restriction endonucleases BamH I/Xho I,were constructed for prokaryotic expression. These chimeric genes were induced by1mmol/L IPTG (final concentration), digested with restriction enzymes and sequenced. The chimeric proteins were analyzed by SDS-PAGE and Western blot.(2)animal experiment:Fifty mice were randomly divided into5groups:A of groups:a negative control group, B of groups:an asthma group, C of groups:an immunotherapy group of Der f1, D of groups:an immunotherapy group of Der f1B, E of groups:an immunotherapy group of Der f1A. On the1st,7th and14th day, the mice in the asthma group, immunotherapy group of Der f1, immunotherapy group of Der f1B and immunotherapy group of Der f1A were injected intraperitoneally with the extract of D. farina3times to sensitize; and on the21st day, the atomized inhalation was carried out for7days. In the control group, phosphate buffer solution (PBS) was applied for sensitization and inhalation. In the immunotherapy groups, Der f1, Der f1Band Der f1A were applied to carry out the specific immunotherapy respectively for30min before the inhalation. Then respectively:the leukocytes in the bronchoalveolar lavage fluid (BALF) were numbered and the pathological sections of lung tissues were observed; IL-5and IFN-y in BALF and spleen cell culture supernatants (SSCS) as well as the specific IgE, IgG2a in the sera were detected.Results (1)After digestion by restriction enzymes and sequencing, the recombinant vectors were constructed successfully. The specific bands for chimeric proteins were visible by SDS-PAGE and Western blot, suggesting that these proteins were purified successfully. Further analyses were performed for IgE-binding properties of Der f1A and Der f1B to sera from patients sensitized to house dust mite, and the fused protein was also purified on a large scale; After treatment of the asthma models on specific immunotherapy basis using Der f1Aand Der f1B as vaccine.(2) In Der f1A group, Der f1B group and Der f1groups of mice lung inflammation significantly reduced than the asthma group. In Der f1A group, Der f1B group and Der f1groups of white blood cell counts in bronchoalveolar lavage fluid and the number of eosinophils was significantly decreased(P<0.01), but the Der f1A group and Der f1B group compared with the Der f1groups had no significant difference(P>0.05). IL-5ELISA method for the determination of SSCS and BALF in mice and IFN-levels; the results showed immune treatment group and asthma group comparison, IL-5BALF and SSCS levels decreased significantly (P<0.01), IFN-gamma content increased significantly (P<0.01). In the IL-5and IFN-gamma of levels that the Der f1A group and Der f1B group compared with the Der f1groups had no significant difference (P>0.05). With the immunotherapy of allergen in serum specific IgE antibody concentrations, the Der f1A group and Der f1B group compared with the Der f1groups had no significant difference (P>0.05).The levels of IgG2a, the Der f1A group and Der f1B group compared with the Der f1groups had no significant difference (P>0.05)Conclusions Two chimeric proteins are expressed successfully, which contain T-/B-cell epitopes of Der f1and provide a basis for specific immunotherapy.These fused proteins as vaccines in specific immunotherapy for the asthmatic murine models can effectively alleviate allergic inflammation of airway and lung in the mice. small molecular dust mite allergen and low toxicity of1kinds of allergens were successfully constructed, and lays a foundation for diagnosis and treatment of allergic asthma.