Specific Immunotherapy Of A Murine Allergic Asthma Model To Dust Mite With Der F 1 Conjugated To SEB As The Immunomodulatory Adjuvant

Background: Allergic diseases,especially allergic asthma,are clinically common diseases.If neglected,the condition can persist for a lifetime.According to statistics,there are as many as many as 300 million people with asthma in the world,of which China accounts for at least 10%.At present,allergen-specific immunotherapy is the most effective causative treatment for allergic diseases.Dust mites are the most frequently exposed allergen in human indoor environment.Allergic asthma caused by Dermatophagoides farina with super sensitization ability deserves attention.Dust mites allergic asthma allergen-specific immunotherapy(AIT)is mainly treated with dust mite crude extract.Traditional dust mite immunotherapy is mainly treated with dust mite crude extract.It contains numerous unwanted impurities and often accompanies side effects.Therefore,it is of great significance to explore more effective treatments for specific immunotherapy.Staphylococcal enterotoxin B(SEB)is an entertoxin secreted by the Staphylococcus aureus.Previous research demonstrated that SEB could induce tolerance in allergic mice at relatively low doses.We therefore hypothesize that SEB could help to boost AIT efficacy through its immunomodulatory effects.Objective: Der f 1,the most important allergen of dust mite,was conjugated to SEB to make a fusion protein Der f 1-seb,which was highly expressed and purified to homogeneity.Cell experiments were conducted to explore the optimal concentration and the initial mechanism of inducing immune tolerance.Animal model experiments were conducted to verify the efficacy of desensitization therapy.Methods: 1)high concentrations of recombinant protein Der f 1-seb and Der f 1 and SEB proteins were obtained by purification with Ni column using prokaryotic expression system.2)Cell experiment: the expression of the phenotype Foxp3 and TGF-? and the expression of IL-10 were detected in the isolated culture of BMDC.The expression of Treg and the expression of TGF-? and IL-10 were detected by co-culture of BMDC and spleen cells.3)Animal experiment: an animal model of dust mite allergic asthma was established by using dust mite with alum adjuvant.The corresponding antibodies and cytokines were determined Western Blot,flow cytometry and other methods.Results: The result of double enzyme digestion indicated that the plasmid was successfully constructed and the recombinant proteins Der f 1-SEB,Der f 1 and SEB were obtained by the prokaryotic expression system.Der f 1-SEB can up-regulate the expression of Foxp3,TGF-? and IL-10 in BMDC.Regulatory T cells were also differentiated from T cells co-cultured with BMDC.In animal experiments,HE staining of lung tissue,cell flap of alveolar lavage fluid,Western Blot results of lung tissue and supernatant results of spleen cells all showed that Der f 1-seb had a good therapeutic effect on asthma.Conclusion: the fusion protein Der f 1-seb can effectively alleviate the symptoms of dust mite allergic asthma in mice.