Prokaryotic Expression,immunogenicity And Sensitization Mechanism Of Dermatophagoides Farinae Acidic Ribosomal P2 Protein

Background: In recent years,the incidence of allergic diseases has been increasing.Allergic diseases are further divided into allergic asthma,atopic dermatitis,rhinitis and the like.Allergic asthma is caused by indoor inhalation allergens.The most common indoor inhalation allergens are dust mites,especially dust mites and house dust mites.At present,a variety of allergen proteins have been cloned and expressed from the dust mites by molecular biology methods,among which the dust mites acidic ribosomal protein P2(ALT)is a newly discovered dust mite allergen.In this study,we cloned the dust mites acidic ribosomal P2 protein by E.coli prokaryotic expression system,and obtained higher purity recombinant ALT protein by affinity chromatography,and identified its immunogenicity,and then passed the animal.A mouse model was established to confirm that the recombinant acidic ribosome P2 protein is a novel allergen,and the sensitization mechanism of ALT was explored by cell experiments.Objective: To clone and express the dust mites ALT protein and identify its immunogenicity and sensitization mechanism.Methods:(1)The ALT gene of Dust mites allergen was synthesized by genetic engineering technology,and then ligated with p ET-32 a vector and transferred into Escherichia coli E.coli BL21 to induce expression,and purified by affinity chromatography to obtain protein;(2)Indirect immunoenzyme-linked assay(ELISA),clinical skin prick test(SPT)to detect the immunogenicity of recombinant protein ALT;(3)animal model confirmed the sensitization of recombinant ALT protein;(4)Cell and animal experiments explored the sensitization mechanism of ALT protein.Results:(1)SDS-PAGE results showed that the molecular weight of recombinant ALT protein was about 27 k Da.(2)Clinical skin prick test showed that 3 of the 20 dust mite allergic patients were allergic to recombinant ALT,the positive rate was 15%;ELISA results showed that the recombinant protein ALT can specifically bind to serum Ig E antibody in patients with dust mite allergies,and the positive rate is 20%;(3)Animal experiments showed that recombinant ALT protein can cause allergic asthma symptoms in mice,imbalance of Th1/Th2 in the body,and elevated Th2 type cytokines such as IL-4 and IL-13,leading to allergies;(4)In vitro cell experiments confirmed that ALT can activate the PI3 K / Akt pathway to induce the increase of IL-13 expression,thereby up-regulating the expression of JAK-1 and leading to phosphorylation of STAT-6,which induces the characteristics of Th2 regulatory factors GATA-3 and Th2.The expression of characteristict cytokines causes an imbalance of Th1/Th2,which leads to allergies.Conclusion: The allergenic dust mites recombinant protein ALT was cloned,expressed and purified,and the sensitization of recombinant ALT was confirmed by cell experiments and animal experiments,indicating that ALT is a new allergen.