Cloning And Expression Of House Dust Mite Allergen Der P 37 And The Allergic Asthma Mechanism

Background : Dust mite is a common indoor allergen in the world.Their secretions,excretions,and skin shell fragments float in the air in the form of dust particles,which is an important allergen causing allergic diseases.The dust mites that can cause allergic reactions in human body are mainly divided into several categories at home and abroad,of which Dermatophagoides farinae and Dermatophagoides pteronyssinus are the most common.The allergen composition of house dust mite is very rich,including many secondary allergens in addition to main allergens,their biological characteristics and sensitization mechanism are not clear.In this study,we cloned and expressed the house dust mite allergen Der p 37 protein using the E.coli prokaryotic expression system,and obtained a higher purity concentration of recombinant Der p 37 protein by affinity chromatography and identified its immunogenicity.Then we established mouse models to confirm that Der p 37 protein is a new allergen,and the sensitization mechanism of Der p 37 was explored by transcriptome sequencing.Objective: To clone and express the house dust mite allergen Der p 37 protein,identify its immunogenicity,and explore its sensitization mechanism.Methods:(1)Using prokaryotic expression system to clone and express,and purified the purified Der p 37 by affinity chromatography;(2)Bioinformatics was used to analyze its homology,and the immunogenicity of recombinant protein Der p 37 was detected by ELISA and western blot using the patients’ serum;(3)The sensitization of the recombinant Der p 37 protein was confirmed by animal models;(4)Transcriptome sequencing was used to explore the sensitization mechanism of Der p 37 protein..Results:(1)The recombinant house dust mite allergen Der p 37 protein with high concentration and purity was successfully cloned,expressed and purified by E.coli prokaryotic expression system.The ELISA and western blot experiments with serum from patients with dust mite allergies proved that Der p 37 has sensitization,and the binding rate with serum is about 16.7%,it is a minor allergen of house dust mite;(2)By constructing a mouse model of Der p 37 allergic asthma,Der p 37 can increase specific IgE in the mouse serum,increase the expression level of IL-4,and significantly increase the level of IL-17.At the same time,neutrophils increased in mouse bronchoalveolar lavage fluid.Lunghistopathological sections showed infiltration of inflammatory cells and hypersecretion of mucus in mice,indicating that Der p 37 protein could induce allergic asthma in mice;(3)Transcriptome sequencing analysis showed that there were up-regulated or down-regulated changes in several genes between Der p 37 sensitized mice and normal blank mice,and most of the genes were immune-related,among which IL-17 gene was significantly different.KEGG pathway analysis also showed that Der p 37 significantly altered the IL-17 signaling pathway,confirming that the mechanism of Der p 37 induced allergic asthma in mice was related to IL-17 and Th17.Conclusion: Der p 37,an allergen of house dust mite,was successfully cloned and purified by prokaryotic system.The recombinant Der p 37 protein was proved to induce allergic asthma through animal experiments.The sensitization mechanism of Der p 37 protein was related to IL-4 and Th2 as well as IL-17 and Th17,indicating that Der p 37 was a new secondary allergen of dust mite.