Identification Of Der F 23 And Der F 39 As New Allergens Of Dermatophagoides Farina And Preliminary Study On The Sensitization Mechanism Of Aspergillus Fumigatus Rnase T2

The house dust mites(HDM)are common allergen sources worldwide.They are directly related to human allergic diseases and are the main allergens causing allergic diseases.As so far,32 of the 37 internationally recognized HDM allergen groups have been identified in Dermatophagoides farina(Der f).Our team sequenced the dust mite genome and transcriptome by the fourth-generation nanopore sequencing technology and discovered new allergens Der f 23 and Der f 39.Der f 23 is another major allergen of dust mites and has the same allergenicity as house dust mites Der p23.Der f 39 is a homologous protein of Troponin C.Troponin C is a protein essential for calcium to regulate bone and myocardial contraction and is one of the most important allergen families.In addition,we also found a protein that promotes the Th2 response in Aspergillus fumigatus,which is RNase T2.It promotes macrophage M2 polarization and stimulates the increase of regulatory T cells(Tregs),thereby promoting Th2 response.Objective: To obtain Der f 23 and identify its allergenicity by bioinformatics analysis;To compare the difference in allergenicity between Der f 23 and Der p 23;To express Der f 39 recombinantly by prokaryotic expression system and identify it Allergenicity;Recombinant expression of Aspergillus fumigatus RNase T2 by prokaryotic expression system and renaturation;To explore the stimulation of RAW264.7 macrophages and Tregs by Aspergillus fumigatus RNase T2,and to explore its sensitization mechanism.Methods: The dust mites RNA was extracted and reverse transcribed into c DNA to construct an expression vector.The expression and purification of Der f 23 and Der f39 were carried out by prokaryotic expression system;Confirmed the allergenicity of Der f 23 by ELISA,Western Blot,Dot Blot,hexosaminidase release test,and pathological observation of lung pathology;Analyzed and compared the Der f 23 and Der p 23 sequences by bioinformatics,and explored the Ig E binding ability differencebetween Der f 23 and Der p 23 by ELISA,Western Blot,Dot Blot and other experiments;Allergenicity of Der f 39 was confirmed by ELISA,Western Blot,Dot Blot and other experiments.Expression,purification and renaturation of Aspergillus fumigatus RNase T2 by prokaryotic expression system;Stimulation of RNase T2 to RAW264.7 macrophages in vitro;Stimulation of RNase T2 to mouse Tregs in vitro;Stimulation of RNase T2 to mouse Tregs in vivo.Result:Part 1: Successfully cloned,expressed and purified Der f 23 with prokaryotic expression system,and Der f 23 is about 26 k Da;Demonstrated that Der f 23 has Ig E binding activity both in vivo and in vitro,and named it as the main allergen of dust mites in the WHO/IUIS nomenclature;Demonstrated that the P2 region of Der f 23 can affect the Ig E binding capacity of Der f 23;Successfully cloned,expressed and purified Der f 39 with prokaryotic expression system,and Der f 39 is about 18 k Da;Demonstrated that Der f 39 has Ig E binding activity and named it in the WHO/IUIS allergen named tissue.Part 2: Successfully cloned,expressed and purified Aspergillus fumigatus RNase T2 by prokaryotic expression system;Successfully renatured RNase T2;Demonstrated that RNase T2 promotes M2 polarization on RAW264.7 macrophages;Demonstrate that RNase T2 stimulates the growth of Tregs cells both in vivo and in vitro.Conclusion: Successfully expressed Der f 23 and named it in the WHO/IUIS nomenclature;Proved that the P2 region of Der f 23 can affect the Ig E binding ability of Der f 23;Successfully expressed Der f 39 and WHO/IUIS nomenclature;Successful expression of Aspergillus fumigatus RNase T2 and its renaturation;Proved that RNase T2 has a role in promoting Th2 response.