| The house dust mite(HDM)species Dermatophagoides pteronyssinus(Der p)and Dermatophagoides farinae(Der f)are major sources of indoor inhaled allergens and highly correlated with the development of allergic diseases.Detection of allergen-specific immunoglob?lin(Ig)E is an important method for diagnosing IgE-mediated allergic diseases.The sensitivity of the indirect IgE-ELISA method against allergen extracts is limited by interference from high IgG titers and low quantities of effectual allergen components in extracts.To overcome these limitations,a novel capture IgE-ELISA based on recombinant Der f 1/Der f 2 fusion protein(rDer f 1/2)was developed to improve the sensitivity of serum IgE detection in patients with dust mite allergy.The previous work of the research team has completed the sequencing and assembly of the dust mites genome and transcriptome by NGS(second-generation)sequencing technology.We found that a group 24 Der f allergen,namely Der f 24,was a ubiquinol cytochrome C reductase binding(UQCRB)protein homologue.The homology of the 24 th component of the house dust mites,Der p 24,and the 24 th component of the dust mites,Der f 24,was 97%,and it was identified that Der p 24 is a major house dust mite allergen component.The World Health Organization and the International Union of Immunological Societies(WHO/IUIS)Allergen Nomenclature Subcommittee named the house dust mite 24 th protein as Der p 24(http://www.allergen.org).There are many mechanisms by which allergens cause allergic diseases,such as Der p 1 and Der p 2 are allergic to protein function,and Der p 24 is a cytochrome-binding protein,wheather through its whole molec?lar and biological functions,or directly from the epitope of a short amino acid sequence without intact biological activity.The identification of key epitopes is very important for the diagnosis and treatment of allergic diseases,providing new ideas for the development of dust mite vaccines.The research team used Nanopore sequencing technology(also known as fourthgeneration sequencing)to sequence and analyze the dust mite gene data,and completed the assembly of high-quality dust mite genome map.The house dust mite allergen Der f 37 was found and identified as the homolog of Der p 37(Petrotrophic like protein domain).Highquality Dermatophagoides farinae genome data can further promote research on dust mite species.Objective: 1.To reduce the interference of high IgG titer,developed a capture IgEELISA method based on recombinant rDer f 1/2 fusion protein to improve the sensitivity of serum IgE detection in patients with dust mite allergy,it has important significance in diagnosis and treatment of allergic diseases;2.Analyze and identify the Der p 24 IgE epitopes to explore the mechanism of diseases caused by dust mite allergens;3.Analyze the genome map of dust mites using nanopore sequencing technology,perform genomic function annotation,and assemble dust mites mitochondrial;4.The allergen database was compared with the Dermatophagoides farinae genome data to identify new allergen components of dust mites.Methods: Allergenicity analysis of the Der f 1/2 fusion protein.1.Construction of pET28a(+)-Der f 1/2 expression vector,expressed in prokaryotic system.IgE-Western blot,IgE-Dot blot and indirect IgE-ELISA methods were used to detect the IgE activity of rDer f1/2 fusion protein.2.To establish Capture IgE-ELISA method,which was compared with indirect ELISA.Identification of Der p 24 IgE epitopes.1.IgE-binding activity was detected by IgEWestern blot.According to the amino acid sequence of Der p 24,the amino acid sequences of Der p 24 N-terminal and C-terminal related truncated proteins were designed,and used the prokaryotic system cloning and expressing the relevant proteins to determine the key epitopes;2.In vitro experiments,identifying the binding activity of epitope peptides to serum IgE in allergic patients by IgE-ELISA,IgE-western blot,IgE-dot blot and other immunological methods;3.Animal experiments in vivo,Der p 24 key epitope peptides were used to sensitized Balb / c mice,detect whether mice can produce sIgE and IgG;cell experiments,to verify whether Der p 24 IgE epitope peptides can promote the release of hexosaminidase from mast cells;sensitized animal lungs tissue sections were subjected to HE staining to compare the degree of allergic inflammatory response.The fine map analysis of the Dermatophagoides farinae genome.1.Artificially purify dust mites and extract DNA;2.The genomic data of Dermatophagoides farinae were assembled and the genomic map was drawn.3.The new allergens were found in comparison with the Dermatophagoides farinae database and was expressed and purified used in prokaryotic system and was identified the allergenicity by western blot method.Results: Allergenicity analysis of the Der f 1/2 fusion protein.1.rDer f 1/2 is expressed in the form of inclusion bodies;rDer f1/2(24/28,85.8%)showing higher sIgE binding activity than rDer f1(21/28,75.0%))and rDer f2(22/28,78.6%)in dust mite allergic patients;2.In sIgE assay,the OD value of Capture IgE-ELISA(71/71,100%)was higher than the indirect ELISA(68/71,95.8%)(P < 0.01).Identification of Der p 24 IgE epitopes.1.Determination of the key N-terminal epitopes of Der p 24 in house dust mite: successfully constructed a high-efficiency expression vector of Der p 24 truncated proteins,obtained recombinant inclusion proteins with purity greater than 95%;res?lts of IgE Western blot showed that recombinant protein rDer p 24,Der p 24(1-98aa),Der p 24(1-78aa)and Der p 24(1-58aa)were strongly positive in serum of patients,while Der p 24(33-118aa)and Der p 24(21-118aa)were negative for serum from patients with dust mite allergies.2.In vitro assay: successfully designed and synthesized the Nterminal polypeptide sequences,multiple immunological methods(IgE-ELISA,IgE Western blot,IgE Dot blot),the Der p 24 N-terminal 32 amino acid residue sequence showed strong IgE binding activity with dust mite allergic patients and did not react with healthy individuals.3.In vivo assay of animal models,Der p 24 N-terminal 32 amino acid residues can produce high titer sIgE antibodies in mice,leading to significant inflammatory response in mouse lung tissues and promoting release of hexosaminidase by mast cell line RBL-2H3.4.The IgE binding activity of the 32 amino acid residues at the N-terminus of Der p 24 is stronger than that of the linear Der p 1 and Der p 2 IgE epitopes alone,and there is a significant difference.The fine map analysis of the Dermatophagoides farinae genome.1.Using the Nanopore technology to assemble a high-quality The fine map analysis of the Dermatophagoides farinae genome map:(1)extracted DNA without contaminants,no bands;(2)genome assembly without heterozygous and contaminated sequences,obtained 10,575 genes for genome annotation,and the assembly quality assessment software BUSCO integrity showed that the Dermatophagoides farinae had the highest genome and gene set quality in the published arachnids,with a completeness of 97.40%,ixodes scap?laris 78.80%,stegodyphus mimosarum was 81.20%,and tetranychus urticae was 92.40%;(3)Nanopore technology and NGS technology gene set were compared,the number of nanopore gene set is 10,575,the degree of completeness was 95.70%,and the length of gene transcript is 3,544.04 bp,the number of exons is 425.85 bp;the number of NGS gene sets is 16,467,the degree of completencess is 84.80%,the length of the gene transcript is 2,358.56 bp,and the number of exons is 414.37 bp;4 the assembly results are corrected,and the mitochondria with a length of 17,442 bp are assembled sequences,the number of chromosomes is closer to the level of the dust mites themselves.2.BLAST(Basic Local Alignment Search Tool)was compared and found to have 76% homology with Der p 37,tentatively named Der f 37,GenBank accession number MK419030.1.It has been identified that it has strong IgE binding activity to serum of patients with dust mite allergy.Conclusion: 1.Established a new capture IgE-ELISA method to improve the sensitivity of detection;2.The IgE epitope of Der p 24 of house dust mite allergen 24 is mainly located at the N-terminal 32 amino acid residue region.The results of this study may have important theoretical significance for the development of diagnostic and therapeutic agents for dust mite allergic diseases;3.High quality dust mite genome obtained by Nanopore sequencing technology;4.Screening and identification of new dust mite allergen Der f 37. |
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