Primary Identification Of Dust Mite New Allergen And Analysis Crystal Structure Of Der F23

Dust mites are the most widely distributed allergens in the world,which are highly related to the occurrence of asthma and are the most major allergens in the world.Dermataatophagoides pteronyssinus?Der p?and Dermatophagoides farinae?Der f?are the main mite species that induce allergic reactions.At present,37 species of Der f allergens have been isolated and identified,all of which are proteins.In the previous work,our research team completed the second generation genome and transcriptome sequencing of D.farinae and the preliminary assembly of its map,and discovered a new allergen,ubiquinone cytochrome c reductase binding protein?UQCRB?homologue,named Der f24.However,there is a rich microbial flora in the intestine of dust mites,which affects the purity of the dust mite genome extraction.Therefore,there is need a genome extraction method that reduces the number of microbial flora to improve the quality of the genome map assembly of the dust mites.In addition,the use of novel methods to screen dust mite new allergen components for the pathological understanding of dust mite allergic diseases,diagnosis and treatment has a substantive guiding significance.Our research team found that Der f23 has a strong allergenicity,and the protein structure can be resolved through crystallization to deepen the understanding of major allergens and enrich the theoretical basis for the development of allergies to dust mites.Objective:1.To establish a high-purity DNA dust mite genome extraction method:isolation and extraction of DNA from dust mites for DNA sequencing;to compare the differences in the number,content,and species of microorganisms in dust mite eggs and dust mites;third generation DNA sequencing of dust mite transcriptome;2.Screening dust mites and new allergen components:establish a dust mites cDNA library;blast with WHO/IUIS allergen database for homology;prokaryotic expression system for expression and purification of recombinant protein and identification of allergenicity;3.Der f23 protein structure analysis:recombinant protein crystal exploration and structural analysis.Methods:1.Culture and purification of dust mite species,isolation of soybean meal,eggs,and dust mites;identification of dust mite species by PCR;2.Isolation of RNA from dust mites,reverse transcription into cDNA,and detection of cDNA quality;3.Dust mite eggs genome sequencing;4.Randomly selected 1 million dust mites egg genome and dust mite genome sequences,compared with the NCBI nt database to analyze the differences in microbial content between the two genomes;5.Dust mite transcriptome was compared with the WHO/IUIS allergen database to blast for the dust mites transcriptome sequences;6.Eligible DNA sequences were extracted from the cDNA library,expressed and purified,and the new allergen was identified by WB and ELISA;7.Der f23 protein expression and purification,exploration of crystallization conditions,and analysis of crystal statistics.Results:1.Successfully established a high-purity DNA dust mite genome extraction method:?1?.Nested PCR identification of artificially cultivated dust mite is pure dust mite without house dust mite pollution,and the isolated dust mite DNA without soy flour DNA pollution;?2?.Successful isolation of dust mites and dust mites eggs,dust mites eggs extracted DNA sequencing preliminary splicing to obtain 79 MB size genome;?3?.Dendrobium dust group genome bacteria accounted for 22.62%,mainly proteobacteria?19.24%?,Bacteroides?2.31%?,hard-walled bacteria?0.78%?,eukaryotes accounted for 76.90%,mainly arthropods?52.83%?,chordate?5.74%?,nematodes?4.51%?,etc.;dust mite eggs genome bacteria accounted for 1.55%,mainly proteobacteria?0.58%?,bacteroides?0.63%?,hard-walled bacteria?0.18%?,eukaryotes accounted for 98.34%,mainly arthropods?69.55%?,notochord?6.99%?,nematodes?5.66%?,etc.The overall dust mite egg genome was 93%lower than the dust mite genome and the eukaryotic content was increased by 27%.2.Homology comparison method was used to screen the dust mite new allergen components:?1?.Dust mite RNA was successfully extracted and reverse transcribed into cDNA.The cDNA mass was qualified by agarose gel electrophoresis and Agilent determination,and a cDNA library of mite dust mites was established;?2?.Dust mite transcriptome was compared with WHO/IUIS allergen database to obtain 31 DNA sequences,and 11 strong IgE-reactive sequences were screened;?3?.Specific primers were designed based on 11 potential allergen DNA sequences,successfully from dust 6genes were amplified from the cDNA library and expressed and purified by prokaryotic expression system.?4?.Specific IgE binding activity was determined by immunoblot,dot blot,and ELISA.A new dust mite allergen was primary identified.3.Der f23 protein structure analysis:?1?.The optimal crystallization conditions of PEG/ION 44?0.2M MgCl₂,0.1 Na₃C₆H₅O₇·2H₂O pH 5.0,2%PEG 2000?were selected through multiple crystallization conditions;?2?.Der f 23 C terminal?PCKFYICSNWEAIHKSCPGNTRWNEKELTCT?growth protein crystal?PDB accession number:5ZJL?,1.7?resolution,size 0.2 mm×0.1 mm×0.1 mm,contains two?-sheets and one?-structure.Conclusion:1:The bacteria content of dust mite eggs is extremely low,which is90%lower than that of dust mite bodies genome.2:A new dust mite allergen was preliminarily identified by biological information technology.3:The C terminal protein structure of Der f23 was initially analyzed.