中国免疫学杂志2024，Vol.40Issue(8)：1744-1748,1754,6.DOI:10.3969/j.issn.1000-484X.2024.08.027
粉尘螨变应原Der f 36重组蛋白制备及其与Der p 36的交叉反应性研究
Preparation of Der f 36 and its cross-reactivity with Der p 36
摘要
Abstract
Objective:To prepare recombinant protein of group 36 allergen of Dermatophagoides farinae(Der f 36),to deter-mine its immunogenicity and bioinformatics analysis was performed.Methods:Nucleic acid sequence coding for Der f 36 was obtained and artificially synthesized,pET-28a(+)was inserted to construct pET-28a(+)-Der f 36 plasmid and transformed into BL21(DE3)receptor cells.After expressed and purified in BL21(DE3),recombinant allergen rDer f 36 was obtained,recombinant protein rDer f 36 was identified by SDS-PAGE and Western blot.Serum IgE binding rates of rDer f 36 was determined by IgE-ELISA.Cross-reactivity between rDer f 36 and rDer p 36 was detected by IgE-ELISA inhibition assay.HNEpC cells were cultured with rDer f 36 for 24 h and cytokines were detected.Bioinformatics softwares were used to analyze physicochemical properties and structures of Der f 36 and Der p 36.Results:Coding gene for Der f 36 was obtained with a total length of 690 bp,whose molecular weight was 25.6 kD;serum IgE binding rate of rDer f 36 was 42.1%by IgE-ELISA;IgE-ELISA inhibition assay showed that rDer p 36 had 40%(8/20)inhibition rate(>50%)for rDer f 36,with an average inhibition rate of 52.98%.Compared with control group,HNEpC cells cultured with rDer f 36 showed that IL-6,IL-8,IL-33,IL-25 and TSLP expressions had increased;bioinformatics analyses show that sequence consistency of Der f 36 and Der p 36 was 77.63%,with similar physicochemical properties.Secondary structure analysis showed that both of them contained α helix,β-turn and random coils,and content of random coils was the highest;structure of C2 domains was also highly overlapping(RMSD=0.046).Conclusion:Recombinant allergen rDer f 36 is successfully prepared with good immunogenicity,laying foundation for diagnosis and treatment of dust mite allergen single component.High similarity in physical and chemical properties,secondary structure,and tertiary structure between Der f 36 and Der p 36 determines their cross reactivity.关键词
Der f 36/免疫原性/生物信息学/Der p 36Key words
Der f 36/Immunogenicity/Bioinformatics/Der p 36分类
医药卫生引用本文复制引用
任雅宁,廖媛芬,周冬梅,崔玉宝,顾晓虹,周鹰,张健..粉尘螨变应原Der f 36重组蛋白制备及其与Der p 36的交叉反应性研究[J].中国免疫学杂志,2024,40(8):1744-1748,1754,6.基金项目
国家自然科学基金(81971511,31572319,31272369) （81971511,31572319,31272369）
无锡市太湖人才计划高端人才项目(2020THRC-GD-7) （2020THRC-GD-7）
无锡市科技局"太湖之光"科技攻关项目(Y20212006) （Y20212006）
无锡市卫生计生科研重大项目(Z201701). （Z201701）
