重组粉尘螨变应原rDer f 27制备及其活性鉴定

Preparation and activity identification of recombinant allergen rDer f 27 from Dermatophagoides farina

目的:制备粉尘螨变应原第27组分(Der f 27)重组蛋白,鉴定其免疫活性。方法:构建pET-28a(+)-Der f 27质粒,转化至E.coli BL21(DE3)感受态细胞,经诱导表达和纯化后,获得重组变应原rDer f 27。IgE-ELISA和IgE-Western blot检测rDer f 27与粉尘螨变应性鼻炎患者血清IgE结合率,分别将变应性鼻炎患者PBMC、人支气管上皮细胞BESA-2B与rDer f 27共孵育24 h后测定细胞因子表达;生物信息学软件分析Der f 27的理化性质和结构。结果:成功构建pET-28a(+)-Der f 27质粒并转化至BL21(DE3)细胞,经IPTG诱导表达和纯化后,SDS-PAGE和Western blot在约48 kD处见特异性条带;IgE-ELISA和IgEWestern blot检测rDer f 27与粉尘螨变应性鼻炎患者血清IgE结合率分别为39.5%和45.5%;与对照组相比,变应性鼻炎患者PBMC与rDer f 27共孵育后,IL-6和IL-8表达升高(P<0.05);BESA-2B细胞与rDer f 27共孵育后,IL-10和TGF-β表达降低,IL-17A和IL-23A表达升高(P<0.05);生物信息学结果显示Der f 27属于丝氨酸蛋白酶抑制物家族,具有此家族通用结构及功能,二级结构主要为α螺旋(42.62%)和无规则卷曲(35.60%)。结论:成功制备了具有较好免疫反应性和免疫原性的重组变应原rDer f 27,并揭示其为变应性鼻炎的重要变应原之一。

Objective:To prepare recombinant protein of group 27 allergen of Dermatophagoides farinae(Der f 27),and to determine its immunoactivity.Methods:pET-28a(+)-Der f 27 plasmid was constructed and inserted into E.coli BL21(DE3)cells.After being expressed and purified,recombinant allergen rDer f 27 was obtained.IgE binding rates of rDer f 27 with sera from patients with allergic rhinitis induced by Dermatophagoides farinae was determined by ELISA and Western blot.PBMC from patients with allergic rhinitis and BESA-2B cells were cultured with rDer f 27 for 24 h,respectively,and cytokine expression was measured.Bioinformatics softwares were used to analyze physicochemical properties and structures of Der f 27.Results:pET-28a(+)-Der f 27 plasmids were prepared successfully and transformed into BL21(DE3)cells.After expressed and purified with PTG,SDS-PAGE and Western blot identified a band about 48 kD.IgE binding rates of rDer f 27 were 39.5%and 45.5%with sera from patients with allergic rhinitis allergic to Dermatophagoides farinae by IgE-ELISA and IgE-Western blot,respectively.Compared with control group,IL-6 and IL-8 expressions were increased in PBMC from patients with allergic rhinitis being cultured with rDer f 27(P<0.05);expressions of IL-10 and TGF-βwere decreased in BESA-2B cells being cultured with rDer f 27,while IL-17A and IL-23A expressions were increased.Bioinformatics analysis showed that Der f 27 belong to serine protease inhibitor family and had universal structure and func⁃tion of this family.Secondary structure of Der f 27 was mainly composed ofα-helix(42.62%)and random coil(35.60%).Conclusion:Recombinant allergen rDer f 27 has been prepared successfully with good immunoreactivity and immunogenicity,becoming one of important allergens of allergic rhinitis.