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

目的:制备粉尘螨变应原第36组分(Der f 36)重组蛋白,鉴定其免疫原性并进行生物信息学分析。方法:获得Der f 36核酸序列并进行人工合成,插入pET-28a(+)载体,制备pET-28a(+)-Der f 36质粒,转化至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 36,SDS-PAGE和Western blot鉴定rDer f 36重组蛋白,IgE-ELISA检测其血清IgE结合率,IgE-ELISA抑制实验检测rDer f 36与rDer p 36的交叉反应性;HNEpC细胞与rDer f 36共孵育24 h后测定细胞因子;生物信息学软件比较Der f 36与Der p 36的理化性质和结构。结果:得到了Der f 36的编码基因,全长690 bp,相对分子质量为25.6 kD;IgE-ELISA鉴定rDer f 36血清IgE结合率为42.1%;IgE-ELISA抑制实验结果显示rDer p 36对rDer f 36的抑制率>50.00%[40.00%(8/20)],平均抑制率为52.98%。HNEpC细胞与rDer f 36共孵育后,IL-6、IL-8、IL-33、IL-25和TSLP表达均高于对照组。生物信息学分析显示Der f 36和Der p 36的序列一致性为77.63%,理化性质较为相似;二级结构分析结果显示二者均含有α螺旋、β转角和无规则卷曲,且无规则卷曲含量最高;C2结构域显示高度重叠(RMSD=0.046)。结论:成功制备了rDer f 36重组蛋白,且具有免疫原性,为粉尘螨变应原单组分诊断及治疗奠定了基础。Der f 36与Der p 36的理化性质、二级结构和三级结构高度相似,决定其具有交叉反应性。

Objective:To prepare recombinant protein of group 36 allergen of Dermatophagoides farinae(Der f 36),to determine 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 containedαhelix,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.