粉尘螨重组变应原Der f8的表达、纯化和免疫学鉴定

Expression,Purification and Immunological Identification of Recombinant Allergen Der f8in Dermatophagoides Farinae

目的获得大量粉尘螨重组变应原Der f8蛋白,检测其免疫原性。方法合成粉尘螨第8组变应原(Der f8)基因,将其连接至pET-32a载体,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达重组Der f8蛋白,圆二色谱初步检测该蛋白二级结构,以粉尘螨过敏患者血清作为一抗,经免疫印迹法(Western-blotting)方法分析Der f8的免疫学特性。结果重组工程菌经IPTG诱导后,高效表达出Der f8蛋白,为可溶性蛋白。SDS-PAGE结果显示,表达产物分子质量约为45ku。Der f8重组蛋白二级结构中,α螺旋34.5%,β折叠12.6%,无规则卷曲53.9%。表达产物经亲和层析纯化后,Western Blot印迹有明显条带显示。结论纯化后的Der f8具有较高的纯度和较强的免疫学活性,为粉尘螨过敏反应性疾病的特异性诊断和治疗及进一步的实验研究奠定了基础。

Objective To obtain a lot of recombinant allergen Der f8 protein from Dermatophagoides farinae,and to detect its immunogenicity.Methods The gene encoding group 8 allergen of Dermatophagoides farinae （Der f8）was synthesized and introduced into pET-32 vector.The expression of recombinant Der f8 protein was induced by IPTG.The secondary structure of Der f8 was detected by circular dichroism spectrum.The immunological characteristics were detected by Western blot assay using the patient’s sera as primary antibody.Results After IPTG induction, the engineering bacteria expressed soluble recombinant Der f8 protein.SDS-PAGE showed a band at 45 ku.The secondary structure of Der f8 included 34.5% of α-helix,12.6% of β-sheet and 53.9% of random coil.After purification by affinity chromatography,Western blot detection of the expression product showed obvious strip.Conclusion The purified Der f8 has high purity and immunological activity.It can lay a foundation for the specific diagnosis,treatment and further experimental studies of dust mite allergy disease.