户尘螨Ⅱ类变应原Der p2 T细胞表位融合基因的克隆和原核表达  

Cloning and Prokaryotic Expression of Fusion Gene of Group Ⅱ Allergen Der p2 T Cell Epitope from Dermatophagoides pteronyssinus

作　　者：段彬彬[1] 宋红玉[1] 李朝品[1] 

机构地区：[1]皖南医学院医学寄生虫学教研室,芜湖241000

出　　处：《中国寄生虫学与寄生虫病杂志》2015年第4期264-268,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.81270091;30872367)~~

摘　　要：目的 原核表达、纯化户尘螨（Dermatophagoides pteronyssinus）主要变应原Der p2的T细胞表位融合肽。 方法 将已报道的户尘螨Der p2中编码4个T细胞表位（T1-T4）的核苷酸序列以T1-T2-T3-T4的方式连接，人工合成为嵌合基因，命名为Der p2 T。PCR扩增目的基因Der p2 T，将纯化的扩增片段克隆至pET-28a（＋）载体，构建原核重组表达质粒pET-28a（＋）-Der p2 T，并进行双酶切验证。大量诱导表达含pET-28a（＋）-Der p2 T的E. coli BL-21菌株，异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，经Ni-NTA亲和层析获得纯化重组蛋白，并进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹（Western blotting）分析。ELISA法检测重组Der p2 T细胞表位融合肽对户尘螨过敏患者血清IgE抗体的结合能力。 结果 双酶切结果表明，构建了原核重组表达质粒pET-28a（＋）-Der p2 T。SDS-PAGE分析结果显示，获得相对分子质量（Mr）为10 000的重组Der p2 T细胞表位融合肽。Western blotting分析结果显示，纯化了Der p2的T细胞表位融合肽。Der p2 T细胞表位融合肽对粉尘螨哮喘患者的血清IgE结合能力［（37.70±9.89）μg/ml］较Der p2显著降低［（85.89±9.63）μg/ml］（P＜0.01）。 结论 制备Der p2 T细胞表位融合肽。与Der p2相比，重组Der p2 T细胞表位融合肽对户尘螨过敏患者血清IgE抗体的结合能力明显降低。Objective To express and purify the T cell epitope fusion peptide of the major allergen Der p2 from Dermatophagoides pteronyssinus. Methods Nucleotide sequences reported to encode four T-cell epitopes（T1-T4） of Der p2 of D. pteronyssinus were linked in the rank of T1-T2-T3-T4. In this way, the chimeric gene was synthesized, named as Der p2 T. The gene of Der p2 T was amplified by PCR, purified, and cloned into the pET-28a（＋） vector, forming the prokaryotic recombinant expression vector pET-28a（＋）-Der p2 T. This formation was verified by double digestion. The pET-28a（＋） -Der p2 T vector was transfected into E. coli strain BL-21, and its expression was induced by addition of IPTG. The recombinant protein was purified and collected by Ni-NTA affinity chromatography, and prepared for SDS-PAGE and Western blotting analysis. ELISA was used to evaluate the binding ability of Der p2 T cell epitope fusion peptide to serum IgE from patients with house dust mite allergy. Results Double digestion results confirmed the construction of the pET-28a（＋）-Der p2 T vector. SDS-PAGE revealed the expression of recombinant Der p2 T cell epitope fusion peptide with Mr of 10 000. Western blotting confirmed the purification of Der p2 T cell epitope fusion peptide. The binding ability of Der p2 T cell epitope fusion peptide to serum IgE from patients with house dust mite allergy [（37.70±9.89） μg/ml] decreased significantly in comparison to that of Der p2 [（85.89±9.63） μg/ml]（P〈0.01）. Conclusion The Der p2 T cell epitope fusion peptide is prepared, and its binding ability to serum IgE from patients with house dust mite allergy significantly decreases than that of Der p2.

关 键 词：户尘螨 Ⅱ类变应原 T细胞表位 融合肽 蛋白质印迹 

分 类 号：R384.4[医药卫生—医学寄生虫学]