经MHCⅡ通路的屋尘螨1类变应原T细胞表位融合肽疫苗载体的构建与表达

摘要

目的：构建经MHCⅡ通路的屋尘螨1类变应原Der p 1的T细胞表位肽疫苗重组载体。方法分别合成TAT、IhC和含编码Der p 1的3段T细胞表位的融合核苷酸序列，用特异性引物PCR扩增相应的基因片段，分别用相应的双酶切后，用T4 DNA连接酶连接形成TAT-IhC-Der p 1-3T融合基因，并插入至原核表达载体pET-28a(+)中，构建重组原核表达载体pET-28a(+)-TAT-IhC-Der p 1-3T，Bam HⅠ和XhoⅠ进行双酶切和测序鉴定。重组载体转化大肠杆菌E. coli BL21（DE3）菌株，IPTG诱导后，经SDS-PAGE电泳分析和Western blot验证，纯化TAT-IhC-Der p 1-3T蛋白后进行IgE结合试验。结果双酶切和测序结果表明，成功构建了pET-28a-TAT-IhC-Der p 1-3T重组原核表达载体；SDS-PAGE电泳分析显示TAT-IhC-Der p 1-3T可诱导表达；Western blot检测表明该融合蛋白纯化成功；IgE结合试验表明TAT-IhC-Der p 1-3T结合屋尘螨过敏病人血清IgE的能力强于Der p 1变应原（P<0.001）。结论成功构建了可表达经MHC通路的编码Der p 1的3段T细胞表位的重组pET-28a-TAT-IhC-Der p 1-3T载体，纯化的TAT-IhC-Der p 1-3T具有较强的IgE结合能力，从而为后续经MHC通路的特异性免疫治疗奠定基础。%Objective To construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway. Methods The nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET-28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p 1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay. Results The recombinant plasmid pET-28a-TAT-IhC-Der p 1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing and the expression of the recombinant protein TAT-IhC-Der p 1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p 1. Conclusion We successfully constructed a recombinant expression vector pET-28a-TAT-IhC-Der p 1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.