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

Construction of a vector encoding T-cell epitopes of Dermatophagoides pteronyssinus major allergen group 1 as a vaccine delivered by MHC class II pathway

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、Ih C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-Ih C-Der p 1-3T融合基因,并插入至原核表达载体p ET-28a(+)中,构建重组原核表达载体p ET-28a(+)-TATIh C-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-Ih C-Der p 1-3T蛋白后进行Ig E结合试验。结果双酶切和测序结果表明,成功构建了p ET-28a-TAT-Ih C-Der p 1-3T重组原核表达载体;SDS-PAGE电泳分析显示TAT-Ih C-Der p 1-3T可诱导表达;Western blot检测表明该融合蛋白纯化成功;Ig E结合试验表明TAT-Ih C-Der p 1-3T结合屋尘螨过敏病人血清Ig E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p ET-28a-TAT-Ih C-Der p 1-3T载体,纯化的TAT-Ih C-Der p 1-3T具有较强的Ig E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, Ih C 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-Ih C-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector p ET28a（＋） to construct the recombinant plasmid p ET- 28a（＋）- TAT- Ih C- 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 TATIh C- Der p 1- 3T was detected by SDS- PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using Ig E- binding assay. Results The recombinant plasmid p ET- 28a- TAT- Ih C- Der p 1- 3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing and the expression of the recombinant protein TAT-Ih C-Der p 1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger Ig E- binding ability than Der p 1. Conclusion We successfully constructed a recombinant expression vector p ET-28a-TAT-Ih C-Der p 1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong Ig E-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.