Construction and expression of Dermatophagoides pteronyssinus group 1 major allergen T cell fusion epitope peptide vaccine vector based on the MHC II pathway

卷:32

Li, Chaopin ; Zhao, Beibei ; Jiang, Yuxin ; Diao, Jidong ; Li, Na ; Lu, Wei

关键词: Dermatophagoides pteronyssinus;    Fused peptide vaccine;    Major group 1;    Allergen;    Prokaryotic expression;    T cell epitope;    IgE-binding assay;   

DOI  :  10.3305/nh.2015.32.5.9613

学科分类：食品科学和技术

【 摘 要 】

Backgound and aims: Dermatophagoides peteronyssinus is one of the important house dust mites responsible for allergic asthma that can be tentatively managed by specific immunotherapy. The present study was 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 p1-3T 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 p1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing, and the expression of the recombinant protein TAT-IhC-Der p1-3T was induced in E. con. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p1. Conclusion: we successfully constructed the recombinant expression vector pET-28a-TAT-IhC-Der p1-3T expressing a T-cell epitope vaccine delivered by MHC 11 pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.