Construction of a vector encoding T-cell epitopes of Dermatophagoides pteronyssinus major allergen group 1 as a vaccine delivered by MHC class II pathway
10.3969/j.issn.1673-4254.2015.02.04
- VernacularTitle: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
- Author:
Beibei ZHAO
1
;
Yuxin JIANG
;
Jidong DIAO
;
Na LI
;
Wei LU
;
Chaopin LI
Author Information
1. 皖南医学院医学寄生虫学教研室
- Keywords:
Dermatophagoides pteronyssinus;
fused peptide vaccine;
major group 1 allergen;
prokaryotic expression;
T cell epitope;
IgE-binding assay
- From:
Journal of Southern Medical University
2015;(2):174-178
- CountryChina
- Language:Chinese
-
Abstract:
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.
