Construction of a vector encoding T-cell epitopes of Dermatophagoides pteronyssinus major allergen group 1 as a vaccine delivered by MHC class II pathway.
- Author:
Beibei ZHAO
1
;
Yuxin JIANG
;
Jidong DIAO
;
Na LI
;
Wei LU
;
Chaopin LI
Author Information
- Publication Type:Journal Article
- MeSH: Allergens; immunology; Animals; Antigens, Dermatophagoides; immunology; Arthropod Proteins; immunology; Base Sequence; Cloning, Molecular; Cysteine Endopeptidases; immunology; Dermatophagoides pteronyssinus; Epitopes, T-Lymphocyte; Escherichia coli; Gene Expression; Genes, MHC Class II; Genetic Vectors; Plasmids; Polymerase Chain Reaction; Recombinant Proteins; immunology; Vaccines; immunology
- From: Journal of Southern Medical University 2015;35(2):174-178
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway.
METHODSThe 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.
RESULTSThe 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.
CONCLUSIONWe 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.