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


CONCLUSIONS
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.
Department of Medical Parasitology, Wannan Medical College, Wuhu 241002, China.E-mail: 626486060@qq.com.
Beibei
B
Zhao
Yuxin
Y
Jiang
Jidong
J
Diao
Na
N
Li
Wei
W
Lu
Chaopin
C
Li
D000485:Allergens / Q000276:immunology; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000276:immunology*; D060829:Arthropod Proteins / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000276:immunology*; D040002:Dermatophagoides pteronyssinus; D018984:Epitopes, T-Lymphocyte; D004926:Escherichia coli; D015870:Gene Expression; D005802:Genes, MHC Class II; D005822:Genetic Vectors; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology; D014612:Vaccines / Q000276:immunology*
D000485:Allergens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D018984:Epitopes, T-Lymphocyte; D011994:Recombinant Proteins; D014612:Vaccines; D003546:Cysteine Endopeptidases; C451162:Dermatophagoides pteronyssinus antigen p 1