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.