Construction of a vector encoding T-cell epitopes of Dermatophagoides pteronyssinus major allergen group 1 as a vaccine delivered by MHC class II pathway / 南方医科大学学报
Journal of Southern Medical University
;
(12): 174-178, 2015.
Article
in Chinese
| WPRIM
| ID: wpr-239217
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
Recombinant Proteins
/
Cysteine Endopeptidases
/
Allergens
/
Vaccines
/
Base Sequence
/
Gene Expression
/
Polymerase Chain Reaction
/
Cloning, Molecular
/
Genes, MHC Class II
Limits:
Animals
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2015
Type:
Article
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