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- Title
CONSTRUCTION AND EXPRESSION OF DERMATOPHAGOIDES PTERONYSSINUS GROUP 1 MAJOR ALLERGEN T CELL FUSION EPITOPE PEPTIDE VACCINE VECTOR BASED ON THE MHC II PATHWAY.
- Authors
Chaopin Li; Beibei Zhao; Yuxin Jiang; Jidong Diao; Na Li; Wei Lu; Li, Chaopin; Zhao, Beibei; Jiang, Yuxin; Diao, Jidong; Li, Na; Lu, Wei
- Abstract
<bold>Unlabelled: </bold>Backgound and aims: Dermatophagoides peteronyssinus is one of the important house dust mites responsible for allergic asthma that can be tentatively managed by specific immunotherapy. The present study was 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.<bold>Methods: </bold>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 p1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay.<bold>Results: </bold>the recombinant plasmid pET-28a-TAT-IhCDer p1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing, and the expression of the recombinant protein TAT-IhC-Der p1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p1.<bold>Conclusion: </bold>we successfully constructed the recombinant expression vector pET-28a-TAT-IhC-Der p1-3T 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.
- Subjects
DERMATOPHAGOIDES pteronyssinus; ALLERGENS; T cells; EPITOPES; PEPTIDES; VACCINES; ANIMAL experimentation; ANTIGENS; CELL physiology; COMPARATIVE studies; DNA probes; GENES; IMMUNOGLOBULINS; RESEARCH methodology; MEDICAL cooperation; PROTEINS; PROTEOLYTIC enzymes; RESEARCH; EVALUATION research
- Publication
Nutrición Hospitalaria, 2015, Vol 32, Issue 5, p2274
- ISSN
0212-1611
- Publication type
journal article
- DOI
10.3305/nh.2015.32.5.9613