title: CONSTRUCTION AND EXPRESSION OF DERMATOPHAGOIDES PTERONYSSINUS GROUP 1 MAJOR ALLERGEN T CELL FUSION EPITOPE PEPTIDE VACCINE VECTOR BASED ON THE MHC II PATHWAY.
abstract: 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.
  
  
  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 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medical Parasitology, School of Medicine, Anhui University of Science & Technology (Huainan), Anhui. Department of Medical Parasitology, Wannan Medical College (Wuhu), Anhui.. cpli001@126.com.
authors.forename: Chaopin
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Medical Parasitology, Wannan Medical College (Wuhu), Anhui.. 626486060@qq.com.
authors.forename: Beibei
authors.initials: B
authors.lastname: Zhao
authors.affiliation: Department of Medical Parasitology, Wannan Medical College (Wuhu), Anhui.. 377151816@qq.com.
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Department of Medical Parasitology, Wannan Medical College (Wuhu), Anhui.. 453727957@qq.com.
authors.forename: Jidong
authors.initials: J
authors.lastname: Diao
authors.affiliation: Department of Medical Parasitology, Wannan Medical College (Wuhu), Anhui.. 1129910161@qq.com.
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.affiliation: Department of Respiratory Disease, Yi Ji Shan Hospital of Wannan Medical College (Wuhu), China.. 806842729@qq.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Lu
mesh_terms: D000485:Allergens / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000096:biosynthesis* / Q000235:genetics*; D060829:Arthropod Proteins / Q000096:biosynthesis* / Q000235:genetics*; D002459:Cell Fusion; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics*; D017931:DNA Primers; D018984:Epitopes, T-Lymphocyte / Q000235:genetics*; D005802:Genes, MHC Class II / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D022223:Vaccines, Subunit / Q000096:biosynthesis* / Q000235:genetics*; D014614:Vaccines, Synthetic / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D000485:Allergens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D017931:DNA Primers; D018984:Epitopes, T-Lymphocyte; D022223:Vaccines, Subunit; D014614:Vaccines, Synthetic; D007073:Immunoglobulin E; D003546:Cysteine Endopeptidases; C451162:Dermatophagoides pteronyssinus antigen p 1