title: [Cloning and Prokaryotic Expression of Fusion Gene of Group II Allergen Der p2 T Cell Epitope from Dermatophagoides pteronyssinus].
abstract: OBJECTIVE
  To express and purify the T cell epitope fusion peptide of the major allergen Der p2 from Dermatophagoides pteronyssinus.
  
  
  METHODS
  Nucleotide sequences reported to encode four T-cell epitopes (T1-T4) of Der p2 of D. pteronyssinus were linked in the rank of T1-T2-T3-T4. In this way, the chimeric gene was synthesized, named as Der p2 T. The gene of Der p2 T was amplified by PCR, purified, and cloned into the pET-28a (+) vector, forming the prokaryotic recombinant expression vector pET-28a (+)-Der p2 T. This formation was verified by double digestion. The pET-28a (+)-Der p2 T vector was transfected into E. coli strain BL-21, and its expression was induced by addition of IPTG. The recombinant protein was purified and collected by Ni-NTA affinity chromatography, and prepared for SDS-PAGE and Western blotting analysis. ELISA was used to evaluate the binding ability of Der p2 T cell epitope fusion peptide to serum IgE from patients with house dust mite allergy.
  
  
  RESULTS
  Double digestion results confirmed the construction of the pET-28a (+)-Der p2 T vector. SDS-PAGE revealed the expression of recombinant Der p2 T cell epitope fusion peptide with M, of 10,000. Western blotting confirmed the purification of Der p2 T cell epitope fusion peptide. The binding ability of Der p2 T cell epitope fusion peptide to serum IgE from patients with house dust mite allergy [(37.70±9.89) µg/ml] decreased significantly in comparison to that of Der p2 [(85.89±9.63) µg/ml] (P<0.01).
  
  
  CONCLUSIONS
  The Der p2 T cell epitope fusion peptide is prepared, and its binding ability to serum IgE from patients with house dust mite allergy significantly decreases than that of Der p2.
authors.forename: Bin-bin
authors.initials: BB
authors.lastname: Duan
authors.forename: Hong-yu
authors.initials: HY
authors.lastname: Song
authors.forename: Chao-pin
authors.initials: CP
authors.lastname: Li
mesh_terms: D000485:Allergens; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D040002:Dermatophagoides pteronyssinus; D018984:Epitopes, T-Lymphocyte; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D011994:Recombinant Proteins
chemical_list: D000485:Allergens; D018984:Epitopes, T-Lymphocyte; D011994:Recombinant Proteins