关键词: 户尘螨, Ⅱ类变应原, T细胞表位, 融合肽, 蛋白质印迹

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 Mr 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）.  Conclusion  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.

Key words: Dermatophagoides pteronyssinus, Group 2 allergen, T cell epitope, Fusion peptide, Western blotting