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).
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.
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J Colloid Interface Sci
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State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Center for Analytical Sciences, College of Chemistry, Nankai University, Tianjin 300071, China; National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Electronic address:
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