Analysis of T-cell epitopes of Der f3 in Dermatophagoides farina

Abstract

House dust mites (HDM) are most important indoor allergens for humans. Der f3, one of the potent allergens with allergenicity, is derived from Dermatophagoides farina (D. farinae), and exhibits strong allergenicity that was confirmed in our previous work. The current study was undertaken to determine the localization of T-cell epitope of Der f3. We initially developed the T-cell fraction from BALB/c mice sensitized with recombinant Der f3 to determine the T-cell epitopes in the murine models, and performed T cell proliferation assay with 25 synthetic overlapping peptides of Der f3. The results indicated that T-cell reactive region of murine were assigned on amino acid range 41-60, 101-120, 161-180 and 201-220, respectively. In addition, we did T-cell proliferation experiment, respectively using the 4 murine T-cell epitope peptide and the human T-cell lines from three patients allergic to mite allergens in order to verify homogenous T-cell epitopes in humans. The results indicated that the amino acid sequences of 41-60, 101-120 and 161-180 had induced T cell proliferation in humans, yet 201-220 failed to. These findings suggest that T-cell epitope in Der f3 is located in the amino acid sequences of 41-60, 101-120 and 161-180, respectively. T-cell epitope localization detected in our study may provide a basis for development of animal therapeutic model and peptide vaccine for asthma.

Keywords: Allergen; Der f3; T-cell epitope; asthma; house dust mite.

Figure 1

25 synthetic overlapping peptides of Der f3.

Figure 2

SDS-PAGE and Western blotting analysis of Der f3. (A) Purified recombinant proteins (0.5 mg each) were run on a 12.5% polyacrylamide gel, and then visualized by CBB staining. M: Molecular weight marker; 1: BL21 with Der f3 but without IPTG; 2: Small dose of Der f3 expression; 3: Purified Der f3. (B) Immunoblot analysis of purified recombinant allergens Der f3. After completion of SDS-PAGE (as shown in A), fractionated protein on the gel was transferred onto a PVDF filter, and then stained with human anti-Der f3 serum and rabbit anti-human IgG antibody respectely. M: Rainbow marker simultaneously transferred onto the filter. 1: Purified Der f3.

Figure 3

Stimulation index of 25 overlapping synthetic peptides to T-cell lines of asthma mice. *, SI>2.

Figure 4

Changes of IL-2 levels in the cultured supernatants from the T-cell lines of asthma mice. *, vs PBS group, P<0.05.

Figure 5

Changes of IFN-γ levels in the cultured supernatants from the T-cell lines of asthma mice. *, vs PBS group, P<0.05.

Figure 6

Changes of IL-4 levels in the cultured supernatants from T-cell lines of asthma mice. *, vs PBS group, P<0.05.

Figure 7

Changes of IL-5 levels in the cultured supernatants from the T-cell lines of asthma mice. *, vs PBS group, P<0.05.