Abstract:

Objective To clone and express must mite allergen Der f 32 protein and identify its antigenicity and its activation on dendritic cellline (DC2.4). Methods The DNA coding for allergen Der f 32 of dust mite was synthesized by genetic engineering technology, and cloned into expression vector pET-24a(+), and then transformed into Escherichia coli BL21 for expression. The expressed recombinant Der f 32 protein(rDer f 23)with His-tag was purified by nickel affinity chromatography and analyzed by SDS-PAGE and Western blotting with sera from patients allergic to dust mite. The rDer f 32 was used for skin prick test to detect its antigenicity in patients with allergic to dust mite. The rDer f 32 was co-cultured with mouse-derived DC2.4 for 24 h, flow cytometry was used to detect the expression of cell surface costimulatory molecules(CD40, CD83 and CD80)on the DC2.4. The DC2.4 cells were incubated with different amount of rDer f 32(10, 20, 30, 40, 50, 60 μg/ml)for 48 h and the expression of T cell immunoglobulin and mucin 4(TIM4)on DC2.4 was detected by Western blotting. Results The SDS-PAGE results showed that the rDer f 32 was successfully expressed in E. coli BL21 with a relative molecular mass of about 35 000. Clinical skin prick test showed that 6 of the 42 patients allergic to dust mite were positive with a positive rate of 14.2%. Western blotting showed that rDer f 32 was specifically recognized by IgE in sera of patients with positive in skin test. The results of flow cytometry showed that the expression levels of CD40, CD83 and CD80 were 24.5%, 9.6% and 24.6% on DC2.4 after being co-cultured with rDer f 32, which was significant higher than that of DC2.4 co-cultured with PBS(11.9%, 3.1% and 15.6%)(P < 0.01), however lower than that of the positive control group with stimulation of Der f 1(38.2%, 15.0%, and 39.1%)(P < 0.05). The results of Western blotting showed that the expression of TIM4 was increased in DC2.4 cells after being co-cultured with rDer f 32 at a dose-dependent pattern, with highest expression when Der f 32 was added at 50 μg/ml. The relative density of TIM4 expression was 1.112. Conclusion The dust mite allergen Der f 32 was successfully cloned and expressed as recombinant protein which could be recognized by the IgE in sera of patients with allergic to dust mite. rDer f 32 could stimulate DC maturation with increased expression of surface costimulatory molecules and TIM4.

Key words: Dust mites, Der f 32, Immunogenicity, Dendritic cell