[Prokaryotic expression of chimeric gene derived from the group 1 allergens of dust mites and bioactivity identification].

OBJECTIVE

To express a chimeric gene R8 derived from the group 1 allergens of dust mites using prokaryotic expression system and detect their bioactivities.

METHODS

PCR amplification was performed using specific primers of Derf1 gene and the pUCm-T recombinant plasmid containing the R8 chimeric gene as a template. The PCR products were inserted into the pET28a(+) empty vector after double digestion using restriction endonuclease BamH I and Xho I, respectively. The recombinant plasmid was transferred into E. coli line BL21 and induced by 1 mmol/L isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The expressed product was detected by SDS-PAGE and the target protein was purified. IgE binding assay of the purified protein R8 was detected by ELISA using dust mite allergic patient sera. For determining immunogenicity of R8 protein, 75 BALB/c mice were randomly divided into 5 groups, namely PBS (negative control), rDer f 1 group and rDer p 1 group (positive groups), R8 group and asthma group. The mice were treated with dust mite extract at 0, 7, 14 day by intraperitoneal injection of allergens (100 jl, 0.1 .tg/jl) and inhaled challenge as aerosol (0.5 microg/ml, 30 min/d) on day 21 for 7 days. Before inhalation in immunotherapy groups at 25-27 day, specific allergen immunotherapy was performed using rDerf 1, rDerp 1 and R8 allergens respectively. Mice in negative control group were treated with PBS all the time. Twenty-four hours after the last challenge, mice in every group were sacrificed. The bronchoalveolar lavage fluid (BALF) was collected. ELISA was used to detect the level of interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) in BALF.

RESULTS

SDS-PAGE analysis revealed that chimeric gene R8 was expressed with a band of approximately M(r) 35 000. Compared with groups of rDerf 1 and rDer p 1 [(80.44 +/- 15.50) and (90.79 +/- 10.38) microg/ml, respectively], IgE binding capacity of the protein R8 (37.03 +/- 12.46) microg/ml was statistically lower (P < 0.001). The level of IFN-gamma in sera of R8 group [(343.43 +/- 38.79) pg/ml] was higher than that of the PBS and asthma groups [(393.93 +/- 50.68) and (208.44 < or = 46.11)pg/ml, respectively] (P < 0.01), but no statistical difference to that of the rDerf 1 and rDer p 1 groups (P > 0.05). IL-4 level in R8 group was lower markedly than the others (P < 0.05 or P < 0.01).

CONCLUSION

Chimeric protein R8 derived from the group 1 allergens of dust mites has been expressed with low allergenicity and high immunogenicity.