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PROKARYOTIC EXPRESSION AND BIOACTIVITY EVALUATION OF THE CHIMERIC GENE DERIVED FROM THE GROUP 1 ALLERGENS OF DUST MITES.

BACKGROUND
we successfully reconstituted the gene from group 1 allergens of dust mites, and obtained a body of shuffled genes. In order to verify the prediction on the chimeric gene, we tentatively cloned R8 into the vector that was prokaryoticly expressed, purified and assessed for its bio-activities.


METHODS
the expressed product was detected by SDSPAGE and the target protein was purified. The purified protein R8 was detected by ELISA. 75 BALB/c mice were divided into 5 groups, namely PBS, rDer f1, rDer p1, R8 and asthma group. The mice were treated with dust mite allergens at 0, 7, 14 day by intraperitoneal injection and inhaled challenge as aerosol on day 21 for 7 days. Specific allergen immunotherapy was performed using rDer f1, rDer p1 and R8 allergens respectively. The level of IFN and IL- 4 in BALF was detected by ELISA.


RESULTS
the chimeric gene R8 was expressed with a band of approximately Mr 35000. Compared with groups of rDer f 1 and rDer p 1 [(80.44±15.50) and (90.79±10.38) μg/ml respectively], IgE binding capacity of the protein R8 (37.03±12.46) μg/ml was statistically lower (P<0.001). The level of IFN 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±46.11) pg/ml respectively] (P<0.01), but no statistical difference to that of the rDer f 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).


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