한국산 집먼지진드기 주알레르겐의 분리 및 합성

Abstract

Background: Purified major allergens of house dust mite are essential for evaluation of the allergic mechanism in molecular basis and development of new modalities of immunemodulation. Objective: In this study, we aimed to purif group 1 and group 2 allergens from Dermatophagoides pteronyssinus (Dp). In addition, cDNAs corresponding to Der pI and II in Korean Dp were isolated and recombinant Der p1 and Der pII were synthesized. Materials and method: Der pI allergen was purified by ammonium sulfate precipitation, anion -exchange column chromatography, and gel filtrat,ion chromatography. Der pII allergen was purified by anion exchange chromatography, gel filtration chromatography, and a preparative isoelectric focusing method. Results: Eight hundred ug of Der pI and 50 ug of Der pII were obtained from 100 g of culture medium and 1 g of mite bodies, respectively. The purities of these allergens were confirmed by SDS PAGE and the strong reactivity to the patient sera was identified. In order to produce a recombinant allergens, poly(A) RNA from house dust mites were isolated and used for cDNA synthesis by RT PCR. The cDNA was inserted into prokaryotic expression vector and the vectors were transformed into E. coli. A little amount of recombinant Der pI protein was produced due to the low solubility, and 1.2 mg of recombinant Der pII was produced from 1 L of E. coli culture medium. The antigenicity of Der pI was relatively weak, however, Der pII showed a strong antigenicity. Amino acid sequence of the amplified cDNA deduced from DNA sequences of Der pII showed 6 different variants. The variation of amino acid sequences suggests the possibility of high incidence of mutation of Der pII protein. Conclusion: A simplified method for the purification of Der pI and Der pII was developed. Recombinant allergens will be useful for the diagnosis and treatment of allergy with lower costs.