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RAPD PCR분석에 의한 국내 피부 사상균속 분류 및 동정
이영선,유재일,최연화,주형렬,김봉수,김동한 대한의진균학회 1998 대한의진균학회지 Vol.3 No.2
Backgound: Dermatophytoses are infections of keratinized tissues, that is, the epidermis, hair and nails, caused by a group of specialized fungi, the dermatophytes. Laboratory diagnoses of dermatophytes such as Trichophyton, Microsporum and Epidermophyton are made by microscopic examination and in vitro culture but they are either time consuming or lacking specificity. Objective: In order to develop and apply more rapid and precise diagnostic tests for fungal pathogens to facilitate the improved identification of dermatophytes, we investigated random amplified polymorphism DNA for classification and identification of dermatophytes. Methods: Amplification reactions were performed in volumes of 50㎕ containing 10mM Tris-HCl (pH 8.3), 50mM KCI, 1.5mM MgCl_2 0.01% (w/v), gelatin, 200mM dNTP mixture, 50pM primer, raq polymerase (0.025units/㎕), DNA 0.O01㎕. The optimal condition for PCR was 2 cycles (denaturing 94℃ 2min, annealing 33℃ 2min, extention 72℃ 4miu), 40 cycles, and extention (72℃ 10min). Results: RAPD showed interspecies polymorphism in T. rubum, T. mentagrophytes, T. tonsurans, T. violaceum, M. ferrugineum, M. canis, M. gypseum, M. audouinii and E. floccosum, but it had identical patterns in intraspecies. Conclusion: It was confirmed that RAPD PCR analysis with optimal conditions is a fast, economical and reproducible method for identification and classification of dermatophytes isolates.
Trichophyton rubrum으로부터 세포외 단백분해효소의 분리 정제 및 특성 연구
김동한,이영선,유재일,최연화,주형렬,김봉수,김기상,김정애 대한의진균학회 1997 대한의진균학회지 Vol.2 No.1
Background: Trichophyton rubrum is the most common dermatophyte isolated from human and has ability to invade the tissues such as stratum corneum, nail and hair. The potential role of proteinases as virulence factors of T. rubrum has been discussed at length. Objective: As a first step towards assessing its virulence role, we report on the purification and characterization of proteinase from T. rubrum isolate culture filtrates. Methods: An extracellular serine proteinase has been purified from culture filtrates of Trichophyton rubrum HP-9 by ultrafiltration, gel filtration chromatography, and affinity column chromatography. Azocoll and keratin azure were employed as the substrates of enzyme activities. Peak of proteolytic activity was analyzed by gelatin co-polymerized gel electrophoresis. Results: The molecular weight of the purified enzyme was approximately exhibited to 14.0 kDa on sodium dodecyl sulfate polyarcylamide gel electrophoresis (SDS-PAGE). The optimum pH and molality of 14.0 kDa proteinase activity was 6.0 and 100mM, respectively. The activity was inhibited by serine proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). The proteinase degraded gelatin, collagen type VI, and keratin from human epidermis but not hemoglobin. Conclusion: The 14,000 Mr extracellular serine proteinase purified from T. rubrum NP-9 culture filtrates has neutral pH optimum 6.0 and activities against gelatin, collagen type VI, and keratin.