KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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In vivo Biological Function of a Fibrinolytic Enzyme after Oral Adminstration

혈전용해효소의 경구투여에 의한 생체 내 작용

  • Lee, Young-Hoon (Department of Microbiological Engineering, Jinju National University) ;
  • Lee, Sung-Ho (Environmental Biotechnology National Core Research center, Gyeongsang National University,) ;
  • Park, Ki-Hoon (Department of Applied Life Science, Gyeongsang Natinal University) ;
  • Choi, Young-Ju (Department of Food and Nutrition, Silla University) ;
  • Lee, Sang-Won (Department of Microbiological Engineering, Jinju National University) ;
  • Kim, Cheol-Ho (Department of Microbiological Engineering, Jinju National University) ;
  • Cho, Soo-Jeong (Department of Microbiological Engineering, Jinju National University) ;
  • Gal, Sang-Wan (Department of Microbiological Engineering, Jinju National University)
  • 이영훈 (진주산업대학교 미생물공학과) ;
  • 이성호 (경상대학교 환경생명국가 핵심연구센터) ;
  • 박기훈 (경상대학교 응용생명과학부) ;
  • 최영주 (신라대학교 식품영양학부) ;
  • 이상원 (진주산업대학교 미생물공학과) ;
  • 김철호 (진주산업대학교 미생물공학과) ;
  • 조수정 (진주산업대학교 미생물공학과) ;
  • 갈상완 (진주산업대학교 미생물공학과)
  • Published : 2006.12.30
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Abstract

A fibrinolytic enzyme gene (BCF-1) was subcloned to the pEB vector which is high expression vector in the Bacillus host. The enzyme was purified by using FPLC after ammonium sulfate precipitation. The enzyme was oral-administrated to the rat and checked the bleeding time, blood clotting time and fibrinolytic effect of the serum. In the bleeding time retardation test, it was longer about 1.7 fold in the feeding rat than without feeding. The serum of rat feeded with the enzyme had the fibrinolytic activity from 1 hour to 3 hours after oral-administration. After 3 hours from feeding, the fibrinolytic activity was decreased gradually. Also blood clotting time after bleeding was longer than that of control rat. The enzyme could be detected at band of 30,000 Da in the blood by western blotting. The enzyme was not harmful to the all internal organs of the rats. Taken together, the enzyme originated from B. subtilis BB-1 can be a candidate to develop the drug for thrombosis, arteriosclerosis and myocardial infarction.

Bacillus subtilis BB-1 (KFCC l1344P)으로부터 분리된 혈전용해효소 유전자 (BCF-1)를 대량발현 벡터인 pEB 벡터에 크로닝하여 순수분리 된 혈전용해효소를 rat 경구 투여하여 출혈시간, 혈액의 응고, serum의 혈전용해능 등에 대한 in vivo 실험을 실시하였으며, 혈전용해효소의 단회경구투여에 따른 독성을 검사하였다. 효소의 경구투여에 따른 rat의 출혈시간에서는 대조군에 비하여 모든 경구 투여군에서 출혈시간이 유의적으로 약 1.75배 이상 길게 나타남을 확인하였고(P<0.05), 혈액의 출혈시간 또한 활발히 진행됨을 관찰하였다. 혈액으로부터 분리된 serum의 혈전용해작용 있어서는 경구투여 후 1시간부터 채혈한 혈액 내에서 혈전용해효소의 활성이 검출되기 시작하여 3시간째까지 높은 활성을 보였으며 4시간째부터 서서히 활성이 감소하는 것을 확인하였고 혈액의 응고 역시 대조군에 비하여 경구 투여군에서 상당히 지연되는 것을 알 수 있었다. Western blot에 의한 효소 검출에서는 경구 투여군에서 30,000 Da 크기의 단일밴드를 확인하였으며, 혈전용해효소의 rat에 대한 단회경구투여 독성실험에서 중량의 변화, 장기의 이상여부, 사망률 등에서 어떠한 이상이나 병변이 발견되지 않았다. 이상의 결과로 동물실험을 통한 혈전용해 효소의 경구투여에 의한 작용을 혈액 내에서 확인 할 수 있었으며, 본 효소의 단회 경구투여 시의 독성은 전혀 없음을 확인할 수 있었다.

Keywords

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