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국내 분리 Bacillus anthracics의 Plasmid pXO1과 pXO2의 유전적 다양성 분석
문성희,유천권,오희복,성원근,전정훈,유재연,이상섭,Mun, Sung-Hee,Yoo, Cheon-Kwon,Oh, Hee-Bok,Seong, Won-Keun,Chun, Jeong-Hoon,Yu, Jae-Yon,Lee, Sang-Seob 대한미생물학회 2003 Journal of Bacteriology and Virology Vol.33 No.4
We compared genetic variations in virulence mega plasmids pXO1 and pXO2 of twenty-seven Bacillus anthracis strains from Korean patients and environmental samples together with those of Bacillus anthracis Sterne, Pasteur and A2012 standard strains. Genetic variations were analyzed in twenty-three variable regions (ten and thirteen variable-number tandem repeats and insertion/deletions in pXOl and pXO2, respectively). The pXO1 plasmids were classified into 7 groups and pXO2 plasmids to 12 groups. Discrete phylogenic lineages could be differentiated between environmental and clinical strains by UPGMA (unweighted pair group method with average) method. In addition, clinical strains showed more variations than environmental isolates. The pXO2 plasmid appeared genetically more unstable than pXO1. A general plasmid genotype could be suggested for Korean soil isolates since they mostly clustered into a representative group.
유전자 cloning에 의한 Bacillus subtilis의 fibrinolytic enzyme 활성 변화
이홍석,유천권,이철수,강상모 한국산업미생물학회 2000 한국미생물·생명공학회지 Vol.28 No.1
구조유전자와 enhancer 유전자를 cloning 하여 fibrinolytic enzyme을 생산하는 B. subtilis K-54와 J-10의 fibrinolytic enzyme의 생산 능력을 높이기 위하여 이 유전자를 포함하는 재조합 vector를 만들어 형질전환하여 그 생산능의 변화를 조사하였다. aprN과 enhancer 유전자인 prtR 유전자의 primer를 제작하여 PCR한 결과, B. subtilis J-10주에서는 aprN과 prtR 유전자 band를 확인하였고 염기서열 분석결과 자체 promoter를 갖는 완전한 유전자임을 확인하여 cloning에 사용하였다. Cloning된 두 유전자를 가지고 B. subtilis J-10과 B. subtilis K-54를 형질전환하기 위해 E. coli/B. subtilis shuttle vector에 cloning하여 재조합 vector를 제작하였다. 그 결과 aprN 유전자를 갖는 pAPR2, prtR 유전자를 갖는 pENC2, 두 유전자 모두 가지는 pFLA1을 만들었으며 B. subtilis J-10과 K-54를 형질전환하여 재조합된 Bacillus속의 균주를 만들어 fibrinolytic activity의 생산량을 조사하였다. pAPR2과 pFLA1로 형질전환하였을 경우 각각 약 27.3 와 16%의 활성 증가를 보였으나 pENC2의 경우 활성 증가를 보이지 않았다. pENC2로 분리주인 B. subtilis K-54를 형질전환하였을 경우 약 5배 정도의 활성 증가를 보여 효소의 생산이 증가하였음을 확인할 수 있었다. The transformation of Bacillus subtilis K-54 and J-10 was carried out with constructed vectors containing structure and enhancer genes of aprN and prtR, to increase their fibrinolytic enzyme activity, Bands for the aprN and prtR genes were indentified from B. subtilis J-10 by PCR that was carried out with the constructed primers for the genes. In addition, the gene fragments contained promoter site based on the results of analysing their nucleotide sequence. The two gene fragments, aprN and prtR, obtained by the PCR, were, then, inserted to vector such as T-vector and E.coli/Bacillus shuttle vector. The constructed vector were designated as pAPR2 (aprN) pENC2(prtR) and pFLA1 (aprN and prtR), respectively. the constructed vector was used for transformation of the strains of B. subtilis J-10 and B. subtilis K-54 and the fibrinolytic activity of the transformed strains was investigated. The introduction of the vector, pAPR2 and pFLA1, resulted in the increase of fibrinolytic enzyme activity in B. subtilis J-10 by 27.3 % and 16 %, respectively. However, the introduction of pENC2 to B. subtilis J-10 did not seem to induce increase of the enzyme activity. The strain of B. subtilis K-54 transformed with pENC2 showed an increased fibrinolytic activity by 5 folds compared with that of the original strain of B. subtilis K-54.
Bacillus subtilis K-54가 생산하는 Fibrinolytic enzyme의 혈전생성 및 스트레스에 미치는 영향
이홍석,이철수,유천권,서원상,강상모 한국산업미생물학회 2000 한국미생물·생명공학회지 Vol.28 No.1
B. subtilis K-54에서 생산된 fibrinolytic 효소의 혈전증과 스트레스에 대한 효과를 mouse를 이용하여 조사하였다. 부분 정제된 효소의 역가를 4 PCU (Protein Casein Unit)로 조정한 후 매일 1회씩 쥐에게 3일간 경구 투여하였다. 효소가 투여된 쥐에 인공적으로 collagen과 epinephrine을 이용하여 혈전을 유발시킨 후 생존 여부를 관찰한 결과 쥐의 수명이 대조군에 비하여 연장됨을 확인하였다. 또한 효소를 투여 한 group의 5-hydroxyindoleacetatic acid(HIAA) 수치가 감소하지 않아 이 효소는 스트레스 자체에는 영향을 미치지 못하는 것으로 판단된다. 간 및 뇌 조직의 과산화 지질 값 및 혈액내의 antiplasmin, Activated Partial Thromboplastin Time(APTT)과 Protrombin Time(PT) 값의 경우 효소를 투여한 스트레스군의 수치가 대조군에 비하여 낮았다. 그러나 단백질 분해의 경우에는 각 군당 별 차이를 나타내지 않았다. The effect of fibrinolytic enzyme produced from Bacillus subtilis K-54 on the thrombosis and stress in vivo was investigated. Each partially purified fibrinolytic enzyme of 4 protein casein unit was administered orally for 3 days before intravenously injection with collagen and epinephrine. In the mice group administered with the enzyme, an increased life span of mice was observed in comparison with that of control. The result suggest that the enzyme may prevent the formation of thrombos in vivo. Administration of the enzyme did not influence to stress itself because 5-hydroxyindoleacetatic acid concentration of brain in the mice group with stress did not decreased after the administration of the enzyme. The value of lipid peroxide (LPO) of the liver and brain cells in the group treated with the enzyme was lower than that of control. However, protein degradation (PD) value showed no significant difference between treatment and control groups. In addition, the value of activated partial thromboplastin time (APTT), protrombin time (PT) and antiplasmin in blood were higher in the stress group than that of the enzyme treated group.