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유영도,한문희,김지영,Yoo, Young-Do,Han, Moon-Hi,Kim, Ji-Young 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.2
프로트롬빈 두단계측정법에서 프로트롬빈을 트롬빈으로 전환시키는데 Echis carinatus독으로부터 분리된 프로트롬빈 활성화 효소인 프로트롬빈을 트롬빈으로 약 30분이내에 거의 전환시켰다. 생성된 트롬빈의 활성은 반응시간이 20시간 경과하여도 안정하게 유지되었다. 한편 프로트롬빈을 혈장-혈청 혼합물을 이용하여 트롬빈으로 전환시켰을 때는 약 9분정도 경과되면 생성된 트롬빈의 활성이 감소함을 보여주었다. 트롬빈 효소의 기질로써 피브리노젠과 합성핵원체 기질을사용하여 프로트롬빈을 측정할 수 있는 농도의 범위를 조사하였다. Clotting assay에서 프로트롬빈 측정 가능범위는 2 ${\mu}g/ml$-100 ${\mu}g/ml$이었으며 chromogenic assay에서는 0.2 ${\mu}g/ml$-16 ${\mu}g/ml$이었다. 프로트롬빈을 활성화시키지 않고 직접 측정하기위하여 효소를 이용한 면역측정법 (ELlSA)을 개발하였다. lndirect ELlSA방법에 의해서는 최소 40 ng/ml, sandwich ELlSA를 사용하여 소 혈장의 프로트롬빈 농도를 측정 한 결과 224 ng/ml이었으며 within-run은 9.3%, between-run은 15.8%의 변화를 보여주었다. In a two-stage assay of prothrombin, prothrombin was activated to thrombin by ecarin, the prothrombin activator from the venom of Echis carinatus, in the absence of calcium ions and phospholipid. The activation was almost complete within 30 minutes and the thrombin generated was not inhibited by the ecarin activation mixtures for a incubation time up to at least 20 hours, while it was inhibited by the absorbed plasma- serum mixture after 9-minute incubation. Two different substrates, fibrinogen and a synthetic chromogenic substrate, were used for assays of the thrombin activity and compared in their sensitivies in detection of prothrombin. The standard curve was linear between 2 ${\mu}g/ml$ of prothrombin concentration in the clotting assay, and between 0.2 ${\mu}g/ml$ and 16 ${\mu}g/ml$ in the chromogenic assay. In addition we developed the enzyme-linked-immunosorbent assays for the direct measurement of prothrombin. The minimum detectable concentrations of prothrombin by the indirect ELISA and the sandwich ELISA were 40 ng/ml and 3 ng/ml, respectively. The prothrombin level in bovine plasma was determined to be 224 ng/ml by the sandwich ELISA. The reliability of the sandwich ELISA was demonstrated by the within runs and between runs tests, which were 9.3% and 15.8%, respectively.
포로트롬빈의 측정시스템 : 프로트롬빈 활성화 효소로써 ecarin 을 사용한 두단계 측정법과 효소를 이용한 면역측정법 ( ELISA )
유영도,한문희,김지영 ( Young Do Yoo,Moon Hi Han,Ji Young Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.2
In a two-stage assay of prothrombin, prothrombin was activated to thrombin by ecarin, the prothrombin activator from the venom of Echis carinatus, in the absence of calcium ions and phospholipid. The activation was almost complete within 30 minutes and the thrombin generated was not inhibited by the ecarin activation mixtures for a incubation time up to at least 20 hours, while it was inhibited by the absorbed plasma-serum mixture after 9-minute incubation. Two different substrates, fibrinogen and a synthetic chromogenic substrate, were used for assays of the thrombin activity and compared in their sensitivies in detection of prothrombin. The standard curve was linear between 2 ㎍/㎖ of prothrombin concentration in the clotting assay, and between 0.2 ㎍/㎖ and 16 ㎍/㎖ in the chromogenic assay. In addition we developed the enzyme -linked -immunosorbent assays for the direct measurement of prothrombin. The minimum detectable concentrations of prothrombin by the indirect ELISA and the sandwich ELISA were 40 ng/㎖ and 3 ng/㎖, respectively. The prothrombin level in bovine plasma was determined to be 224 ng/㎖ by the sandwich ELISA. The reliability of the sandwich ELISA was demonstrated by the within runs and between runs tests, which were 9.3% and 15.8%, respectively.
E . coli 에서 사람 프리트롬빈 2 cDNA 의 크로닝과 발현
최은화,김용주,김종명,홍효정,한문희,김지영 ( Eun Hwa Choi,Yong Joo Kim,Jong Myoung Kim,Hyo Jeong Hong,Moon Hi Han,Ji Young Kim ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2
A 0.6 kbp of 3`-portion of human prothrombin cDNA was isolated from a liver cDNA library of λgt 10 by in situ plaque hybridization. A cDNA fragment corresponding to the N-terminal region of prethrombin 2 was obtained from another cDNA library synthesized by reverse transcription of human liver mRNA using a specific oligonucleotide as a primer. The 48 bp cDNA sequences starting from the first nucleotide to the AvaI site of human prethrombin 2 cDNA was chemically synthesized such that prethrombin 2 can be expressed directly in E. coli expression vector system. The open reading frame(ORF) for human prethrombin 2 was cloned in pUC18 by combining the synthetic fragment and the cDNA fragments. Expression plasmids were constructed in which the ORF for human prethrombin 2 was inserted into the site downstream from the thermoinducible leftward promoter (P_L) and the cII ribosome binding site of bacteriophage λ. Upon temperature induction, the plasmids expressed 34,000 dalton polypeptide, which was accumulated to an amount corresponding to 5-10% of the total bacterial proteins. The 34,000 dalton polypeptide was identified as human prethrombin 2 by SDS-gel electrophoresis and Western blot analysis.
송기방,김경태,김지영,이상기,한문희,Song, Ki-Bang,Kim, Kyong-Tai,Kim, Ji-Young,Rhee, Sang-Ki,Han, Moon-Hi Korean Society for Biochemistry and Molecular Biol 1987 한국생화학회지 Vol.20 No.2
B형 간염 바이러스 표면항원 유전자가 효모 GAL10, ADCI 혹은 GAL1-ADCI double promoter 조절하에 발현되도록 재조합 플라즈미드를 제조하였다. Double prornoter는 ADCI promoter DNA절편과 GAL1-GAL10 promoter DNA 절편을 결합시킴으로써 만들었다. 효모에서 세가지의 재조합 플라즈미드는 모두 HBsAg을 발현시켰다. GAL promoter가 유도되었을 때 ADCI promoter 보다 3-5 배 많은 HBsAg을 생산하였다. Double promoter 시스템에서 galactose에 의해 전사가 유도되는 것이 확인되었지만 HBsAg 생성량은 유도되기 전의 수준에서 크게 증가되지 않았다. Northern blot에 의해 RNA 분석 결과 윗쪽 GAL promoter 가 유도됨에 따라서 아랫쪽 ADCI promoter 조절하의 전사시작이 저해 되고 있음이 시사되었다. We have constructed plasmids in which transcription of the gene encoding human hepatitis B virus surface antigen(HBsAg) is under the control of GAL10, ADCI or a double promoter consisting of GAL1 and ADCI. Construction of the double promoter was achieved by insertion of a 650 bp fragment of divergent GAL1-GAL10 promoter DNA in the upstream of ADCI promoter. By radioimmunoassay, it was shown that three recombinant plasmids were all capable of producing HBsAg in the host Saccharomyces cerevisiae strains. When induced by galactose, cells synthesized 3-5 fold more HBsAg under GAL promoter control than under ADCI promoter control in an identical plasmid. Production of HBsAg in a system utilizing the double promoter was not increased additively, although it appeared to retain galactose inducibility. Northern blot analysis indicated that induction of the upstream GAL1 promoter caused inhibition of transcription initiation at the downstream ADCI promoter.