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cis - Diamminedichloroplatinum(Ⅱ) 에 의한 pBR322 DNA 의 변성과 구조 변화
양재명,한태룡,구자춘,임창수 한국농화학회 1990 Applied Biological Chemistry (Appl Biol Chem) Vol.33 No.4
E. coli LE392, transformed with CDDP-treated pBR322 DNA, was plated on ampicillin containing media. The number of colonies formed on ampicillin containing agar plate was reduced to undetectable level after trot the DNA with 13.3 μM CDDP. The CDDP- treated pBR322 DNA was susceptible to angle strand DNA specific Sl nuclease and it's migration pattern in agarose gel electrophoresis was changed. These results suggest, that CDDP adduction to pBR322 DNA resulted in denaturation of the double helix and changes in it's conformation which ultimately leads to the inactivation of the ampicillin resistant gene(Received November 25, 1990, Accepted December 22, 1990).
cis - Diamminedichloroplatinum(Ⅱ)(CDDP) 에 의한 불루텅 바이러스 이중가닥 RNA 의 구조변화
양재명 한국농화학회 1991 Applied Biological Chemistry (Appl Biol Chem) Vol.34 No.2
cis-Diamminedichloroplatinum(II)(CDDP), an antitumor drug, did not generate crosslink between bluetongue virus (BTV) capsid protein at moderate concentration. Cesium chloride density gradient centrifugation study revealed that protein-RNA crosslink was not detectable in CDDP treated BTV. CDDP treated BTV ds RNA showed remarkable change in the migration pattern in polyacrylamide gel electrophoresis. These results suggest that the reduction of BTV core associated transcriptase activity is most likely by the CDDP adduction to the genomic ds RNA rather than by the protein-RNA crosslink and/or protein-protein crosslink.
Inhibition of Bluetongue Virus(BTV) Infectivity by cis-Diamminedichloroplatinum(Ⅱ) (CDDP)
Yang, Jai-Myung,Manning, JaRue S. 成均館大學校 科學技術硏究所 1991 論文集 Vol.42 No.1
블루텅 바이러스 감염성에 대한 CDDP의 영향을 조사하였다. 조직배양세포에서 기른 뒤 정제하지 않은 바이러스는 CDDP에 의해 급속히 감염성이 감소하나 완전이 없어지지는 않았다. CDDP를 처리할 때 non-ionic detergent를 같이 넣어 주면 BTV의 감염도가 감지할 수 없을 정도로 낮아진다. CDDP에 의해 감염성이 제거된 바이러스는 왁친으로 사용할 수 있는 가능성이 있다.
cis-Diamminedichloroplatinum(Ⅱ) Induces Con-formational Changes in Bluetongue Virus ds RNA
Yang, Jai-Myung,Manning, JaRue S. 成均館大學校 科學技術硏究所 1991 論文集 Vol.42 No.1
CDDP 처리한 블루텅 바이러스 ds RNA를 전자현미경으로 촬영한 사진은 RNA가 구부러져 짧아졌음을 보여주었다. 같은 RNA를 1% 아가로즈 겔에서 전기영동하면 이동 속도가 빨라진다. 이 실험결과는 CDDP가 ds RNA의 구조를 compact하게 변화시킴을 뜻한다. Form I과 Ⅲ의 pBR 322 DNA를 CDDP로 처리한 뒤 전기영동한 실험 결과는 CDDP가 긴거리의 염기 사이에 crosslink를 잘 형성하지 않음을 암시한다.
Construction of a Recombinant AcNPV Carrying E. coli β-galactosidase
Yang, Jai-Myung,Koo, Ja-Choon 成均館大學校 科學技術硏究所 1990 論文集 Vol.41 No.2
대장균의 β-galactosidase 유전자를 BamHI과 Aha Ⅲ로 절단한 DNA 단편을 pAcI 벡터의 BamHI 자리에 클론하여 pHBG-I을 만들고 야생형 AcNPV DNA와 pHBG-I DNA를 Spodoptera frugiperda 세포에 cotransfection한 뒤 extracellular virus (ECV)를 플라그 검정하여 polyhedral body를 형성하지 않는 재조합 AcNPV를 순수분리하였다. 재조합 AcNPV (β-gal-AcNPV)가 대장균의 β-galactosidase를 지니고 있음은 Southern blot 결과와 X-gal 존재하에 blue plaque이 형성되는 사실로 확인하였다. β-gal-AcNPV가 감염된 SF21 세포내에서 β-galactosidase는 감염된 후 약 18시간 뒤부터 합성되기 시작한다. β-galactosidase 유전자가 polyhedrin 촉진유전자의 제어를 받으므로 β-gal-AcNPV는 polyhedrin 유전자의 발현 조절 기작을 밝혀내는데 유용하게 사용될 수 있다. Plasmid pHBG-I was generated by cloning BamHl-Aha Ⅲ digested fragment of E. coli β-galactosidase into the BamHI site of plasmid pAcI. Wild type AcNPV DNA and supercoiled pHBG-I DNA were cotransfected into Spodoptera frugiperda, an insect cell line, and extracellular virus (ECV) was plaque assayed. A probable recombinant AcNPV from a plaque that did not generate occlusion body was purified three times. Southern blot analysis by using BglI digested 2.1 kb fragment of pHBG-I containing β-galactosidase coding region, as a probe, indicated that the plaque purified recombinant AcNPV carries E. coli β-galactosidase. The β-gal-AcNPV was able to form blue plaques in the presence of X-gal and β-galactosidase was highly produced in SF21 cell after 24 h p. i.
Insect Cell Cultures for Recombinant Protein Production
Park, Young-Min,Yang, Jai-Myung,Chung, In-Sik 경희대학교 유전공학연구소 1989 遺傳工學論文集 Vol.1 No.-
실험실 규모의 배양기에서 곤충세포의 배양을 수행하였다. 회분식 배양에서의 곤충세포의 성장은 serum 의 농도, 다른 영양소, 초기 접종농도, 기계적인 교반과 같은 변수에 의해 영향을 받는 것으로 나타났다. Lactate와 ammonrum은 회분식 배양에서의 말기에 관찰되는 농도에서는 세포의 성장을 저해하는 요인은 아닌 것 같았다. 아울러 유전공학적으로 재조합된 baculovirus를 곤충세포에 감염시켜 재조합 단백질의 생산을 시도하였으며 dilution rate 가 0.006hr^(-1)일 때 반응기당 최대 2800units 의 β-galactosidase가 생산되었다. Insect cell cultures were performed in laboratory-scale vessels. The batch growth of insect cells was affected by such parameters as serum content, other nutrients, seeding density, and mechanical agitation. Lactate and ammonium were not likely to be environmental factors that inhibited cell growth at the concentrations observed at the end of batch cultures. Recombinant protein production by cells infected with a genetically-modified baculovirus was also demonstrated. The maximum β-galactosidase synthesis of 2800units per reactor volume was achieved at the dilution rate of 0.006hr^(-1).
한국형 사람 Calicivirus Replicase 단백의 발현 및 항원성 평가
장미윤,양재명,김경희 대한바이러스학회 1997 Journal of Bacteriology and Virology Vol.27 No.2
In view of the potential of replicase protein as a diagnostic reagent for human caliciviruses (HuCVs), we have cloned and over-expressed this gene from the Snow Mountain-like Korean strains in Escherichia coli as a fusion protein with glutathione S-transferase (GST), and described the preliminary antigenic characterization of the recombinant products. Each 470bp fragment corresponding to highly conserved region of RNA-dependent RNA polymerase was generated by RT-PCR from stools of two diarrheal children, cloned in pMOSBlue T-vector, and subcloned between the EcoRI and Sal I restriction sites of pGEX-4T-3, a GST gene fusion vector, yielding pGCV. This construct expressed a Snow Mountain-like HuCV replicase under the control of the IPTG-inducible tac promoter. An extract prepared by sonication of the E. coli cell inclusion bodies bearing pGCVp products was purified and analyzed by SDS-PAGE. After Coomassie blue staining, it was shown that the recombinant replicase migrated on the gels with an approximate molecular mass of 46.5 kDa, that was subsequently cleaved into a 26 kDa GST fragment and a 20.5 kDa replicase protein upon digestion with thrombin protease. The replicase was recognized on immunoblotting with the sera from symptomatic children with the HuCV-associated diarrhea but not by asymptomatic sera from adults. The results presented the first biological activity of individually expressed HuCV replicase subunit and provided important reagents for diagnosis of HuCV infection.