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- 저자 : 변시명 ( Jong Hwa Kim (검색결과 4 건)
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Rearrangement of Light Chain Gene of Anti-Tac Antibody
김종화,이병룡,김학주,변시명,Kim, Jong-Hwa,Lee, Byeong-Ryong,Kim, Hack-Joo,Byun, Si-Myung 생화학분자생물학회 1987 한국생화학회지 Vol.20 No.3
Anti-Tac 항체를 생산하는 하브리도마 세포인 Hd-2-4-5로 부터 유전자를 분리하고 Bam HI 제한효소를 이용하여 완전 분해한 후 DNA조각을 agarose gel 상에서 크기에 따라 10가지 부분으로 나누였다. 이렇게하여 형성된 각 DNA조각을 Southern blot를 행하므로서 어느 부분이 anti-Tac 항체의 L chain을 가지고 있는지를 확인한 후 in vitro packaging 법과 situ plaque hybridization 방법을 이용하여 L chain을 함유하는 clone을 분리하였다. 이렇게 하여 얻어진 clone으로 부터 pBR 322에 subcloning 실험과 제한효소 지도작성을 통하여 배아세포의 L chain파 비 교함으로써 anti-Tac항체의 L chain의 재배열을 연구했다. 이때 anti-Tac 항체의 L chain은 $J_1-J_2$ cluster가 deletion 되어 있었고 V-J recombination에 의해서 V 부위가 직접 $J_3-J_4-J_5$ cluster에 연결되어 있음을 알 수 있었다. A high molecular weight DNA from a hybridoma cell, Hd-2-4-5, producing anti-tac antibody was digested to completion with BamHI restriction endonuclease. The resulting DNA fragments were fractionated according to the size in 1% preparative agarose gel electrophoresis. After ligation of the fragment with modified Charon 28 and packaging in vitro, DNA fragments carrying the gene sequences coding for the variable region of light chains were detected by hybridization with mouse $J_k$ fragment labeled with $^{32}P$. We have cloned the k-sequence positive BamHI DNA fragments and characterized the cloned sequences by comparing with mouse embryonic light chain. On the basis of the results, it was concluded that the cloned light chain gene underwent somatic rearrangement. The$J_1-J_2$cluster was deleted and the region was directly linked to remaining J cluster by V-J recombination.
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Molecular Cloning of Streptokinase Gene from Streptococcus equisimilis and Its Expression in E. coli
노동출,김종화,박승국,이종우,변시명,Roh, Dong-Chul,Kim, Jong-Hwa,Park, Seung-Kook,Lee, Jong-Woo,Byun, Si-Myung 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.4
The streptococcal genomic DNA digested with Pst I was cloned in E. coli HB101. The overlay technique of casein/plasminogen was used to screen the clones for recombinants carrying the streptokinase gene. The insert size of the plasmid carrying the streptokinase gene was a 2.5, 4.3, and 5.8 Kb, respectively. The restriction maps of all three hybrid plasmids were constructed by digestion with Pst I, Pvu II, Sal I, Hind III, Ava I, BamH I, and Cia I. For the identification of cloned gene, streptokinase was highly purified from S. equisimilis by the methods of gel chromatography and isoelectric focusing and rabbits were immunized with this purified streptokinase. Several lines of evidence, including proof obtained by the immunodiffusion technique, established that the enzyme from E. coli was identical to that from S. equisimilis. In the E. coli cell culture, we found the activity of streptokinase in all three principal locations of the cell. More than 50% were existed in the intracellular space. Streptokinase를 생산하는 균주인 Streptococus equisimilis로 부터 유전자를 분리하고 Pst I으로 분해하여 pBR 322를 vector로 하여 E. coli HB101 대장균에 cloning하였다. Streptokinase를 발현하는 균주를 casein/plasminogen overlay technique를 이용하여 skc를 확인하였다. 이때 2.5 Kb, 4.3 Kb, 5.8 Kb의 DNA 단편이 삽입된 clone이 각각 streptokinase 활성을 보였고 제한 효소 지도를 작성한 결과 2.5 Kb는 4.3 Kb, 5.8 Kb의 일부임을 확인할 수 있었다. 또 streptokinase에 대한 항체를 사용한 immunodiffusion technigue으로 확인한 결과 streptokinase를 생성하는 균주임이 확실하였다. 대장균에서는 streptokinase의 활성이 주요 3부분인 세포내, periplasm, 세포외에 모두 존재 하였고 그 활성은 log phase 후기까지는 배양시간에 따라 비율이 항상 일정하였고, 그 이후에는 periplasm의 활성이 세포외로 나왔으나 세포내에는 50% 이상의 활성이 그대로 존재함을 알 수 있었다.
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Streptococcus equisimillis 로 부터 streptokinae 유전자의 분리와 대장균에서의 발현
노동출,김종화,박승국,이종우,변시명 ( Dong Chul Roh,Jong Hwa Kim,Seung Kook Park,Jong Woo Lee,Si Myung Byun ) 생화학분자생물학회 1986 BMB Reports Vol.19 No.4
The streptococcal genomic DNA digested with Pst I was cloned in E. coli HB101. The overlay technique of casein/plasminogen was used to screen the clones for recombinants carrying the streptokinase gene. The insert size of the plasmid carrying the streptokinase gene was a 2.5, 4.3, and 5.8 Kb, respectively. The restriction maps of all three hybrid plasmids were constructed by digestion with Pst I, Pvu II, Sal I, Hind III, Ava I, BamH I, and Cla I. For the identification of cloned gene, streptokinase was highly purified from S. equisimilis by the methods of gel chromatography and isoelectric focusing and rabbits were immunized with this purified streptokinase. Several lines of evidence, including proof obtained by the immunodiffusion technique, established that the enzyme from E. coli was identical to that from S. equisimilis. In the E. coli cell culture, we found the activity of streptokinase in all three principal locations of the cell. More than 50% were existed in the intracellular space.
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Anti - Tac 항체의 L chain 유전자 재배열
김종화,이병룡,김학주,변시명 ( Jong Hwa Kim,Byeong Ryong Lee,Hack Joo Kim,Si Myung Byun ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.3
A high molecular weight DNA from a hybridoma cell, Hd-2-4-5, producing anti-tac antibody was digested to completion with BamHI restriction endonuclease. The resulting DNA fragments were fractionated according to the size in 1% preparative agarose gel electrophoresis. After ligation of the fragment with modified Charon 28 and packaging in vitro, DNA fragments carrying the gene sequences coding for the varibable region of light chains were detected by hybridization with mouse J_k fragment labeled with ^(32)P. We have cloned the κ-sequence positive BamHI DNA fragments and characterized the cloned sequences by comparing with mouse embryonic light chain. On the basis of the results, it was concluded that the cloned light chain gene underwent somatic rearrangement. The J₁-J₂ cluster was deleted and the region was directly linked to remaining J cluster by V-J recombination.
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