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민소영,박호용,김명희,김정일,Min, So-Young,Park, Ho-Yong,Kim, Myoung-Hee,Kim, Jeong-Il 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.4
인시목 곤충에 독성을 나타내는 B. thuringiensis 가운데서 subspecies kurstaki HD-73으로부터 독소 유전자를 E. coli에 cloning하였다. 먼저 biotin으로 표지된 DNA probe (pC34)로서 전체 plasmid들과 hybridization한 결과, 75 kb plasmid상에 독소유전자가 존재함을 알았다. 75 kb plasmid를 제한효소 BgIII로 절단하여 vector pBR322에 삽입시켜 독소유전자를 포함하는 14.3 kb의 삽입부위를 가지는 clone인 pMK73을 얻었고, 이를 subcloning하여 3.7 kb의 독소유전자만을 포함하는 clone을 얻었다. 이 clone들의 total cell extract들은 흰불나방 유충에 독성을 나타내어 독소유전자의 E. coli에서의 발현을 확인할 수 있었다. Insecticidal crystal protein (CP) gene of Bacillus thuringiensis subsp. kurstaki HD-73 has been identified to be located in a 75-kilobase plasmid by southern hybridization technique using the biotinylated DNA probe (pC34), and cloned into the gram negative bacterium, Escherichia coli. E. coli transform ants harboring full length or truncated (nearly full length) CP genes have expressed them with no significant differences in their larvicidal activity. The crude extracts of E. coli clones were seen to be about 30-fold less potent than that of HD-73, as long as the larvicidal activity against the fall webworm, Hyphantria cunea, is concerned.
민소영,박호용,김명희,김정일,Min, So-Young,Park, Ho-Yong,Kim, Myoung-Hee,Kim, Jeong-Il 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4
인시목 곤충에 독성을 나타내는 B. thuringiensis subsp. kurstaki HD-73의 독소유전자를 함유하고 있는 3.7 Kb NdeI fragment (민 등 1986, 한국생화학회지 19, 363)를 강한 promoter, Ptac을 함유하고 있는 대장균 발현 벡터 pDR 540에 삽입하였다 (pDR 540-cp). Plasmid pDR 540-cp에 존재하는 독소유전자는 tac promoter에 의하여 발현이 되며 동시에 inducer (IPTG) 에 의하여 단백질생성이 조절된다. 적절한 조건하에서 독소단백질이 E. coli 전체 단백질의 20-30%까지 생성이 되는데 이 단백질 (130-Kdal)은 매우 안정되었고 약 24시간 induction 후에는 대부분의 E. Coli가 inclusion body를 형성하였으며, 독소단백질이 inclusion body안에 존재함을 SDS-PAGE와 bioassay로써 확인하였다. A 3.7 kb NdeI fragment (Min et al., 1986, Korean Biochem. J. 19, 363) carrying the insecticidal crystal protein gene of B. thuringiensis subsp. kurstaki HD-73 was recloned under the tac promoter in pDR540, the E. coli expression vector. The 130-Kdal crystal protein expressed in E. coli was very stable and amounted to about 30% of the total cellular protein under optimal conditions, and most of them are contained in the inclusion body.
Bacillus thuringiensis subsp . kurstaki HD - 73 독소유 전자의 cloning 및 E . coli 에서 발현
민소영,박호용,김명희,김정일 ( So Young Min,Ho Yong Park,Myoung Hee Kim,Jeong Il Kim ) 생화학분자생물학회 1986 BMB Reports Vol.19 No.4
Insecticidal crystal protein (CP) gene of Bacillus thuringiensis subsp. kurstaki HD-73 has been identified to be located in a 75-kilobase plasmid by southern hybridization technique using the biotinylated DNA probe (pC34), and cloned into the gram negative bacterium, Escherichia coli. E. coli transformants harboring full length or truncated (nearly full length) CP genes have expressed them with no significant differences in their larvicidal activity. The crude extracts of E. coli clones were seen to be about 30-fold less potent than that of HD-73, as long as the larvicidal activity against the fall webworm, Hyphantria cunea, is concerned.
Tac promoter 를 이용한 Bacillus thuringiensis subsp . kurstaki HD - 73 독소유전자의 E . coli 내에서의 발현증대
민소영,박호용,김명희,김정일 ( So Young Min,Ho Yong Park,Myoung Hee Kim,Jeong Il Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4
A 3.7 kb NdeI fragment (Min et al.,1986,Korean Biochem. J.19, 363) carrying the, insecticidal crystal protein gene of B. thuringiensis subsp. kurstaki HD-73 was recloned under the tac promoter in pDR540, the E. coli expression vector. The 130-Kdal crystal protein expressed in E. coli was very stable and amounted to about 30% of the total cellular protein under optimal conditions, and most of them are contained in the inclusion body.