摘要
以基因组DNA为模板,利用PCR技术从克雷伯氏菌(Klebsiellapneumoniae)中扩增得到含有1,3丙二醇氧化还原酶基因(dhaT)的DNA片段,将其定向连接到克隆质粒pGEM-3zf上,得到重组质粒pGEMdhaT。将此重组质粒转化到受体菌EscherichiacoliDH5α中,通过蓝白斑鉴定挑选出阳性菌株。DNA序列分析表明其基因全长为1185bp。将该片段插入表达载体pSE380,构建成重组子pSE-dhaT,并在E.coliJM109中获得表达,经SDS-PAGE检测,表达产物分子质量与天然纯1,3丙二醇氧化还原酶(DHAT)相同,约为43ku。
Genomic DNA of 1,3-propanediol oxidoreductase was used as a template. A segment containing gene of 1,3-propanediol oxidoreductase from Klebsiella pneumoniae was gained by PCR amplification and cloned into pGEM-3zf to obtain recombinant plasmids pGEM-dhaT,then the plasmid was transformed into Escherichia coli DH5α.The positive strains were obtained by the determination of blue-white plot.It was shown from DNA sequence analysis that the open reading frame of dhaT was 1 185 bp corresponding 395 amino acid residues. When the engineered strain E.coli JM109/pGEM-dhaT was expressed in LB + Amp medium by the induction of IPTG, a band with MW of 43 ku which was similar to pure DHAT was occurred by SDS-PAGE.
出处
《广西农业生物科学》
CAS
CSCD
2004年第2期145-148,共4页
Journal of Guangxi Agricultural and Biological Science
基金
国家"863"高科技资助项目(2003AA001039)
关键词
克雷伯氏菌
1
3-丙二醇氧化还原酶
大肠杆菌
克隆
表达
Klebsiella pneumoniae
1,3-propanediol oxidoreductase
Escherichia coli
cloning
expression
