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枯草芽孢杆菌葡萄糖脱氢酶基因gdh的克隆与表达

发表时间:2008-06-15  浏览量:1856  下载量:571
全部作者: 张梁,龚靓洁,丁重阳,石贵阳
作者单位: 江南大学工业生物技术教育部重点实验室;江南大学生物工程学院
摘 要: 本文运用分子生物学手段,根据文献报道的枯草芽孢杆菌葡萄糖脱氢酶编码序列设计引物,采用常规聚合酶链式反应(polymerase chain reaction, PCR)方法获得葡萄糖脱氢酶基因;然后与克隆载体pUC19连接,转化至大肠杆菌JM109中,筛选阳性克隆转化子,测定其序列并与原序列进行比较分析,获得同源性大于97%的葡萄糖脱氢酶基因;将目的基因与表达载体pET-28a连接后转化至大肠杆菌DE3 codon plus中,获得成功表达,用细胞粗提液测定的葡萄糖脱氢酶活力为0.003 254 U/mg,能够为NADH或NADPH依赖性羰基还原酶的手性催化提供足够的还原力。
关 键 词: 微生物学;枯草芽孢杆菌;葡萄糖脱氢酶;表达
Title: Cloning and expression of glucose dehydrogenase gene gdh in E.coli from Bacillus subtilis
Author: ZHANG Liang, GONG Liangjie, DING Zhongyang, SHI Guiyang
Organization: The Key Laboratory of Industrial Biotechnology, Ministry of Education,Jiangnan University;School of Biotechnology, Jiangnan University
Abstract: In this paper, primers were designed according to the encoding sequence of Bacillus subtilis glucose dehydrogenase and gdh gene was amplified by polymerase chain reaction (PCR) using the primers. Then the PCR product was ligated to plasmid pUC19 and the ligation product was transformed into Escherichia coli JM109. Positive recombinants were obtained through screening by Ampr. The sequence was compared with original sequence in the GenBank, and the alignment result showed 97% homology. Subsequently, the gdh gene was sub-cloned into expression vector pET-28a and transformed into Escherichia coli DE3 codon plus. The gdh gene was expressed successfully in E.coli and the activity of GDH in the cell-free extract of E.coli reached 0.003 254 U/mg.
Key words: microbiology; Bacillus subtilis; glucose dehydrogenase; expression
发表期数: 2008年10月第11期
引用格式: 张梁,龚靓洁,丁重阳,等. 枯草芽孢杆菌葡萄糖脱氢酶基因gdh的克隆与表达[J]. 中国科技论文在线精品论文,2008,1(11):1218-1222.
 
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