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化学学报
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化学学报 ›› 2005, Vol. 63 ›› Issue (18): 1646-1650. 上一篇    下一篇

研究论文

aprE基因表达的分子生物学和微量热法分析

李美菊1，陈向东*,1，刘国生1,3，杨洋1，王小娟1，耿晶1，梅运军1，刘义2，沈萍1，王金晶1   

  1. (1武汉大学生命科学学院 2化学与分子科学学院 武汉 430072)
    (3河南师范大学生命科学学院 新乡 453007)
  • 投稿日期:2004-12-30 修回日期:2005-04-19 发布日期:2010-12-10
  • 通讯作者: 陈向东

Analysis of aprE Gene Expression Employing Microcalorimetric Technique and Molecular Biological Method

LI Mei-Ju1, CHEN Xiang-Dong*, 1, LIU Guo-Sheng1, c, YANG Yang1, WANG Xiao-Juan1, GENG Jing1, MEI Yun-Jun1, LIU Yi2, SHEN Ping1, WANG Jin-J   

  1. (1College of Life Sciences, 2College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072)
    (3College of Life Sciences, Henan Normal University, Xinxiang 453007)
  • Received:2004-12-30 Revised:2005-04-19 Published:2010-12-10
  • Contact: CHEN Xiang-Dong

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利用分子生物学方法(SDS-PAGE和酶活检测法)未检测到所克隆的aprE基因在大肠杆菌中的表达产物(碱性蛋白酶), 而微量热法检测结果发现: 重组菌株的生长代谢产热曲线之间存在明显的差异. 根据这些差异, 分析了该基因的上、下游调控序列对该基因在大肠杆菌中表达的重要作用, 从而进一步对该基因进行了亚克隆, 得到了生物学方法可检测到的表达产物. 由此推测, 微量热技术有可能为检测外源基因表达及其调控, 以及为指导进一步基因工程操作提供一种新的快速灵敏的技术和方法.

关键词: 大肠杆菌, aprE基因, 微量热技术, 基因表达

The expression product of the cloned aprEin E. coli failed to be detected by general molecular biological methods including SDS-PAGE and protease activity determination. However, the results obtained by microcalorimetric technique showed that there were obvious differences among their metabolism heat-out curves. Further analysis suggested that both up-stream and down-stream regulation sequences of the aprE gene could be the key factor for the expression of this gene in E. coli. Based on the information achieved by the analysis of metabolism heat-out curves, the aprE gene segments were sub-cloned again in another plasmid vector with a stronger promoter, and the gene expression product could be detected by general molecular biological method. The results suggested that microcalorimetric technique be a novel, fast and sensitive method to detect the foreign gene expression and regulation, furthermore to provide some suggestions for genetic engineering.

Key words: Escherichia coli, aprE gene, microcalorimetry, gene expression