Screening and cloning of antimicrobial DNA sequences using a vital staining method
Screening and cloning of antimicrobial DNA sequences using a vital staining method
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使用活体染色方法筛选和克隆抗菌 DNA 序列
DOI：
10.1016/j.gene.2008.10.017
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发表时间：
2009-02-01
期刊：
影响因子：
3.5
通讯作者：
Altosaar, Illimar
中科院分区：
文献类型：
--
作者：
Cheng, Xiongying;Liu, Guozhen;Altosaar, Illimar
描述了一种基于细胞活力测定克隆编码细胞毒性分子的基因的新方法。工作假设是编码细胞毒性分子的DNA序列在细菌细胞中的表达将导致细胞死亡或损伤，并且损伤或死亡细胞的分离可以导致鉴定负责使宿主细胞衰弱的DNA序列。我们通过在大肠杆菌细胞中分离众所周知的抗微生物Puroindoline B基因来验证这一概念。我们进一步证明了使用这种方法从cDNA表达文库中分离编码抗菌剂的DNA的可行性。序列分析和生物测定表明，分离的克隆编码先前表征的抗菌蛋白（AMP），蛋白质以前没有表征为AMP，以及新的抗菌肽。此外，还鉴定了含有核糖体蛋白编码cDNA的克隆。因此，该方法也可用于鉴定对维持细菌细胞活力重要的宿主基因。(C)2008 Elsevier B.V.保留所有权利。
A novel method for cloning of genes coding for cytotoxic molecules based on a cell viability assay is described. The working hypothesis is that expression of DNA sequences coding for cytotoxic molecules in bacterial cells will lead to cell death or impairment, and the isolation of the impaired or dead cells could lead to identification of DNA sequences responsible for debilitating the host cells. We verified this concept by isolating the well known antimicrobial Puroindoline b gene in Escherichia coli cells. We further demonstrated the feasibility to use this approach for isolating DNA encoding for antimicrobials from cDNA expression libraries. Sequence analysis and bioassay indicated that the isolated clones encoded previously characterized antimicrobial proteins (AMPs), proteins not previously characterized as AMPs, as well as novel antimicrobial peptides. In addition, clones harboring ribosomal protein encoding cDNA were also identified. Therefore, this method could also be used to identify host genes important in maintaining bacterial cell viability. (C) 2008 Elsevier B.V. All rights reserved.