Screening and cloning of antimicrobial DNA sequences using a vital staining method
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- 作者：Xiongying Cheng ; Guozhen Liu ; Gongyin Ye ; Haijiao Wang ; Xiaojing Shen ; Kechun Wu ; Jiahua Xie ; Illimar Altosaar
- 关键词：Antimicrobial protein ; Antimicrobial peptide ; DNA library screening ; Expressional assay ; Vital assay ; Antibiotic discovery ; Bacterial inhibition
- 刊名：Gene
- 出版年：2009
- 出版时间：1 February 2009
- 年：2009
- 卷：430
- 期：1-2
- 页码：132-139
- 全文大小：554 K
文摘
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.