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- Title
Characterization of a MutantListeria monocytogenesStrain Expressing Green Fluorescent Protein.
- Authors
Ling-Li Jiang; Hou-Hui Song; Xue-Yan Chen; Chun-Lin Ke; Jing-Jing Xu; Ning Chen; Wei-Huan Fang
- Abstract
To construct a recombinant strain ofListeria monocytogenesfor the expression of heterologous genes, homologous recombination was utilized for insertional mutation, targeting its listeriolysin O gene (hly). The gene encoding green fluorescent protein (GFP) was used as the indicator of heterologous gene expression. The genegfpwas inserted intohlydownstream from its promoter and signal sequence by an overlapping extension polymerase chain reaction, and was then cloned into the shuttle plasmid pKSV7 for allelic exchange with theL. monocytogeneschromosome. Homologous recombination was achieved by growing the electro-transformedL. monocytogenescells on chloramphenicol plates at a non-permissive temperature. Sequencing analysis indicated correct insertion of the target gene in-frame with the signal sequence. The recombinant strain expressed GFP constitutively as revealed by fluorescence microscopy. The mutant strainL. monocytogenes hly-gfplost its hemolytic activity as visualized on the blood agar or when analyzed with the culture supernatant samples. Such insertional mutation resulted in a reduced virulence of about 2 logs less than its parent strainL. monocytogenes10403s as shown by the 50%-lethal-dose assays in the mouse and embryonated chicken egg models. These results thus demonstrate that mutatedL. monocytogenescould be a potential carrier for the expression of heterologous passenger genes or could act as an indicator organism in the food industry.
- Publication
Acta Biochimica et Biophysica Sinica, 2005, Vol 37, Issue 1, p19
- ISSN
1672-9145
- Publication type
Academic Journal
- DOI
10.1093/abbs/37.1.19