单核细胞增生性李斯特菌fbpa基因敲除菌株的构建

Construction of fbpA-deletion Mutant of Listeria Monocytogenes

目的构建单核细胞增生性李斯特菌fbpa基因敲除菌株。方法克隆fbpa及其上、下游基因,构建其载体质粒;通过酶切反应将上、下游基因分别重组到载体质粒中,形成同源重组质粒;同源重组质粒电转入细菌内,进行同源重组;采用PCR、Western blot鉴定敲除菌株。结果单核细胞增生性李斯特菌fbpa基因敲除菌株基因组DNA无fbpa基因片段,且无FbpA蛋白表达。结论成功构建单核细胞增生性李斯特菌fbpa基因敲除菌株。

Objective To construct an fbpA-deletion mutant of Listeria monocytogenes. Methods The fbpA gene and its upstream, downstream genes of Listeria monocytogenes were cloned into plasmid pCR II. The upstream and downstream fragments were ligated into the pAULA using restriction enzyme as pAULA-△fbpA. To achieve allelic exchange, pAULA-△fbpA was introduced into Listeria monocytogenes by electroporation, The mutant was confirmed by PCR and Western blot. Results The tbpA gene was not detected in genome of fbpA- deletion mutant of Listeria monocytogenes,and FbpA was not expressed in /bpA-deletion mutant of Listeria monocytogenes. Conclusion The/bpA-deletion mutant of Listeria monocytogenes was constructed successfully.