单增李斯特菌prfA基因缺失菌株的构建及其生物学特性鉴定

Construction and Biological Activity of a Strain of Listeria monocytogenes Deleted for Positive Regulatory Factor A Gene(prfA)

单核细胞增生性李斯特菌(Listeria monocytogenes,Lm)是一种风险较高的食源性致病菌,其绝大多数毒力基因的表达均受到由prfA基因编码的PrfA(positive regulatory factorA)蛋白全部或部分调控。使用同源重组的方法敲除Lm野生菌株EGDe的prfA基因,并通过测定生长曲线、毒力基因表达和侵袭Caco-2细胞能力等探讨单缺失菌株EGDe-ΔprfA生物学特性。通过测定生长曲线显示prfA敲除株和野生型EGDe二者生长状态无差异;运用实时定量荧光聚合酶链式反应检测EGDe-ΔprfA的主要毒力基因表达,结果显示plcA、plcB毒力基因的表达量分别上升至原来的2.5倍和2倍,inlA、inlB、inlC、actA、vip等均呈下降趋势,prfA基因表达量趋向于零;侵袭Caco-2细胞结果显示EGDe-ΔprfA侵袭数为野生株的1/5。该基因缺失菌株的构建及生物学特性研究对食源性致病菌EGDe致病机理研究具有重要意义并且提供了重要材料。

Among the known Listeria strains, only Listeria monocytogenes （Lm） can cause human disease. The expression of most virulence genes is regulated fully or partially by positive regulatory factor A （PrfA） protein encoded by prfA gene.In this study, we adopted homologous recombination method to knock out the prfA gene of the wild strain EGDe, and investigated the biological characteristics of EGDe-ΔprfA by testing growth state, virulence gene expression and Caco-2 cell invasion. There was no difference in the growth status of EGDe-ΔprfA and EGDe as demonstrated by the growth curves. Real time quantitative PCR results showed that the expression levels of plcA and plcB genes increased by 2.5 times and twice, respectively, while the expression levels of inlA, inlB, inlC, actA and vip declined. The expression level of prfA gene was nearly zero. The invasion of Caco-2 cells by EGDe-ΔprfA was one fifth as high as that by the wild strain.The gene-deleted strain can provide an important tool for studying the pathogenic mechanism of the foodbome pathogen Listeria monocytogenes.