单核细胞性李斯特菌基因dal的敲除及其生物学特性初步分析  

Knockout of dal Gene and Its Effect on Listeria monocytogenes

作　　者：曾海娟[1] 刘武康 谢曼曼[1] 丁承超 董庆利[1] 刘箐[1] 

机构地区：[1]上海理工大学医疗器械与食品学院,上海200093

出　　处：《食品科学》2017年第22期48-53,共6页Food Science

基　　金：国家自然科学基金面上项目(31371776)

摘　　要：在单核细胞性李斯特菌(Listeria monocytogenes)野生株EGDe act A及inl B双基因缺失株(EGDeΔact AΔinl B)的基础上,利用同源重组的方法进一步构建了缺失营养基因dal的菌株(EGDe Δact AΔinl BΔdal),并对该缺失菌株生长状态、毒力基因表达水平、生物被膜的形成量及细胞侵袭等方面作进一步分析。结果显示,37℃摇床培养6 h后,缺失株的菌浓度显著低于EGDe Δact AΔinl B(P<0.001),培养基中补充D-丙氨酸的缺失株生长速率与亲本株相比无显著差异;实时荧光定量聚合酶链式反应结果显示,缺失株的sig B基因表达水平变化最明显(P<0.01),约下调90%;缺失株生物被膜形成量显著增加(P<0.05),培养基补充D-丙氨酸后缺失株生物被膜的生成量与亲本株相比无差异;对Coca-2细胞的侵袭无影响,表明该基因对细菌生长能力及生物被膜形成具有重要的调控作用,并不影响菌株对细胞的侵袭力。此缺失株的构建为进一步研究基因dal的功能提供了理论支持。In this study, the act A and inl B double gene deletion mutant（EGDe Δact AΔinl B） of Listeria monocytogenes wild-type（WT） strain EGDe was used as the parent to delete the dal gene by homologous recombination technology, and the biological characteristics of the dal-deficient mutant such as growth capacity, virulence gene expression, cell invasion and biofilm formation were further studied. Growth curves showed that the concentration of the new mutant strain was significantly lower than that of EGDe Δact AΔinl B after 6 h of shaking culture at 37 ℃ （P 〈0.001）, but there was no difference in the growth rates of the parental and the mutant strains when D-alanine was added to the medium. Quantitative real-time-PCR showed that the sig B gene expression level of the mutant strain was changed most significantly（P〈 0.01） and down-regulated by 90% compared with EGDe Δact AΔinl B. The biofilm formation of the mutant strain increased compared with EGDe Δact AΔinl B, but this difference did not exist when D-alanine was added to the medium for the mutant. There was no significant difference in Caco-2 cells invasion ability compared with EGDe Δact AΔinl B. The results indicated that the dal gene played an important regulatory role in the growth and biofilm formation of bacteria and did not affect the ability of cell invasion. The construction of this deletion strain can provide a tool for further study of the function of the dal gene.

关 键 词：单核细胞性李斯特菌 基因敲除 生长能力 细胞侵袭 生物被膜 

分 类 号：TS201.3[轻工技术与工程—食品科学]