Abstract: 【Objective】 This experiment was aimed to study the effects of 6-phospho-β-glucosidase encoded by the Lm4b_02324 gene on the biological characteristics of Listeria monocytogenes,and to lay a foundation for the study of the pathogenic mechanism of Listeria monocytogenes.【Method】 In this study,overlapping extended PCR technique was used to construct the Lm4b_02324 gene deletion strain (Lm928ΔLm4b_02324) and Lm4b_02324 gene complement strain (CLm928ΔLm4b_02324) of stable genetic wild type Lm928 through continuous passage.The growth characteristics of Lm928,Lm928ΔLm4b_02324 and CLm928ΔLm4b_02324 were analyzed by detecting D_{600 nm} values of different culture time and drawing growth curves.The mice were injected intraperitoneally with three strains of different concentrations,and the clinical symptoms after infection were observed.The lethal dose 50% (LD₅₀) of the three strains to the mice was calculated by Koch’s calculation method.Lm928,Lm928ΔLm4b_02324 and CLm928ΔLm4b_02324 were infected with multiplicity of infection (MOI) as 10,respectively.The adhesion and invasion ability and intracellular survival ability of each strain were analyzed by detecting the number of bacteria in cells at different time after infection.DNA of wild and deletion strains was extracted.Primers of 9 virulence related factors,such as prfA,mpl and hly,were used to detect their expressions by Real-time quantitative PCR.【Result】 PCR identification results showed that gene deletion strain Lm928ΔLm4b_02324 (wild strain:2 517 bp,deletion strain:1 209 bp) and the complement strain CLm928ΔLm4b_02324 (1 301 bp,consistent with the band of wild strain) were successfully obtained,and the deletion strain did not appear back mutation phenomenon after 15 generations.The results of growth curve showed that there was no significant difference in the growth status of wild strain,deletion strain and complement strain.LD₅₀ determination results showed that the LD₅₀ of the deletion strain was significantly lower than that of the wild strain (P＜0.05).The results of adhesion and invasion test showed that the intracellular survival number of the deletion strain was significantly or extremely significantly higher than that of wild strain and complement strain (P＜0.05 or P＜0.01).Within 12 h after infection with HBMEC cells,the intracellular viable bacteria number of the deletion strain was higher than that of wild strain,and the difference was significant (P＜0.05).Real-time quantitative PCR results showed that the deletion of Lm4b_02324 gene significantly decreased the expression of prfA,inlA and plcA genes(P＜0.05),and the expressions of hly,actA,mpl and inlC were extremely significantly decreased (P＜0.01).【Conclusion】 The results of this study showed that the deletion of Lm4b_02324 gene could increase the mortality of Listeria monocytogenes in mice and enhance its intracellular proliferation ability,which laid a foundation for the study of the role of Lm4b_02324 gene in the pathogenesis of Listeria monocytogenes.

Key words: Listeria monocytogenes; deletion strains; 6-phospho-β-glucosidase; intracellular survival