目的 构建单核细胞增生李斯特菌LLO-InlP缺失株,探究LLO、InlP基因对单核细胞增生李斯特菌生物学特性的影响.方法 通过温度敏感型(Ts)质粒载体基因敲除法,构建单核细胞增生李斯特菌LM681△InlP、LM681△LLO-InlP基因缺失株,通过检测不同时间的OD600nm值绘制生长曲线分析LM681、LM681△InlP、LM681△InlP-△LLO三株菌的体外生长能力;以感染复数MOI值为10感染细胞,测定LM681、LM681△InlP、LM681△InlP-△LLO对人绒毛膜滋养层细胞(HTR-8/SV40)、人绒毛膜癌细胞(JEG-3)黏附侵袭能力;动物水平上测定小鼠半数致死量、感染后的脑、肝、脾载菌量.结果 成功获得片段大小为1 125 bp的缺失株LM681△InlP、LM681△InlP-△LLO(亲本株LM681为2 292 bp);生长曲线结果显示,各菌OD600值无统计学差异;3株菌体外感染HTR-8/SV40细胞、JEG-3细胞黏附侵袭试验结果显示,LM681△InlP、LM681△InlP-△LLO 较 LM681 对 HTR-8/SV40 细胞、JEG-3 细胞黏附侵袭率均极显著降低(HTR-8/SV40 细胞 FLM681△InlP-LM681=102.792、FFLM681△InlP-△LLO-LM681=128.459,均 P＜0.01,JEG-3 细胞 FLM681△InlP-LM681=203.755、FLM681△InlP-△LLO-LM681=81.279,均 P＜0.01);LM681亲本株的LD5.为106.78,LM681△InlP缺失株的LD5.为107.45,在攻毒剂量范围LM681△InlP-△LLO不存在LD50,LM681△InlP-△LLO较LM681的LD50提高了 6.78个对数数量级,LM681△InlP-△LLO组织载菌量整体较亲本株LM681降低.结论 LLO、InlP基因缺失在动物感染水平和体外培养细胞感染水平上均极大程度的降低了 LM的的毒力,且InlP基因对LM681菌株黏附侵袭滋养层细胞具有一定意义,为研究InlP在单核细胞增生李斯特菌致病中的作用奠定基础.
A Listeria monocytogenes LM681 △LLO-InlP gene deletion strain was constructed through the temperature sen-sitive(Ts)plasmid vector gene knockout method,and the OD600 nm values at different times were plotted to analyze the growth curves indicating the in vitro growth ability of three strains of bacteria:LM681,LM681 △ InlP,and LM681 △ InlP-△LLO.Cells were infected with each strain at an MOI value of 10.The adhesion and invasion ability were detected in human cho-rionic trophoblast cells(HTR-8/SV40)and human choriocarcinoma cells(JEG-3).At the animal level,the median lethal dose(LD50)in mice,and the bacterial loads in the brain,liver and spleen after infection were measured to explore the effects of the LLO and InlP genes on the biological characteristics of Listeria monocytogenes.The missing strain LM681 with a fragment size of 1 125 bp △ InlP,LM681 △ InlP-△ LLO(parent strain LM681 with 2 292 bp)was successfully obtained.The growth curve results indicated no significant difference in OD6oo values among bacteria.The deletion of LLO and InlP genes greatly decreased the virulence of LM in both animal infection and in vitro culture cell infection models.The InlP gene facilitates the LM681 strain's adherence to,and invasion of,trophoblast cells,thus laying a foundation for studying the role of InlP in the pathogenesis of Listeria monocytogenes.
晋瑞婕;曾东东;秦赫;邓秋艳;任静静;蒋建军;王鹏雁
石河子大学动物科技学院,石河子 832000
畜牧业
单核细胞增生李斯特菌;内化素P;溶血素O;生物学特性
Listeria monocytogenes;endothelin P;hemolysin O;biological characteristics
《中国人兽共患病学报》 2024 (003)
224-230 / 7
国家自然科学基金(No.32260895、No.32160833、No.32160834) Supported by the National Natural Science Foundation of China(No.32260895,No.32160833,No.32160834)
10.3969/j.issn.1002-2694.2024.00.035
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