| 食源性单增李斯特菌inlA/inlB/inlC基因缺失株的构建及其生物学特性分析 |
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| 引用本文： | 任静静,杨铭伟,陈云飞,尹华,王欣雨,李蓓蓓,蒋建军,王鹏雁.食源性单增李斯特菌inlA/inlB/inlC基因缺失株的构建及其生物学特性分析[J].中国畜牧兽医,2019,46(1):45-53. |
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| 作者姓名： | 任静静 杨铭伟 陈云飞 尹华 王欣雨 李蓓蓓 蒋建军 王鹏雁 |
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| 作者单位： | 石河子大学动物科技学院, 石河子 832003 |
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| 基金项目： | 国家自然科学基金项目（31260606） |
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| 摘 要： | 为探究内化素inlA/inlB/inlC基因对单增李斯特菌(Listeria monocytogenes,Lm)生物学特性的影响,本研究采用融合PCR方法构建Lm681 inlC基因缺失突变体,并构建pKSV7-△inlC穿梭载体,将其转化Lm681-△inlAB感受态细胞,利用温度(42℃)和氯霉素(10μg/mL)抗性双重压力来实现同源重组,筛选同源重组子进行鉴定并研究其部分生物学特性。结果显示,PCR和测序结果证实成功构建了3基因缺失株(Lm681-△inlABC),且缺失株的生长特性与野生株相比无明显差异,溶血特性与野生株保持一致;小鼠感染试验显示,野生株Lm681、Lm681-△inlAB和Lm681-△inlABC对小鼠的致死率分别为80%(8/10)、60%(6/10)和40%(4/10),对小鼠的LD50分别为4.36×10~4、1.35×10~6和2.95×10~7 CFU,且Lm681-△inlABC在肝脏、脾脏及脑组织中的定植能力极显著低于野生株(P<0.01)。研究结果表明,inlA/inlB/inlC基因对Lm致病性发挥具有一定的作用,为深入研究inlX基因介导Lm入侵宿主细胞过程中的作用机制提供了科学依据。
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| 关 键 词： | 单增李斯特菌 内化素基因 基因突变株 生物学特性 |
| 收稿时间： | 2018-07-09 |
Construction and Characterization of inlA/inlB/inlC Genes Deleted Mutant Strain of Food-borne Listeria monocytogenes |
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| REN Jingjing,YANG Mingwei,CHEN Yunfei,YIN Hua,WANG Xinyu,LI Beibei,JIANG Jianjun,WANG Pengyan.Construction and Characterization of inlA/inlB/inlC Genes Deleted Mutant Strain of Food-borne Listeria monocytogenes[J].China Animal Husbandry & Veterinary Medicine,2019,46(1):45-53. |
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| Authors: | REN Jingjing YANG Mingwei CHEN Yunfei YIN Hua WANG Xinyu LI Beibei JIANG Jianjun WANG Pengyan |
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| Institution: | College of Animal Science and Technology, Shihezi University, Shihezi 832003, China |
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| Abstract: | In order to evaluate the effect of inlA/inlB/inlC genes on biological characteristics of Lm,the inlC gene deletion mutant was constructed by fusion PCR method,and then pKSV7-△inlC shuttle vector was constructed,which was transformed into competent cells Lm681-△inlAB.Homologous recombination was conducted using temperature (42℃)and chloramphenicol (10 μg/mL) resistance to achieve the inlC gene deletion strain,and then the homologous recombination was screened for identification and some biological characteristics were studied.PCR and sequencing results confirmed that Lm681-△inlABC was successfully obtained.The results showed that the growth characteristics of the deletion mutants were the same as the wild-type strain Lm681 and there was no significant difference.The hemolysis activity of the mutant strains were consistent with wild-type strain Lm681.In the mouse infection experiment,the mortality rate of wild-type strain Lm681,Lm681-△inlAB and Lm681-△inlABC were 80%(8/10),60%(6/10) and 40%(4/10),respectively.Results of mice virulence determination showed that the median lethal dose of wild-type strain,Lm681-△inlAB and Lm681-△inlABC were 4.36×104,1.35×106 and 2.95×107 CFU,respectively.Furthermore,the colonization ability of the inlA/inlB/inlC genes deleted strain was extremely significantly lower than wild-type strain in liver,spleen and brain (P < 0.01).Collectively,these results indicated that inlA/inlB/inlC genes might play an important role in pathogenicity of Lm infection and provided scientific basis for the study on the mechanism in host cell invasion by Lm. |
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| Keywords: | Listeria monocytogenes internalin gene gene mutant biological characteristics |
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