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

摘要

In order to evaluate the effect of inlA/inlB/inlCgenes on biological characteristics of Lm, the inlCgene 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 (10μg/mL) resistance to achieve the inlCgene 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) and40％ (4/10) , respectively.Results of mice virulence determination showed that the median lethal dose of wild-type strain, Lm681-△inlAB and Lm681-△inlABC were 4.36×10`4, 1.35×106 and 2.95×107 CFU, respectively.Furthermore, the colonization ability of the inlA/inlB/inlCgenes 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/inlCgenes 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.%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4.36×104、1.35×106和2.95×107 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.