单增李斯特菌(LM90)inlA单基因及inlB/inlA双基因缺失株的构建及其生物学特性初步研究

Construction and characterization of LM90-ΔinlA and LM90-ΔinlB/ΔinlA mutant strains of Listeria monocytogenes

为探究内化素inlA、inlB基因缺失对单核细胞增生性李斯特菌(LM)生物学特性的影响,本研究采用同源重组技术构建了inlA基因缺失株(LM90-inlA)和inlB、inlA双基因缺失株(LM90-ΔinlB/ΔinlA),并对其生物学特性进行了初步研究。结果显示:PCR和测序结果证实成功构建了目的基因(inlA、inlB)缺失株;菌落PCR和测序结果显示2种缺失株在体外连续培养20代过程中均能稳定遗传;生长特性实验表明2种缺失株与亲本株生长差异无统计学意义(t_(LM90/LM90-ΔinlA=0.34),p>0.05;t_(LM90/LM90-ΔinlB/ΔinlA)=0.324,p>0.05;t_(LM90-ΔinlA/LM90-ΔinlB/ΔinlA)=0.008,p>0.05)且inlA缺失后2缺失株对小鼠的LD_(50)分别升高了0.64、1.26个对数数量级。研究结果表明inlA基因和inlB基因对LM90的部分生物学特性具有一定的调控作用,这对于阐明LM毒力因子的致病机理提供了参考依据,也为研究内化素介导LM穿越血脑屏障(BBB)的作用机制奠定了科学基础。

In order to evaluate the effect of inlA and inlB gene on biological characteristics of Listeria monocytogenes. Based on homologous recombination technology, we constructed deletion mutants LM90-AinlA and LM90-AinIB/AinlA of LM. The results showed that the deletion mutants were constructed successfully and they had good genetic stability through continuous passage to 20 generations in vitro. The growth characteristics of the two deletion mutants were the same as the parent strain LM90 and there were no significant difference （tLM90-/LM90-ΔinlB-ΔinlA=0.34, p〉0.05; tLM90/LM90-ΔinlA=0.324, p〉0.05; =0.008, p〉0.05）. Moreover, the median lethal dose of LM90-AinlA and LM90-AinlB/AinlA were increased 0.64 and 1.26 logarithmic order of magnitudes to that with LM90 respectively. Collectively, these results indicated that the inlA and inlB gene could regulate some biological characteristics of LM90, which would provide scientific evidence for clarifying the pathogenesis of LM virulence factor and provide a scientific foundation to study the mechanism of LM penetrating Blood Brain Barrier （BBB） into brain mediated by InlA and InlB.