单核细胞增生李斯特菌NTSNΔ0287缺失株的构建及其生物学特性

Construction and Characterization of Listeria monocytogenes NTSNΔ0287 Deletion Strain

内化素蛋白家族是一类重要的表面蛋白,其与单核细胞增生李斯特菌(Listeria monocytogenes,Lm)对宿主的黏附侵袭作用密切相关。Lm NTSN_0287基因编码含有信号肽序列和LRR重复结构域的内化素家族蛋白,其作用机制有待于阐明。通过荧光定量PCR的方法,发现该基因在Lm侵袭Caco-2细胞时上调表达,达到体外表达量的1 300倍。为明确该基因的生物学特性,研究利用同源重组技术构建了LmNTSNΔ0287缺失株。与野生株相比,NTSNΔ0287缺失株对Caco-2细胞的黏附与侵袭能力均显著降低,对HeLa细胞的黏附能力下降,侵袭能力增加。经口服和尾静脉途径接种BALB/c小鼠,NTSNΔ0287缺失株在脾脏和肝脏中的载菌量均降低,但其LD_(50)与野生株相比无明显差异。因BALB/c小鼠缺乏人所特有的细胞表面内化素受体E-cad,并不是最佳实验动物模型,下一步将选用同时具有内化素受体E-cad和Met的沙鼠做进一步研究。

The internalized protein family is an important class of surface proteins, which is closely related to the adhesion and invasion of Listeria monocytogenes （Lm） to the host. Lm NTSN_0287 gene encodes a family of proteins containing the signal peptide sequence and the LRR repeat domain, and its mechanism of action remains to be elucidated. It was found that this gene was up-regulated when Lm was invaded into Caco-2 cells by FQ-PCR, reaching 1 300-fold in vitro. In order to clarify the biological characteristics of this gene, in this study, the strain Lm NTSNA0287 was constructed by homologous recombination technique. Compared with the wild strain, the adhesion and invasion ability of NTSNA0287 to Caco-2 cells were significantly decreased, the adhesion ability to HeLa cells was decreased,while the invasive ability was increased. The BALB/c mice were inoculated by oral and tail vein routes. The results showed that the amount of the bacteria in the spleen and liver decreased, and the LD50 of NTSN△0287 was not significantly different from the wild strain. Because BALB/c mice is lack of human-specific cell surface receptor E-cad, it may be not the best experimental animal model, we will use both endogenous hormone receptor E-cad and Met gerbils to make further research.