结核分枝杆菌higBA基因对细菌应激反应及胞内感染免疫机制的研究

Effect of Mycobacterium tuberculosis higBA on Bacterial Stress Response and Intracellular Infection and Immunity

目的探究结核分枝杆菌(Mycobacterium tuberculosis,Mtb)higBA基因对细菌应激反应及胞内感染免疫的作用。方法从Mtb H37Rv基因组上扩增获得目的基因,与载体连接后电转化入耻垢分枝杆菌(Mycobacterium smegmatis,Ms)构建重组菌,对空载菌Ms_vec和重组菌Ms_higBA进行应激实验和Raw264.7小鼠巨噬细胞感染实验,检测细菌菌落形成单位(CFU)和细胞因子白介素(interleukin,IL)-1β、IL-6、IL-10、IL-12p40,干扰素(interferon,IFN)-γ,肿瘤坏死因子(tumor necrosis factor,TNF)-α和诱导型一氧化氮合酶(inductible nitric oxide synthase,iNOS)的转录水平变化。结果成功构建Ms_higBA重组菌。应激实验结果表明higBA确能提高细菌在体外培养特定条件下的生存能力。胞内感染实验证明higBA能提高细菌在巨噬细胞内存活能力,影响细胞因子的转录水平。结论Mtb的higBA基因在细菌应激反应和胞内感染免疫中发挥了作用。

Objective To investigate the effect of Mycobacterium tuberculosis(Mtb)higBA on bacterial stress response and intracellular infection and immunity.Methods The target gene amplified from Mtb H37Rv genome was cloned to the vector and then transferred to Mycobacterium smegmatis(Ms)to construct a recombinant strain.Stress response experiment and Raw264.7 mouse macrophage infection was carried out with Ms_higBA,the recombinant strain,and Ms_vec,the vector strain.Tests were conducted to measure bacterial colony forming unit(CFU)and transcriptional levels of cytokines,including interleukin(IL)-1β,IL-6,IL-10,IL-12p40,interferon(IFN)-γ,tumor necrosis factor(TNF)-α,and inducible nitric oxide synthase(iNOS).Results The recombinant strain,Ms_higBA,was constructed successfully.According to the findings of the stress response experiment,higBA could indeed enhance bacterial survival under certain conditions of in vitro culture.Intracellular infection experiment demonstrated that higBA enhanced bacterial survival in macrophages and influenced the transcriptional level of cytokines.Conclusion The higBA genes from Mtb play a role in bacterial stress response and intracellular infection and immunity.