【摘要】 为了进一步了解Rv1140蛋白酶在结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染中的功能，试验采用同源重组方法构建MTB H37Ra rv1140基因缺失菌株(ΔRv1140菌株)及MTB H37Ra rv1140基因回补菌株(ΔRv1140+Rv1140菌株)，并采用PCR和Southern-blot方法进行鉴定;绘制MTB H37Ra菌株、ΔRv1140菌株和ΔRv1140+Rv1140菌株的生长曲线;利用H37Ra菌株感染巨噬细胞后采用实时荧光定量PCR(q PCR)方法检测感染后rv1140转录水平;利用MTB H37Ra菌株、ΔRv1140菌株和ΔRv1140+Rv1140菌株感染小鼠腹腔巨噬细胞，采用q PCR方法检测炎性细胞因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)基因的转录水平及胞内定殖情况。结果表明:成功构建出ΔRv1140菌株及ΔRv1140+Rv1140菌株; Rv1140蛋白酶的缺失并不影响H37Ra的生长;与感染前相比，H37Ra菌株感染巨噬细胞第48,72小时，rv1140基因的mRNA相对表达量极显著升高(P<0.001);感染U937细胞第24,48,72小时，rv1140基因的mRNA相对表达量显著或极显著升高(P<0.05或P<0.01、P<0.001)。与ΔRv1140菌株感染小鼠腹腔巨噬细胞相比，感染H37Ra菌株和ΔRv1140+Rv1140菌株第12小时，IL-1β mRNA相对表达量极显著升高(P<0.001)，第6,12小时后，H37Ra菌株和ΔRv1140+Rv1140菌株感染的IL-6和TNF-α mRNA相对表达量极显著升高(P<0.01或P<0.001)。说明Rv1140蛋白酶不仅参与调节宿主免疫反应，还与MTB在巨噬细胞内的定殖能力呈现负相关的关系。更多还原

【Abstract】 To further elucidate the function of Rv1140 protease in Mycobacterium tuberculosis( MTB) infection,the study employed homologous recombination to construct MTB H37 Ra rv1140 gene knockout strains( ΔRv1140 strains) and MTB H37 Ra rv1140 gene complementation strains( ΔRv1140+Rv1140 strains),which were identified via PCR and Southern-blot analysis. Growth curves were plotted for the H37 Ra,ΔRv1140,and ΔRv1140+Rv1140 strains. Following infection of macrophages with the H37 Ra,post infection rv1140 transcription levels were detected using real-time quantitative PCR( q PCR). Mouse peritoneal macrophages were infected with the H37 Ra,ΔRv1140,and ΔRv1140+Rv1140 strains,and the transcriptional levels of inflammatory cytokines interleukin-1β( IL-1β),interleukin-6( IL-6),and tumor necrosis factor-α( TNF-α),as well as alongside intracellular colonization,were detected by q PCR. The results indicated both the ΔRv1140 strain and the ΔRv1140+Rv1140 strain were constructed successfully. Compared with the H37 Ra strain,deletion of the Rv1140 protease did not affect H37 Ra growth. Compared with that before infection,the mRNA relative expression level of the rv1140 gene significantly increased at 48 and72 hours post infection in H37 Ra-infected macrophages( P<0. 001); in U937 cells,the mRNA relative expression of rv1140 was significantly or highly significantly upregulated at 24,48,and 72 hours post infection( P< 0. 05 or P< 0. 01 or P< 0. 001). Compared to ΔRv1140 strain infection,both H37 Ra strain and the ΔRv1140+Rv1140 strain induced a highly significant increase in the mRNA relative expression of IL-1βat 12 h post infection( P<0. 001). At 6 and 12 hours post infection,the mRNA relative expression levels of IL-6 and TNF-α were significantly elevated in cells infected with H37 Ra and ΔRv1140+Rv1140( P<0. 01 or P< 0. 001). These findings indicated that the Rv1140 protease not only participated in regulating the host immune response but also exhibited a negative correlation with the intracellular colonization ability of MTB in macrophages.更多还原