结核杆菌CRISPR-associated Csm4(Rv2820c)诱导iNOS表达对耻垢杆菌胞内存活的影响  

Effect of the Expression of iNOS Induced by Mycobacterium tuberculosis CRISPR-associated Csm4(Rv2820c)on Intracellular Viability of Mycobacterium smegmatis

作　　者：翟小倩 鲍朗 罗涛 彭璇 孙长峰 杨国平 ZHAI Xiao-qian;BAO Lang;LUO Tao;PENG Xuan;SUN Chang-feng;YANG Guo-ping(Laboratory of Infection and Immunity, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu 610041, China)

机构地区：[1]四川大学华西基础医学与法医学院感染免疫研究室,成都610041

出　　处：《四川大学学报（医学版）》2018年第3期319-324,共6页Journal of Sichuan University(Medical Sciences)

基　　金：国家传染病重大专项(No.2012ZX10003008)资助

摘　　要：目的研究Csm4蛋白在重组耻垢分枝杆菌(Mycobacterium smegmatis,MS)中的表达及其影响胞内存活的机制。方法 PCR扩增Csm4基因,构建重组pMV261-Csm4穿梭表达质粒,将重组质粒和空白质粒电穿孔进MS生成重组菌MS_Csm4和对照重组菌MS_V,采用Western blot对重组菌Csm4蛋白表达进行检测。体外培养观察MS_Csm4和MS_V生长曲线,并检测活性氮、活性氧环境下的集落形成单位(CFU)变化。MS_Csm4和MS_V重组菌感染人源巨噬细胞THP-1,计数CFU反映胞内存活,实时荧光定量PCR检测诱导性一氧化氮合酶基因(inducible nitric oxide synthase,iNOS)表达。硝酸还原酶法检测一氧化氮(nitric oxide,NO)释放指标。结果 Csm4蛋白在MS_Csm4中成功表达,且其表达不影响MS_Csm4的生长情况;MS_Csm4在体外活性氮、氧环境中CFU下降,与MS_V相比,差异有统计学意义(P<0.05);重组菌MS_Csm4作用于THP-1细胞后诱导iNOS表达上调,促进NO的释放以及减少胞内生存,与MS_V相比,差异有统计学意义(P<0.05)。结论重组菌MS_Csm4不能耐受体外活性氮、氧压力环境,可诱导宿主细胞iNOS表达上调,促进NO释放,从而影响其胞内生存能力。Objective To determine how Csm4 protein expression affects intracellular survival of Mycobacterium smegmatis（MS）.Methods Csm4 gene was amplified by PCR to construct pMV261-Csm4 shuttle expression plasmid.The Csm4 protein expression in MS_Csm4 was detected by Western blot after electroporation of the recombinant plasmid into MS.The growth kinetics of MS_Csm4 in vitro and the influence of reactive N,O species on the growth of MS_Csm4 were observed.The intracellular survival of MS_Csm4 and expressions of inducible nitric oxide synthase gene（iNOS）and nitric oxide production（NO）were detected after infection with THP-1 macrophages.Results Csm4 protein was successfully expressed in MS_Csm4,which did not affect the growth of the recombinant MS.Reactive N,O species decreased MS_Csm4 colony forming unit（CFU）in vitro.THP-1 increased the expression of iNOSand NO production and decreased intracellular survival of MS_Csm4.Conclusion Recombinant MS_Csm4 is susceptible to reactive N,O species in vitro.THP-1 promotes NO release and thus discourages intracellular survival of MS.

关 键 词：结核Csm4基因 重组耻垢分枝杆菌 活性氮、氧环境 胞内生存 

分 类 号：R378.911[医药卫生—病原生物学]