结核杆菌Pup－蛋白酶体系统与结核杆菌持留性的相关性研究

摘要

目的：本研究通过诱导结核杆菌在低氧环境中进入持留状态，比较分析不同时间点、不同环境下结核杆菌Pup、Dop、PafA和Mpa 基因的表达水平差异，探讨研究Pup－蛋白酶体系统对结核杆菌持留性的调控作用。方法：本实验以结核杆菌国际标准强毒株H37Rv菌株（以下简称为H37Rv菌株）为研究对象，在低氧、有氧环境下进行培养，分别提取不同时间点每组样本菌株的总RNA，并进行纯度鉴定。运用SYBR GreenⅠ实时荧光定量PCR的方法，分别检测各组结核杆菌菌株的Pup、Dop、PafA和Mpa 基因的表达；比较分析不同时间点、不同环境下的各结核杆菌菌株Pup、Dop、PafA和Mpa基因的表达水平差异。结果：在不同时间点对低氧环境下菌株Pup、Dop、PafA和Mpa的表达水平进行检测，Pup、Dop、PafA和Mpa基因的相对表达水平：低氧环境下，与0 d比较，Pup基因在4 d、7 d、10 d分别上调1．66、2．43、2．76倍，Dop基因在2 d、4 d、7 d、10 d分别上调1．38、1．91、2．54、3．28倍，PafA基因在2 d、4 d、7 d、10 d分别上调1．22、1．75、2．37、2．67倍，Mpa 基因在4 d、7 d、10 d分别上调1．66、2．21、2．63倍（ P＜0．05）；以有氧环境组做对照，低氧环境下，相同时间点，Pup 基因在4 d、7 d、10 d分别上调1．85、2．81、2．93倍，Dop基因在2 d、4 d、7 d、10 d分别上调1．20、1．76、2．01、3．01倍，PafA基因在2 d、4 d、7 d、10 d分别上调1．22、1．57、2．29、2．29倍，Mpa 基因在2 d、4 d、7 d、10 d分别上调1．16、1．58、2．16、2．69倍（ P＜0．05）。结论：低氧环境下，不同时间点，Pup、Dop、PafA和Mpa 基因的相对表达量存在差异；以有氧环境组作对照，相同时间点，低氧环境下与有氧环境下Pup、Dop、PafA和Mpa 基因的相对表达量也存在差异。 Pup－蛋白酶体系统对结核杆菌持留性具有一定的调控作用。%Objective:To explore the regulation of prokaryotic ubiquitin-like protein ( Pup )-proteasome system on the persistence of Mycobacterium tuberculosis by inducing Mycobacterium tuberculosis to being persistence state under hypoxia conditions and then analyzing the difference on the expression levels of Pup,Dop,PafA and Mpa gene at various time and different conditions. Methods:The total mRNA of the international standard virulent strains of Mycobacterium tuberculosis(H37Rv),which were cultured under hypoxia and aerobic conditions, were extracted from each group at various time and purity of the mRNA were identified.The expression of Pup,Dop,PafA and Mpa genes of M.tuberculosis strains in each group were quantified by SYBR Green I qRT-PCR,which aimed at finding the difference among the expression of Pup,Dop,PafA and Mpa genes at various time and different conditions.Results:The expression levels of Pup, Dop, PafA and Mpa genes in Mycobacterium tuberculosis under hypoxia conditions were measured at various times.The expression levels of Pup,Dop,PafA and Mpa genes:compared with the 0 d,the expression of the Pup gene was up-regulated 1.66,2.43 and 2.76-fold at 4 d,7 d,10 d respectively(P<0.05);the expression of the Dop gene was up-regulated 1.38, 1.91,2.54 and 3.28-fold at 2 d,4 d,7 d,10 d respectively(P<0.05);the expression of the PafA gene was up-regulated 1.22,1.75, 2.37 and 2.67-fold at 2 d,4 d,7 d,10 d respectively( P<0.05);the expression of the Mpa gene was up-regulated 1.66,2.21 and 2.63-fold at 4 d,7 d,10 d respectively(P<0.05).Take the aerobic conditions as control,under hypoxic conditions with the same culture time,the expression of the Pup gene was up-regulated 1.85,2.81 and 2.93-fold in 4 d,7 d,10 d respectively(P<0.05);the ex-pression of the Dop gene was up-regulated 1.20,1.76,2.01 and 3.01-fold in 2 d,4 d,7 d,10 d,respectively( P<0.05);the expression of the PafA gene was up-regulated 1.22,1.57,2.29 and 2.29-fold in 2 d,4 d,7 d,10 d,respectively(P<0.05);the expression of the Mpa gene was up-regulated 1.16,1.58,2.16 and 2.69-fold in 2 d,4 d,7 d,10 d,respectively(P<0.05).Conclusion:Under hypoxic conditions,there were significant differences on the expressions of Pup, Dop, PafA and Mpa genes at various times;what′s more, significant differences on the expressions of Pup,Dop,PafA and Mpa genes exist between hypoxic and aerobic conditions at the same time,Prokaryotic ubiquitin-like protein( Pup)-proteasome system plays a regulatory role on M.tuberculosis′s persistence.