泛素结合酶UBE2T基因在布鲁氏菌感染过程中的作用

Role of ubiquitinase-binding enzyme UBE2T gene in Brucella infection

[目的]探讨泛素结合酶E2T(UBE2T)在布鲁氏菌感染过程中的功能。[方法]构建UBE2T基因过表达载体pLVX-puro-UBE2T,对其进行PCR和EcoRⅠ/BamHⅠ双酶切验证,并将其转染小鼠巨噬细胞RAW264.7,利用实时荧光定量PCR(qRT-PCR)检测UBE2T基因过表达效果。设计并合成3条UBE2T基因沉默表达的siRNA(UBE2T-379-siRNA、UBE2T-599-siRNA和UBE2T-661-siRNA),将其转染小鼠巨噬细胞RAW264.7,利用qRT-PCR检测UBE2T基因沉默表达效果。采用四甲基偶氮唑盐(MTT)试验,检测UBE2T沉默或过表达对RAW264.7细胞活性的影响。利用qRT-PCR,检测M5-90感染对RAW264.7细胞UBE2T基因相对表达量的影响,以及对过表达或沉默表达UBE2T基因的RAW264.7细胞中含pyrin结构域NOD样受体家族3(NLRP3)和半胱氨酸天冬氨酸蛋白水解酶1(Caspase-1)基因相对表达量的影响。用布鲁氏菌M5-90菌株感染过表达和沉默UBE2T基因的RAW264.7细胞,用活菌计数法检测UBE2T沉默或过表达对RAW264.7细胞内布鲁氏菌增殖的影响,利用相应的试剂盒检测细胞乳酸脱氢酶(LDH)水平、半胱氨酸天冬氨酸蛋白水解酶3(Caspase-3)活性及白细胞介素6(IL-6)、IL-18、IL-1β和γ-干扰素(IFN-γ)等细胞因子的水平。[结果]成功构建了UBE2T基因过表达载体pLVX-puro-UBE2T,过表达效果良好。3条siRNA中,UBE2T-661-siRNA的干扰效果最佳,其对UBE2T基因的沉默效率为84%。UBE2T沉默或过表达对RAW264.7细胞活性无影响。M5-90感染可提高RAW264.7细胞UBE2T的表达量;沉默UBE2T可提高M5-90介导的NLRP3和Caspase-1的表达,而过表达UBE2T会抑制M5-90介导的NLRP3表达,提高M5-90介导的Caspase-1表达。沉默表达UBE2T可抑制胞内M5-90的增殖,而过表达UBE2T可促进胞内M5-90的增殖。沉默表达UBE2T可抑制M5-90介导的LDH水平,提高Caspase-3活性及IFN-γ、IL-6和IL-1β水平;而过表达UBE2T可提高M5-90介导的LDH水平,抑制Caspase-3的活性及IFN-γ、IL-1β水平。[结论]泛素结合酶UBE2T对胞内布鲁氏菌增殖具有正调控作用。

[Objective]This study aimed to investigate functions of ubiquitin binding enzyme E2 T(UBE2 T)during Brucella infection.[Method]The pLVX-puro-UBE2 T vector of overexpression of UBE2T gene was constructed and verified by PCR and EcoRⅠ/BamHⅠdouble enzyme before being transfected into mouse macrophage RAW264.7 cells.The effects of overexpression of UBE2T gene were detected by real-time fluorescence quantitative PCR(qRT-PCR).Three silence siRNAs of UBE2T gene(UBE2T-661-siRNA,UBE2T-379-siRNA and UBE2T-599-siRNA)were designed and synthesized,and transfected into mouse macrophage RAW264.7 cells.The effects of silence of UBE2T gene were also detected by qRT-PCR.The effects of overexpression and silence of UBE2T gene on activity of RAW264.7 cells were measured by MTT assay.The effects of M5-90 infection on the relative expression of pyrin domain NOD-like receptor family 3(NLRP3)and cysteine aspartic proteolytic enzyme 1(Caspase-1)in RAW264.7 cells of overexpression and silence of UBE2T gene and on the relative expression of UBE2T in normal RAW264.7 cells were then determined.After RAW264.7 cells of overexpression and silence of UBE2T gene were infected with Brucella M5-90,the effects of overexpression or silence of UBE2T gene on intracellular proliferation of M5-90 were measured by living bacteria counting method.The levels of lactate dehydrogenase(LDH)and activity of cysteine aspartic acid proteolytic enzyme 3(Caspase-3)and levels of interleukin 6(IL-6)and IL-18,IL-1β,andγ-gamma interferon(IFN-γ)were measured by corresponding kits.[Result]The pLVX-puro-UBE2 T vector of overexpression of UBE2T gene was successfully constructed with good overexpression.Among the 3 siRNAs,UBE2T-661-siRNA had the best interference effect and its silence efficiency of UBE2T gene was 84%.The overexpression or silence of UBE2T gene had no effect on activity of RAW264.7 cells.The expression of UBE2 T in RAW264.7 cells with infection of M5-90 was increased.The expression of M5-90-mediated NLRP3 and Caspase-1 was increased by silence of UBE2T gene,while the expression of M5-90-mediated NLRP3 was inhibited and that of M5-90-mediated Caspase-1 was increased by overexpression of UBE2T gene.The intracellular proliferation of M5-90 was inhibited by silence of UBE2T gene,while that was promoted by overexpression of UBE2T gene.The level of M5-90-mediated LDH was inhibited and M5-90-mediated activity of Caspase-3 and levels of IFN-γ,IL-6 and IL-1βwere increased by silence of UBE2T gene.The level of M5-90-mediated LDH were increased and activity of Caspase-3 and levels of IFN-γand IL-1βwere inhibited by overexpression of UBE2T gene.[Conclusion]Ubiquitin binding enzyme UBE2 T had positive regulatory effects on intracellular proliferation of Brucella.