重组TACE前肽蛋白对小鼠内毒素血症抑制作用的实验研究

Inhibitory effects of the recombinant TACE pro-domain on endotoxemia in mice

目的建立小鼠内毒素血症模型,观察肿瘤坏死因子前体转换酶(TACE)前肽结构域对其催化结构域的自身抑制作用,为人工干预炎症过程提供依据和手段。方法首先利用DNA重组技术分别构建含前肽结构域和催化结构域的重组载体:用PCR方法扩增出TACE的胞外结构域(T1300)和前肽结构域(T591),并克隆至载体pET-28a(+)中,转化至大肠杆菌BL21,经IPTG诱导表达出带有His-tag的目的蛋白,两者均为包涵体,变性复活后经Ni2+-NTA亲和层析柱对表达的重组蛋白进行纯化。对纯化后的蛋白进行Western印迹分析。构建TACE信号肽+前肽结构域(T648)的真核表达质粒(pEGFP-N1/T648),将其瞬时转染HeLa细胞,通过观察绿色荧光蛋白的表达观察pEGFP-N1/T648转染后在细胞中分布情况。建立小鼠内毒素血症模型组,前肽重组蛋白治疗组和PBS对照组,通过流式细胞术检测小鼠腹腔巨噬细胞mTNF-α的表达,并取肝,肺,肾组织行HE染色,观察纯化的前肽蛋白对炎症的抑制作用。结果成功构建了pEGFP-N1/T648真核表达载体,在体外转染HeLa细胞,可见荧光主要分布在细胞膜上。流式细胞技术证明重组前肽蛋白可明显对抗LPS诱发的sTNF-α分泌增加,使小鼠腹腔巨噬细胞膜表面mTNF-α增加,常规病理切片HE染色显示该蛋白可降低LPS诱发的小鼠肝,肾,肺组织的炎症反应。结论TACE前肽重组蛋白降低了sTNF-α的分泌,对炎症有明显的抑制作用,为抗炎药物的设计和改造提供了新的依据和方法。

Objective To study the effects of tumor necrosis factor-α(TNF-α)converting enzyme(TACE) inhibitors on TNF-α secretion and develop an approach to interfere inflammation processes.Methods The cDNA coding full-length ectodomain (T1300) and pro-domain(T591) of TACE were amplified by PCR.The expression plasmid was constructed and transformed into E.coli BL21(DE3).After Ni2+-NTA resin affinity chromatography,the recombinant T591 protein was obtained and assayed by Western bloting. The plasmid PEGFP-N1 was used to generate the expression plasmid with the signal peptide and pro-domain(designated as T648). The expression plasmids were used to transfect the HELA cells.The Kunming mice were divided into the endotoxic shock model,TACE pro-domain protein cure group,and PBS control group.The mTNF-α of the macrophage cells was detected by FCM and the histopathology HE section was performed to study the inflammatory changes of liver,kidndy,and lung.Results Thebrightest green fluorescence was mostly seen in the membrane of Hela cell transfected with the recombinant plasmid pEGFP-N1/T648.FCM indicated the recombinant pro-domain protein could inhibit the TACE activity,which made the sTNF-α decreased and mediated the accumulation of TNF-α on the surface of macrophage cells.HE staining proved the recombinant protein could decrease the inflammatory response of liver,kidney,and lung. Conclusion The recombinant pro-domain protein is an effective antagonist of TACE and inhibite the sTNF-α release,which indicates TACE is an novel target for inflammat-ion theraphy.