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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 establish mouse model of endotoxemia and observe its inhibitory effect on the catalytic domain of pro-peptide domain of tumor necrosis factor precursor converting enzyme (TACE), and to provide basis and means for artificially interfering with the inflammatory process. Methods Recombinant vectors containing the propeptide domain and the catalytic domain were constructed by DNA recombinant technology. The extracellular domain (T1300) and propeptide domain (T591) of TACE were amplified by PCR and cloned into the vector pET -28a (+). The recombinant protein was transformed into E.coli BL21 and induced by IPTG to express the target protein with His-tag, both of which were inclusion bodies. After denaturation, the recombinant protein was expressed by Ni2 + -NTA affinity chromatography Protein was purified. The purified protein was subjected to Western blot analysis. The eukaryotic expression plasmid (TEGFP-N1 / T648) of TACE / T648 was constructed and transfected into HeLa cells transiently. The expression of green fluorescent protein (GFP) was observed and the expression of pEGFP-N1 / T648 Cell distribution. Mouse model of endotoxemia, pro-peptide recombinant protein treatment group and PBS control group were established. The expression of mTNF-α in mouse peritoneal macrophages was detected by flow cytometry. HE staining was performed on liver, lung and kidney , Observe the inhibitory effect of the purified propeptide protein on inflammation. Results The eukaryotic expression vector pEGFP-N1 / T648 was successfully constructed and transfected into HeLa cells in vitro. Fluorescence was found mainly in the cell membrane. Flow cytometry showed that recombinant propeptide protein could significantly increase the secretion of sTNF-α induced by LPS and increase mTNF-α on the surface of peritoneal macrophages in mice. HE staining showed that the protein could reduce LPS-induced mouse liver , Kidney, lung tissue inflammation. Conclusion The recombinant prokaryotic TACE protein can reduce the secretion of sTNF-α and inhibit the inflammation. It provides a new basis and method for the design and modification of anti-inflammatory drugs.