重组TACE前肽对自身TACE活性的抑制作用

Inhibition of the recombinant TACE pro-domain to TACE

目的以脂多糖（LPS）刺激人单核细胞型淋巴瘤细胞株THP-1为模型，观察重组肿瘤坏死因子α转换酶（TACE）前肽结构域对催化TACE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重组技术分别构造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Ni^2＋ -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 blot分析。LPS分时相刺激THP-1细胞后，用细胞学技术观察纯化的T591在蛋白质水平上对TACE的表达和活性的影响。结果TACE重组前肽蛋白能显著抑制TACE的活性，减少分泌型TNF-α（sTNF-α）分泌，抑制率达57％。结论TACE重组前肽抑制了TACE的活性，降低了sTNF-α的分泌，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 effect of tumor necrosis factor-α （TNF-α） converting enzyme （TACE） inhibitors on TNF-α secretion and to develop an approach to interfere inflammation processes. Methods The cDNA coded for full-length ectodomain （T1300） and pro-domain（T591） of TACE was 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 blot. The experiment in vitro was carried out on THP-1 cell lines stimulated by LPS. The inhibition of recombinant protein to TACE was detected by ELISA and immunohistochemical technique. Results The recombinant pro-domain protein inhibited 57% of TNF-α secretion and mediated the accumulation of TNF-α on the surface of THP-1 cell. Conclusion The recombinant pro-domain protein is an effective antagonist of TACE and inhibite the secretary TNF-α release, which indicates that TACE is a novel target for inflammation therapy.