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目的:构建肿瘤坏死因子相关凋亡诱导配体(TRAIL)高表达工程菌株,以获得具有生物学活性的重组TRAIL蛋白。方法:合成TRAIL基因,转到大肠杆菌中,IPTG诱导获得成功表达;进一步优化,增加可溶形式的蛋白质。利用硫酸铵对发酵液进行沉淀,经Ni柱、阴离子和阳离子交换柱层析,最终获得蛋白质纯品,对其进行体外活性检测。结果:经过IPTG诱导后,TRAIL达到菌体总蛋白的30%;经过优化,可溶形式蛋白达到55%。纯化获得纯度高于95%的TRAIL样品,体外测活结果表明:TRAIL蛋白能有效抑制多种肿瘤细胞增殖,抑制率达到70%～92%。结论:重组TRAIL以可溶形式得到高效表达,且具有较好的生物学活性,为其开发成药用蛋白奠定基础。
OBJECTIVE: To construct an engineered strain with high expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to obtain a recombinant TRAIL protein with biological activity. Methods: The TRAIL gene was synthesized, transferred to E. coli, and induced by IPTG. The recombinant protein was further optimized to increase the soluble protein. Fermentation broth was precipitated with ammonium sulfate, and purified by Ni column, anion and cation exchange column chromatography. The pure protein was obtained finally and its activity was tested in vitro. Results: After induction with IPTG, TRAIL reached 30% of the total bacterial proteins; after optimization, the soluble protein reached 55%. The purity of TRAIL was higher than 95%. The in vitro activity of TRAIL protein showed that TRAIL protein could effectively inhibit the proliferation of many tumor cells with the inhibition rate of 70% -92%. CONCLUSION: Recombinant TRAIL is highly expressed in soluble form and has good biological activity, laying a foundation for its development into a medicinal protein.