茶树菇Agrocybe aegerita新泛素偶联酶蛋白表达及抗肿瘤活性研究

Expression and Antitumor Activity of a Novel Ubiquitin Conjugating Enzyme from the Medicinal Fungus Agrocybe aegerita

大型真菌茶树菇活性蛋白组分Yt在动物模型中被发现具有抗肿瘤及免疫调节活性。为研究其蛋白组分,构建新鲜蘑菇子实体c DNA文库,对c DNA序列进行测序筛选,获得新型真菌泛素偶联酶(Ub-conjugating enzyme,UBC)的c DNA序列(Gen Bank登录代码:JQ362395),分析其含有UBC家族的保守残基。使用原核表达p ET系统表达和纯化重组UBC蛋白,得率是10 mg/L大肠杆菌培养液。在细胞培养中加入重组UBC蛋白将显著改变细胞形态,通过流式细胞仪发现UBC(1.8、3.6、7.2μM)均能诱导He La细胞凋亡,AnnexinⅤ+/PI-百分比由对照组的1.4%上调至10.7%、15.8%和20.3%,呈浓度依赖性。本文是首次报道真菌UBC具有抗肿瘤活性。该结果暗示,真菌泛素系统成员可能通过改变细胞基本生理功能,成为潜在的候选药物,值得探讨。

The active protein component Yt from macrofungi Agrocybe aegerita has been found to have antitumor and immune regulation activity. For the investigation of the proteins, a cDNA library of the Agrocybe aegerita fruiting body was constructed and screened. The cDNA of a novel fungal Ub-conjugating enzyme (UBC) was identified (Genbank accession code: JQ362395), and sequence analysis revealed that it contained the conserved residues and belonged to the UBC family. Recombinant UBC was expressed using the pET system and purified, and obtained 10 mg from 1 L E coli. culture media. Exogenously applied UBC (1.8, 3.6 and 7.2 μM) in cell cultures altered HeLa cell morphology and induced cell apoptosis determined by flow cytometry, and the percentage of AnnexinV+/PI- cells was increased from 1.4% in control to 10.7%, 15.8% and 20.3%, which was concentration dependent. This is the first report on the effects of a fungal UBC on cancer cells, which suggests that the members of the fungal ubiquitin system might alter fundamental cellular processes and require further investigation. © 2015, South China University of Technology. All right reserved.