人Pokemon蛋白锌指结构域的表达与纯化.docVIP

  人 Pokemon 蛋白锌指结构域的表达与纯化# 杨予涛，徐志卿** （首都医科大学基础医学院神经生物学系） 5 10 摘要：目的：原核表达 Pokemon 基因的锌指结构域，纯化获得 GST-Zinc finger 的融合蛋白。方法：以人胶 质瘤 T98G 细胞的 cDNA 为模板，利用 PCR 扩增带有 BamHI 和 Sal I 酶切位点的人 Pokemon 基因的锌指结 构域，然后将其克隆到 pGEX-4T-1 原核表达载体中。将正确的重组载体转入大肠杆菌 BL21（DE3），用 IPTG 诱导表达，再利用 MagneGST particles 亲和纯化 Zinc finger 融合蛋白，最后通过 Western blot 鉴定此 融合蛋白。结果：成功构建 pGEX-4T-1-Zinc finger 原核表达载体；30条件下，0.2 mmol/L 的 IPTG 能诱 导出大量的可溶性 GST-Zinc finger 蛋白；经 MagneGST particles 纯化的 GST-Zinc finger 蛋白可被识别 Pokemon 锌指结构域的抗体特异识别。结论：成功在大肠杆菌中表达了 GST-Zinc finger 蛋白，纯化的 GST-Zinc finger 蛋白可用于后续的生物学研究。 关键词：Pokemon; 锌指结构域；原核表达；蛋白纯化 中图分类号：R34 15 Prokaryotic expression and purification of zinc finger domain of human Pokemon protein Yang Yutao, Xu Zhiqing  20 25 30 35 40 (School of basic medical sciences , Capital medical university,100069) Abstract: Objective: To express human Zinc finger domain of Pokemon gene in prokaryotic cells and purify the GST-Zinc finger fusion protein. Methods: The segment of zinc finger domain of Pokemon gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-1.The recombinant plasmid pGEX-4T-1-Zinc finger was transformed into E.coli BL21（DE3）and exogenous protein was induced by IPTG. After purification using MagneGST particles,the GST-Zinc finger fusion protein was further identified by Western blot. Results: The recombinant plasmid pGEX-4T-1-Zinc finger was constructed successfully. When BL21(DE3) cells transformed with pGEX-4T-1-Zinc finger were cultured at 30℃ and induced with 0.2 mmol/L IPTG,GST-Zinc finger protein was obtained in a large quantity in supernatant. The purified GST-Zinc finger was further identified specifically by Pokemon antibody. Conclusion: GST-Zinc finger fusion protein was successfully expressed and prufied, and it could be used for further study of the function of Pokemon. Key words: Pokemon; Zinc finger; prokaryotic expression; protein purification 0 引言 Pokemon，即 POK 红系髓性致癌因子(POK erythroid myeloid Ontogenic factor )，也被称 为 LRF、FBI-1