traf3ip3基因胞外区原核表达及表达谱分析.doc

人类TRAF3IP3基因的表达谱分析、原核表达及亚细胞定位研究 王洋1，王沂1，余源1，刘青1，毛焕2，熊艳杰2，张秀军1△ （河北联合大学1生命科学学院，2附属医院病理科，唐山 063000） 摘要 目的：克隆TRAF3IP3基因，原核表达TRAF3IP3蛋白的膜外区，明确其组织表达谱和亚细胞定位。方法：RT-PCR检测TRAF3IP3基因在人体各组织的表达情况；克隆该基因胞外区部分并构建原核表达载体，经IPTG诱导在大肠杆菌Rosetta（DE3）中表达并通过镍柱纯化重组蛋白；克隆TRAF3IP3 ORF区基因并构建真核亚细胞定位质粒，转染人胚肾HEK 293细胞，激光共聚焦显微镜观察融合蛋白的亚细胞定位。结果：RT-PCR证实该基因在被检的10种组织中均有表达，其中脾、骨髓、淋巴结、胃表达水平较高；成功克隆了TRAF3IP3 ORF区基因和膜外区基因，并分别构建成为荧光定位质粒和原核表达质粒；SDS和western blot证实TRAF3IP3胞外区在大肠杆菌Rosetta（DE3）中得到高效表达，并通过镍柱纯化得到了高纯度重组蛋白；激光共聚焦显微镜观察证实TRAF3IP3蛋白主要定位于核膜，在核周呈颗粒状分布。结论：成功克隆、表达并纯化了TRAF3IP3蛋白的膜外区部分，TRAF3IP3基因在淋巴细胞富集的器官呈高表达，可能参与免疫系统的发育、成熟及调控，其表达产物在细胞内主要定位于核膜及核周胞浆。 Q343.1　 文献标识码：A Gene-expression profile, prokaryotic expression and subcellular localization of human TRAF3IP3 gene WANG Yang, WANG Yi, YU Yuan, LIU Qing, MAO Huan, XIONG Yanjie, ZHANG Xiujun College of Life Sciences, Hebei Union University，Tangshan 063000, China Department of pathology, Hebei United University Affiliated Hospital, Tangshan 063000, China Abstract Objective: To prokaryotic express TRAF3IP3 extramembrane fragment and make clear the tissue expression profile and subcellular localization. Methods: Detect the mRNA expression of TRAF3IP3 in several human tissues by RT-PCR. Clone the extramembrane fragment of TRAF3IP3 and construct prokaryotic expression vector. The recombinant protein was obtained by IPTG induction. Clone the full-length TRAF3IP3 cDNA and construct subcellular location plasmid. The recombinant plasmid is transfected into HEK 293 cells. Subcelluar location of TRAF3IP3 was detected by confocal laser scanning microscopy. Results: RT-PCR demonstrated TRAF3IP3 mRNA was expressed in all detected human tissues and highly expressed in spleen, bone marrow, lymph node and stomach. SDSand western blot showed that the expressed product of TRAF3IP3 extramembrane fragment was a fusion protein about 62kD. Highly purified recombinant TRAF3IP3 protein was obtained by Ni-affinity chromatography. Confocal laser scanning microscopy showed TRAF3IP3 protein