人体TRIP15蛋白的纯化结晶及其在人脐静脉内皮细胞表达的研究

发布时间：2018-01-06 06:13

本文关键词：人体TRIP15蛋白的纯化结晶及其在人脐静脉内皮细胞表达的研究　出处：《昆明医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：人体甲状腺激素受体相互作用蛋白15(thyroid hormone receptor interacting protein15, TRIP15)也被称为CSN2、COPS2、SGN2、ALIEN,是核受体家族的选择性转录共抑制子。TRIP15参与构成COP9信号复合体,并与涉及细胞周期调控、DNA损伤修复的多种分子相互作用,影响细胞的增殖、分化和凋亡,进而与肿瘤、内分泌疾病和脂质代谢紊乱的发病相关。但迄今为止,仍未得到TRIP15的三维结构,也没有其在人脐静脉内皮细胞株(human umbilical vein endothelial cells, HUV-EC-C)表达情况的报道。本论文将对TRIP15的克隆表达、纯化结晶及其在HUV-EC-C的表达情况进行研究。本论文工作由两部分组成。1.TRIP15蛋白质的克隆表达、纯化结晶：采用PCR从人肝细胞cDNA文库获得人TRIP15基因1332bp的全长编码区序列。此片段经BamH I及XhoI双酶切后连接到同样经双酶切处理的带有GST标签的pGEX-6P-1载体上,转染大肠杆菌。经IPTG诱导,蛋白质以部分可溶形式表达,经过亲和层析、分子筛、超滤等手段得到适于结晶的电泳纯蛋白质。应用气相扩散悬滴法筛选结晶条件并进行结晶条件的优化。将晶体进行X射线晶体衍射实验,但该晶体衍射能力很弱；2.通过RT-PCI及Western Blot技术,证实TRIP15在HUV-EC-C中存在表达,为后续TRIP15在HUV-EC-C中的功能研究(如RNAi及基因过表达)奠定了基础。本论文的创新性体现在： 1.构建了人体TRIP15在大肠杆菌中的表达载体,获得了该蛋白质表达纯化及初步结晶的实验条件； 2.在mRNA及蛋白质水平证实了TRIP15在HUV-EC-C中存在表达。
[Abstract]:Thyroid hormone receptor interacting protein15. Trips 15) is also known as CSN2 / COPS2 / SGN2Alen. TRIP15, a selective transcriptional co-suppressor of nuclear receptor family, is involved in the formation of COP9 signal complex and interacts with many molecules involved in cell cycle regulation and repair of DNA damage. It affects cell proliferation, differentiation and apoptosis, which is related to tumor, endocrine disease and lipid metabolism disorder. However, up to now, the three-dimensional structure of TRIP15 has not been obtained. There was no umbilical vein endothelial cells in human umbilical vein endothelial cell line. In this paper, the cloning expression, purification and crystallization of TRIP15 and its expression in HUV-EC-C were studied. This thesis consists of two parts. 1. Cloning and expression of TRIP15 protein. Purified crystallization: the full-length encoding region of human TRIP15 gene 1332bp was obtained from human hepatocyte cDNA library by PCR. This fragment was amplified by BamH. I and XhoI were digested and ligated to pGEX-6P-1 vector with GST tag. After transfection with E. coli, the protein was expressed in partially soluble form after IPTG induction, and then the molecular sieve was obtained by affinity chromatography. The electrophoretic pure protein suitable for crystallization was obtained by ultrafiltration. The crystallization conditions were selected by gas phase diffusion suspension method and the crystallization conditions were optimized. The crystal was tested by X-ray crystal diffraction. But the diffraction ability of the crystal is very weak. 2.The expression of TRIP15 in HUV-EC-C was confirmed by RT-PCI and Western Blot. It lays a foundation for the functional study of TRIP15 in HUV-EC-C, such as RNAi and gene overexpression. The innovation of this thesis lies in: 1. The expression vector of human TRIP15 in Escherichia coli was constructed, and the experimental conditions for the expression, purification and primary crystallization of the protein were obtained. 2. The expression of TRIP15 in HUV-EC-C was confirmed at the level of mRNA and protein.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R3411;R363

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