人甲状腺激素受体相互作用蛋白15的初步结晶实验及其在人组织表达的研究

Preliminary Crystallization of Human Thyroid Hormone Receptor Interacting Protein 15 and Its Expression in Human Tissue

目的:构建人甲状腺激素受体相互作用蛋白15(thyroid hormone receptor interacting protein15,TRIP15)的原核表达载体,在大肠杆菌表达并纯化、结晶表达产物;证实TRIP15在人脐静脉内皮细胞株、血小板、二尖瓣、胆囊和胶质瘤存在表达。方法:以人肝细胞cDNA文库为模板,通过PCR扩增TRIP15的全长编码区序列,双酶切后连接到pGEX-6P-1载体上,转化E.coli BL21(DE3)菌株,通过亲和层析及分子筛对表达产物进行纯化,采用气相扩散悬滴法筛选并优化结晶条件,通过X射线晶体衍射技术检测晶体衍射;采用RT-PCR和Western blot研究TRIP15在人脐静脉内皮细胞株、血小板、二尖瓣、胆囊和胶质瘤的表达情况。结果:成功构建了TRIP15的原核表达载体,获得了电泳纯TRIP15蛋白,得到了蛋白质晶体,但衍射能力很弱;通过RT-PCR证实其在人脐静脉内皮细胞株、血小板、二尖瓣、胆囊和胶质瘤存在表达。结论:成功构建了TRIP15的原核表达载体、建立了表达纯化策略并获得了初步结晶的实验条件,为最终解析其三维结构奠定了基础;初步证实TRIP15在人脐静脉内皮细胞株、血小板、二尖瓣、胆囊和胶质瘤存在表达,为相关研究的开展奠定了基础。

Objective： To construct prokaryotic expression vector of the full length human thyroid hormone receptor interacting protein 15 （hTRIP15）, to establish the strategy to express, purify and crystallize hTRIP15 ; To determinate its expression in human umbilical vein endothelial cells line （HUV-EC-C）, platelet, mitral valve, gall bladder and glioma. Methods： Full length coding sequence of hTRIP15 was amplified from human hepatocyte cDNA library by PCR and cloned into pGEX-6P-1 vector after digested with BamH I and Xho I. GST- hTRIP15 was expressed in E. coli BL21 （ DE3 ）, and then protein was purified by affinity chromatography and gel filtration chromatography. Hanging drop vapor diffusion method was employed to screen and optimize the crystallizing condition. Crystal structure determination was tried by X-ray crystal diffraction technique. Further, the expression of hTRIP15 in HUE-EC-C, platelet, mitral valve, gall bladder and glioma was evaluated by RT- PCR and Western blot. Results： Recombinant hTRIP15 was successfully expressed in E. coli and purified. The preliminary crystallization condition was determined using the hanging-drop vapor-diffusion method. TRIP15 mRNA was determined by RT-PCR in human HUV-EC-C, platelet, mitral valve, gall bladder and glioma, and its expression in HUV-EC-C was further confirmed by Western blot. Conclusion： The strategy of cloning, expressing and purifying the hTRIP15 was established. The preliminary crystallization condition was obtained. It provides experimental data for final tertiary structure determination. The expression of hTRIP15 was preliminarily determined in HUV-EC-C, platelet, mitral valve, gall bladder and glioma. It provides experiment data for further function study.