【作者】 孙其昌；

【导师】 吴士良； 周迎会；

【作者基本信息】 苏州大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 目的:β1,3半乳糖基转移酶(β3GalT7)是一个新型的人类糖基转移酶,属于β3糖基转移酶大家族。本文旨在对其催化功能进行研究。同时,在此基础上,对该酶与肿瘤的关系进行初步的研究。方法:1.将pGEM-T载体中的目的基因亚克隆至原核表达载体pGEX-6p-1,转化BL21大肠杆菌,IPTG诱导表达融合蛋白,表达产物复性后用亲和层析柱进行纯化后利用高效液相色谱(HPLC)进行酶活检测。2.克隆β3GalT7编码区的基因并构建其真核真义和反义表达载体,并运用脂质体介导将此重组载体转染中度表达该基因的人SGC7901细胞株,和较高表达β3GalT7的A549细胞株,并通过荧光显微镜,激光共聚焦显微镜,RT-PCR,western印迹等方法对其进行了初步的功能研究。结果:1.HPLC结果表明β3GalT7具有β3GnT糖基转移酶家族的催化活性。2.通过激光共聚焦观察转染了β3GalT7的SGC7901细胞,发现β3GalT7编码的蛋白大部分在胞浆,而核膜上观察到有一小部分。转染了β3GalT7的SGC7901细胞其蛋白表达水平增加, MMP2、MMP14的蛋白表达水平亦有所上升,同时转染了反义β3GalT7的A549细胞其MMP2和MMP14的蛋白表达量随着β3GalT7的被抑制而成正相关下降。结论:初步证明β更多还原

【Abstract】 Object: UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase polypeptide 7 is a new member of glycosyltransferases which belongs to the beta-3 glycosyltransferase family. This thesis aims to the catalytic activity ofβ3GalT7. Simutaneously, study on the relationship between theβ3GalT7 and the tumor cell on the base of the that. Methods: 1.The cDNA gene encodingβ3GalT7, which was amplified from plasmid pGEM-T-T7 by PCR, was subcloned into prokaryotic expression vector pGEX-6p-1, the recombinant plasmid was subsequently transformed into Escherichia coli BL21. Then a fusion protein was expressed after the induction by IPTG. After testified by western blot, we purified the expression products by affinity chromatography. After renaturation, its enzyme activity was assayed by High Performance Liquid Chromatography (HPLC). 2. Theβ3GalT7 fragment was subcloned to the eukaryotic plasmid and transfected to the SGC7901 and A549 cell lines. With fluorescence microscope, laser confocal microscope, RT-PCR, Western blot, the preliminary functions ofβ3GalT7 in the tumor更多还原