beta-1，4-半乳糖基转移酶基因的克隆及表达

【作者】 吴文英；

【导师】 龚兴国；

【作者基本信息】 浙江大学 ， 生物物理学， 2002， 硕士

【摘要】 β-1，4-半乳糖基转移酶(β-1，4-galaetosyltransferase，β-1，4-GT)是一种Ⅱ型膜结合糖蛋白，通常认为是一种管家基因产物，是至今研究最多的糖基转移酶之一。β-1，4-GT利用尿苷二磷酸半乳糖(UDP-galactose，UDP-Gal)作为激活的糖供体，把半乳糖基转移到N-多糖复合物末端的N-乙酰基葡萄糖胺上。它不仅存在高尔基体膜上，而且整合于细胞质膜上，并表现不同的生物学功能。 本论文采用PCR技术，以pMGT-239／2615为模板克隆获得了β-1，4-半乳糖基转移酶(β-1，4-galactosyltransferase，β-1，4-GT)基因，测定了克隆获得的DNA片段，与从Genbank中调取的基因序列是基本一致的，只是在12位点处核苷酸T突变为G，但由于CGT和CGG均编码氨基酸Arg，所以并不影响β-1，4-GT基因的阅读框架。这种改变是否是PCR扩增引起的，尚难定论。然后，我们将β-1，4-GT基因重组到原核表达载体pGEX-4T-2中，转化大肠杆菌，用IPTG诱导表达后提取总蛋白进行Western印迹反应，检测表达结果为阳性。用IPTG诱导表达的获得β-1，4-GT与GST(谷胱甘肽转移酶)的融合蛋白，经Western blot验证与预想的一致。这是国内首次在原核中融合表达β-1，4-GT。 细胞质膜上的β-1，4-GT与精子的受精作用、胚胎形成、内分泌、神经元细胞的迁移、癌细胞的转移、表皮细胞的增殖及自体免疫性疾病等方面都有着密切的关系。可通过化学修饰或定点突变等方法来研究其结构与功能的关系。通过β-1，4-GT的定点突变改变其功能，从而以此为靶标进行药物筛选。经基因工程改良的β-1，4-GT蛋白质分子，往往具有一些新的特性，与天然β-1，4-GT相比，更适于用作筛选药物的靶标。我们的研究工作为日后对β-1，4-GT进行更广泛深入的研究奠定了基础。更多还原

【Abstract】 β-1,4-Galactosyltransferase-I,β-1 4-Gal-Tl, catalyzes the transfer of galactose (Gal) from UDP-Gal to the N-acetylglucosamine (GlcNAc) residue present at the non-reducing terminal end of glycans of glycoproteins and glycolipids, producing (3-1,4 linked galactosylated glycan. hi addition to GlcNAc as an acceptor, the enzyme can also use other sugars, such as N-acyl substituted glucosamines and N-acetyl-D-mannosamine, as acceptors.Although the genes for all β-1,4-Galactosyltransferase family are located at different chromosomal loci, the evolutionary relationship is indicated by a 30-55% amino acid identity. Moreover, they have hi common the localization in the Golgi apparatus, the topology of type II membrane proteins, and the transfer of galactose from UDP-galactose onto the C4-hydroxyl group of GlcNAc residues.The β-1,4-GT is the most extensively studied enzyme among many kinds of glycosyltransferases. β-1,4-GT seems to play a multifunctional role hi normal cell physiology and has been associated with Sperm-egg binding, cell-cell recognition, cell migration on basal lamina and neurite extension, embryonic maturation, rheumatoid arthritis, and cell development.hi this paper,it is studied on expression of theβ-1,4-GT gene. Theβ-1,4-GT gene was got from the plasmid pMGT-239/2615 through PCR techniques, and cloned into the pGEMT vector. Its sequence was determined by DNA sequencing system. It was then subcloned into the pGEX-4T-2 expression vector, and expressed hi E.coli BL(DE3) after transformation and IPTG induction. The constructed plasmid containing GST-GT fused gene gave a high level of expression of the fused gene. Western blot analysis indicated that the fusion protein could specifically bind to anti-GST antibody. The SDS-PAGE analysis showed that the weight of the fusion protein is correct.更多还原