半乳糖基转移酶的克隆表达.docVIP

2017 届毕业设计（论文） 题 目：半乳糖基转移酶的克隆与表达 专 业： 药物制剂 班 级： 药剂1306 姓 名：林碧琦 指导老师：钦松 起讫日期：2017年1月－2017年6月 2017年 6 月 半乳糖基转移酶的克隆表达 摘 要 半乳糖基转移酶是一种催化酶，它可以将活性半乳糖残基从核苷二磷酸半乳糖中转移给糖基受体分子。半乳糖基转移酶都以UDP-Gal为糖基供体，由于形成的二糖键的不同也就有α1，3-半乳糖基转移酶、β1，3-半乳糖基转移酶、β1，4-半乳糖基转移酶等数十种不同的半乳糖基转移酶。本论文中我们以β1，4-半乳糖基转移酶进行克隆与表达。 本文采用PCR技术以巨大芽孢杆菌的基因组为模板克隆出β1，4-半乳糖基转移酶基因，测定克隆获得的DNA片段，与从基因数据库中调取的基因一致。将pcr扩增获得的目的基因BMGA2构建到pet28a载体中，得到重组表达载体pet28a-GA2，再把载体转化到感受态细胞，蛋白胶验证，最后优化表达条件。 关键词：半乳糖基转移酶 克隆表达 表达条件优化  A clonal expression of galactosyltransferase Abstract Galactosyltransferase is the enzyme that catalyzes the transfer of the active semi lactose resi due from the caramate half lactose to the glycogen receptor molecule.Galactose base - galactose transferase with UDP as glycosyl donors, are due to the different form the disaccharide keys hav -e alpha 1, 3 - galactosyl transferase,beta 1, 3 - galactosyl transferase, beta 1, 4 - galactosyltransf -erase and so on dozens of a lot of different galactosyl transferase.In this paper, we clone and exp -ress the enzyme, beta 1, 4-galactosyltransferase. In this passage ,we use PCR technology.Pet28a was used to clone the beta 1,4-galactogenic transferase gene.The DNA fragments obtained from cloning were identical to those obtained fro -m the genetic database.The gene BMGA2, that is the target gene for amplification of PCR, was built into the pet28a carrier, which was reprogrammed to express the expression, pet28a-ga2.And then convert it into the e. coli BL21 sensor -y cell, and verify that the recombinant is correct and then express.Finally, the express -ion condition is optimized. Key Words: galactosyltransferase;cloning and expression; the optimization of expression condition 目录 摘 要 I Abstract Ⅱ 第一章 文献综述 1 1.1 前言 1 1.2 半乳糖基转移酶的研究进展 1 1.2.1 两类β1,4-半乳糖基转移酶 1 1.2.2 β1,4-半乳糖基转移酶的分离与克隆 2 1.2.3 β1,4-半乳糖基转移酶的化学结构 2 1.2.4 β1,4-半乳糖基转移酶的理化性质 3 1.2.5 β1,4-半乳糖基转移酶的生理学功能及应用前景 3 第二章 半乳糖基转移酶的克隆与表达 5 2.1 实验材料 5