论文总字数：17257字

摘 要

C118P是C118的前药，是一种新型微管蛋白抑制剂，具有独特的抗肿瘤机制，结构新颖，具有自主知识产权，作用机制明确、抗肿瘤效果明显、抑瘤谱广、体外稳定性高，有较好的成药性和安全性。

糖苷酶在水相介质中的水解活性较强，而在非水相介质中比水相介质中具有更高的糖苷合成效率，但是存在着糖苷酶在有机溶剂体系内不太稳定和催化合成效率较差的缺陷，因此，开发高效且耐亲水有机溶剂的酶类是合成糖苷化合物的关键因素。

本实验从实验室菌库中筛选得到了糖基化转移酶ZS4，先提取相应的菌株和质粒，用细菌通用引物扩增糖基转移酶的基因，基因片段和载体pET-28a的质粒进行双酶切，所得的重组菌在转化后的感受态大肠杆菌内进行诱导表达，电泳、测序。用该糖基转移酶于非水相体系中全细胞高效催化二苯乙烯类抗肿瘤药的生物转化。

关键词：C118P 糖基转移酶 非水相体系 生物转化 天然产物

Biotransformation of C118P

——clone and expression of glycosyltransferase

Abstract

C118P is the prodrug of C118. It is a novel antitubulin with unique and clear anti tumor mechanism, novel structure, independent intellectual property rights, obvious anti tumor effect, broad antibacterial spectrum and high stability. It has good druggability and safety.

Glycosidases has a strongly hydrolytic activity in the aqueous phase and higher glycoside synthesis efficiency in nonaqueous media, but the stability of the enzyme gave limitation to the glycosides prepared by the glycosides acquired effectively. In addition, some reported glycosides prepared by the glycosidases are both low in efficiency and concentration of glycoside products. Thus, exploitation of high activity, low-cost, and organic solventstable glycosidase would be an effective way to prepare glycosides.

The experiment screens the glycosyltransferase ZS4 from the laboratory strains library. Firstly, extract corresponding strains and plasmid, then amplify glycosyltransferase’s gene with universal bacterial primers. Put the gene fragment and the vector pET-28a’s plasmid into double enzyme digestion. The recombinant strain was induced to express in feeling E.coli which has been transformed, and the results were obtained by electrophoresis and sequencing.The biotransformation of C118P is catalyzed by whole cell in high efficiency in non-aqueous system by using the glycosyltransferase .

Key Words: C118P;glycosyltransferase;Non-aqueous media;biotransformation；natural products

目 录

摘要 Ⅰ

ABSTRACT Ⅱ

第一章 文献综述 1

1.1 CA4及C118简介 1

1.1.1 CA4及C118结构 1

1.1.2 CA4及C118抗肿瘤机制 1

1.2非水相酶催化体系和非水相酶结构 2

1.2.1水互溶有机溶剂单相体系中天然产物的生物转化…………………2

1.2.2液-液两相体系(水-有机溶剂两相体系)中天然产物的生物转化.......2

1.2.3固-液两相体系中天然产物的生物转化………………………………...2

1.3天然产物活性组分的糖基化修饰 3

1.3.1糖基化修饰的影响…………………………………………………..………3

1.3.2 CA4糖基化修饰的目的和现状........………………………………...…....3

1.4耐有机溶剂糖苷酶及酶解法………………………………………………...……..3

1.5本课题的主要研究目的与研究意义 4

第二章 糖基转移酶的克隆表达 5

2.1 实验材料 5

2.1.1实验仪器 5

2.1.2实验试剂………………..……………………………………………………...6

2.1.3菌株与质粒………………………………..…………………………………...7

2.1.4培养基 7

2.2 实验方法 8

2.2.1细菌DNA的提取 8

2.2.2糖基化转移酶的PCR扩增 8

2.2.3目的基因与载体的连接………………….……………………………………..9

2.2.4感受态细胞制备…………………………………….......................................9

2.2.5转化……………………………………..……………………………………..10

2.2.6菌落PCR法鉴定阳性克隆与测序…………………………….……......10

2.2.7表达载体的构建………………………………………..……………….......11

2.2.8糖基化转移酶在大肠杆菌中的诱导表达……………………….……..11

2.2.9 SDS-PAGE聚丙烯酰胺凝胶电泳检测………………….…....………...12

2.2.10测序………………………………………..…………………………………13

2.3 结果与讨论 13

2.3.1 糖基化转移酶的PCR扩增 13

2.3.2 糖基化转移酶基因表达载体的构建 13

2.3.3 糖基化转移酶在大肠杆菌中的诱导表达…………...…….…………..14

2.4本章小结 15

第三章 结论与展望 16

3.1 结论 16

3.2 展望 16

参考文献 17

致 谢 19

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