人CYP17A1酶的表达、纯化及抑制剂筛选模型的建立

论文总字数：19281字

摘 要

Abstract ………………………………………………………………………………………… Ⅰ

1.前言 ……………………………………………………………………………………………1

1.1 前列腺疾病 ……………………………………………………………………………1

1.2 阉割抵抗性前列腺癌 …………………………………………………………………1

1.3 17-α-羟化酶/17,20-裂解酶（CYP17） …………………………………………………2

2. 研究背景及进展 ………………………………………………………………………………3

2.1 CYP17酶抑制剂 ………………………………………………………………………3

2.2 雄激素受体抑制剂 ……………………………………………………………………3

2.3 本文的研究目的和内容 …………………………………………………………………4

3.CYP17A酶的表达和纯化 ………………………………………………………………………4

3.1 实验对象和方法 ………………………………………………………………………5

3.2 实验材料和仪器 ………………………………………………………………………5

3.3 实验过程 ………………………………………………………………………………7

4.实验结果 ………………………………………………………………………………………8

4.1 目的基因的设计、优化、合成与克隆 ………………………………………………8

4.2 诱导、表达与纯化 ……………………………………………………………………11

4.3二氢睾酮转化实验 ……………………………………………………………………13

5.结语 …………………………………………………………………………………………14

致谢 ……………………………………………………………………………………………14

参考文献（References） ………………………………………………………………………15

人CYP17A1酶的表达、纯化及酶活测定

摘要：前列腺癌是男性生殖系统常见的恶性肿瘤，具有显著的发病率和死亡率。当发展为阉割抵抗性前列腺癌 (Castration resistant prostate cancer, CRPC)的时候，常规治疗策略效果较差，其临床治疗变得更为困难。近年来，随着对阉割抵抗性前列腺癌的研究，一批具有新型作用机制的药物已经问世，CYP17抑制剂就是其中之一。由CYP17A1基因编码的细胞色素P450酶(CYP)17是细胞色素P450超家族的重要成员之一。它的本质为17α羟化酶和17,20-裂解酶，在体内雄激素的催化合成过程中起着关键作用。抑制CYP17，可以降低肿瘤细胞内雄激素的浓度，因而CYP17抑制剂成为了CRPC治疗的关键手段。本设计的课题是使用真核和原核表达系统进行人CTP17A1基因的表达，对表达产物CYP17A1酶进行纯化，建立CYP17酶的活性分析方法和CYP17抑制剂的高通量筛选模型。本文将对前列腺癌治疗的研究进展做一综述，介绍CYP17抑制剂研究的背景、研究目的及研究意义，同时介绍以大肠杆菌工程菌为载体表达人类CYP17A1基因，及对表达产物CYP17膜蛋白进行分离纯化，建立CYP17A1酶的活性分析方法和CYP17A1酶抑制剂的高通量筛选模型。

关键词：CYP17A1酶；表达；纯化；抑制剂筛选

Expression and Purification and Activity Assaying of Human CYP17A1 enzyme

Abstract:The prostatic cancer (PC) is one of the most frequently diagnosed kind of male reproductive system cancers. The incidence and death rate of this kind of cancer stays in a high position despite the medical developments. At first, PC is hormone-dependent, which could be effectively treated by either surgical or chemical castration. However, when progressed to a castration-insensitive phase, the conventional treatments are no more useful. As the research on the castration resistant prostate cancer (CRPC) progressed, a series of new medicine came out. The CYP17 inhibitors are one of them.

The cytochrome P450 17α-hydroxylase/17,20-lyase(CYP17) is a member of cytochrome P450 superfamily, and was encoded by the gene CYP17A1. It plays a key role in androgen biosynthesis, thus its inhibition can lead to a sharp decrease of androgen level in the tumor cells. As a result, the inhibitors of CYP17 have great potential to become useful medicine on dealing with PC.

In this article, the main achievements of the prostatic cancers research and the progresses made on the CYP17 inhibitors will be overviewed. What's more, the method of expressing the human CYP17A1 gene using the E.coli engineering bacteria and the separation and purification of the CYP17 membranes protein are demonstrated.

Key words: CYP17A1 enzyme; expression; purification; inhibitor screening

1.前言

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