人CYP17A1酶在动物细胞中的瞬时表达

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摘 要

前列腺癌是男性泌尿生殖系统最常见的恶性肿瘤之一，其发展的主要机制为细胞内雄激素受体受到雄激素的持续刺激。CYP17A1酶是人体内催化雄激素合成的关键酶，在前列腺肿瘤的发生、发展和转移中起重要作用。通过抑制CYP17A1酶的活性来降低肿瘤细胞内雄激素浓度已成为治疗前列腺癌的关键手段。而要筛选安全有效的CYP17A1抑制剂必须先大规模获得CYP17A1酶。目前，已有研究者成功在大肠杆菌中表达该酶，但后续研究表明，该重组酶的活性低于人体内的天然酶，且表达量不高，表达效果不理想。因此，本研究选择改用中国仓鼠卵巢(CHO)细胞作为宿主细胞，构建CYP17A1的真核表达体系，以大规模获得具有良好生物活性的表达产物CYP17A1酶。这有利于今后进行进一步的蛋白结构与功能研究，也为建立CYP17A1酶抑制剂的高通量筛选模型打下了基础。

方法：构建人CYP17A1酶的真核表达载体，双酶切反应及测序检测目的基因；将该重组质粒转染到CHO细胞中，筛选获得稳定细胞株；并进行诱导表达，提取表达的蛋白质，经镍柱亲和层析分离提纯，SDS-PAGE电泳检测是否有目的蛋白条带，并利用高效液相色谱法进行酶活分析测定。

结果：构建了人CYP17A1酶的真核表达载体pEF6-CYP17，双酶切检测得到了预期大小的条带，测序得到的结果与目的基因序列一致，表明重组质粒构建成功；经转染筛选获得了稳定CHO细胞株，提取诱导表达的蛋白并经镍柱纯化后，获得了分子量为55.88kD的目的蛋白；转化实验表明该酶具有羟基化活性

结论：本实验成功构建了人CYP17A1酶的真核表达载体pEF6-CYP17，并获得了可大规模表达人CYP17A1酶的稳定CHO细胞株，分离提纯得到了目的蛋白，且其具有良好的17α羟化酶活性。

关键词：人CYP17A1酶；中国仓鼠卵巢（CHO）细胞；真核表达

The expression of human CYP17A1 enzyme in CHO cells

Student: Ru Tian supervisor:Xudong Xu

The Medical School of Southeast University, Nanjing 210009

ABSTRACT

Prostate cancer is one of the most common malignancies in the male genitourinary system. The main mechanism of its development is that the androgen receptor in the cells is stimulated continuously by androgen. CYP17A1 enzyme is a key enzyme in the synthesis of androgen in the human body, playing an important role in the occurrence, development and metastasis of prostate cancer. Reducing androgen levels in tumor cells by inhibiting the activity of CYP17A1 enzyme has become a key means of treating prostate cancer. To screen safe and effective CYP17A1 inhibitors, we’d better obtain CYP17A1 enzyme on a large scale. At present, researchers have successfully expressed the enzyme in E.coli, but follow-up studies have shown that the activity of the recombinant enzyme is lower than the natural enzyme in the human body, and the expression level is not high, so the expression is not ideal. Therefore, this study uses Chinese hamster ovary (CHO) cells as host cells to construct a eukaryotic expression system of CYP17A1 to obtain large-scale expression product with good biological activity, which is conducive to further studies about protein structure and functional in the future and lays a foundation for the establishment of high-throughput screening model of CYP17A1 enzyme inhibitors.

Methods: The eukaryotic expression vector of human CYP17A1 enzyme was constructed, and the target gene was detected by double digestion and gene sequencing. The plasmid was transfected into CHO cells to obtain stable cell lines. Then we induced the cells to express protein. After that, the study chose affinity Ni-NTA column to purify the protein and SDS-PAGE electrophoresis to detect the target protein band. At last we used the high performance liquid chromatography for enzyme activity analysis.

Results: The eukaryotic expression vector pEF6-CYP17 of human CYP17A1 was constructed and we got the expected band by double digestion. The results of gene sequencing were consistent with the sequence of target gene, indicating that the recombinant plasmid was constructed successfully. The results of SDS-PAGE electrophoresis showed that there was a correct band of 55.88 kD. The results of HPLC showed that the enzyme had good activity of 17α hydroxylase.

Conclusion: The eukaryotic expression vector pEF6-CYP17 of human CYP17A1 enzyme was successfully constructed and a stable CHO cell line for expressing human CYP17A1 enzyme was obtained. The expressing product was purified by Ni-NTA column and we obtained the target protein. The analysis of enzyme activity showed that the target protein had good biological activity.

Key words: human CYP17A1 enzyme; Chinese hamster ovary (CHO) cells; eukaryotic expression

目 录

第一章 综述 1

1.1 CYP17A1酶 1

1.2 CYP17A1酶的抑制剂 1

1.3 CYP17A1酶的大肠杆菌体外重组表达系统 2

1.4 展望 2

1.5 本研究的主要目的和内容 3

第二章 实验材料与方法 3

2.1实验材料 3

2.1.1 基因 3

2.1.2 质粒和细胞株 3

2.1.3 主要试剂及试剂盒 3

2.1.4 实验中配置的主要试剂 4

2.1.5 主要仪器设备 5

2.2 实验方法 5

2.2.1 真核表达载体的构建 5

2.2.2 细胞培养 9

2.2.3 真核表达载体转染CHO细胞及筛选 9

2.2.4 细胞总蛋白的提取 10

2.2.5 变性条件下Ni-NTA 柱纯化目的蛋白 10

2.2.6 表达蛋白的SDS-PAGE凝胶电泳 11

2.2.7 粗酶液转化孕酮活性分析 12

第三章 结果与讨论 12

3.1 人CYP17A1基因的优化与合成 12

3.2 真核表达载体的构建 15

3.3 真核表达载体的检测 16

3.4 CHO细胞中目的基因的检测 17

3.5 CYP17A1在CHO中的表达及其检测 17

3.6 粗酶液转化孕酮活性分析 20

第四章 总结与展望 21

参考文献 23

致 谢 25

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