论文总字数：22290字

摘 要

癌症是目前严重影响人类身体健康的主要疾病之一。从细胞凋亡角度看,肿瘤的发生是由于凋亡受阻所致。TRAIL在癌症治疗中有着巨大的优势和潜力，但它在肿瘤治疗过程中仍然存在很多问题，如在体内的循环半衰期很短、死亡受体激活率低和肿瘤部位滞留时间短等，这严重阻碍了其在肿瘤治疗中更进一步的应用。为了实现TRAIL的功能改善，我们选择疏水蛋白（HFB）作为融合蛋白的配体，基质金属蛋白酶-9（MMP9）酶切序列作为连接片段，设计合成了TRAIL-MMP9-HFB融合蛋白。该融合蛋白可以通过增大体积使得TRAIL的半衰期增长，同时在肿瘤组织中TRAIL能够通过肿瘤组织中过表达的MMP9酶的作用从融合蛋白中分离出来发挥作用，而在其他正常组织中以无杀伤活性的形式存在。文章主要针对疏水蛋白的表达情况和包载疏水性药物阿霉素（DOX）的能力进行了简单的考察。实验发现疏水蛋白I类的EAS和II类的HFBII均能够通过修饰Nusa融合标签后在大肠杆菌表达系统顺利表达，且产量较高，纯度可观。同时可使用超声破碎的方法将DOX包载入疏水蛋白中。实验结果表明选择的疏水蛋白具有优秀的包载疏水性药物的能力，对于延长药物的半衰期有可以预见的应用。此外，良好的PDI和较小的粒径也为在药物递送中应用增强渗透和滞留效应（EPR）带来了可能。

关键词： TRAIL 疏水蛋白 肿瘤 纯化 表达

Expression, Purification and Study on Anti-tumor Activity of Hydrophobin Polypeptides Fused with TRAIL

Abstract

Cancer is one of the major diseases that seriously affects human health today. From the perspective of apoptosis, the occurrence of malignant tumor is due to the obstruction of natural apoptosis. Even though TRAIL has been verified with great advantages and potential in cancer therapy, there are still many problems needed to be solved, such as short circulating half-life in vivo, low death receptor (DR) activation rate and short residence time in tumor sites, which seriously hinders its further application in tumor therapy. In order to achieve the functional improvement of TRAIL, we choose hydrophobin (HFB) as the ligand and the cleavage site of matrix metalloproteinases 9 (MMP9) as the linker to design and synthesize TRAIL-MMP9-HFB fusion protein, which can efficiently prolong the half-life of TRAIL through increasing its volume. Besides, TRAIL can be separated from fusion protein by over-expressed MMP9 in tumor tissue to perform normal function , and remains inactive state in other healthy tissues. In this paper, the expression of hydrophobin and the capability of carrying hydrophobic drug such as doxorubicin (DOX), were investigated. The experiment result found that both EAS of class I hydrophobins and HFBII of class II hydrophobins can be successfully expressed in the Escherichia coli (E. coli) expression system with high yield and considerable purity by modifying the Nusa fusion tag. At the same time, the DOX can be loaded into HFBII proteins by ultrasonic fragmentation. And the experimental results show that the selected hydrophobin has excellent capability of entrapping hydrophobic drugs, and has predictable application for prolonging the half-life of drugs. In addition, smaller PDI and particle size of TRAIL-MMP9-HFBs also make it possible to apply enhanced permeation and retention effect (EPR) in drug delivery.

Key words: TRAIL; Hydrophobin; Tumor; Purification; Expression

目录

摘要 I

Abstract II

目录 IV

第一章 文献综述 1

第二章 疏水蛋白I类和II类的表达与表征 11

2.1 材料及仪器 11

2.1.1 实验试剂 11

2.1.2 实验仪器 12

2.2 实验方法 13

2.2.1固体培养基和液体培养基的制备 13

2.2.2 BCA蛋白标准曲线的绘制 13

2.2.3 疏水蛋白EAS和HFBII的表达制备 13

2.2.4 验证疏水蛋白HFBII的自组装特性 14

2.3 结果与讨论 14

2.3.1 BCA蛋白标准曲线 14

2.3.2 疏水蛋白EAS和HFBII的表达 15

2.3.3 验证疏水蛋白HFBII的自组装特性 16

2.4实验小结 18

第三章 总结与展望 19

3.1 课题总结 19

3.2 课题展望 19

参考文献 20

致谢 24

第一章 文献综述

癌症严重威胁着人类的生命，是目前影响人类健康的主要疾病之一。据世界卫生组织报告显示，2000年全球新发癌症人数多达1000万，其中死亡人数620万，当前患病人数达2200多万，预计到2020年每年新发癌症人数将达1500多万，死亡人数1000万，如今患病人数将高达3000多万。我国卫生部统计资料指出，从20世纪70年代到现在，我国的癌症发病率一直呈上升趋势。由此可见，癌症正成为新世纪人类第一杀手。

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