蝎子是地球上系统发育最古老的动物之一，已经存活了 4 亿多年。其毒液被用作防御机制或固定和消化猎物，是酶和非酶成分的复杂混合物，具有特定的病理生理功能，可以作为药物发现中新的主要成分的宝贵来源。

东亚钳蝎（Buthus Martensii Karsch）是亚洲地区分布最多的蝎亚科，全蝎作为传统中药已有两千多年的用药历史。其主要药效成分蝎毒作为复杂的多肽混合物在结构方面研究相对薄弱，活性机制尚不明确。为了获取蝎肽单体的空间结构，本研究采用HPLC技术，对蝎毒的活性成分进行分离，并结合大肠杆菌重组表达技术和核磁共振方法，解析多肽的三维空间结构，为蝎毒多肽的活性机制研究提供物质和结构基础。

主要研究内容和结果如下：

1、采用离子交换色谱、反相高效液相色谱技术，建立了东亚钳蝎蝎毒活性单体的分离纯化方法。其中对蝎毒多肽BmK-M10通过基质辅助激光解析电离-飞行时间质谱仪（MALDI-TOF/TOF）结合搜库获得多肽序列，为后续研究提供了物质基础。

2、根据东亚钳蝎cDNA库的BMK-M10信息，使用PCR扩增技术设计目的基因，进行Nco1和Hind III双酶切后插入表达载体pET-32a(+)中，经单克隆鉴定和测序工作筛选正确转化的大肠杆菌培养。本法得到的重组蛋白部分可溶，部分形成在沉淀里，推测聚集形成错误折叠的包涵体，1 L菌液培养物共获得约10mg重组BMK-M10。本研究确定了大肠杆菌系统可以作为BMK-M10的获取途径，为核磁共振波谱法研究BMK-M10结构提供了条件。

3、通过M9培养基获得进行¹⁵N标记的重组蛋白，采用核磁共振方法解析多肽的空间结构，为证明BMK-M10蛋白的结构与活性机制关系提供了科学依据。

4、通过SPR技术，明确了蝎肽BMK-M10可与人源重组凝血因子X有所结合，影响抗凝通路。

Scorpions are one of the oldest phylogenetic animals on Earth, having survived for more than 400 million years. Their venom is a complex mixture of components with specific pathophysiological functions and can serve as a valuable source of new major ingredients in drug discovery.

Buthus Martensii Karsch is the most widely distributed scorpion subfamily in Asia. Scorpion has been used as traditional Chinese medicine for more than two thousand years.  In order to obtain the spatial structure of the scorpion peptide monomer, in this study we used HPLC technology to separate the active components of scorpion venom, and used prokaryotic expression technology to produce peptide with high yield to provide material basis for the subsequent research.

The main research content and results are as follows:

By using ion exchange chromatography and reversed-phase HPLC as separation and purification method, the peptide sequence of the scorpion venom peptide BmK-M10 was obtained by MALDI-TOF/TOF and database searching.

2. According to the information of BMK-M10 from cDNA library, the target gene was designed by PCR amplification technology, and inserted into the expression vector pET-32a(+) , the strain BL21(DE3) was used to produce the peptide. A total of about 10 mg of recombinant BMK-M10 was obtained in 1 L of bacterial culture. This study confirmed that the Escherichia coli system can be used as a way to obtain rBMK-M10, which makes the study of 3D structure of ¹⁵N-rBMK-M10 by NMR possible.

3. The ¹⁵N-labeled recombinant protein was obtained through the M9 medium, and the spatial structure of the peptide can be analyzed by NMR, which provided a scientific basis for proving the relationship between the structure and the activity mechanism of the BMK-M10 protein.

4. Through SPR technology, it is clear that the scorpion peptide BMK-M10 can be combined with human recombinant coagulation factor X to affect anticoagulation.

摘要 I
Abstract II
目录 III
主要符号对照表 1
第一章 绪论 2
1.1蝎毒的研究概述 2
1.1.1蝎毒的化学成分 2
1.1.2蝎肽的成分分类 2
1.1.3蝎肽的药理学作用 7
1.1.4活性多肽分离纯化的研究概述 8
1.2大肠杆菌表达系统的研究概述 9
1.2.1表达策略的选择 10
1.2.2表达载体和宿主菌的选择 10
1.2.3偏好大肠杆菌密码子的优化 10
1.2.4重组蛋白表达可溶性的优化 11
1.2.5重组蛋白的纯化方法 12
1.2.6大肠杆菌表达系统的优缺点 14
1.3 结构生物学的研究概况 14
1.3.1 X-Ray晶体学 14
1.3.2 核磁共振波谱法 15
1.3.3 冷冻电子显微学 16
1.4 血管栓塞性疾病 16
1.2.1 概述 16
1.2.2 凝血通路及凝血因子X 17
1.5 本课题的研究方向及立题依据 18
第二章 东亚钳蝎BMK M10的分离鉴定 20
2.1 序言 20
2.2 实验材料 20
2.2.1 材料与试剂 20
2.2.2 仪器与设备 20
2.3 实验方法 21
2.3.1 蝎毒多肽的分离纯化 21
2.3.2 质谱分析和氨基酸测序 22
2.4 实验结果 22
2.4.1 BMK-M10的纯化 22
2.4.2 氨基酸序列鉴定 23
2.5 讨论 24
2.6 小结 25
第三章 重组Bmk-M10的表达纯化与鉴定 26
3.1序言 26
3.2实验材料 26
3.2.1材料与试剂 26
3.2.2 仪器与设备 27
3.3实验方法 28
3.3.1培养基及常用试剂的配制 28
3.3.2重组rBMK-M10多肽的原核表达与纯化 29
3.3.3 重组多肽Toxin BmKaTX15的鉴定 32
3.4结果与分析 32
3.4.1密码子的优化调整 32
3.4.2 重组多肽纯化结果 33
3.4.3 重组多肽鉴定结果 34
3.5讨论 35
3.6小结 35
第四章：重组蛋白rBMK-M10的三维结构探索 37
4.1 序言 37
4.2 材料与方法 38
4.2.1 材料与试剂 38
4.2.2 仪器与设备 38
4.3 实验方法 39
4.3.1 培养基配方 39
4.3.2 原核表达15N标记重组多肽rBMK-M10 40
4.3.3 15N标记的重组多肽rBMK-M10的鉴定 42
4.4 实验结果 42
4.4.1 重组多肽表达及纯化结果 42
4.4.2 重组多肽表达产物的鉴定 43
4.5 讨论 44
4.6 小结 44
第五章：BMK-M10的靶点结合验证 45
5.1 序言 45
5.2 材料与设备 45
5.2.1 材料与试剂 45
5.2.2 仪器设备 45
5.3 实验方法 46
5.4 实验结果 46
5.5 讨论 47
5.6 小结 48
总结与展望 49
参考文献 50
致谢 55
北京大学学位论文原创性声明和使用授权说明 56
学位论文答辩委员会名单 57
个人简介 58

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