论文总字数：19795字

摘 要

在各种不同的载体中，肽一直受到重视，特别是，细胞穿膜肽（CPPs），因为它们能够允许大分子在全身体内多种组织内给药，并且具有100％的诱导能力穿透给定细胞群体。目前，CPPs也有被用于治疗人类疾病的临床前案例。在过去几年里，我们已经发现最普遍的CPPs（TAT47-57，YGRKKRRQRRR）可以携带外源性大分子蛋白质进入大脑用于治疗阿尔茨海默氏病的，帕金森氏病和抑郁症。

同时CPPs可用于脑外源蛋白质的传输，它们不能与细胞特异性结合因为每次CPPs在一个不同类型细胞中效果是不同的。这里所所说的CPPs的主要缺陷是他们缺乏细胞特异性，并且这种限制阻碍了CPPs广泛前景的应用。

在蛋白运输中使用的肽如能靶向到特定细胞或组织将具有重要的相关性。大自然中存在一些嗜神经生物，比如狂犬病毒和破伤风病毒。这些天然的嗜神经生物肽似乎保持着高度的细胞特异性的能力，因此我们试图在药物递送领域中使用它。例如，从破伤风中得到的破伤风病毒C-段（TTC）可被用作分子的工具，专门针对运动神经元和动物实验中用于缓解神经系统疾病治疗药物分子的运输。

关键词：RDP CPPs 质粒构建 蛋白表达

Expression and purification of RDP cPPs

ABSTRACT

Among the various carriers, interest has been focusedon peptide, particularly, cell-penetrating peptides(CPPs), because they are able to allow macromolecular translocation within multiple tissues in vivo upon systemic administration, and have the capability of inducing cell penetration within 100% of cells of a given cell culture population. Currently, CPPs have also been used to treat preclinical models of human disease. In the past years, we have reported that the one of the most popular CPPs (TAT 47–57, YGRKKRRQRRR) can carry the exogenous macromolecular proteins into brains for treatment of Alzheimer’s disease,Parkinson’s disease and depress.

While CPPs provide a means for brain delivery of exogenous proteins, they can not deliver them with cell specificity since CPPs are effective in a very large number of different cell types. Herein lays one of the major drawbacks of CPPs,their lack of cell specificity, and this limitation hampers the most promising applications of CPPs .

The use of peptides in the delivery of proteins would have a major relevance if they were able to target specific cells or tissues. There lie neurotropic organisms in the nature, such as rabies virus and Clostridium tetani. These naturally occurring peptide derived from neurotropic organisms seems to keep their ability of the highly cell specificity, so they are attempted to use in the field of drug delivery. For example,tetanus toxin C-fragment (TTC) derived from tetanus has been exploited as a molecular tool to specifically target motor neurons and has significantly alleviated symptoms in animal models of neurological diseases upon delivery of therapeutic molecules .

Rabies virus is highly neurotropic, and its glycoprotein is known to be the only protein component of the viral envelope that mediates viral entry into host cells. The sequence analysis reveals that the 330–357 amino acid sequence of glycoprotein is the key nerve binding region of the virus. Thus, we assumed the peptides derived from the rabies virus glycoprotein (RDPs) of sequence 330–357 had the similar cell specificity. In our previous study, we have reported that one of the RDPs can deliver the macromolecular protein into the hippocampus of brain, while the other RDP with different amino acid sequence was totally lack of delivery function. Here we showed that another RDP (39 amino acid residue, KSVRTWNEIIPSKGCLRVGGRCHPH VNGGGRRRRRRRRR) had the ability of targeted delivery proteins with different molecular weight and pI into the CNS. Furthermore, a potential therapeutic protein, brain-derived neurotrophic factor (BDNF), fused to RDP could be used for treatment of mouse experimental stroke.

Key Words ：RDP CPPs Plasmid construction Protein expression

目 录

摘 要.............................................................Ⅰ

ABSTRACT ......................................................Ⅱ

第一章 文献综述..................................................1

1.1选题背景和依据...............................................1

1.2穿膜肽的定义.................................................2

1.3穿膜肽的分类.................................................2

1.4穿膜肽的跨膜机制.............................................3

1.5穿膜肽的应用.................................................4

1.6穿膜肽蛋白的表达.............................................4

第二章 实验材料和仪器..................................... 6

2.1实验材料.....................................................6

2.2实验仪器.....................................................7

第三章 试验方法.................................................7

3.1质粒构建.....................................................7

3.1.1 载体选择..............................................7

3.1.2 目标基因分析..........................................7

3.1.3 引物设计与合成........................................7

3.1.4 cDNA第一链合成........................................7

3.1.5 PCR...................................................7

3.2核蛋白基因克隆...............................................8

3.2.1 PCR产物割胶回收纯化...................................8

3.2.2 连接..................................................8

3.2.3 感受态的制备（CaCl2法）...............................8

3.2.4 转化..................................................8

3.2.5 重组质粒筛选、鉴定.....................................9

3.2.6 DNA琼脂糖凝胶电泳.....................................9

3.3核蛋白基因表达载体构建......................................10

3.3.1 PCR纯化产物双酶切....................................10

3.3.2 双酶切产物纯化.......................................10

3.3.3 表达载体质粒碱法制备与抽提...........................10

3.3.4 表达质粒pET32a( )双酶切..............................11

3.4质粒转化....................................................12

3.4.1 诱导表达.............................................12

3.4.1.1 重组质粒转化到表达菌BL21.......................12

3.4.1.2 诱导表达和表达条件优化.........................12

3.4.2 目标蛋白检测.........................................12

3.4.2.1 染料法蛋白含量测定.............................12

3.4.2.2 SDS-PAGE电泳检测...............................13

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