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1、第一章 前 言 2 摘 要 I 摘摘 要要 穿膜肽（cell-penetrating peptides，CPPs）是一类具特殊功能的多肽分子， 一般由 30 个以下的氨基酸残基构成，具有生物膜穿透功能，还可携带其它分子 甚至超分子颗粒进入细胞中。正是因为这种特殊的功能，它们被看作生物活性 分子有效的细胞内转运工具，具有广泛的应用前景。 其中，TAT 是最先被发现并证实的具穿膜功能的多肽分子。大量的实验研 究表明，TAT 是一类细胞毒性较低但运输能力较强的活性多肽。 为了确认 TAT 具有的穿膜和携带生物大分子能力，我们引入了绿色荧光蛋 白（GFP）作为标记因子，使 tat-gfp 基因串联于原
2、核表达载体 pET28a，在大肠 杆菌 （E.coli） BL21 中进行 IPTG 诱导表达。 利用蛋白带有的 His 标签进行 Ni-NTA 亲和层析柱纯化，得到了融合蛋白 TAT-GFP。然后我们利用得到的蛋白分别进 行了体外试验和体内实验。 通过在激光共聚焦显微镜下直接观察与 BHK-21 细胞 孵育一定时间后的细胞中 GFP 的自发荧光， 考察了 TAT 携带 GFP 在体外培养细 胞中的穿膜特性及细胞定位，证明其可迅速穿透细胞膜而广泛分布于细胞的各 个部分，尤其是细胞核中；对照组的 GFP 则无穿膜功能。体内实验表明，在通 过尾静脉注射给药 2h 后，即可在小鼠的各个主要器官和组织
3、中观察到明显的荧 光出现，表明融合表达的 TAT 在活体内也具有广泛的穿膜活性。 在以上 TAT 功能研究基础上，我们对其应用进行了初步的探索，这一工作 主要着眼于真核细胞的保护方面。目前细胞的保护主要通过细胞保护剂和低温 来实现，但现有的很多保护剂对细胞有伤害或难以达到理想的效果。海藻糖是 近年研究最为广泛的细胞保护剂之一，它对细胞基本无害且保护效果显著，但 在应用上一个主要的问题是，细胞内海藻糖很难达到足够的浓度以发挥功效。 我们期望通过 TAT 的应用为解决这一难题提供新的思路。 首先，我们通过 PCR 技术获得了 E.coli DH5 菌株的海藻糖合成酶基因-6- 磷酸海藻糖合成酶基因
4、(otsA)和 6-磷酸海藻糖磷酸酯酶基因(otsB)，并将二者 分别与tat基因实现串联、 融合表达。 利用纯化的蛋白进行体外酶催化反应， HPLC 穿膜肽 TAT 融合蛋白的构建、表达及其应用研究 II 检测初步证实了两融合蛋白在体外可催化底物形成海藻糖，具有一定的酶催化 活性。由此提示，通过 TAT 增加细胞内海藻糖合成酶的含量有可能提高海藻糖 浓度，从而增加细胞抗性。 总之，通过原核表达我们获得了融合形式的 TAT-GFP 蛋白，利用此蛋白证 明 TAT 在体内和体外都具有穿膜活性；通过 TAT 短肽所进行的细胞保护方面的 初步探索，为提高细胞内海藻糖负载提供了新的思路。 关键词关键词
5、： 穿膜肽 融合表达 海藻糖 细胞保护 ABSTRACT III Abstract Cell-penetrating peptides (CPPs) are short peptides of less than 30 amino acids that are able to penetrate cell membrane and translocate different cargoes into cells. The only common feature of these peptides appears to be that they are amphipathic and net p
6、ositively charged. The mechanisms of cell translocation are not known yet, but in many cases, translocation can be partially mediated by endocytosis. Cargoes successfully transported by CPPs range from small molecules to proteins and supramolecular particles. CPPs are novel vehicles for the transloc
7、ation of bioactive molecules into cells, whose properties make them potential delivery agents, of interest for future use. TAT is the first identified peptide that can rapidly translocate over membrane into the cytoplasm in 1988 by Green et al. Plenty studies have indicated TAT has low intrinsic tox
8、icity and strong penetrating-ability. In order to study the property and localization of cell-penetrating peptide TAT, we introduced GFP as a reportor. Tat and gfp gene were tandem cloned into vector pET28a. Then pET28a-tat-gfp recombinant was transfected into BL21. The recombinant protein TAT-GFP w
9、as expressed induced by IPTG. The purified protein was incubated with BHK-21 for different times to observe its location in cell by laser scanning confocal fluorescence microscope. At the same time, we injected TAT-GFP and control GFP into mice by caudal vena. After some time, we dissected the mice
10、and got the main organs into paraform to fix them. After 24h, we make section with the fixed tissue samples to study the disposition of protein. Cell test indicated that TAT-GFP could penetrate plasm membrane and distribute extensively in BHK-21 cell, exceptionally in nucleus. Otherwise the control
11、protein GFP can not enter the BHK-21 cell. Animal test implicated TAT could take GFP into the many organs and tissues of mice after ip 2h, even passing through blood brain barrier. Based on preceding works, we carried out the exploratory development on application of TAT and our research focused on
12、its cytoprotective activity. Most 穿膜肽 TAT 融合蛋白的构建、表达及其应用研究 IV traditional methods relies on cryopreservation protocols that include the addition of 1.02.0 M of penetrating cryoprotectants (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol. These agents may damage cells or achiev
13、e bad results. However, small carbohydrate sugars, especially trehalose, have an exceptional ability to stabilize and preserve proteins, viruses, and bacteria as well as an excellent ability to form stable glasses. But a critical question is that its hard for trehalose to achieve efficient concentra
14、tion in cells. So we hope to solve these problems with TAT. First of all, we amplified otsA(coding the Trehalose-6-phosphate synthetase) and otsB(coding Trehalose-6-phosphate phosphatase)genes from E.coli DH5 genome by PCR. Then otsA and otsB gene were tandem cloned individually into vector pET28a-t
15、at. Then pET28a-tat-otsA/otsB recombinant was transfected into BL21 and induced by IPTG. Purified fusion proteins was used in vitro reaction system and enzyme activity was measured by HPLC. According to above test, we presume that trahalose concentration can be raised through increasing level of tre
16、halose synthetases by TAT and this may increase cell resistance. In summary, we got the fusion protein TAT-GFP by prokaryotic expression, and proved its penetrating-ability. we confirmed TAT can be used in cytoprotection research. This work also offered a new approach to promote trehalose content in cells. Key words: cell-penetrating peptides, fusion expression, trehalose, cytoprotection 目 录 V 目目 录录 摘摘 要要 . I ABSTRACT III 目目 录录 V 缩略语索引缩略语索引 . VIII 第第 1 章章 前前 言言 . 1 1.1 穿膜肽的研究进展 . 1 1.1.1
