| 题名 | 雨蛙皮肤分泌液中两种活性蛋白结构和功能的研究 |
| 作者 | 游德文 |
| 学位类别 | 博士 |
| 答辩日期 | 2009-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 赖仞 |
| 关键词 | 华西雨蛙 Kunitz 型丝氨酸蛋白酶抑制剂 神经毒素 干细胞自我更新支持因子 内环境稳定 |
| 其他题名 | Studying on the structure and function of two bioactive proteins secreted from skin of the tree frog, Hyla annectans |
| 中文摘要 | 在本研究中我们首次从雨蛙皮肤分泌液中分离得到了一种神经毒素（命名为Anntoxin）和一种干细胞自我更新支持因子（命名为AnSF）。随后，我们通过构建雨蛙皮肤cDNA文库，利用特异引物筛选到Anntoxin和AnSF的cDNA编码序列，前者的Gene Bank登录号为FJ598043，后者还在等待分配登录号。Anntoxin具有60个氨基酸，是一种Kunitz类型的丝氨酸蛋白酶抑制剂，构建Anntoxin的3D-NMR溶液结构，证实Anntoxin不同于有三对二硫键（键组合模式：1-6，2-4，3-5）的Kunitz类型丝氨酸蛋白酶抑制剂，它只有两对二硫键（组合模式：1-4，2-3）。AnSF具有123个氨基酸，在C端具有和Calmadolin同源的两个EF手指结构，能够支持人类胚胎干细胞（hESC）和猴神经干细胞（rNSC）的自我更新。 为了进行Anntoxin的生物活性和结构分析，我们在体外成功表达了Anntoxin，获得了大量的重组Anntoxin（rAnntoxin）。经过生物活性分析，rAnntoxin和天然分离到的Anntoxin生物活性相当，都具有很强的胰蛋白酶抑制剂活性。Anntoxin是一种Kunitz类型的丝氨酸蛋白酶抑制剂，和来源于芋螺（Cone Snail）的神经毒素Conkunitzin-S1，黑色眼镜蛇毒液（black cobra, Dendroaspis polylepis polylepis）的树突毒素δ-DaTX或蛋白酶抑制剂K分别具有32.8%和36.7%的相同序列，和鱼类（fish）来源的Stonustoxin也有一定的同源性。利用膜片钳技术分别检测Anntoxin对大鼠背根神经节（rat DRG）上Na+通道，K+通道，Ca2+通道的作用，结果证明Anntoxin对河豚毒素敏感（TTX-S）的钠离子通道（Nav）有较强的抑制活性，对K+通道，Ca2+通道作用不明显。随后我们在非洲爪蟾卵母细胞上表达几种典型和常用于测试对亚型K+通道作用的Kv1.1，Kv1.2，Kv1.3，Kv2.1和Kv4.2，Kv4.3，Anntoxin对这些亚型K+通道上的K+电流都没有明显影响。我们成功构建了Anntoxin的3D-NMR溶液结构（NMR号：PDB ID 2KCR，BMRB ID 16094），证实Anntoxin具有典型的Kunitz结构，由反向平行的β–折叠片和α–螺旋及转角组成梨形结构。利用RT-PCR，WesternBlot以及ELISA技术，发现在皮肤、脑、肝、胃和肠中都能检测Anntoxin mRNA转录，但只在皮肤、脑、肝和胃中有蛋白表达，表达量分别为29.5、5.39、4.80和2.02微克/克鲜重，可以看出Anntoxin在皮肤中大量表达，是皮肤分泌液中非常重要的组成部分。因为皮肤是雨蛙接触外界的第一屏障，雨蛙的生存环境中存在很多潜在威胁，比如微生物、吸血昆虫、鸟类、爬行动物、哺乳动物等，所以Anntoxin有可能是雨蛙适应环境的重要化学武器，于是我们测试了Anntoxin对甜菜夜蛾幼虫（Laphygma exigua Hubner）、水蛇（Enhydris plumbea）、鹌鹑（Coturnix coturnix）、昆明小鼠（Kunming mice）的急性毒性，其LD50分别为50，450，2500和3000微克/千克体重，说明在华西雨蛙皮肤中大量表达的Anntoxin对几类潜在天敌确实有较强的杀灭作用。 为了检测AnSF的生物学活性，我们在体外成功表达了AnSF，获得了大量rAnSF。设计三个浓度梯度10、100和500ng/ml，把AnSF和hESC共培养，发现在10~100 ng/ml浓度时对hESC的自我更新有支持作用；设计三个浓度梯度10、100和500ng/ml，把AnSF和rNSC共培养，发现在10ng/ml时对rNSC的自我更新有较强的支持作用。在超过500ng/ml高浓度时，AnSF对hESC和rNSC都有明显的细胞毒性作用，对rNSC的毒性作用更明显。利用RT-PCR技术，我们检测了雨蛙的皮肤、肌肉、肝脏、胰脏、胃、肠、心脏和脑，AnSF只在皮肤中有少量表达。这表明AnSF可能只参与雨蛙皮肤干细胞库的维持，保持皮肤内环境稳定，因为蛙类的皮肤细胞要负责产生大量活性物质参与先天免疫和抗氧化等重要的生理活动，需要经常更新，而AnSF的存在可能保证雨蛙皮肤干细胞库容量稳定，不断分化出各种成熟的皮肤细胞来使皮肤能够得到足够和及时的更新，保证其功能的正常行使。所以AnSF是维持华西雨蛙皮肤内环境稳定的重要物质。 |
| 英文摘要 | We have separated one neurotoxin, named as Anntoxin, and one stem cell self-renewal promoting factor, named as AnSF, from the skin secretions of Hyla annectans annectans (Jerdon) firstly. And then we get the encoding cDNA sequences of Anntoxin and AnSF in the constructed cDNA library through designing special primers of Anntoxin and AnSF. The Anntoxin cDNA sequence’s Genebank number is FJ598043, and the AnSF cDNA sequence is not defined now. Anntoxin has 60 amino acid residues, and has a classical Kunitz solution structure through Tridimensional Nuclear Magnetic Resonance (3D-NMR) test. But Anntoxin has only two (motif: 1-4, 2-3) out of the three (motif: 1-6, 2-4, 3-5) disulfide bridges in the normal Kunitz protease inhibitors. AnSF has 123 amino acid residues, and it can promote human embryonic stem cells (hESCs) and rhesuss monkey nervous stem cells (rNSCs) self-renewal. Getting enough protein to test its biology functions in vivo and in vitro and analyze its solution structure through 3D-NMR, we expressed Anntoxin in vitro successfully. The rAnntoxin has nearly similar bio-activities to native Anntoxin separated from the skin secretions of H. annectans. It can inhibit the hydrolysis activity of tripsin to its substrate strongly too. Through 3D-NMR the solution structure of Anntoxin is constructed, which proves Anntoxin has classical Kunitz structure though it only possesses two out of three disulfide bonds as normal Kunitz serine protease inhibitors, and the NMR number is PDB ID 2KCR and BMRB ID 16094. Anntoxin has similar sequence of 32.8% and 36.7% with Conkunitzin-S1, originating from Cone Snail, and δ-DaTX, separated from the venom of black cobra (Dendroaspis polylepis polylepis), respectively. It also has some homology to Stonustoxin from fish. Using patch-clamp technology, we test the effect of Anntoxin on voltage-gated ion channels such as K+ channel, Na+ channel, and Ca2+ channel on rat dorsal root ganglion (DRG). It is proved that Anntoxin has strong inhibition effect on the tetrodotoxin sensitive (TTX-S) Na+ channel, and little effect on K+ channel and Ca2+ channel. Then we test the effect of Anntoxin on sub-type K+ channels such as Kv1.1, Kv1.2, Kv1.3, Kv2.1, Kv4.2 and Kv4.3, expressed on Xenopus laevis oocytes, and we get negative result. According to the result of RT-PCR, WesternBlot, and enzyme-linked immunosorbent assay (ELISA), though mRNA of Anntoxin can be detected in skin, brain, stomach, liver and intestine (RT-PCR), there are 29.5, 5.39, 4.80, and 2.04 μg/g wet fresh tissues (WT) expressed in the skin, brain, stomach, and liver, respectively (ELISA). This shows that Anntoxin is expressed abundant in skin, ten folds more than in other tissues. Because skin is the most important interface between the toad internal milieu and the outer environment, and acts as the first barrier against microorganism, blood-sucking insects, birds, snakes, and other mammals, highly expressed Anntoxin in the skin may furnish skin with a strong chemical weapon to fulfill its functions. As the result of acute toxicity of Anntoxin to the third instar worm of Laphygma exigua Hubner, Enhydris plumbea, Coturnix coturnix, and Kunming Mice, the LD50 is 50, 450, 2 500, and 3 000 μg/kg bodyweight, respectively. It shows that Anntoxin can kill these potential natural enemies in H. annectans’ environment. Through the same method as obtaining rAnntoxin, we get recombinated AnSF (rAnSF). After released from the fusion His-tagged protein by formic acid, the rAnSF is centrifuged in Amicon Ultra-4 Centrifugal device (10K, 30K), and then the retenate solution is uploaded in C8 (30×0.46 cm), and through RP-HPLC we get highly pure AnSF. AnSF is cocultured with human embryonic stem cells (hESCs) and rhesus monkey nervous stem cells (mNSCs), respectively, at three concentrations of, i.e. 10, 100, and 500 ng/ml. We found that between the 10 and 100 ng/ml AnSF can sustain hESCs self-renew, so do 10 ng/ml AnSF to mNSCs. High concentration (500 ng/ml) of AnSF has heavy cyto-toxicity to both hESCs and mNSCs. It is investigated from the RT-PCR result, tested in skin, muscle, liver, pancreas, stomach, intestine, heart, and brain, only skin expresses a little AnSF. This shows AnSF may act as key factor to sustain the skin stem cell pool, keeps homeostasis in the skin, and provides enough skin stem cell to differentiate into mature skin cells, which need to be changed quickly, because skin should secrete many peptides and proteins involved in the innate immunity system and other physiological processes. |
| 语种 | 中文 |
| 公开日期 | 2010-10-22 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6319]  |
| 专题 | 昆明动物研究所_动物毒素室 |
| 推荐引用方式 GB/T 7714 | 游德文. 雨蛙皮肤分泌液中两种活性蛋白结构和功能的研究[D]. 北京. 中国科学院研究生院. 2009. |
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