| 题名 | 特异性抑制Nav1.6通道的少棘蜈蚣毒素的分离纯化及结构与功能的研究 |
| 作者 | 刘杰 |
| 学位类别 | 硕士 |
| 答辩日期 | 2014-05 |
| 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 | 少棘蜈蚣 毒素 Nav1.6通道 疼痛 |
| 其他题名 | Purification, structure-function analysis of a selective Nav1.6 inhibitor from centipede（Scolopendra) |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 蜈蚣在中国作为传统中药治疗疾病已有千年历史，如治疗中风偏瘫、中风、癫痫、破伤风、百日咳、结核病、烧伤烫伤和急性心脏病等疾病。尽管人们经常遇到蜈蚣并遭遇其伤害，但有关蜈蚣毒液的组成及作用方面却少有深入研究。Nav1.6通道在病理性疼痛中扮演着重要作用，用Nav1.6通道作为镇痛药物筛选靶标也少见报道。 目前，我们从少棘蜈蚣毒液中分离到一种多肽毒素，该毒素能抑制大鼠背根神经节细胞（DRG）上河豚毒素敏感性（TTX-S）钠离子通道，进一步研究表明该多肽能选择性抑制Nav1.6通道，而对其他的钠离子通道亚型的活性较弱或无活性。首先通过凝胶过滤和反相高压液相色谱从少棘蜈蚣毒液中分离纯化得到μ-SLPTX-Ssm4a并用Edman降解法测定μ-SLPTX-Ssm4a的N-端序列，再根据N-端序列从少棘蜈蚣毒腺cDNA文库中克隆得到了编码多肽前体的cDNA序列。它的成熟肽包含50个氨基酸和三对二硫键，分子量为5532.4 Da。最后应用芴甲氧羰基（Fmoc）固相化学合成的方法合成μ-SLPTX-Ssm4a线性肽，经谷胱甘肽法氧化复性后测定其生物学功能。μ-SLPTX-Ssm4a选择性抑制Nav1.6通道，其IC50为0.46 μM，在高浓度下对Nav1.7通道也有一定的抑制作用，IC50为10.7 μM。小鼠镇痛模型试验表明μ-SLPTX-Ssm4a对醋酸、热辐射、福尔马林和冷刺激诱导的疼痛具有良好的镇痛作用，但其镇痛效果不如吗啡明显。本研究确定Nav1.6通道能够作为镇痛药物的筛选靶点并从少棘蜈蚣毒液中筛选出特异性抑制Nav1.6通道的多肽，有希望成为新型镇痛药物的先导分子。 |
| 英文摘要 | In China, the centipede has been used for thousands of years as a traditional Chinese medicine to treat disorders, such as stroke-induced hemiplegia, apoplexy, epilepsy, tetanus, whooping cough, tuberculosis, scald burns, and pyocutaneous disease. Despite their abundance and oftenpainful encounters with humans, little is known about the composition and function of centipede venom. Nav1.6 channel plays a key role in neuropathicpain, but it was rarely reported as a target for screening of analgesic drugs. Currently, we isolated a peptide toxin from Scolopendra subspinipes mutilans venom, it can inhibit the tetrodotoxin-sensitive (TTX-S) sodium channels in rat dorsal root ganglion (DRG), further research shows that the it can selectively inhibit Nav1.6 channels, and has weak or no activity to other sodium channel subtypes. Firstly, a polypeptide named as μ-SLPTX-Ssm4a was purified from from Scolopendra subspinipes mutilans venoms by gel filtration and RP-HPLC and Edman degradation method was used for the determination of its N-terminal amino acid sequence. Secondly, according to the N-terminal sequence, cDNA sequence encoding the precursor of μ-SLPTX-Ssm4a was cloned from a cDNA library of Scolopendra subspinipes mutilans venom gland. The mature peptide was composed of 50 amino acid residues with a molecular mass of 5532.4 Da and contained three pairs of disulfide bonds. Finally, linear peptide of μ-SLPTX-Ssm4a was synthesized using a solid-phase chemical approach with Fmoc-protected amino acids. It was refolded by glutathione oxidation and was subjected to test its biological functions. μ -SLPTX-Ssm4a selectively inhibited Nav1.6 channels, the IC50 was 0.46 μM, and also had inhibition on Nav1.7 channel at high concentrations, the IC50 was 10.7 μM. Several animal models of persistent inflammatory and neuropathicpain showedμ-SLPTX-Ssm4a has a strong analgesic function in acetic acid-induced writhing test, tail-flick test, the formalin-induced paw licking and cold plate test, but the analgesic effect of morphine was significantly better. This study establishes Nav1.6 channel could be a target for screening of analgesic drugs and u-SPTX-Ssm4a may be a promising lead molecule for the development of novel analgesics targeting Nav1.6 channel. |
| 语种 | 中文 |
| 公开日期 | 2014-06-18 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7905]  |
| 专题 | 昆明动物研究所_动物毒素室 |
| 推荐引用方式 GB/T 7714 | 刘杰. 特异性抑制Nav1.6通道的少棘蜈蚣毒素的分离纯化及结构与功能的研究[D]. 北京. 中国科学院研究生院. 2014. |
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