肩突硬蜱神经毒素ISTX-I的结构与功能研究
文献类型：学位论文
| 作者 | 张美玲 |
| 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 | 硬蜱 神经毒素 原核表达 电压门控钠离子通道 二硫键 |
| 其他题名 | Study on the structure and function of ISTX-I, a neurotoxin from hard tick(Ixodes scapularis) |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 硬蜱是专性吸血的体外寄生虫，是仅次于蚊子的人与动物疾病第二大传播媒介，其能传播多种病原体，引起人和动物莱姆病、巴贝斯虫病和斑点热等一系列疾病。硬蜱唾液腺还能分泌多种活性物质抵抗宿主的免疫反应，也能释放毒素导致宿主麻痹，而目前国内还没有关于硬蜱唾液腺神经毒素方面的研究报道。本论文对肩突硬蜱（Ixodes scapularis）神经毒素ISTX-I的结构和功能进行了一系列研究。 我们利用大肠杆菌pET表达系统成功地对肩突硬蜱神经毒素ISTX-I进行了原核表达，经过镍柱亲和层析、甲酸切割、Sephadex G-50、AKTA Resource Q阴离子交换层析和RP-HPLC分离纯化，得到了多肽ISTX-I的纯品。经MALDI-TOF-MS检测，其分子量为4825.3 Da，与理论分子量一致。该神经毒素ISTX-I有6个半胱氨酸，形成3对二硫键。采用部分还原、Edman降解测序和胰蛋白酶酶切法确定毒素ISTX-I的二硫键配对方式为Cys3-Cys15（C1-C4）、Cys9-Cys29（C2-C5）和Cys14-Cys39（C3-C6）。 采用全细胞膜片钳技术，检测了ISTX-I对电压门控钾离子、钙离子和钠离子通道的作用。结果显示，ISTX-I对背根神经节（Dorsal root ganglion, DRG）上电压门控钾离子通道、钙离子通道和河豚毒素不敏感型（Tetrodoxin-resistance, TTX-R）钠离子通道无明显作用，但对DRG神经元河豚毒素敏感型（Tetrodoxin-sensitive, TTX-S）钠离子通道有明显的抑制作用，其IC50值为2.47 ± 1.47 μM。进一步检测ISTX-I对电压门控钠离子通道亚型（Nav1.1～Nav1.7）的活性，发现ISTX-I选择性抑制Nav1.7（IC50值为1.60 ± 0.18 μM）而对Nav1.1、Nav1.2、Nav1.3、Nav1.4、Nav1.5和Nav1.6无明显作用。对DRG神经元TTX-S钠离子通道和电压门控钠离子通道Nav1.7的激活和失活动力学分析表明，ISTX-I不能影响DRG神经元TTX-S钠离子通道和电压门控钠离子通道Nav1.7的激活动力学，但能使两者的稳态失活曲线分别向超极化方向偏移11.22 mV和6.11 mV，降低两者的 失活阈值，缩短该通道开放时间。 本论文首次在肩突硬蜱唾液腺中发现了一种电压门控钠离子通道Nav1.7抑制剂（ISTX-I）并对其二硫键结构进行了解析。本实验结果将为蜱毒素的研究提供新思路，也为解释蜱瘫形成的机制提供一定理论依据。 |
| 英文摘要 | Ticks are obligate haematophagous ectoparasites and second only to mosquitoes as vectors of pathogens causing human and animal disease. They are capable of transmitting a variety of pathogens involved in some infectious diseases, such as human lyme disease, babesiosis, spotted fever and so on. The salivary glands of ticks can secrete a diversity of active components to resist the host immune response and can also release toxins, result in paralysis of the infested host. At present, there are few reports about the neurotoxins from salivary glands of hard ticks. In this thesis, a series of experiments were carried out to study the structure and function of neurotoxin ISTX-I from Ixodes scapularis. A neurotoxin ISTX-I from Ixodes scapularis was successfully expressed by the pET expression system in E.coli. After Ni-chelating affinity chromatography, formic acid cutting, Sephadex G-50, AKTA Resource Q anion exchange chromatography and RP-HPLC, we got purified ISTX-I polypeptide. By MALDI-TOF-MS detection, its molecular weight is 4825.3 Da, consistent with the theoretical molecular weight. The neurotoxin ISTX-I has six cysteines, which have formed three pairs of disulfide bond. The disulfide linkage of ISTX-I was determined between Cys3-Cys15, Cys9-Cys29 and Cys14-Cys39, adopting a 1-4, 2-5 and 3-6 disulfide bond pattern, by means of partial reduction, Edman degradation sequencing and trypsin digestion. The effects of ISTX-I on voltage-gated K+, Ca2+ and Na+ channels were investigated by whole-cell patch clamp technique. The results revealed that ISTX-I had no effect on voltage-gated potassium channels, calcium channels and TTX-R sodium channels of dorsal root ganglion. However it can obviously inhibit TTX-S sodium channels of DRG neurons and voltage-gated sodium channels Nav1.7, the IC50 value was 2.47 ± 1.47 μM. The further detection of its activity on the subtypes of voltage-gated sodium channels (Nav1.1-Nav1.7), found that ISTX-I selectively inhibit Nav1.7 (IC50 = 1.60 ± 0.18 μM), but it had no significant effect on Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5 and Nav1.6. The analysis of its effects on the activation and inactivation kinetics of TTX-S sodium channels and voltage-gated sodium channels Nav1.7 showed that ISTX-I could not affect their activation curves, but it shifted their inactivation curves. The inactivation curves of TTX-S sodium channels and voltage-gated sodium channels Nav1.7 were shifted for about 11.22 mV and 6.11 mV in hyperpolarizing direction, respectively. This reduced the threshold of inactivation of the channels and shortened the opening time of the channels. This paper found a voltage-gated sodium channels Nav1.7 inhibitor (ISTX-I) from the salivary glands of Ixodes scapularis for the first time and its disulfide bond pattern was identified. The results will provide new ideas for the research of tick toxins and a certain theoretical basis as to explaining the forming mechanism of tick paralysis. |
| 语种 | 中文 |
| 公开日期 | 2014-06-18 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7907]  |
| 专题 | 昆明动物研究所_动物毒素室 |
| 推荐引用方式 GB/T 7714 | 张美玲. 肩突硬蜱神经毒素ISTX-I的结构与功能研究[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：昆明动物研究所
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