CN1032427C - 虎纹捕鸟蜘蛛毒素及其制备方法 - Google Patents
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Abstract
“虎纹捕鸟蛛毒素-I”为一新型哺乳动物神经毒素，它是从我国虎纹捕鸟蛛中经反相和离子交换高效液相色谱提纯的，其化学结构为由33个氨基酸残基以特定的顺序组成的多肽，含有三对链内二硫键。它能不可逆地阻断哺乳动物神经肌肉接头传递，可作为工具试剂应用于神经生物学和药理学研究中。
Description
所属技术领域：生物化学与分子生物学技术。
现有技术：天然生物毒素在神经生物学、神经递质受体和离子通道等基础研究中已成为十分重要的工具试剂，同时在药理学、临床医学等领域也有重要应用。蜘蛛毒是天然动物毒素的一个重要领域，由于其含量少和采毒的相对困难，对它的研究和开发没有如蛇毒和蝎毒那样深入和广泛。目前国内外已做过分离纯化工作的蜘蛛毒有漏斗蜘蛛(A.robustus和A.aperta)、平甲蛛(L.reclusa)、黑寡妇蛛(L.tredecimgutatus)以及我国的穴居狼蛛(L.singoriansis)等，但只有很少数分离纯化的毒素完成了化学结构测定。虎纹捕鸟蜘蛛(Selenocosmia huwena)是最近在我国发现的蜘蛛新种，本专利发明人摸索出一种新的有效采取其毒液的方法，并通过反相与离子交换高效液相色谱分离纯化了虎纹捕鸟蛛毒素-I，完成了其化学结构测定，从其化学结构分析它是一种不同于任何已知动物毒素(包括已知蜘蛛毒素)的一种新的神经毒素。
发明的目的：本研究开发的目的是从蜘蛛毒中分离出在神经生物学、药理学和临床医疗学的基础研究和应用中有价值的新的神经毒素分子。上述huwentoxin-I毒素的化学结构和性质证明它符合我们原定研究的目的。
发明的内容和方案：虎纹捕鸟蜘蛛是在我国云南广西交界的山区发现的新种，从野外捕来之后在实验室饲养1-2年之后进行采毒。
1.粗毒的采取；2.粗毒的分离纯化； 3.进行药理学性质和生理活性测定等。
特点与应用：
上述huwentoxin-I的化学结构不同于目前已知的所有天然毒素的结构，经过检测，证明其为一新的神经毒素，不与已知其他毒素同源。在目前已知化学结构的蜘蛛毒素中它是首个确定含有三对二硫键的毒素。
由于该毒素能不可逆阻断神经肌肉接头传递，因而可以作为一种试剂工具应用在胆碱能的突触传递和突触后膜受体的神经生物学研究中，同时在药理学研究中也有应用前景。
实施例：
(1)毒液的采集和huwentoxin-I的分离纯化：
将3-4根长为4厘米内径为1.5毫米的透明塑料软管捆成一束作为诱物，用大镊子从侧面夹住蜘蛛胸部，蜘蛛便张开螯爪，这时将软管束诱物送入螯爪下，它即用触肢抱住软管束，将螯爪有力地刺入软管内射出毒液。由于每只蜘蛛的两个螯爪通常并不同时射毒，而且每次每个螯爪不一定射出全部毒液，因而上述过程需进行多次。射毒后从螯爪下慢慢抽出软管束，用微量注射器将其中的毒液抽出，经冰冻干燥后得到的白色(略带黄色)干粉即为粗毒。
将1-3毫克上述粗毒溶于200微升双蒸水中，上样到事先用0.1％三氯乙酸平衡好的C4反相色谱柱中(美国Waters公司出品，Deltapak，C4300A，30×0.46cm)，用0％-70％乙腈梯度洗脱，柱温45℃，时间120分钟，流速0.7ml/min。洗脱结果共分到约23个紫外吸收峰(220nm)(见附图-1)，其中的A峰即含有huwnetoxin-I，收集A峰洗脱液，冰冻干燥后溶于双蒸水中，上样到事先用0.02M磷酸钠缓冲液(pH6.6)平衡的WCX-1离子交换高效液相色谱柱中(日本岛津公司，5×0.4cm)，用0％-45％1M乙酸钠(pH7.0)溶液梯度洗脱，时间30分钟，洗速0.8ml/min，结果得到两个紫外吸收峰(280nm)(见附图-2)，其中第二个峰即为huwentoxin-I。将第二峰收集液用国产YWG-C18柱脱盐后，冰冻干燥即为纯净的产品。用上述方法得到的huwentoxin-I用SDS凝胶电泳和等电点聚焦电泳测试皆为一条区带。
huwentoxin-I经MilliGen 6600型蛋白质顺序分析仪多次测定，以及系统二硫键定位法测定出其化学结构如下：
上述化学结构除得到氨基酸组成分析、羧肽酶Y降解分析实验的验证外，另外在质谱仪上做了质谱分析，质谱测出其准确分子量为3749.3±1，与按顺序分析结果计算得出的分子量3750完全吻合，并确证6个半肽氨酸残基是以三对二硫键形式存在。
用小白鼠膈神经-膈肌标本研究了huwentoxin-I的药理学性质。将神经肌肉标本放在标本槽内，槽内温度维持在30-32℃，用台氏液灌流，并通以95％CO2和5％CO2的混合气体，50-70个气泡/分钟，并用电子刺激器产生的方波脉冲通过吸力电极刺激神经或直接刺激肌肉，脉冲宽度0.2毫秒，刺激频率为每分钟12次，用半导体应变片张力传感器将肌肉收缩的机械能转化为电信号，经放大后用笔描记录仪记录。待收缩稳定后，换用浓度为1×10-5g/ml的huwentoxin-I(用台氏液配制)灌流标本，观察对肌肉收缩的影响。结果表明在上述浓度的毒素作用下，肌肉收缩幅度逐渐减小，最后停止在舒张状态，但对直接刺激仍能产生正常的收缩反应，表明神经肌肉接头传递被阻断(见附图3)。在五例标本中，接头传递完全阻断所需时间13.4＋1.3分钟，冲洗半小时不能恢复，说明阻断是不可逆的。如用筒箭毒与huwentoxin-I的混合液灌流标本，则可防止阻断作用的出现，表明毒素的作用在突触后，与箭毒有竞争性关系。
huwnetoxin-I对小鼠的毒性实验结果为腹腔注射LD50为0.70mg/kg体重，脑内注射LD50为9.40μg/kg。
上述结果证明huwnetoxin-I是一种较强的哺乳动物神经肌肉接头传递的不可逆阻断剂。
附图说明：
图1虎纹捕鸟蛛毒素经C4反相高效液相色谱分离的图谱
其中A峰为虎纹捕鸟蛛毒素-I(HWTX-I)所在峰
图2含有虎纹捕鸟蛛毒素-I的组份经WCX-1离子交换柱的进一步分离纯化
其中第二峰为纯净的HWTX-I
图3虎纹捕鸟蛛毒素-I对小鼠膈神经膈肌的生理活性实验
1.未加毒前的肌肉收缩反应，A为间接刺激，B为直接刺激
2.加入浓度为10-5g/ml HWTX-I后的肌肉收缩反应，A、B、C、D分别为加毒后8、10、12、14分钟
3.加毒传递阻断后肌肉对直接刺激的反应
附氨基酸化学结构式简写符号文字说明：
NH2：氨基端；A：丙氨酸；C：半胱氨酸；K：赖氨酸；G：甘氨酸；V：缬氨酸；F：苯丙氨酸； D：天冬氨酸；T：苏氨酸；P：脯氨酸；N：天冬酰氨；E：谷氨酸；R：精氨酸；S：丝氨酸；H：组氨酸；W：色氨酸； L：亮氨酸；COOH：羧基端。
三对二硫键以S-S表示二硫键。
Claims (2)
1.一种虎纹捕鸟蜘蛛毒素，其特征在于氨基酸序列结构如下：
它是由33个氨基酸残基组成的多肽毒素，含有三对二硫键。
2.权利要求1的虎纹捕鸟蜘蛛毒素的制备方法，其特征在于所述方法包括：
1)毒液采集：用特制的软管作诱物送入虎纹捕鸟蜘蛛螯爪下，使蜘蛛毒液射出，然后用微量注射器将毒液抽出冰冻干燥后得到白色干粉即为粗毒；
2)粗毒的纯化：
将上述1)所述粗毒1-3毫克溶于200微升双蒸水中，上样到事先用0.1％三氯乙酸平衡好的C4反相色谱柱中，用0％-70％乙腈梯度洗脱，柱温45℃，时间120分钟，流速0.7ml/min，洗脱结果共分到约23个紫外吸收峰(220nm)其中A峰含有虎纹捕鸟蜘蛛毒，收集A峰洗脱液，冰冻干燥后溶于双蒸水中，上样到事先用0.02M磷酸钠缓冲液(pH6.6)平衡的WCX-1离子交换高效液相色谱柱中，用0％-45％1M乙酸钠(pH7.0)溶液梯度洗脱30分钟，流速0.8ml/min，得到两个紫外吸收峰(280nm)，其中第二个峰为虎纹捕鸟蜘蛛毒素，将第二峰收集液用YWG-C18柱脱盐后，冰冻干燥为纯净的毒素，纯净毒素用SDS凝胶电泳和等电点聚焦电泳测试为一条区带。
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| WO2008077277A1 (fr) * | 2006-12-25 | 2008-07-03 | Graduate School At Shenzhen, Tsinghua University | Procédé de préparation d'un lyophilisat d'araignée et son utilisation dans des médicaments ou des alicaments |
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| CN100383250C (zh) * | 2004-12-06 | 2008-04-23 | 北京大学 | 一种表达虎纹捕鸟蛛毒素-i的方法及其专用载体 |
| CN101233838B (zh) * | 2008-02-04 | 2010-07-21 | 湖北大学 | 一种诱导蜘蛛产生抗菌活性物质的方法 |
| CN101845099A (zh) * | 2010-04-23 | 2010-09-29 | 中国药科大学 | 一种长效镇痛肽及其应用 |
| CN103655631A (zh) * | 2013-12-01 | 2014-03-26 | 大理学院 | 抑制真菌生长的络新妇属蜘蛛有效部位的制备及其用途 |
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