CN102050874B - 虎纹捕鸟蛛毒素-xvi的制备方法 - Google Patents
虎纹捕鸟蛛毒素-xvi的制备方法 Download PDFInfo
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Abstract
本发明公开了一种虎纹捕鸟蛛毒素-XVI,是从虎纹捕鸟蛛粗毒中经离子交换和反相高效液相色谱分离提纯得到的,其一级结构由39个氨基酸残基组成,含有6个半胱氨酸并形成三对二硫键,分子量为4437.4Da;该毒素纯化冻干粉的理化性状为白色或类白色疏松体,无气味,极易溶解于水,水溶液近于无色透明;对大鼠背根神经元N-型钙通道的半数最大抑制浓度为60nmol/L,是一种很强的选择性作用于N-型钙通道的抑制剂。
Description
技术领域
本发明涉及一种虎纹捕鸟蛛毒素。
背景技术
痛觉是一种与组织损伤或潜在的损伤相关的不愉快的情感体验和主观感觉,它是感觉神经系统的功能,是机体自我保护的一种反射机制。多肽类钙通道抑制剂广泛存在于蜘蛛、芋螺和蝎子等有毒动物的毒液中,具有开发成治疗人类疾病的镇痛新药的应用前景。例如,ω-芋螺毒素MVIIA是研究比较清楚的脊椎动物神经元N-型钙通道阻断剂,通过堵塞其受体孔道而达到目的。ziconotide(合成的ω-芋螺毒素MVIIA)作为一种治疗严重慢性疼痛的新型非阿片类新药已于2004年被美国食品和药物管理局获准出售。因此,全世界都将精力转移到开发不以阿片类受体为靶点的强效镇痛药。
发明内容
本发明旨在于提供一种能明显抑制哺乳动物N-型钙通道的虎纹捕鸟蛛毒素-XVI的制备方法.
本发明虎纹捕鸟蛛毒素-XVI的制备方法,其特征在于包括如下步骤:
(1)阳离子交换柱层析, 称取粗毒溶于双蒸水并离心,取上清液进样,在配备有486紫外检测器的蛋白质纯化系统上,采用Waters CM填料,使用Waters P-1 型阳离子交换柱进行阳离子交换层析;采用四元梯度洗脱:(A)0.1mol/L 磷酸二氢钠,(B)0.1 mol/L磷酸氢二钠,(C) 1.0 mol/L 氯化钠;(D) 双蒸水,其中A液和B液用来调节洗脱液的pH值,采用氯化钠梯度洗脱;在280 nm波长室温检测并收集所有被洗脱峰,找出目的峰后再进一步进行反相脱盐处理;
(2)收集目的峰,脱盐纯化在高效液相色谱工作站上进行,采用分离柱为Phenomenex C18柱,先用100% ddH2O冲洗20 min,将混在样品中的盐分洗涤干净之后,用乙腈溶液进行梯度洗脱。流速为3.0 mL/min,检测波长为280/215 nm,柱温为室温,收集每个洗脱峰,用质谱鉴定所含成分的分子量,找出含有HWTX-XVI的洗脱峰并进行冷冻干燥;
(3)目的样品再在高效液相色谱工作站或者反相HPLC纯化系统上进行纯化,分离柱为Phenomenex C18柱,收集洗脱峰,并用MALDI-TOF质谱仪鉴定样品的纯度,获得虎纹捕鸟蛛毒素-XVI;
(4)对步骤(3)获得的虎纹捕鸟蛛毒素-XVI,进行序列测定,其氨基酸序列如下:
Cys Ile Gly Glu Gly Val Pro Cys Asp Glu Asn Asp Pro Arg Cys Cys Ser Gly Leu Val Cys Leu Lys Pro Thr Leu His Gly Ile Trp Tyr Lys Ser Tyr Tyr Cys Tyr Lys Lys。
本发明方法提供的这种虎纹捕鸟蛛毒素-XVI(HWTX-XVI)其一级结构由39个氨基酸残基组成,其中含6个半胱氨酸并形成三对二硫键,分子量为4437.4 Da,该毒素纯化冻干粉的理化性状为白色或类白色疏松体,无气味,极易溶解于水,水溶液近于无色透明;对大鼠背根神经元N-型钙通道的半数最大抑制浓度为60 nmol/L, 它是一种很强的选择性作用于N-型钙通道的抑制剂,具有低毒性和可逆性,能有效抑制哺乳动物N-型钙通道,有希望成为开发治疗N-型钙通道相关疾病的先导分子。
附图说明
图1是虎纹捕鸟蛛粗毒阳离子交换HPLC图谱。箭头表示目的峰,纵坐标表示洗脱峰在280 nm的吸收值,横坐标表示洗脱时间。
图2是虎纹捕鸟蛛粗毒目的阳离子交换峰反相HPLC图谱。“*”表示目的峰,纵坐标表示各个洗脱峰在280 nm下的吸收值,横坐标表示洗脱时间。
图3是HWTX-XVI的反相HPLC图谱。
图4是HWTX-XVI的MALDI-TOF质谱图谱。
图5是1 μM HWTX-XVI对大鼠输精管收缩的影响。1 μM HWTX-XVI能快速抑制低频率电刺激诱导的大鼠输精管收缩,而且这种抑制作用是可逆的。
图6是HWTX-XVI抑制大鼠输精管收缩的浓度依从性。浓效曲线均用Hill方程进行拟合得出IC50。方程y=1-(1-f max)/(1 + ([x] /IC50)nH)中,x代表毒素浓度,IC50代表毒素半数有效抑制浓度,n H 是Hill常数。
图7是10μM HWTX-XVI对低电压激活钙电流的影响。
图8是10μM HWTX-XVI能明显抑制高电压激活钙电流。
图9是HWTX-XVI不能进一步阻断GVIA-不敏感的电流。
图10是在细胞外液中存在HWTX-XVI时,GVIA不能抑制电流。
图11是MVIIA可阻断部分HWTX-XVI不敏感钙电流。
图12是HWTX-XVI对钙通道电流-电压关系(I-V curve)的影响。
图13是HWTX-XVI抑制N-型钙通道的浓度依赖性。图中每个点的数据来自于5~8个实验细胞,用平均值±标准误(mean±S.E.)来表示。浓度曲线均用Hill方程进行拟合得出IC50。方程y=1-(1-f max)/(1 + ([x] /IC50)nH)中,x代表毒素浓度, IC50代表毒素半数有效抑制浓度,n H 是Hill常数。
图14是HWTX-XVI抑制N-型钙通道的时间依从性。
具体实施方式
1、实验:
1.1虎纹捕鸟蛛粗毒的分离纯化:
虎纹捕鸟蛛粗毒的分离纯化分三步进行:(1)阳离子交换柱层析,在Waters650E色谱系统上进行,层析柱子采用Waters P-1 型阳离子交换柱(10 mm×100 mm)。称取10 mg 粗毒,溶于1 mL 双蒸水,并于台式高速离心机(国产)上离心10 min(转速为10,000 rpm),沉淀不溶物。取上清液进样,在配备有486紫外检测器的Waters 650 E高级蛋白质纯化系统上,采用Waters CM (300 nm)填料,使用常压自装柱(10 mm × 100 mm)进行阳离子交换层析。采用四元梯度洗脱:(A)0.1mol/L 磷酸二氢钠;(B)0.1 mol/L磷酸氢二钠; (C) 1.0 mol/L 氯化钠;(D) 双蒸水(ddH2O)。其中A液和B液用来调节洗脱液的pH值,采用氯化钠梯度洗脱。在280 nm波长室温检测并收集所有被洗脱峰,找出目的峰后再进一步进行反相脱盐处理。脱盐纯化在Waters 515 pump & Empower高效液相色谱工作站,2487检测器上进行。采用分离柱为Phenomenex C18柱(4.6 mm × 250 mm)。一次进样200~300 mL。先用100% ddH2O(含0.1%TFA)冲洗20 min,将混在样品中的盐分洗涤干净(紫外检测吸收值为零)之后,用乙腈(含0.1%TFA)溶液进行梯度洗脱。流速为3.0 mL/min,检测波长为280/215 nm,柱温为室温。收集每个洗脱峰,用质谱鉴定它们所含成分的分子量,找出含有HWTX-XVI的洗脱峰并进行冷冻干燥。最后,目的样品再在Waters 515 pump & Empower高效液相色谱工作站,2487 检测器,或者反相HPLC纯化系统(Waters公司,Alliance 2690 HPLC & Millennium32 高效液相色谱工作站),996 PDA检测器上进行纯化。分离柱:为Phenomenex C18柱(4.6 mm × 250 mm);洗脱液分别为:A液(0.1% TFA/H2O)、B液(0.1% TFA/CAN),流速为1.0 mL/min,检测波长为280/215 nm,柱温箱温度为40℃。收集洗脱峰,并用MALDI-TOF质谱仪鉴定样品的纯度,目的样品的冻干粉末置于-20℃冰箱储存备用。
1.2 MALDI-TOF质谱分析和氨基酸序列测定:
MALDI-TOF质谱分析在美国应用生物系统公司生产的Voyager-DETM STR型的MALDI-TOF Mass(Matrix-assisted Laser desorption/ionization time-of-flight)质谱仪上进行。用50℅乙腈、50℅水、0.1℅TFA混合液制备CCA(α-cyano-4-hydroxycinnamic acid) 基质(5mg/mL),然后分别取 1.0μl样品与5μl CCA基质液混合,再取0.5μl混合液在质谱仪的样品盘上分别点样,室温下自然风干后测定各样品的分子量。采用反射模式,离子源加速电压为20 kV,N2激光波长337nm,脉冲宽度3ns,离子延迟提取150ns,真空度4×10-7 Torr,质谱信号单次扫描累加100次,正离子模式。
氨基酸序列分析是应用Edman降解原理,在 Perkin Elmer Procise 491A型气相测序仪(美国Applied Biosystem公司产品)上进行的。一般不直接使用天然毒素进行测序,而选用经碘乙酰胺烷基化修饰后的毒素肽作为测序样品,因为没有经碘乙酰胺修饰的半胱氨酸(cysteine)在214 nm波长检测下没有吸收值,观察不到信号,不便于准确定位多肽中半胱氨酸的位置,而经修饰的半胱氨酸的PTH-CM-Cys出峰信号明显,在线HPLC检测能够得到多肽序列中半胱氨酸的位置。根据毒素分子量大小推测其含有氨基酸残基的个数,然后设定实际测序循环数,即1 个空白循环+1 个标准循环+氨基酸残基数目。
1.3生物活性测定:
1.3.1大鼠输精管实验:
大鼠输精管标本制备方法:大鼠经断头处死后立即取出长约2厘米的输精管,并置于预先用95% O2/ 5% CO2 饱和的克氏液(mM:NaCl 119.0,KCl 4.7,CaCl2 2.5,MgSO4 1.2,NaHCO3 2.5,KH2PO4 1.2, glucose 11,EDTA 0.026,pH 7.3)中, 轻柔的挤出输精管的内容物。将输精管的两端分别与32 oC恒温的5 ml 浴槽的底部和换能器连接, 立即将标本置于浴槽中, 通过1g 砝码定标输精管的拉伸长度。 通过分布于浴槽两端的电极向输精管施加脉冲电场刺激 (波宽: 0.14 ms,强度: 100V,周期:15 s)诱导输精管收缩。 在平衡30min 后进行药理学试验。通过测定HWTX-XVI阻断电刺激诱导的输精管收缩的时间,确定HWTX-XVI 的生物学活性。
1.3.2大鼠背根神经节细胞的急性分离培养:
挑选出生4周左右、体重120~200 g的SD大白鼠,断颈处死后,用剪子迅速取出脊椎并剪成2~3段,再沿与肋骨平面垂直的方向将椎管剪开,并浸泡在盛有少量培养液的烧杯中;用镊子撕破附在椎管内壁上的一层黏膜,暴露位于椎管和肋骨的交汇处的背根神经纤维。在胸腰椎段,可挑选18个左右并置入盛有2 mL培养液的培养皿中;在解剖显微镜下,用维娜斯剪和尖头镊子分离出神经节。剥离包在节外的絮状物和轴索后,放入盛有约0.5 mL培养液的培养皿中。用吸管吸去液体,将分离好的所有神经节剪碎,越碎越好。剪碎之后,转入消化液,在34℃、震荡频率110 rpm的环境中进行酶解消化反应20 min。期间每隔10 min取出用移液枪吸打数次;向消化液中加入酶的抑制剂,终止酶解处理过程。无菌操作将消化得到的细胞液转入离心管中进行离心(800 rpm、5 min),去上清,加入8 mL含10%小牛血清的长期培养液。 重悬细胞后分成3~4皿,放入培养箱(5%CO2、95%空气)中,37℃培养3~4 h贴壁。使用Ba2+作为Ca2+的电荷替代物,钙通道电流(ICa)的大小通过测得IBa值来确定。实验中的细胞外液(in mM): 160 TEA-Cl, 10 HEPES, 2 BaCl2, 10 glucose,和 200nM TTX, 用TEA-OH调节 pH到7.4;细胞内液(in mM): 120 CsCl, 5 Mg-ATP, 0.4 Na2-GTP, 10 EGTA, 20 HEPES-CsOH,调节pH到7.2。
1.3.3膜片钳电生理活性实验:
膜片钳实验均在室温(25±1℃)进行,采用全细胞膜片钳技术。挑选质膜光滑可见、胞质均匀的DRG细胞作为实验细胞。电流记录通过全细胞膜片钳技术利用放大器EPC9(HEKA公司, German)在电脑上进行。计算机记录和分析系统采用Pulse+Pulsefit 8.0软件。玻璃电极管为硼硅酸盐玻璃毛细管(南京泉水教学实验器材厂)。玻璃电极两步拉制而成,经抛光仪(Narishige,Japan)抛光后电极尖端直径约为3 μm,充灌电极液后电极电阻为1-3MΩ。膜片钳实验要在室温条件下进行,整个实验过程中温度的变动最多上下不超过2℃。采用SigmaPlot 9.0软件分析实验结果。
2、实验结果
2.1 HWTX-XVI的分离纯化
二维色谱是一种非常有效的分离纯化的方法,通过阳离子交换HPLC以及反相HPLC两步分离,我们从虎纹捕鸟蛛粗毒中成功分离到HWTX-XVI。图1是虎纹捕鸟蛛粗毒的阳离子交换HPLC图谱,在280 nm波长下检测,可观察到8个非常明显的洗脱峰,其中第2个峰是目的峰,经MALDI-TOF质谱鉴定该峰内含有多种组分。收集此峰后,在Waters 515 pump & Empower高效液相色谱工作站上进行脱盐处理和反相HPLC分离纯化,所得图谱见图2,出现多个主峰。收集目的峰并冷冻干燥后,在Alliance系统上进行再次反相HPLC(见图3)。图中显示含HWTX-XVI洗脱峰为单一峰,通过质谱鉴定纯度达到98%以上。质谱结果表明为纯度较高的目标毒素虎纹捕鸟蛛毒素-XVI(huwentoxin-XVI,HWTX-XVI),分子量4437.4 Da(见图4)。HWTX-XVI的序列测定在491-A测序仪上进行。
2.2 HWTX-XVI对整体动物和大鼠输精管的影响
小鼠腹腔注射大剂量(5 mg/kg体重)的HWTX-XVI 后,未观察到异常的生理反应。同样美洲蜚蠊腹腔注射200 μg/g HWTX-XVI 也未发现异常的生理反应。与空白对照相反,1 μM HWTX-XVI能快速抑制低频率电刺激诱导的大鼠输精管收缩,而且这种抑制作用是可逆的,用空白溶液冲洗标本后,输精管的收缩能在数分钟后恢复到对照水平(图5)。我们进一步检测了不同浓度HWTX-XVI对大鼠输精管收缩的抑制作用,发现HWTX-XVI对大鼠输精管收缩的抑制呈浓度依赖性,它的半数有效抑制浓度(IC50)是85 ± 6 nM(图6)。上述结果表明HWTX-XVI能抑制大鼠输精管交感神经末梢上的N-型钙通道。
2.3 HWTX-XVI对大鼠背根神经细胞电压门控钙通道的影响
我们选用大鼠背根神经神经元作为研究对象检测了HWTX-XVI对大鼠DRG细胞电压门控钙通道的作用。根据生理和药理学特性电压门控钙通道可分为N、L、P/Q、R、T型。根据其通道激活电压阈值可分为两类:高电压激活(HVA)钙通道和低电压(LVA)激活钙通道。其中HVA钙通道包括:N-,L-,P/Q-,R-型钙通道,而T-型钙通道属于LVA钙通道。10 μM HWTX-XVI对低阈值激活钙通道没有明显影响(见图7), 但能抑制约45%的高电压激活钙通道(见图8),表明HWTX-XVI能抑制部分高电压激活钙通道。为了研究HWTX-XVI对于HAV钙通道的选择性, 我们采用各种HAV 钙通道的专一性抑制剂分离电流, 如GVIA是一种ω-芋螺毒素,专一性阻断N-型钙电流, 而nifedipine能专一性抑制L-型钙电流,而剩余电流通过加入Ni2+阻断。在本实验中,细胞外液中加入3 μM GVIA能够阻断43.6±3.8%的N-型钙电流,继续加入10 μM nifedipine 可进一步抑制41±4.7%的L-型钙电流,剩余的钙电流为P/Q-型和R-型,可被Ni2+完全抑制。在细胞外液加入3 μM GVIA阻断N-型钙电流后,加入10 μM HWTX-XVI对于剩余的电流没有抑制作用,而加入nifedipine和 Ni2+时电流能被完全抑制(图9);与之类似,在细胞外液中先加入10 μM HWTX-XVI后,继续加入3 μM GVIA对于HWTX-X-不敏感的电流没有影响(图10)。这一结果表明HWTX-XVI选择性阻断GVIA-敏感的N-型钙电流。另一种芋螺毒素MVIIA(ω-conotoxin MVIIA),是一种既能抑制N-型钙离子通道,同时也能抑制P/Q-型钙离子通道的抑制剂。采用相同方法比较了HWTX-XVI与MVIIA的抑制作用, 在先加入10 μM HWTX-XVI后,3 μM MVIIA能够继续阻断部分剩余电流,这与MVIIA的相关研究结果是一致的, MVIIA在较高浓度能够部分阻断P/Q 型钙电流(图11),由此推断HWTX-XVI对于HAV钙通道选择性高于MVIIA。在细胞周围加入10 μM的HWTX-XVI,再以相同的去极化脉冲诱导电流-电压关系曲线,HWTX-XVI不影响钙电流的起始激活电压、最大电流激活电压和逆转电位,说明毒素与通道的相互作用没有改变通道对离子通透的选择性(图12)。
鉴于HWTX-XVI专一性阻断N-型钙电流, 在本实验中GVIA被用于分离N-型电流,即每一细胞在测试的最后都加入3 μM GVIA完全阻断N-型电流,以此作为N-型钙电流被100%抑制,而不同浓度的HWTX-XVI阻断强度与它相比得出相对的阻断比率, 通过这种方法我们获得了HWTX-XVI阻断N-型钙电流的浓效曲线(图13)。HWTX-XVI抑制N-型钙电流具有浓度依赖性,它的半数有效抑制浓度(IC50)是60 ± 5 nM(图13)。HWTX-XVI抑制N-型钙电流呈现时间依赖性,10 μM HWTX-XVI能够迅速阻断N-型电流(τon = 28.3±2.3 s),但要相对慢于MAIIA和GVIA的阻断速率(τon分别为17.4±3.1 s 和15.4±2.2s)。在HWTX-XVI完全阻断N-型电流后,通过外液灌流2 min内能够恢复至对照的92%(τoff = 64.8±3.2 s),MVIIA完全阻断通过4 min的灌流能够恢复约41%的电流, 而GVIA的阻断效应几乎不能恢复(图14)。
总之,在本研究中我们从虎纹捕鸟蛛毒液分离和鉴定到了一种新型的钙通道毒素。HWTX-XVI能够阻断大鼠DRG神经元上GVIA敏感的N-型钙通道,它的低毒性和可逆性使之有希望成为开发治疗N-型钙通道相关疾病的先导分子。
虎纹捕鸟蛛毒素-XVI的氨基酸序列为:
Cys Ile Gly Glu Gly Val Pro Cys Asp Glu Asn Asp Pro Arg Cys Cys Ser Gly
1 5 10 15
Leu Val Cys Leu Lys Pro Thr Leu His Gly Ile Trp Tyr Lys Ser Tyr Tyr Cys
20 25 30 35
Tyr Lys Lys。
SEQUENCE LISTING
<110> 湖南师范大学, 国防科学技术大学
<120> 虎纹捕鸟蛛毒素-XVI
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 39
<212> PRT
<213> 虎纹捕鸟蛛(Ornithoctonus huwena)
<400> 1
Cys Ile Gly Glu Gly Val Pro Cys Asp Glu Asn Asp Pro Arg Cys Cys Ser Gly
1 5 10 15
Leu Val Cys Leu Lys Pro Thr Leu His Gly Ile Trp Tyr Lys Ser Tyr Tyr Cys
20 25 30 35
Tyr Lys Lys
Claims (1)
1.一种虎纹捕鸟蛛毒素-XVI的制备方法,其特征在于包括如下步骤:
(1)阳离子交换柱层析, 称取粗毒溶于双蒸水并离心,取上清液进样,在配备有486紫外检测器的蛋白质纯化系统上,采用Waters CM填料,使用Waters P-1 型阳离子交换柱进行阳离子交换层析;采用四元梯度洗脱:(A)0.1mol/L 磷酸二氢钠,(B)0.1 mol/L磷酸氢二钠,(C) 1.0 mol/L 氯化钠;(D) 双蒸水,其中A液和B液用来调节洗脱液的pH值,采用氯化钠梯度洗脱;在280 nm波长室温检测并收集所有被洗脱峰,找出目的峰后再进一步进行反相脱盐处理;
(2)收集目的峰,脱盐纯化在高效液相色谱工作站上进行,采用分离柱为Phenomenex C18柱,先用100% ddH2O冲洗20 min,将混在样品中的盐分洗涤干净之后,用乙腈溶液进行梯度洗脱,流速为3.0 mL/min,检测波长为280/215 nm,柱温为室温,收集每个洗脱峰,用质谱鉴定所含成分的分子量,找出含有HWTX-XVI的洗脱峰并进行冷冻干燥;
(3)目的样品再在高效液相色谱工作站或者反相HPLC纯化系统上进行纯化,分离柱为Phenomenex C18柱,收集洗脱峰,并用MALDI-TOF质谱仪鉴定样品的纯度,获得虎纹捕鸟蛛毒素-XVI;
(4)对步骤(3)获得的虎纹捕鸟蛛毒素-XVI,进行序列测定,其氨基酸序列如下:
Cys Ile Gly Glu Gly Val Pro Cys Asp Glu Asn Asp Pro Arg Cys Cys Ser Gly Leu Val Cys Leu Lys Pro Thr Leu His Gly Ile Trp Tyr Lys Ser Tyr Tyr Cys Tyr Lys Lys。
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