CN114010656A - 壳寡糖二价金属配合物作为水母毒素蛋白酶抑制剂的用途 - Google Patents
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Abstract
本发明公开了一种壳寡糖二价金属配合物作为水母毒素蛋白酶抑制剂的用途，壳寡糖二价金属配合物在溶解后可释放金属离子，可以作为水母毒素蛋白酶抑制剂，应用于水母蜇伤的治疗。本发明将具有抗菌抗炎、活化机体细胞、促进损伤组织修复能力的壳寡糖和能抑制毒素酶活性的二价金属离子结合，为研制水母蜇伤外用治疗药剂提供了新的思路。
Description
技术领域
本发明涉及海洋生物技术领域，特别涉及壳寡糖二价金属配合物作为水母毒素蛋白酶抑制剂的用途。
背景技术
水母作为刺胞动物，拥有用于捕猎或防御的刺细胞以及成分复杂的生物毒素，加之水母活动于不同深度沿海开阔水域的习性，经常会与人类发生意外接触并导致水母蜇伤事件频发，严重威胁着公共健康安全。水母蜇伤曾经多发生于地中海地区以及澳洲，但近年来，由于气候变化、海水富营养化等原因，全世界范围内水母爆发式增长，大西洋东北部以及我国沿海地区也成为了水母蜇伤的重灾区，仅秦皇岛市每年收治的蜇伤游客就达上千例。水母蜇伤症状主要以红疹、水疱等皮肤局部反应为主，并伴随着灼伤感的疼痛和刺痒。目前广泛应用的蜇伤处理措施，如小苏打水冲淋以及参考其它皮炎的抗炎、抗过敏治疗等，其效果并不理想，甚至加重病情。
水母毒素是由多种生物活性分子组成的复杂混合物，其成分具有显著的多样性，包括低分子量的非蛋白化合物，如组胺，以及高分子量的复合蛋白，包括酶、成孔毒素和神经毒素等。其中，酶类成分中的蛋白酶和酯酶是水母毒素的主要成分。如中国海域常见的白色霞水母中，其毒素中的金属蛋白酶种类占总蛋白种类的67.24％。相关活性研究表明金属蛋白酶会降解基底膜蛋白，并且对内皮细胞产生毒性，引起出血、骨骼肌损伤、肌坏死等症状，在蜇伤引起的局部反应中起着重要作用。因此水母毒素蛋白酶抑制剂是治疗水母皮炎的潜在良药。
发明内容
为解决上述技术问题，本发明提供了壳寡糖二价金属配合物作为水母毒素蛋白酶抑制剂的用途，从毒性的抑制与皮肤组织损伤的修复两方面，为治疗水母蜇伤提供新的研究思路。
为达到上述目的，本发明的技术方案如下：
壳寡糖二价金属配合物作为水母毒素蛋白酶抑制剂的用途，所述壳寡糖二价金属配合物的结构式如式(Ⅰ)所示：
式(Ⅰ)中，M＝Cu或Zn，n＝2-8。
上述方案中，所述壳寡糖二价金属配合物的平均分子量为500-2500Da。
优选地，所述壳寡糖二价金属配合物的平均分子量为900-1000Da。
上述方案中，所述壳寡糖二价金属配合物与水母毒素蛋白的质量比为0.5:1-10:1。
通过上述技术方案，本发明提供的壳寡糖二价金属配合物作为水母毒素蛋白酶抑制剂的用途具有如下有益效果：
(1)本发明提供了壳寡糖二价金属配合物的新用途，壳寡糖二价金属配合物作为水母毒素蛋白酶抑制剂，应用于水母蜇伤的治疗。
(2)本发明将可抑制蛋白酶酶活性的金属离子与具有抗菌抗炎、可促进创伤组织再生修复等特点的壳寡糖进行结合，从毒性的抑制与皮肤组织损伤的修复两方面，为治疗水母蜇伤提供新的研究思路。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1为本发明所公开的不同浓度的壳寡糖Cu(Ⅱ)配合物与水母毒素共同孵育后，配合物抑制毒素蛋白酶活性的明胶酶谱图，NnV代表实验组中的阳性对照，即水母毒素未与壳寡糖Cu(Ⅱ)配合物孵育情况下对明胶的降解。
图2为本发明所公开的不同浓度的壳寡糖Zn(Ⅱ)配合物与水母毒素共同孵育后，配合物抑制毒素蛋白酶活性的明胶酶谱图，NnV代表实验组中的阳性对照，即水母毒素未与壳寡糖Zn(Ⅱ)配合物孵育情况下对明胶的降解。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。
本发明提供了壳寡糖二价金属配合物作为水母毒素蛋白酶抑制剂的用途，该壳寡糖二价金属配合物的结构式包括如式(Ⅱ)所示的壳寡糖Cu(Ⅱ)配合物和如式(Ⅲ)所示的壳寡糖Zn(Ⅱ)配合物。
式(Ⅱ)和式(Ⅲ)中，n＝2-8。
壳寡糖Cu/Zn(Ⅱ)配合物抑制水母毒素蛋白酶活性通过明胶酶谱法测定。
1、壳寡糖Cu/Zn(Ⅱ)配合物制备方法：
称取1.00g的壳寡糖溶于10mL去离子水中，室温搅拌溶解。另外称取6.2mmol的CuSO4/Zn(NO3)2溶于10ml去离子水中，将CuSO4/Zn(NO3)2溶液缓慢滴加到壳寡糖水溶液中，搅拌反应3h。反应结束后加入过量无水乙醇使产物沉淀，并进行反复离心、无水乙醇洗涤，最后得到的沉淀用透析袋(截留分子量大于500Da)透析72h。将透析后的溶液进行冷冻干燥，即得到产物壳寡糖Cu/Zn(Ⅱ)配合物。使用时用PBS溶液(20mM,pH7.4)溶解为相应浓度的配合物溶液。壳寡糖Cu/Zn(Ⅱ)配合物的平均分子量为1000Da。
2、2mg/mL水母毒素溶液制备方法：
取新鲜水母自溶后的液体分别用60目和100目的分样筛过滤，收集过滤液，3000g离心15min，收集下层沉淀。沉淀用20mM的PBS(pH7.4)重悬，4℃预冷12000g离心10min，反复洗2-3次即得到较为纯净的刺丝囊。离心好的刺丝囊加入5倍质量的PBS溶液重悬，用berbin均质器破碎(15min，8000rpm，3×30s，间歇30s)，之后将破碎后得到的混合液4℃预冷，12000g离心30min，得到的上清液即为水母毒素溶液，采用福林酚法以牛血清白蛋白(BSA)为标准测得水母毒素溶液蛋白浓度为18mg/mL，放置于-80℃保存。取用时4℃解冻，并按比例进行稀释得到以蛋白浓度计为2mg/mL的水母毒素溶液。
3、蛋白酶孵育缓冲液(1×Assay buffer)配置方法：
称取6.057gTris溶于500mL去离子水，用0.1mol/L的稀盐酸调节pH为8.8，之后定容至1000mL。再加入5.844gNaCl和CaCl2至溶液并充分溶解，4℃保存备用。
4、2.5％TritonX-100(v/v)漂洗液配置方法：
量取25mLTritonX-100，加入800mL 1×Assaybuffer轻轻搅拌使Triton充分溶解，最后加去离子水定容至1000mL，4℃保存备用。
5、抑制试验：
分别取60μL的壳寡糖Cu/Zn(Ⅱ)配合物溶液(20mg/mL、10mg/mL、6mg/mL、2mg/mL、1mg/mL)与60μl的水母毒素溶液(2mg/mL)在室温下混合孵育30min，壳寡糖Cu/Zn(Ⅱ)配合物与水母毒素(以蛋白计)的质量比分别为10:1、5:1、3:1、1:1、0.5:1。同时设置不添加壳寡糖Cu/Zn(Ⅱ))配合物溶液的空白对照组(NnV)。孵育完成后加入40μL 4×非变性蛋白上样缓冲液充分混合，加20μL样品溶液至10％SDS-Page胶(含2mg/mL明胶底物)的上样孔道，置于冰浴中进行电泳。起始电压设为80V，待上样缓冲液所示指示带进入分离胶后，调节电压至120V，电泳时间约2h。
电泳完成后取出凝胶，浸泡于2.5％Triton X-100漂洗液中，置于摇床低速漂洗2遍，每次30min。再将Page胶浸泡于Assay buffer中，置于摇床低速漂洗2遍，每次30min。最后将Page胶浸泡于Assay buffer中于37℃环境过夜存放。次日用R-250考马斯亮蓝染液浸泡Page胶3h进行染色，再用脱色液(甲醇：水：乙酸＝5：4：1)对凝胶进行脱色。脱色至肉眼可见透明清晰条带后，进行拍照，未被毒素中酶成分降解的明胶呈深蓝色，通过比较各泳道明胶条带颜色的深浅，分析水母毒素中蛋白酶活性是否被壳寡糖Cu/Zn(Ⅱ)配合物所抑制，结果见图1/图2。
阳性对照对应泳道(NnV)在63-180kDa范围内分布有不同明亮程度的条带，表明水母毒素中含有可降解胶内明胶底物的蛋白酶(R-250考马斯亮蓝染液无法对被蛋白酶降解后的明胶底物进行染色)，且该类酶的分子量的分布较广。图中，Marker代表标准蛋白，kDa为蛋白分子量单位。
如图1所示，当水母毒素与20mg/ml的壳寡糖Cu(Ⅱ)配合物孵育后，各条带均完全变暗，表明壳寡糖Cu(Ⅱ)配合物完全抑制了水母毒素蛋白酶的活性。
当水母毒素与10mg/ml的壳寡糖Cu(Ⅱ)配合物孵育后，泳道中除180kDa处的条带之外，其余条带均完全变暗，表明壳寡糖Cu(Ⅱ)配合物已完全抑制了除个别大分子量外的水母毒素蛋白酶的活性。
当水母毒素与6mg/ml的壳寡糖Cu(Ⅱ)配合物孵育后，泳道中除180kDa处的条带之外，其余条带均完全变暗，表明壳寡糖Cu(Ⅱ)配合物已完全抑制了除个别大分子量外的水母毒素蛋白酶的活性。
当水母毒素与2mg/ml的壳寡糖Cu(Ⅱ)配合物孵育后，泳道中各条带较阳性对照组相比均变暗，尤其是在100kDa左右，表明壳寡糖Cu(Ⅱ)配合物抑制了水母毒素蛋白酶的活性。
当水母毒素与1mg/ml的壳寡糖Cu(Ⅱ)配合物孵育后，泳道中各条带较阳性对照组相比均变暗，尤其是在100kDa左右，表明壳寡糖Cu(Ⅱ)配合物抑制了水母毒素蛋白酶的活性。
如图2所示，当水母毒素与20mg/ml的壳寡糖Zn(Ⅱ)配合物孵育后，除了180kDa处，其余条带均完全变暗，表明壳寡糖Zn(Ⅱ)配合物完全抑制了除个别大分子量外的水母毒素蛋白酶的活性。
当水母毒素与10mg/ml的壳寡糖Zn(Ⅱ)配合物孵育后，泳道中除180kDa处的条带之外，其余条带均显著变暗，表明壳寡糖Zn(Ⅱ)配合物已显著抑制了不同分子量水母毒素蛋白酶的活性。
当水母毒素与6mg/ml的壳寡糖Zn(Ⅱ)配合物孵育后，泳道中各条带较阳性对照组相比均变暗，表明壳寡糖Zn(Ⅱ)配合物抑制了水母毒素蛋白酶的活性。
当水母毒素与2mg/ml的壳寡糖Zn(Ⅱ)配合物孵育后，泳道中各条带较阳性对照组相比均变暗，表明壳寡糖Zn(Ⅱ)配合物抑制了水母毒素蛋白酶的活性。
当水母毒素与1mg/ml的壳寡糖Zn(Ⅱ)配合物孵育后，泳道中各条带较阳性对照组相比均变暗，表明壳寡糖Zn(Ⅱ)配合物抑制了水母毒素蛋白酶的活性。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (4)
1.壳寡糖二价金属配合物作为水母毒素蛋白酶抑制剂的用途，其特征在于，所述壳寡糖二价金属配合物的结构式如式(Ⅰ)所示：
式(Ⅰ)中，M＝Cu或Zn，n＝2-8。
2.根据权利要求1所述的壳寡糖二价金属配合物作为水母毒素蛋白酶抑制剂的用途，其特征在于，所述壳寡糖二价金属配合物的平均分子量为500-2500Da。
3.根据权利要求1或2所述的壳寡糖二价金属配合物作为水母毒素蛋白酶抑制剂的用途，其特征在于，所述壳寡糖二价金属配合物的平均分子量为900-1000Da。
4.根据权利要求1或2所述的壳寡糖二价金属配合物作为水母毒素蛋白酶抑制剂的用途，其特征在于，所述壳寡糖二价金属配合物与水母毒素的质量比为0.5:1-10:1，其中水母毒素以其中的蛋白计。
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