CN106432461B - Vdac蛋白质在利用草酸钠或氯化钙抑制细胞生长中的应用 - Google Patents
Vdac蛋白质在利用草酸钠或氯化钙抑制细胞生长中的应用 Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
本发明通过实验证明了VDAC3蛋白质对草酸或钙盐的亲和力。草酸钙等有机酸钙的形成可降低钙浓度，钙浓度的降低和很硬的、不溶的有机酸钙盐等可导致痉挛等相关现象。根据动物痉挛、拒食、无法站立、易被捕捉、水禽不肯下水、海鸟惧怕痉挛朝大陆飞等抑制性行为活动和异常现象可准确预报大陆或海上地震；因此本发明还提供VDAC3多肽在地震预测中的应用，从而建立简便有效的地震预报方法，对预防和减轻全国乃至全世界的自然灾害大有裨益。
Description
技术领域
本发明涉及生物技术领域，更具体地，涉及VDAC蛋白质在利用草酸或钙盐抑制细胞生长与地震预测中的应用。
背景技术
VDAC在人、鼠等哺乳动物中存在3种亚型。动物VDAC蛋白一级结构的氨基酸序列具有较大差异性，但碱性氨基酸是高度保守的。小鼠VDAC3蛋白中有超过12%的碱性氨基酸残基。通过对50个来自于不同哺乳动物且高度同源的VDAC3氨基酸序列比对，分析得知VDAC3中碱性氨基酸的多态性与动物感知地震的能力具有正相关性——含有碱性氨基酸数量越多的动物对地震的感知越敏感。
VDAC的电压依赖性表现在通过电压控制通道的开关状态，进而调节离子的传导效率。一般情况下，当跨膜电压在30mV至80mV范围内时，VDAC1和VDAC2通道具有阳离子选择性，处于低通透状态，只有小的阳离子可以通过；跨膜电压低于30mV时，VDAC通道具有阴离子选择性，处于高通透开放状态，小的阳离子和较大的阴离子可以通过。与VDAC1和VDAC2不同的是，当膜电压高于80mV时VDAC3通道还不会关闭，并且VDAC3是唯一在70mV膜电压时会表现出在通道开关间快速转换的通道。线粒体外膜上的VDAC在正常情况下处于半开启状态，只有阳离子可以通过，阴离子不能通过。在完全开放的状态下，准许一些阴性代谢分子通过，如ADP、ATP、柠檬酸盐、琥珀酸盐等。当 VDAC 开放时，Ca2+可经 VDAC 流入线粒体，导致线粒体肿胀、线粒体通透性转换孔开放，凋亡发生。
预测地震的方法主要分三种：地震地质法、地震统计法和地震前兆法。地震地质法是分析地震地质背景、构造体系的活动性及新构造运动的强度；地震统计法是通过统计地震活动的空间、时间、强度的变化来达到以震报震的目的；地震前兆法是根据地震前兆监测设施所提供的资料（如地下水观测、地形变观测、电磁场观测），分析异常数据来预报地震。这三种方法通常相互结合并互相补充，但依然达不到及时和准确预报地震的目的。全国，甚至全世界因无法及时准确预测地震来临，每年都会带来巨大的人员伤亡，并造成数以千亿元计的经济损失。
当动物感知较强电场后，阴离子通道VDAC1和VDAC2停止运输阴离子并开始转运阳离子，VDAC3阴离子通道仍能运输阴离子，故可能形成很硬、不溶的有机酸钙盐等，伴随着胞内钙离子的减少导致动物痉挛抽筋。动物的非特异的电压依赖型阴离子通道蛋白质VDAC富有带正电荷的碱性氨基酸，易对电压产生反应。由于地震前震区会有压电反应，有机酸钙在地震感知、痉挛可能起关键作用，从而可能准确预报地震。
发明内容
本发明所要解决的技术问题是克服现有技术存在的上述缺陷，提供VDAC蛋白质在利用草酸或钙盐抑制细胞生长中的应用。
本发明的第二个目的是提供VDAC蛋白质在地震预测中的应用。
本发明的目的是通过以下技术方案予以实现的：
VDAC3多肽在利用草酸或/和钙盐在抑菌方面的应用，所述VDAC3多肽的序列如SEQID NO：1～7所示。
本发明通过实验证明了VDAC3多肽对草酸或/和钙盐的亲和力；即可以转运草酸或/和钙盐。
优选地，本发明所述菌为真菌或/和细菌。
具体地，所述真菌可以是酿酒酵母，细菌为大肠杆菌。
具体地，所述应用是通过下述步骤实现的：
S1. 用无菌水溶解各合成的VDAC3多肽，然后用液体培养基将各种肽分别稀释到不同的终浓度；
S2. 稀释菌液，将菌液与S1获得的不同浓度的肽溶液点样于96孔板中，适宜条件培养一段时间，用酶标仪在492nm处测OD值。根据测量的OD值计算出各种肽在不同浓度、不同培养基中对大肠杆菌或酿酒酵母的抑菌率。
具体地，S1是将肽稀释到40μM 、80μM、160μM和320μM。
具体地，当S2所述菌液是大肠杆菌时，菌液的浓度为5*104cfu/ml，当S2所述菌液是酿酒酵母时，菌液的浓度为1*103cfu/ml。
具体地，当S2所述菌液是大肠杆菌时，是在37℃恒温箱培养大肠杆菌20小时，当S2所述菌液是酿酒酵母时，是在30℃恒温箱培养酿酒酵母21小时。
本发明还提供VDAC3多肽在利用草酸和/或钙盐方面的应用，所述VDAC3多肽的序列如SEQ ID NO：1～7所示。
草酸钙等有机酸钙的形成可降低钙浓度，已知钙浓度的降低等可导致痉挛等相关现象。根据动物痉挛、拒食、无法站立、易被捕捉、水禽不肯下水、海鸟惧怕痉挛朝大陆飞等抑制性行为活动和异常现象可准确预报大陆或海上地震。
因此本发明还提供VDAC3多肽在地震预测中的应用，所述VDAC3多肽的序列如SEQID NO：1～7所示；具体是将SEQ ID NO：1～7所示的多肽注射入动物体内，这些多肽可以吸引带负电荷的有机酸根，有机酸钙的产生和钙浓度降低会导致动物痉挛。地震前因为应力有压电反应，会使3种VDAC全长蛋白质产生相应反应。
与现有技术相比，本发明具有以下有益效果：
本发明通过实验证明了VDAC3蛋白质对草酸或钙盐的亲和力。不溶且很硬的草酸钙等有机酸钙的形成可降低钙浓度，钙浓度的降低和很硬的、不溶的有机酸钙盐等可导致痉挛等相关现象。根据动物痉挛、拒食、无法站立、易被捕捉、水禽不肯下水、海鸟惧怕痉挛朝大陆飞等抑制性行为活动和异常现象可准确预报大陆或海上地震；因此本发明还提供VDAC3多肽在地震预测中的应用，从而建立简便有效的地震预报方法，对预防和减轻全国乃至全世界的自然灾害大有裨益。
附图说明
图1为检测7种VDAC3多肽在不同培养基对大肠杆菌的抑菌率；误差线显示一个标准差。
图2为检测8种VDAC3多肽在不同培养基对酿酒酵母的抑菌率；误差线显示一个标准差；来自于人VDAC3通道的R8肽基本无抑制，其少一个碱性氨基酸故活性减弱。
具体实施方式
下面结合说明书附图和具体实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的简单修改或替换，均属于本发明的范围；若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1 检测7种肽对大肠杆菌MG1655的抑制率
用无菌水溶解各合成肽，使肽的储存液浓度为20mM。然后用4种相应液体培养基将各种肽分别稀释至四个终浓度形成肽溶液，分别为40μM、80μM、160μM、320μM。稀释大肠杆菌菌液至4*105cfu/ml终浓度。按照表2将菌溶液和肽溶液点样于96孔板中，每个点样孔设置3个平行对照。根据之前确定的大肠杆菌培养时间，将点好样的96孔板放于37℃恒温箱培养20小时后，用酶标仪在492nm处测OD值。根据测量的OD值计算出各种肽在不同浓度、不同培养基中对大肠杆菌的抑菌率。计算公式为：
抑菌率%=（a-b）/（c-d）×100%
（其中a、b、c、d对应的是表2中不同点样方案测得的OD值）
本实施例用到的4种相应液体培养基分别为LB培养基、含12mM柠檬酸钠的无盐LB液体培养基、含12mM草酸钠的无盐LB液体培养基、含12mM氯化钙的无盐LB液体培养基，所有培养基的pH均调为7.0。
结果如图1，可以看出，不同浓度的肽溶液在草酸钠培养基和柠檬酸钠培养基一般具有较高抑菌率，在氯化钙培养基有弱的抑菌率。在对照LB培养基抑菌率一般不高或促进生长。
实施例2 检测8种肽对酿酒酵母菌INVSc1的抑制率
用无菌水溶解各合成肽，使肽的储存液浓度为20mM。然后用4种相应液体培养基将各种肽分别稀释至四个终浓度形成肽溶液，分别为40μM、80μM、160μM、320μM。稀释酿酒酵母菌液至1*103cfu/ml终浓度。按照表3将菌溶液和肽溶液点样于96孔板中，每个点样孔设置3个平行对照。根据之前确定的酵母菌培养时间，将点好样的96孔板放于30℃恒温箱培养21小时后，用酶标仪在492nm处测OD值。根据测量的OD值计算出各种肽在不同浓度、不同培养基中对酵母菌的抑菌率（抑菌率计算同实施例1）。
本实施例用到的4种相应液体培养基分别为YPD培养基、含12mM柠檬酸钠的YPD液体培养基、含12mM草酸钠的YPD液体培养基、含12mM氯化钙的YPD液体培养基。
结果如图2，在含有草酸钠和氯化钙的培养基中，多个肽有中等或一定抑菌率，来自于人VDAC3通道的R8肽基本无抑制。K1、K2、K3、R4来自于老鼠VDAC3；K6来自于褐家鼠（沟鼠、白尾鼠、大家鼠或挪威鼠），比K3多一个带正电荷的赖氨酸，在YPD培养基活性要高一些；该试验表明老鼠VDAC3蛋白质的短肽片段对草酸盐和钙离子具有一定亲和力。
SEQUENCE LISTING
<110> 中山大学
<120> VDAC蛋白质在利用草酸或钙盐抑制细胞生长与地震预测中的应用
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Claims (2)
1.VDAC3多肽联合草酸钠在抑菌方面的应用，其特征在于，所述VDAC3多肽的序列如SEQID NO：1～7中任一条所示，所述菌为大肠杆菌MG1655和/或酿酒酵母菌INVSc1。
2.VDAC3多肽联合氯化钙在抑菌方面的应用，其特征在于，所述VDAC3多肽的序列如SEQID NO：1～7中任一条所示，所述菌为大肠杆菌MG1655和/或酿酒酵母菌INVSc1。
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| WO2007113837A2 (en) * | 2006-04-06 | 2007-10-11 | Ben-Gurion University Of The Negev | N-terminal vdac variants and uses thereof |
| WO2010125519A1 (en) * | 2009-04-27 | 2010-11-04 | Centre National De La Recherche Scientifique (Cnrs) | Vdac3-s as a cell marker |
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