CN1775284A - 降低血液粘度的试剂 - Google Patents
降低血液粘度的试剂 Download PDFInfo
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- CN1775284A CN1775284A CNA2005100740806A CN200510074080A CN1775284A CN 1775284 A CN1775284 A CN 1775284A CN A2005100740806 A CNA2005100740806 A CN A2005100740806A CN 200510074080 A CN200510074080 A CN 200510074080A CN 1775284 A CN1775284 A CN 1775284A
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- Prior art keywords
- alanine
- glycine
- aspartic acid
- glutamic acid
- valine
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Abstract
一种降低血液粘度的试剂,其含有来源于纳豆枯草芽孢杆菌的蛋白质,并且从氨基末端顺序包括第一结构氨基酸序列,第二结构氨基酸序列,第三结构氨基酸序列和第四结构氨基酸序列,从而降低全血的粘度。
Description
背景技术
本发明涉及降低血粘度试剂,其降低全血的粘度。
长久以来,纳豆已经成为日本传统发酵食物,从1600s到目前,并且作为允许日本人有效摄取大豆蛋白质的食物是杰出的。例如,纳豆具有比大豆更高的可消化性。这是因为作为大豆的主要成分的蛋白质、脂肪、淀粉等被纳豆枯草芽孢杆菌(Bacillus subtilis natto)分解成氨基酸、脂肪酸和葡萄糖,并且增强了摄入时的可消化性。
包含在纳豆中的大多数维生素B家族缓和身体功能并缩短疲劳恢复时间。纳豆包含大量的维生素B2,并预期用于恢复视力疲劳。纳豆中维生素K的含量在食品中是最高的。预期纳豆提高血钙代谢,如预防骨质疏松症和改善血液凝结能力。
近年来,已阐明了纳豆的多种功能,并且作为保健食品,极大关注纳豆的作用。已经增加了纳豆的消费。具体地,称为纳豆激酶的含纳豆的酶作为纤维蛋白溶解试剂和血栓溶解试剂是有效的,如日本专利公开号61-162184(参考文献1),3-168082(参考文献2)和6-153977(参考文献3)中所公开的生产方法,理化性质和生化性质。
纳豆激酶作为血栓溶解试剂或血栓形成抑制剂已受到极大关注,如在日本专利公开号1-180834(参考文献4)和8-208512(参考文献5)中所述。这些参考文献公开了与含有纳豆激酶作为活性成分、安全、容易进入的试剂代替目前在临床领域用于血栓的试剂即溶解纤维蛋白试剂、血小板聚集抑制剂例如尿激酶、链激酶和组织纤溶酶原激活物相关的技术。
日本专利公开号2001-352929(参考文献6)公开了以下述方式生产的纳豆加工食品。在缺乏发酵纳豆独特香味的情形中,从透析的培养基中提取纳豆独特的活性成分,在所述培养基中将纯化自大豆的豆蛋白粉末与纳豆枯草芽孢杆菌一起培养。根据参考文献6的方案,使用这种加工的食品能延迟血液凝固时间,并抑制血栓形成,除了使用纳豆激酶的溶解血栓作用以外。
如上所述,来源于纳豆的产品具有各种作用,例如除了溶解血栓作用以外,延迟血液凝固时间和抑制血栓形成。本发明人发现在参考文献6中公开的纳豆加工食品的除了上述作用以外的新功能。
发明内容
发明概述
本发明的一个目的是提供一种通过使用来源于纳豆的产品具有新功能的试剂。
为了实现本发明的上述目的,提供了一种降低血液粘度的试剂,其基本上由来源于纳豆枯草芽孢杆菌的蛋白质组成,其从氨基末端顺序包括第一结构氨基酸序列,第二结构氨基酸序列,第三结构氨基酸序列和第四结构氨基酸序列,所述第一结构氨基酸序列具有丙氨酸,苏氨酸,天冬氨酸,甘氨酸,缬氨酸,谷氨酸,色氨酸,天冬酰胺,缬氨酸,天冬氨酸,谷氨酰胺,异亮氨酸,天冬氨酸,丙氨酸,脯氨酸,赖氨酸,丙氨酸,色氨酸,丙氨酸,亮氨酸,甘氨酸,酪氨酸,天冬氨酸,甘氨酸,苏氨酸,甘氨酸,苏氨酸,缬氨酸,缬氨酸,丙氨酸,丝氨酸,异亮氨酸,天冬氨酸,苏氨酸,甘氨酸,缬氨酸,谷氨酸,色氨酸,天冬酰胺,组氨酸,脯氨酸,丙氨酸,亮氨酸,赖氨酸,谷氨酸,赖氨酸,酪氨酸,精氨酸,甘氨酸,酪氨酸,天冬酰胺,脯氨酸,谷氨酸,天冬酰胺,脯氨酸,天冬酰胺,谷氨酸,脯氨酸,谷氨酸,天冬酰胺,谷氨酸,甲硫氨酸,天冬酰胺,色氨酸,酪氨酸,天冬氨酸,丙氨酸,缬氨酸,丙氨酸,甘氨酸,谷氨酸,丙氨酸,丝氨酸,脯氨酸,酪氨酸,天冬氨酸,天冬氨酸,亮氨酸,丙氨酸,组氨酸,甘氨酸,苏氨酸,组氨酸,缬氨酸,和苏氨酸,所述第二结构氨基酸序列具有丙氨酸,苯丙氨酸,丝氨酸,谷氨酸,天冬氨酸,甘氨酸,甘氨酸,苏氨酸,天冬氨酸,丙氨酸,天冬氨酸,异亮氨酸,亮氨酸,谷氨酸,丙氨酸,甘氨酸,谷氨酸,色氨酸,缬氨酸,和亮氨酸,所述第三结构氨基酸序列具有天冬氨酸,丙氨酸,谷氨酸,甘氨酸,天冬酰胺,脯氨酸,组氨酸,脯氨酸,谷氨酸,甲硫氨酸,丙氨酸,脯氨酸,天冬氨酸,和缬氨酸,所述第四结构氨基酸序列具有缬氨酸,脯氨酸,甘氨酸,谷氨酰胺,丙氨酸,酪氨酸,谷氨酸,天冬氨酸,甘氨酸,色氨酸,和天冬氨酸,从而降低全血的粘度。
附图说明
图1是显示血液粘度测量结果的图表;和
图2是显示血液粘度测量结果的图表。
具体实施方式
优选实施方式的描述
参考附图将描述本发明的实施方案。本发明人已开发了作为加工食品的产品(下文表示为NKCP),其通过有效纯化来自纳豆枯草芽孢杆菌培养的大豆液体培养基的纤维蛋白溶解物质而获得。在该开发过程中,本发明人在研究纳豆的溶解纤维蛋白活性过程中已获得一种新的与溶解血液凝块一样的发现。
下面描述NKCP的生产。将纳豆枯草芽孢杆菌(Takahashi菌株)加入到纯水中,并搅拌以制备悬浮液。将少量悬浮液接种到通过使用标准琼脂培养基制备的琼脂板上。在37℃培养悬浮液24小时,以获得在琼脂板上纳豆枯草芽孢杆菌的菌落。
在具有500ml内部体积的锥形瓶中制备含有1g葡萄糖和4g大豆蛋白粉末作为原料的200ml液体培养基。在高压灭菌器中于120℃灭菌锥形瓶30分钟,以灭菌锥形瓶的内部。大豆蛋白粉末纯化自大豆。通过研磨大豆获得的大豆粉末可用作原料。大豆蛋白包含在原料中就足够了。
用2mm直径的接种环从在琼脂板上培养的纳豆枯草芽孢杆菌的菌落取样纳豆枯草芽孢杆菌一次。将取样的纳豆枯草芽孢杆菌接种到锥形瓶的液体培养基中。将含有其中接种了纳豆枯草芽孢杆菌的液体培养基的锥形瓶在40℃保持开放,并且旋转和振动两天以在锥形瓶中孵育液体培养基。
在具有30L内部体积的小型发酵罐中制备20L含有100g葡萄糖,400g大豆蛋白粉末和可溶性淀粉的液体培养基,从而灭菌液体培养基。另外,将在锥形瓶中孵育的200ml纳豆枯草芽孢杆菌培养基加入到小型发酵罐中的液体培养基中。孵育得到的培养基,同时在42℃鼓泡和搅拌两天。制备三种另外相同的培养基,以获得约50L的大豆蛋白液体培养基。
从得到的大豆蛋白液体培养基中通过离心去除纳豆枯草芽孢杆菌菌体和杂质,向其加入1mol硫酸铵。加入的硫酸铵通过使得蛋白质变成疏水性质而促进聚集,所述蛋白质包括高分子化合物例如分散在发酵溶液中的各种酶。硫酸铵的加入产生新的不溶性物质。这些物质用玻璃纤维滤纸过滤。
将用1mol硫酸铵溶液平衡的疏水性层析树脂浸渍在过滤得到的大豆蛋白液体培养基中。疏水性高分子化合物聚集(附着)在树脂上。在树脂上的化合物浸渍在0.1mol碳酸氢铵溶液中以洗脱附着在树脂上的高分子化合物,从而获得40L浓缩的液体培养基。
使用具有10,000分子量制备范围的超滤膜超滤浓缩的液体培养基,以分离低分子量物质。将浓缩的液体培养基从40L浓缩到10L。向通过超滤膜获得浓缩物中加入10L纯水以获得20L溶液。将得到的溶液再次进行超滤。通过分离低分子量物质将溶液重复浓缩三次,直至体积变为10L。通过这三次浓缩,可以制备发酵的大豆蛋白液体培养基,其中几乎消除了所有的低分子量物质。
发酵的纳豆独特的气味的主要组分是低分子量化合物,例如二甲基吡嗪(分子量:108.14),三甲基吡嗪(分子量:122.17),四甲基吡嗪(分子量:126.20),2-甲基丁酸酯(分子量:102.13),异戊酸(分子量:102.13),和氨水(分子量:17.03)。包含在纳豆中的维生素K2是具有649分子量的烃化合物。另一方面,蛋白质例如酶(如纳豆激酶)是具有几万或更大分子量的聚合物化合物。
通过超滤去除低分子量物质,可以从发酵的大豆蛋白液体培养基中去除几乎所有低分子量物质例如对发酵纳豆独特“气味”组分和维生素K2。最后,将作为稀释剂的2kg乳糖加入到发酵的大豆蛋白液体培养基中,所述培养基通过超滤去除了低分子量物质,冷冻干燥得到的溶液以制备约2.1kg大豆蛋白发酵粉末(NKCP)。
自从约1990年以来,来自纳豆的用作纤维蛋白溶解物质的纳豆激酶已被报道为枯草杆菌蛋白酶(碱性蛋白酶)。纯化如上制备的NKCP的主要活性物质,并通过各种设备分析检验。分析结果显示主要活性物质是34,000道尔顿杆菌肽酶F,其用作由纳豆枯草芽孢杆菌分泌的酶之一,并且不同于以前的纳豆激酶。
为了设置作为NKCP功能食品的标准,除了称为基质分解活性(stroma decomposition activity)的传统替代指数外,通过制备下述测量试剂盒进行定量分析。在制备34,000道尔顿杆菌肽酶F的抗体的基础上,通过酶联免疫吸附测定(ELISA)制备试剂盒。日本食品研究实验室公司已证实这些分析方法是合适的定量分析方法。
为了开发NKCP作为功能食品,在各种动物实验和人临床测试中检验安全性和功效。关于安全性,该发现满足具有健康、劳动和福利部接受的申明的具体健康应用的食品标准,并且在临床剂量内未观察到临床问题。在各种动物实验和各种人临床测试中均证实预期的溶解纤维蛋白活性的功效。在这些实验和测试过程中,在NKCP中不仅发现先前已报道的枯草杆菌蛋白酶对于血栓的溶解纤维蛋白活性,而且发现抑制血栓形成和降低血液粘度的作用。
以下将描述证实了在含该实施方案的NKCP的抗凝剂中降低血液粘度作用。从不吸烟、预先同意的20-22岁的志愿者收集新鲜血液。收集后,立即将新鲜血液倒入测量容器,其中振荡粘度计保持在37℃。在400-500mPa·S的剪切速率(share rate)下随时间测量临床测试数值和血液的血液粘度值。
将样品溶解在生理盐水中,并将1/100样品加入倒入了新鲜血液的测量容器中。在测量粘度的过程中,快速搅拌样品和新鲜血液。在不刺激凝结/溶解纤维系统的条件下,小心收集血液。处理血液使得可以在振荡粘度计中观察到凝结。
可以在无抗凝剂或稀释剂的条件下,在生理温度下同时保证如同血液在实际人体内流动的物理变化,观察通过上述方法测量的人血的粘度。在将血液放置在测量系统中后,血液粘度立即显示平衡状态。在血液凝结反应基础上,在平衡状态后粘度指数级地增加约300sec。另外的详细信息参见参考文献7,8和9。
在另一个血液粘度测量装置例如MC-FAN(Micro Channel array FlowANalyzer)中,用抗凝剂进行测量以防止血液在装置中凝结。当测量与血液中血细胞(大部分血液红细胞)的粘附和变形相关的血液粘度时就足够了。然而,在包括血液的凝结和纤维蛋白溶解的活体条件下,在测量中振荡粘度计比上述装置更好。
当以0.05mg/mL或更大的量向血液中加入NKCP时,延迟了粘度的指数级增加时间。未证实以0.5mg/mL或更大的量增加粘度。以0.25mg/mL的量降低平衡的粘度约10%,以0.5mg/mL的量降低约20%。加入NKCP的血液的凝结症状程度和表现时间不同于加入作为溶解纤维蛋白试剂的组织纤溶酶原激活物或作为抗凝剂的肝素的血液。
测量摄入NKCP后在临床条件下的血液粘度。有8名预先同意的成人受试者。选择6名另外的受试者作为安慰剂处理的对照组。在选择的受试者中,明显患有急性疾病和慢性疾病的急性恶化的人排除在外。
每日剂量是10个片剂,其等同于1,250mg NKCP。在研究的第一天,在早晨将10个片剂与两个米团一起给药。从第二天,在午餐后,每天给药一次10个片剂,持续1周。在研究的第一天,在给药前、给药后105分钟、180分钟和240分钟测量血液粘度。
在测量容器中,在400-500mPa·s的剪切速率下测量血液粘度,所述容器中以如上所述相同的方式将所有血液样品保持在37℃振荡粘度计中。利用在粘度指数级变化前设置的“平衡粘度”测定血液粘度的变化。如表1和图1所示,在180分钟和240分钟观察到血液粘度的降低,而在安慰剂处理组中未观察到血液粘度的变化。
表1
给药NKCP后随时间的变化参数
给药前 | 45分钟 | 105分钟 | 180分钟 | 240分钟 | |
KCP(n=8) | 3.93±0.283 | 3.86±0.243 | 3.80±0.193 | 3.79±0.244 | 3.89±0.205 |
安慰剂(n=6) | 4.10±0.444 | 4.11±0.528 | 4.00±0.513 | 4.09±0.298 | 4.02±0.359 |
数值是8名志愿者的平均±SD。给药前的差异,邓肯氏多重检验(Duncan’sMultiple Test),*:p<0.05,**:p<001。
VIS:在32℃的血液粘度(mPa·s),Ht:血细胞比容(39.8-51.8%)。
使用振荡粘度计测量添加了NKCP的血液粘度,添加了另一种纳豆激酶的血液粘度和无添加剂的血液粘度。确定了图2中的变化。在图2中,添加了NKCP的血液测量结果,添加了另一种纳豆激酶的血液测量结果,和无添加剂的血液测量结果分别表示为圆点,黑方块和黑三角。表示为圆点的添加了NKCP的血液测量结果显示粘度增加少于具有另一种纳豆激酶的血液。因此,在纳豆中包含的酶和蛋白质的情况下基本上未观察到血液粘度的变化。
关于血液粘度测量,参见Masahito Hitosugi等.,“Rheologic Changes inVenous Blood During Prolonged Sitting”,Thrombosis Research 100,409-412页,2000(参考文献7),Masahito Hitosugi等.,“Change in BloodViscosity by Heparin and Argatroban’,Thrombosis Research 104,371-374页,2001(参考文献8),和Masahito Hitosugi,等.,“Change in BloodViscosity with Synthetic Protease Inhibitors”,Journal of PharmacologicalSciences,卷91,334-336页,2003(参考文献9)。
如上所述,依照含有该实施方案的NKCP的降低血液粘度试剂,血液粘度明显降低。预期降低血液粘度作用有助于改善人类的卫生保健和幸福,例如预防用于诱导血栓形成的止血,改善在外周组织例如肌肉中关于增加供给氧和营养物质的活性能力,改善伴随降低外周血管抗性的高血压,预防动脉硬化发展和改善由降低外周血管抗性导致的心力衰竭的病症。
降低血液粘度试剂具有预防血液粘度增加和收集血液后的凝结、改善临床测试精度和便利性的性能,所述凝结导致在利用取样血液的临床测试中的难题。还预期降低血液粘度试剂改善由于抑制在临床检查和处理中使用的工具的血液污染中粘度增加的清洁作用。降低血液粘度试剂可有助于经历治疗和外科手术的组织、和从活体清除的组织及从其中收集的血液的维持和保留。
在下文描述依照该实施方案用作降低血液粘度试剂的NKCP的物化性质的研究结果。通过“16S核糖体DNA的核苷酸序列方法”分析用作产生NKCP的菌株的纳豆枯草芽孢杆菌(Takahashi菌株)的基因序列,其显示与168枯草芽孢杆菌亚种,杆菌菌株99.9%或更高的同源性。
通过TOF-MS(飞行时间质谱,Time Of Flight Mass Spectrometry)测量NKCP的分子量为34,134道尔顿。NKCP的分子量为约34,100道尔顿。为了进行该测量,使用分子筛载体如SuperQ-Toyopeal 650M,Butyl-Toyopearl650M,或Toyopearl HW-40F,通过重力层析重复纯化NKCP样品。在随后的测量中使用以如上所述相同方式纯化的样品。
利用Edman降解法通过自动氨基酸序列分析仪分析NKCP结构,从氨基末端分析85氨基酸序列。当使用赖氨酰基内肽酶有限降解NKCP时获得几个肽片段。利用高压液相色谱纯化这些肽片段。对于纯化的肽片段,利用TOF-MF进行质量分析和利用Edman降解法进行自动氨基酸残基序列分析。获得的NKCP结构如下。
证实NKCP从氨基末端具有结构氨基酸序列(肽片段)例如“ATDGVEWNVDQIDAPKAWALGYDGTGTVVASIDTGVEWNHPALKEKYRGYNPENPNEPENEMNWYDAVAGEASPYDDLAHGTHVT”,“AFSEDGGTDADILEAGEWVL”,“DAEGNPHPEMAPDV”和“VPGQAYEDGWD”。换言之,NKCP是源自纳豆枯草芽孢杆菌从氨基末端具有上述氨基酸序列的蛋白质。在上述序列中,A表示丙氨酸;G-甘氨酸;M-甲硫氨酸;S-丝氨酸;C-半胱氨酸;H-组氨酸;N-天冬酰胺;T-苏氨酸;D-天冬氨酸;I-异亮氨酸;P-脯氨酸;V-缬氨酸;E-谷氨酸;K-赖氨酸;Q-谷氨酰胺;W-色氨酸;F-苯丙氨酸;L-亮氨酸;R-精氨酸和Y-酪氨酸。
从质量分析结果和分子结构的分子量的计算值的综合,Xu-Chu,Wu等“Cloning,Genetic Organization,and Characterization of a Structural GeneEncoding Bacillopeptidase F from Bacillus Subtilis*”,The Journal ofBiological Chemistry卷265,No.12,6845-6850页,1990(参考文献10)显示的枯草芽孢杆菌的基因数据库的综合,和氨基酸序列的易生物切割能力的测定结果,NKCP具有结构序列“ATDGVEWNVDQIDAPKAWALGYDGTGTVVASIDTGVEWNHPALKEKYRGYNPENPNEPENEMNWYDAVAGEASPYDDLAHGTHVTGTMVGSEPDGTNQIGVAPGAKWIAVKAFSEDGGTDADIIEAGEWVLAPKDAEGNPHPEMAPDVVNNSWGGGSGLDEWYRDMVNAWRAADIFPEFSAGNTDLFIPGGPGSIANPANYPESFATVATDINKKLADFSLQGPSPYDEIKPEISAPGVNIRSSVPGQAYEDGWDFTSMAGPHVSAVAALLKQANASLSVDEMEDILTSTAEPLTDSTFPDSPNNGYGHGLVNAFDAVSAVTDGLGK”。
证实NKCP具有丝氨酸蛋白酶活性中心作为同源序列,其包括天冬酰胺残基,组氨酸残基,和丝氨酸残基。这些由用作枯草芽孢杆菌的胞外蛋白酶的杆菌肽酶F证实,如Alan Sloma,等.,“Cloning and Characterization ofthe Gene for an Additional Extracellular Serine Protease of Bacillus Subtilis”,Journal of Bacteriology,卷173,No.21,6889-6895页,Vov.1991(参考文献11)所述。这证实NKCP是一种丝氨酸蛋白酶。
将描述日本发酵传统食品-纳豆的常规生理活性。纳豆比大豆具有更高的消化吸收性。这是因为作为大豆的主要组分的蛋白质,脂肪和淀粉可被纳豆枯草芽孢杆菌分解成氨基酸,脂肪酸和葡萄糖。纳豆在摄入后具有更高的消化吸收效率。大多数包含在纳豆中的维生素B家族平缓身体功能并且恢复疲劳。维生素B2大量包含在纳豆中,并且对于视力疲劳是有效的。纳豆中维生素K2的含量是在食品中最高的。这可预期血液中钙代谢的改善例如改善骨质疏松症和血液凝结。
在纳豆枯草芽孢杆菌降解大豆蛋白的过程中,分泌各种酶例如蛋白酶和淀粉酶,并用作活性酶试剂以改善消化和活化代谢。纳豆激酶可溶解血栓。
将考虑纳豆激酶和NKCP之间作为酶的差异。在纳豆中发现的用作强溶解纤维蛋白酶的纳豆激酶是一种分泌自纳豆枯草芽孢杆菌的丝氨酸蛋白酶,具有20,000道尔顿的分子量。纳豆激酶具有降解血栓和纤溶酶的合成的显色底物的能力。可从纳豆检测除了上述酶之外的具有27,000道尔顿或更高分子量的一些溶解纤维蛋白的酶。
使用生理盐水可容易地从纳豆中提取纳豆激酶,所述纳豆激酶用作具有在纳豆中发现的强溶解纤维蛋白活性的新的溶解纤维蛋白酶。纳豆激酶具有20,000道尔顿的分子量,和8.6的等电点。纳豆激酶不仅分解纤维蛋白,而且分解合成的显色纤溶酶底物(S-2251)。纳豆激酶在中性pH中相对稳定,并且当其被加热到60℃时损失其活性。
在4℃向纳豆中加入10倍生理盐水,搅拌混合物并通过离心分离。得到的上清液中添加80%乙醇以凝胶过滤。浓缩得到的洗脱液,冷冻干燥以制备粗酶级分。制备的粗酶级分具有一些溶解纤维蛋白的酶,所述酶具有尿激酶原激活物活性。在27,000道尔顿或更高分子量发现三个或更多的活性峰。
如上所述,NKCP是获自纳豆枯草芽孢杆菌培养物的溶解纤维蛋白的蛋白的粗略纯化产品。在获自NKCP的Lysim-X条带中具有水解特异性的来源于纳豆枯草芽孢杆菌的蛋白包含预定量的活性蛋白,其分子量和最适pH分别是约34,100道尔顿和9.0。从粗纯化产品中去除维生素K和纳豆枯草芽孢杆菌菌体,所述纳豆枯草芽孢杆菌导致发酵纳豆的独特气味。这具有不同于纳豆激酶的分子量。
以下将描述NKCP对血栓和溶解纤维蛋白系统的作用。证实NKCP具有体外和体内溶解纤维蛋白作用。该作用比可商购的组织纤溶酶原激活物(t-Pa)更适度。NKCP溶解纤维蛋白活性增加的作用机制是由直接的纤维蛋白降解和纤溶酶原激活物抑制因子1(PAI-1)的降解导致的。通过使用振荡粘度计测量还在降解血液粘度中发现NKCP作用机制。
显示了NKCP对组织培养和动物实验的凝结溶解血栓系统作用的检测结果。使用HUVEC细胞和U87-MG细胞,在体外和原位比较了加入NKCP和不加入任何NKCP的纤溶酶原激活物抑制因子1(PAI-1)产物量。48小时时,由HUVEC细胞产生的总PAI-1是700ng/ml,游离的PAI-1增加高达580ng/ml。在0小时和24小时未观察到NKCP关于PAI-1量的影响。
在高NKCP浓度(10μg/ml)时在完全培养基中,对于所有孵育时间,U87-MG细胞的产量受到完全抑制。关于DMEM培养基,PAI-1生产受到完全抑制。将在加入了NKCP的完全培养基和DMEM培养基中PAI-1产物与那些不加入任何NKCP的相比较。在HUVEC细胞中未观察到NKCP的影响,但在U87-MG细胞中观察到低分子量蛋白增加。这表明NKCP的溶解纤维蛋白活性是通过PAI-1降解的纤溶酶原活化功能。
当在收集后立即向血液中添加NKCP时,并使用振荡粘度计测量血液粘度,发现血液粘度被降低。使用无添加的组织纤溶酶原激活物(t-PA)、添加NKCP、和添加肝素作为对照实施例,当进行血液的300sec临床测试时,通过振荡粘度计其显示凝结,发现了通过添加NKCP导致的不是由抗血栓活性和纤溶酶活性引起的溶解纤维蛋白活性。当健康成人摄入250mg-500mg的NKCP持续7天时,观察到血液粘度的降低。
如上所述,依照本发明,由于上述氨基酸序列是获自来源于纳豆枯草芽孢杆菌的蛋白质,通过使用来源于纳豆枯草芽孢杆菌作为源自纳豆的产品提供了一种可以降低血液粘度的新试剂。
Claims (3)
1.一种降低血液粘度的试剂,其特征在于基本上由来源于纳豆枯草芽孢杆菌的蛋白质组成,并且从氨基末端顺序包括第一结构氨基酸序列,第二结构氨基酸序列,第三结构氨基酸序列和第四结构氨基酸序列,从而降低全血的粘度,
所述第一结构氨基酸序列具有丙氨酸,苏氨酸,天冬氨酸,甘氨酸,缬氨酸,谷氨酸,色氨酸,天冬酰胺,缬氨酸,天冬氨酸,谷氨酰胺,异亮氨酸,天冬氨酸,丙氨酸,脯氨酸,赖氨酸,丙氨酸,色氨酸,丙氨酸,亮氨酸,甘氨酸,酪氨酸,天冬氨酸,甘氨酸,苏氨酸,甘氨酸,苏氨酸,缬氨酸,缬氨酸,丙氨酸,丝氨酸,异亮氨酸,天冬氨酸,苏氨酸,甘氨酸,缬氨酸,谷氨酸,色氨酸,天冬酰胺,组氨酸,脯氨酸,丙氨酸,亮氨酸,赖氨酸,谷氨酸,赖氨酸,酪氨酸,精氨酸,甘氨酸,酪氨酸,天冬酰胺,脯氨酸,谷氨酸,天冬酰胺,脯氨酸,天冬酰胺,谷氨酸,脯氨酸,谷氨酸,天冬酰胺,谷氨酸,甲硫氨酸,天冬酰胺,色氨酸,酪氨酸,天冬氨酸,丙氨酸,缬氨酸,丙氨酸,甘氨酸,谷氨酸,丙氨酸,丝氨酸,脯氨酸,酪氨酸,天冬氨酸,天冬氨酸,亮氨酸,丙氨酸,组氨酸,甘氨酸,苏氨酸,组氨酸,缬氨酸,和苏氨酸,
所述第二结构氨基酸序列具有丙氨酸,苯丙氨酸,丝氨酸,谷氨酸,天冬氨酸,甘氨酸,甘氨酸,苏氨酸,天冬氨酸,丙氨酸,天冬氨酸,异亮氨酸,亮氨酸,谷氨酸,丙氨酸,甘氨酸,谷氨酸,色氨酸,缬氨酸,和亮氨酸,
所述第三结构氨基酸序列具有天冬氨酸,丙氨酸,谷氨酸,甘氨酸,天冬酰胺,脯氨酸,组氨酸,脯氨酸,谷氨酸,甲硫氨酸,丙氨酸,脯氨酸,天冬氨酸,和缬氨酸,
所述第四结构氨基酸序列具有缬氨酸,脯氨酸,甘氨酸,谷氨酰胺,丙氨酸,酪氨酸,谷氨酸,天冬氨酸,甘氨酸,色氨酸,和天冬氨酸。
2.依照权利要求1的试剂,其中来源于纳豆枯草芽孢杆菌的蛋白质基本上由大豆蛋白发酵物组成,所述大豆蛋白发酵物通过从含有获自大豆的大豆蛋白的纳豆枯草芽孢杆菌的液体培养基中去除纳豆枯草芽孢杆菌菌体和低分子量组分获得,所述低分子量组分具有比由纳豆枯草芽孢杆菌产生的酶低的分子量。
3.依照权利要求2的试剂,其中所述低分子量组分通过超滤去除。
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CN1857722A (zh) * | 2005-04-30 | 2006-11-08 | 成都地奥九泓制药厂 | 蜡样芽孢杆菌在制备治疗血栓疾病药物中的用途 |
WO2007002572A2 (en) * | 2005-06-24 | 2007-01-04 | N-Zymeceuticals, Inc. | Nattokinase for reducing whole blood viscosity |
SG10201510384UA (en) | 2006-09-13 | 2016-01-28 | Abbvie Inc | Cell culture improvements |
US8911964B2 (en) | 2006-09-13 | 2014-12-16 | Abbvie Inc. | Fed-batch method of making human anti-TNF-alpha antibody |
CA2738499A1 (en) | 2008-10-20 | 2010-04-29 | Abbott Laboratories | Viral inactivation during purification of antibodies |
AU2009347206C1 (en) | 2008-10-20 | 2016-12-08 | Abbvie Inc. | Isolation and purification of antibodies using Protein A affinity chromatography |
CN109666603B (zh) * | 2018-12-19 | 2021-11-26 | 武汉科诺生物科技股份有限公司 | 一种生产低黏度多粘类芽孢杆菌发酵液的方法 |
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JPS61162184A (ja) | 1985-01-10 | 1986-07-22 | Noriyoshi Morimoto | 新規な線溶酵素およびその取得法 |
JP2688603B2 (ja) | 1988-01-13 | 1997-12-10 | 日本ケミカルリサーチ株式会社 | 血栓溶解剤 |
JPH0813271B2 (ja) | 1989-11-27 | 1996-02-14 | 日本ケミカルリサーチ株式会社 | 線溶活性蛋白質およびその製造法 |
JPH06153977A (ja) | 1992-11-16 | 1994-06-03 | Japan Energy Corp | 線溶活性を持つ蛋白質の製造法 |
JP3168082B2 (ja) | 1992-12-24 | 2001-05-21 | キヤノン株式会社 | テープローディング機構 |
JPH08208512A (ja) | 1995-02-03 | 1996-08-13 | Nippon Chem Res Kk | 血栓形成阻害剤 |
US6395889B1 (en) * | 1999-09-09 | 2002-05-28 | Millennium Pharmaceuticals, Inc. | Nucleic acid molecules encoding human protease homologs |
AUPQ387599A0 (en) * | 1999-11-05 | 1999-12-02 | Metabolic Pharmaceuticals Limited | Product and method for control of obesity |
US6866851B1 (en) * | 1999-12-28 | 2005-03-15 | Washington University | GFRα1-RET specific agonists and methods therefor |
JP3881494B2 (ja) * | 2000-04-21 | 2007-02-14 | 株式会社日本生物科学研究所 | 納豆菌培養エキス |
JP3532503B2 (ja) | 2000-06-14 | 2004-05-31 | 大和薬品株式会社 | 加工食品および食品加工方法 |
TWI305778B (en) * | 2001-12-03 | 2009-02-01 | Nishida Teruo | Peptides of an expressing unit for igf-i minimal activity and their pharmaceutical use |
CN1451745A (zh) * | 2002-04-15 | 2003-10-29 | 黑龙江省科学院应用微生物研究所 | 利用枯草芽孢杆菌生产的纳豆激酶及其生产方法和用途 |
US20040043014A1 (en) * | 2002-08-30 | 2004-03-04 | Hiroyoshi Moriyama | Platelet aggregation inhibitor and supplement food effective for inhibiting platelet aggregation |
US7795385B2 (en) * | 2004-12-17 | 2010-09-14 | Bexar Global, Inc. | Use of bombesin/gastrin-releasing peptide antagonists for the treatment of inflammatory conditions, acute lung injury and bipolar disorder |
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2004
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Publication number | Publication date |
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ATE446360T1 (de) | 2009-11-15 |
EP1657304B1 (en) | 2009-10-21 |
CN1775284B (zh) | 2011-01-26 |
JP2006143601A (ja) | 2006-06-08 |
US20090202982A1 (en) | 2009-08-13 |
HK1088849A1 (en) | 2006-11-17 |
KR100709300B1 (ko) | 2007-04-20 |
US20060105071A1 (en) | 2006-05-18 |
US7972835B2 (en) | 2011-07-05 |
CA2527154A1 (en) | 2006-05-16 |
EP1657304A1 (en) | 2006-05-17 |
KR20060055302A (ko) | 2006-05-23 |
TWI284537B (en) | 2007-08-01 |
DE602005017235D1 (de) | 2009-12-03 |
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