CN111100195B - buforin衍生物及其用途 - Google Patents
buforin衍生物及其用途 Download PDFInfo
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- CN111100195B CN111100195B CN201911026170.6A CN201911026170A CN111100195B CN 111100195 B CN111100195 B CN 111100195B CN 201911026170 A CN201911026170 A CN 201911026170A CN 111100195 B CN111100195 B CN 111100195B
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- Prior art keywords
- arg
- gly
- buforin
- lys
- leu
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Abstract
本发明涉及buforin衍生物及其的用途。更具体地，涉及buforin，以及包含该buforin作为有效成分的用于预防或治疗神经性疾病的药物组合物及用于预防或改善皮肤皱纹的组合物。根据本发明的buforin衍生物可通过有效地抑制SNARE复合体的形成，进而抑制膜融合，从而有效地抑制神经递质的释放。因此，含有作为有效成分的buforin衍生物的药物组合物可有效地用于治疗皮肤皱纹、多汗症、各种疼痛性疾病、过敏、颅神经疾病等因神经递质释放过多引起的神经性疾病。
Description
技术领域
本发明涉及buforin衍生物及其用途。更具体地，本发明涉及buforin，以及包含该buforin作为有效成分的用于预防或治疗神经性疾病的药物组合物及用于预防或改善皮肤皱纹的化妆品组合物。
背景技术
SNARE复合体为蛋白复合体，在酵母中由24个或以上的SNARE蛋白组成，而在哺乳类中由60个或以上的SNARE蛋白组成，在介导突触小泡(synaptic vesicle)融合中起关键作用。为了释放神经递质，位于神经末梢的突触小泡与突触前膜(presynaptic membrane)之间的膜需要进行融合。这种调节膜融合的SNARE蛋白通过形成SNARE复合体，以使膜融合，从而释放出神经递质。SNARE复合体的形成在如神经递质的释放等的细胞外排泄中起重要作用。
肉毒杆菌毒素是通过特异性地作用于SNARE蛋白来抑制SNARE复合体形成的代表性物质，被用于通过抑制SNARE复合物的形成来阻断神经传递(美国专利公开号2016-0151466)。具体地，肉毒杆菌毒素B(BoNT/B)通过不可逆地分解syb2(synaptobrevin-2，小突触小泡蛋白-2)来阻断神经传递。由此，可以抑制肌肉运动、交感神经系统或副交感神经系统，从而不仅被用于去除皱纹，还被有效地用于例如皮肤疼痛、肌肉疼痛、背部疼痛、慢性头痛等的各种疼痛疾病，并且还被广泛用于治疗例如多汗症、毛孔扩张症、过敏、颅神经疾病、斜视、胃酸反流疾病等的多种疾病。
但是，肉毒杆菌毒素是一种剧毒物质，具有很高的副作用风险，在实际使用药物时需要格外注意。因此，需要开发一种在有效抑制SNARE复合体的形成的同时，副作用低的新物质。
发明内容
技术问题
本发明的发明人通过确认buforin衍生物能有效地抑制SNARE复合体的形成来抑制与突触小泡间的的膜融合，最终完成了本发明。
技术方案
为了实现上述目的，在一个方面，本发明提供一种用于预防或治疗神经性疾病的药物组合物，其含有作为有效成分的由以下式(I)表示的buforin衍生物：
N端扩展结构域-第一核心结构域-C端扩展结构域(式I)。
在另一个方面，本发明提供一种用于预防或改善皮肤皱纹的化妆品组合物，其含有作为有效成分的由所述式(I)表示的buforin衍生物。
在又一个方面，本发明提供由以下式(II)表示的buforin衍生物：
N端扩展结构域-第二核心结构域-C端扩展结构域(式II)。
有益效果
根据本发明的buforin衍生物可通过有效地抑制SNARE复合体的形成，来抑制膜融合，从而有效地抑制神经递质的释放。因此，含有作为有效成分的buforin衍生物的药物组合物可有效地用于治疗皮肤皱纹、多汗症、各种疼痛性疾病、过敏、颅神经疾病等因神经递质释放过多引起的神经性疾病。
附图说明
图1示出了用于确认buforin-1和SNARE蛋白是否结合的蛋白质拉下实验(pull-downassay)示意图。
图2示出了对buforin-1和SNARE蛋白是否结合的确认结果。
图3示出了用于确认小鼠的SNARE蛋白之间是否结合的蛋白质拉下实验示意图。
图4示出了对buforin-1和小鼠脑组织内的SNARE蛋白是否结合的确认结果。
图5示出了buforin-1对SNARE复合体形成的各阶段所起的影响。
图6示出了buforin-1对SNARE复合体形成的抑制结果。
图7示出了对buforin-1、buforin-2及parasin对SNARE复合体形成的抑制效果的比较。
图8示出了利用酵母液泡和SNARE蛋白进行的膜融合实验示意图。
图9示出了buforin-1对膜融合的抑制效果。
图10示出了在PC-12细胞和HEK-293细胞中buforin-1的分布位置与SNAP-25相同。
图11示出了在PC-12细胞中buforin-1的分布位置与Stx1的分布位置相同。
图12示出了在PC-12细胞中buforin-1对CgA-EAP释放的抑制效果。
图13示出了H2A在PC-12细胞和HEK-293细胞中的分布。
图14示出了用H2A处理小鼠脑组织匀浆(homogenates)后，利用抗组蛋白H2A抗体确认与H2A结合的SNARE蛋白，以确认SNARE蛋白与H2A是否结合。
图15示出了buforin-1及人buforin-1样肽(buforin-1-like human peptide)对SNARE复合体形成的抑制效果。
图16示出了buforin-1及人buforin-1样肽对神经性SNARE-介导膜融合的抑制效果。
图17示出了各种buforin衍生物对SNARE复合体形成的抑制效果的比较。
优选的实施方式
在一个方面，本发明提供一种用于预防或治疗神经性疾病的药物组合物，其含有作为有效成分的由以下式(I)表示的buforin衍生物：
N端扩展结构域-第一核心结构域-C端扩展结构域(式I)。
在所述式(I)中，第一核心结构域为具有SEQ ID NO.1所示的氨基酸序列的多肽；N端扩展结构域为具有SEQ ID No.2所示的氨基酸序列的多肽、或为SEQ ID NO.2的连续缺失了从其第1位氨基酸起沿N端至C端方向的1至15个氨基酸的多肽；C端扩展结构域为具有SEQID NO.3所示的氨基酸序列的多肽、或为SEQ ID NO.3的连续缺失了从其第4位氨基酸起沿C端至N端方向的1至3个氨基酸的多肽。
在本说明书中，术语“第一核心结构域”是指具有buforin-1蛋白的第17至35位氨基酸序列的多肽。全长buforin-1蛋白可以由SEQ ID NO.116所示的氨基酸序列组成。此外，所述第一核心结构域可以由SEQ ID NO.1所示的氨基酸序列组成。
所述N端扩展结构域作为与上述核心结构域的N端相结合的结构域，其可为具有SEQ IDNO.2所示的氨基酸序列的多肽，或为从所述多肽的第1位氨基酸起由C端至N端的方向缺失1、2、3、4、5、6、7、8、9、10、11、12、13、14或15个氨基酸残基的多肽。
在本发明的一个实施例中，所述N端扩展结构域为具有SEQ ID NO.2所示的氨基酸序列的多肽，或为SEQ ID NO.2的缺失了从其第1位氨基酸起沿N端至C端方向的5、10或15个氨基酸残基的多肽。
所述SEQ ID No.2表示为Xaa1 Gly Arg Gly Lys Gln Gly Gly Lys Xaa2 ArgAla Lys Ala Ly sThr，其中Xaa1或Xaa2可分别独立为Se、Ala及Val中的任意一个。在一个实施例中，当所述SEQ ID NO.2的Xaa1为Ala，Xaa2为Val时，可被表示为Ala Gly Arg GlyLys Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr，在又一个实施例中，当所述SEQ IDNO.2的Xaa1为Ser，Xaa2为Ala时，可被表示为Ser Gly Arg Gly Lys Gln Gly Gly Lys AlaArg Ala Lys Ala Lys Thr。
所述C端扩展结构域作为与上述核心结构域的C端相结合的结构域，其可为具有SEQ ID NO.3所示的氨基酸序列的多肽，或为从所述多肽的第4位氨基酸起由C端至N端的方向缺失1个、2个或3个氨基酸残基的多肽。
所述氨基酸残基的缺失可发生在N端扩展结构域和C端扩展结构域中的任意一个结构域中，或者在两端的结构域中均有发生。
所述N端扩展结构域为SEQ ID NO.2的连续缺失了从其第1位氨基酸起沿N端至C端方向的5个、10个或15个氨基酸的多肽，所述C端扩展结构域为SEQ ID NO.3的连续缺失了从其第4位氨基酸起沿C端至N端方向的2个、或3个氨基酸的多肽。
在本发明的一个实施例中，所述buforin衍生物可为具有SEQ ID NO.4至SEQ IDNO.67的氨基酸序列中的任意一个氨基酸序列的buforin衍生物。
在本说明书中，术语“buforin”是指在中华大蟾蜍(Bufo gargarizans)的组蛋白H2A的蛋白水解过程中所生成的源自组蛋白H2A的抗菌肽，其包括buforin-1、buforin-2、parasin等。
由于buforin-1在结构上与Syb2相似，它可以代替Syb2与Stx1及SNAP-25进行结合，来抑制SNARE复合体的形成。肉毒杆菌毒素B(BoNT/B)是一种能切断SNARE蛋白的蛋白酶，其中SNARE蛋白为参与神经递质释放的核心蛋白。肉毒杆菌毒素B通过不可逆地切断SNARE蛋白，尤其是syb2，来抑制神经传递。反之，buforin-1是通过抑制SNARE复合体的形成来抑制神经传递的竞争性抑制剂(competitive inhibitor)。因此，具有与stx1和/或SNAP-25结合的能力的上述式(I)的buforin衍生物可抑制SNARE复合体的形成。因此，上述式(I)的buforin衍生物可有效地用于治疗与SNARE蛋白相关的疾病。
所述组蛋白H2A属于组蛋白的一个分类，所述组蛋白被分为包括H2A在内的五个类，分别为H1、H2A、H2B、H3以及H4。组蛋白的主要功能是调节细胞核中的基因表达。
在本发明的一个实施例中，作为所述药物组合物的有效成分的buforin衍生物，可通过代替v-SNARE蛋白与t-SNARE蛋白进行结合，来抑制SNARE复合体的形成。
在本说明书中，术语“SNARE蛋白”是指形成参与细胞膜中发生的膜融合的巨大SNARE(可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体)复合体的蛋白质。SNARE蛋白通过所形成的SNARE复合体使突触小泡和突触前膜之间的膜进行融合，由此膜融合开启神经递质的释放通道。当进行所述膜融合时，会发生脂质双层的重排，这些膜并非自发融合，而是需要有强大的外力来克服膜之间的排斥力。此时，足以克服膜之间的排斥力的强力就是由SNARE蛋白产生。亦即，在膜融合时，附着于目标膜(target membrane)上的作为Stx1和SNAP-25复合体的t-SNARE，以及附着于小泡上的作为v-SNARE的syb2的相互参与而形成SNARE复合体，并且仅通过此方式才能使得膜进行融合。它们具有像麻花一样的相互扭曲缠绕的结构，并且当所述SNARE蛋白的接合和缠绕过程未被完全执行时，将无法实现膜融合，从而不会发生神经递质的释放。
在本发明的一个实施例中，所述v-SNARE蛋白优选为syb1及syb2，最优选为syb2，但不限于此。
在本发明的一个实施例中，所述t-SNARE蛋白可优选为选自Stx1、Stx2、Stx3、Stx4、Stx5、Stx6、Stx7、Stx8、Stx10、Stx11、Stx12、Stx16、Stx17、Stx18、Stx19及SNAP-25中的一种或多种，最优选为选自Stx1及SNAP-25中的一种或多种，但不限于此。
在本发明的一个实施例中，作为所述药物组合物的有效成分的buforin衍生物可通过抑制SNARE复合体的形成来控制神经递质的释放。
在本发明的一个实施例中，作为所述药物组合物的有效成分的buforin衍生物可通过抑制SNARE复合体的形成，来抑制突触小泡和突触前膜之间的液泡膜融合。在本说明书中，术语“液泡”是指由与细胞壁、细胞含有物同属于后成质的膜包围的巨大的细胞器。
所述神经性疾病可以为选自斜视、颈肌张力障碍、强直性眼睑痉挛、多汗症、毛孔扩张症、肛裂、阴道痉挛、失弛缓症、皮肤疼痛、肌肉痛、腰痛、慢性头痛、颞下颌关节紊乱症、特发性及神经逼尿肌增生、局灶性肌张力障碍、颞下颌关节紊乱症、糖尿病性神经病、颅神经疾病、胃酸反流疾病、声带功能障碍、过敏性疾病、自身免疫性疾病、慢性神经病、面肌肥大症及其组合中的任意一种疾病。
具体地，韩国专利公开号第10-1803201号公开了，当抑制SNARE复合体形成时，通过抑制神经递质释放，可表现出预防或治疗多汗症的效果，韩国专利公开号第10-1729138号公开了，当抑制SNARE复合体形成时，通过阻碍突触小泡和突触前膜之间的膜融合，可表现出毛孔收缩的效果。此外，韩国专利公开号第10-0901074号公开了，当抑制SNARE复合体形成时，通过阻碍神经递质的释放，可缓解皮肤疼痛。本发明的药物组合物可有效地抑制SNARE复合体的形成，因此可以有效地用于预防或治疗如上所述的神经性疾病。
在另一个方面，本发明提供一种用于预防或改善皮肤皱纹的化妆品组合物，其含有作为有效成分的由下式(I)表示的buforin衍生物：
N端扩展结构域-第一核心结构域-C端扩展结构域(式I)。
在所述式(I)中，
第一核心结构域为具有SEQ ID NO.1所示的氨基酸序列的多肽；
N端扩展结构域为具有SEQ ID NO.2所示的氨基酸序列的多肽，或为SEQ ID NO.2的连续缺失了从其第1位氨基酸起沿N端至C端方向的1至15个氨基酸的多肽；
C端扩展结构域为具有SEQ ID NO.3所示的氨基酸序列的多肽，或为SEQ ID NO.3的连续缺失了从其第4位氨基酸起沿C端至N端方向的1至3个氨基酸的多肽。
由所述式(I)表示的buforin衍生物被包含在药物组合物中时，也与上述的内容相同。
现有技术(韩国专利公开号第10-1736775号，韩国专利公开号第10-1673226号，韩国专利公开号第10-1997474号，韩国专利公开号第10-1650020号)公开了，当抑制SNARE复合体形成是，通过阻碍神经递质的释放，可表现出改善皮肤皱纹的效果。
根据本发明的buforin衍生物，是通过有效地抑制SNARE复合体的形成，来抑制液泡的膜融合，从而可有效地抑制神经递质的释放，因此，含有作为有效成分的本发明的buforin衍生物的化妆品组合物通过这种对SNARE复合体的形成的抑制能力，可用作SNARE靶向前药，并且可有效地用于预防和治疗由神经递质释放过多所导致的皮肤皱纹。
在又一个方面，本发明提供由以下式(I)表示的buforin衍生物在预防或治疗神经性疾病中的用途：
N端扩展结构域-第一核心结构域-C端扩展结构域(式I)。
在所述式(I)中，
第一核心结构域为具有SEQ ID NO.1所示的氨基酸序列的多肽；
N端扩展结构域为具有SEQ ID NO.2所示的氨基酸序列的多肽，或为SEQ ID NO.2的连续缺失了从其第1位氨基酸起沿N端至C端方向的1至15个氨基酸的多肽；
C端扩展结构域为具有SEQ ID NO.3所示的氨基酸序列的多肽，或为SEQ ID NO.3的连续缺失了从其第4位氨基酸起沿C端至N端方向的1至3个氨基酸的多肽。
由所述式(I)表示的buforin衍生物与上述的内容相同。
在又一个方面，本发明提供由以下式(I)表示的buforin衍生物在预防或改善皮肤皱纹中的用途：
N端扩展结构域-第一核心结构域-C端扩展结构域(式I)。
在所述式(I)中，
第一核心结构域为具有SEQ ID NO.1所示的氨基酸序列的多肽；
N端扩展结构域为具有SEQ ID NO.2所示的氨基酸序列的多肽，或为SEQ ID NO.2的连续缺失了从其第1位氨基酸起沿N端至C端方向的1至15个氨基酸的多肽；
C端扩展结构域为具有SEQ ID NO.3所示的氨基酸序列的多肽，或为SEQ ID NO.3的连续缺失了从其第4位氨基酸起沿C端至N端方向的1至3个氨基酸的多肽。
由所述式(I)表示的buforin衍生物与上述的内容相同。
在又一个方面，本发明提供由以下式(II)表示的buforin衍生物。
N端扩展结构域-第二核心结构域-C端扩展结构域(式II)
在所述式(II)中，第二核心结构域为具有SEQ ID NO.68所示的氨基酸序列的多肽；N端扩展结构域为具有SEQ ID No.2所示的氨基酸序列的多肽、或为SEQ ID NO.2的连续缺失了从其第1位氨基酸起沿N端至C端方向的1至15个氨基酸的多肽；C端扩展结构域为具有SEQ ID NO.69所示的氨基酸序列的多肽、或为SEQ ID NO.69的连续缺失了从其第3位氨基酸起沿C端至N端方向的1至2个氨基酸的多肽。
在本说明书中，术语“第二核心结构域”是指具有buforin-1蛋白的第17至36位所示的氨基酸序列的多肽。所述全长buforin-1蛋白可以由SEQ ID NO.116的氨基酸序列组成，所述第二核心结构域可以由SEQ ID NO.68所示的氨基酸序列组成。
所述N端扩展结构域作为与上述核心结构域的N端结合的结构域，其可为具有SEQID NO.2所示的氨基酸序列的多肽，或为从所述多肽的第1位氨基酸起由C端至N端的方向缺失1、2、3、4、5、6、7、8、9、10、11、12、13、14或15个氨基酸残基的多肽。
在本发明的一个实施例中，所述N端扩展结构域为具有SEQ ID NO.2所示的氨基酸序列的多肽，或为SEQ ID NO.2的缺失了从其第1位氨基酸起沿N端至C端方向的5、10或15个氨基酸残基的多肽。
所述SEQ ID No.2表示为Xaa1 Gly Arg Gly Lys Gln Gly Gly Lys Xaa2 ArgAla Lys Ala Lys Thr，其中Xaa1或Xaa2可分别独立为Se、Ala及Val中的任意一个。在一个实施例中，当所述SEQ ID NO.2的Xaa1为Ala、Xaa2为Val时，可表示为Ala Gly Arg Gly LysGln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr，在另一个实施例中，当所述SEQ IDNO.2的Xaa1为Ser、Xaa2为Ala时，可表示为Ser Gly Arg Gly Lys Gln Gly Gly Lys AlaArg Ala Lys Ala Lys Thr。
所述C端扩展结构域作为与上述核心结构域的C端相结合的结构域，其可为具有SEQ ID NO.69所示的氨基酸序列的多肽，或为从所述多肽的第3位的氨基酸开始，从C端向N端的方向缺失1个或2个氨基酸残基的多肽。
所述氨基酸残基的缺失可发生在N端扩展结构域和C端扩展结构域中的一个结构域中，或者在两端的结构域中均有发生。
在本发明的一个实施例中，所述buforin衍生物可具有选自SEQ ID NO.70至SEQID NO.114所示的氨基酸序列中的任意一个氨基酸序列。
所述buforin衍生物的特征可在于不具有选自SEQ ID NO.115至SEQ ID NO.117所示的氨基酸序列中的任意一个氨基酸序列。此外，所述buforin衍生物可具有选自SEQ IDNO.119至SEQ ID NO.122所示的氨基酸序列中的任意一个氨基酸序列。
在又一个方面，本发明提供一种用于预防或治疗神经性疾病的药物组合物，其含有作为有效成分的由下式(II)表示的buforin衍生物。
N端扩展结构域-第二核心结构域-C端扩展结构域(式II)
由所述式(II)表示的buforin衍生物与上述的内容相同。
在另一个方面，本发明提供一种用于预防或改善皮肤皱纹的化妆品组合物，其含有作为有效成分的由下式(II)表示的buforin衍生物。
N端扩展结构域-第二核心结构域-C端扩展结构域(式II)
由所述式(II)表示的buforin衍生物与上述的内容相同。
在又一个方面，本发明提供由下式(II)表示的buforin衍生物在预防或治疗神经性疾病中的用途。
N端扩展结构域-第二核心结构域-C端扩展结构域(式II)
由所述式(II)表示的buforin衍生物与上述的内容相同。
在又一个方面，本发明提供由下式(II)的buforin衍生物在预防或改善皮肤皱纹中的用途。
N端扩展结构域-第二核心结构域-C端扩展结构域(式II)
由所述式(II)表示的buforin衍生物与上述的内容相同。
实施例
以下，将结合以下实施例更详细地描述本发明。但以下实施例仅用于说明本发明，本发明的范围不限于此。
[制备例1]神经性SNARE蛋白的表达及纯化
将编码作为SNARE蛋白的Stx1(氨基酸1-265，SEQ ID NO.123)、SNAP-25(氨基酸1-206，SEQ ID NO.124)和syb2(氨基酸1-96，SEQ ID NO.125)的细胞质结构域的基因分别插入于pHis-parallel载体(Addgene公司)中。将这些载体分别导入到大肠杆菌Rosetta 2(λDE3)(安诺伦公司(Novagen))中。将导入后的大肠杆菌(E.coli)在LB培养基中以37℃的温度条件进行培养。当600nm波长下的光密度(O.D)值达到0.4至0.6时，添加浓度为1mM的IPTG(isopropyl-β-D-thiogalactopyranoside，异丙基-β-D-硫代半乳糖苷)。将所述转化后的大肠杆菌，在22℃的温度下进行12小时的培养后，在5000×g的条件下进行30分钟的离心分离。然后，再悬浮于含有2mM的DDT(Dithiothreitol，二硫苏糖醇)及2mM的苯甲基磺酰氟(phenylmethylsulfonyl fluoride)的PBS中。
然后，用超声均化器(Vibra-Cell,Sonics&Materials)对转化后的大肠杆菌进行破碎。在12000×g的条件下进行30分钟的离心分离后，获得上清液。将获得的上清液用Ni2+-次氮基三乙酸色谱柱(密理博公司，Millipore)进行纯化，然后，用含有250mM的咪唑的PBS进行洗脱，以分离出Stx1(氨基酸1-265)、SNAP-25(氨基酸1-206)及syb2(氨基酸1-96)蛋白。用Amicon离心超滤装置(Millipore，默克密理博公司)将分离出的SNARE蛋白分别更换到含有150mM的NaCl及0.5％的正辛基-d-吡喃葡萄糖苷的20mM的Tris-HCl缓冲液中。通过使用SDS-PAGE和考马斯亮蓝染色法(Coomassie Brilliant Blue staining)确认每种SNARE蛋白的纯度为90％以上。
[实施例1]确认buforin-1和SNARE蛋白结合与否
[实施例1.1]确认buforin-1和重组SNARE蛋白结合与否
通过进行如下所述的实验,以确认buforin-1和SNARE蛋白是否结合。
在此，生物素-buforin-1由安利健(anygen)公司合成，生物素-链霉亲和素试剂盒购自赛默飞世尔公司(Thermos Fisher)。具体地，将生物素-buforin-1固定于链霉亲和素珠粒(bead)上，然后用20μM的在所述制备例1中制备的Stx1、SNAP-25或syb2 SNARE蛋白分别对珠粒进行处理，并在4℃的温度下进行6小时的反应。用Tris-HCl缓冲液进行4次洗涤后，用洗脱缓冲液分离结合在珠粒上的蛋白，然后通过SDS-PAGE及蛋白质印迹来确认蛋白是否结合。
其结果确认了buforin-1与Stx1和SNAP-25结合，但未与syb2结合(图1和图2)。
[实施例1.2]确认buforin-1和小鼠的SNARE蛋白之间是否结合
此外，摘取C57/BL6雄性小鼠(12周龄)的脑组织，在含有150mM的NaCl、2mM的CaCl2、1％的Triton X-100(聚乙二醇辛基苯基醚)、蛋白酶抑制剂混合物(Roche)的20mM的Tris(pH7.8)缓冲液中进行匀浆化处理，然后在4℃的温度下，以13,000×g的条件进行30分钟的离心分离。收集上清液并用于蛋白质下拉实验分析，对洗脱的样品使用SDS-PAGE及抗-Stx1(sc-12736)、抗-SNAP-25(sc-376713)、抗-syb2抗体(sc-69706，SantaCruzBiotechnology)，并通过蛋白质印迹进行确认。所有动物实验均在光州科技学院所属动物伦理委员会的监督和批准下进行(GIST-2018-033)。
其结果确认了buforin-1与SNAP-25结合，但未与syb2结合(图3和图4)。由此可确认，buforin-1代替syb2，与作为神经性t-SNARE蛋白的Stx1及SNAP-25进行相互作用。
[实施例2]确认buforin-1对SNARE复合体形成的各阶段所起的影响
通过进行如下所述的实验，以确认buforin-1对SNARE复合体形成的哪个阶段具有抑制作用。
用浓度分别为0μM、3μM、10μM、30μM及100μM的buforin-1分别对Stx1、t-SNARE复合体及完整的SNARE复合体进行处理。其中，所述t-SNARE复合体由浓度为3μM的Stx1蛋白与浓度为3μM的SNAP-25在室温下进行2小时的反应而形成。然后，t-SNARE复合体与buforin-1在4℃温度下进行2小时的反应(图5(b))，将在t-SNARE复合体中添加有Syb2(3μM)的完整的SNARE复合体与buforin-1在4℃温度下进行一晚的反应(图5(C))。通过使用SDS-PAGE和考马斯亮蓝染色法确认各实验条件下SNARE复合体的形成。此时，通过使用浓度计(GS-900,Bio-Rad公司)对与SNARE复合体相对应的条带的浓度进行定量。
其结果如图5所示，使t-SNARE复合体和buforin-1进行反应时，以及使Stx1和buforin-1进行反应时，SNARE复合体的形成受抑制。相反，完整的SNARE复合体完全不受buforin-1处理的影响。由此可确认，当buforin-1抑制SNARE复合体形成时，相较于抑制形成完整的SNARE复合体时的最后阶段，主要对t-SNARE复合体的形成起到抑制作用，或者对Syb2与t-SNARE复合体的结合起到抑制作用。
[实施例3]buforin-1、buforin-2及parasin的比较
对神经性SNARE复合体形成和神经性SNARE-介导膜融合的抑制效果进行实验，以比较作为源自组蛋白H2A的多肽的buforin-1、buforin-2及parasin。
首先，下表1比较了源自组蛋白H2A的各种多肽的序列相似性，并且用红色和下划线的表示序列差异。
【表1】
[实施例3.1]对神经性SNARE复合体形成的抑制效果的比较
通过进行如下所述的实验，以确认buforin-1、buforin-2及parasin对神经性SNARE复合体形成的抑制效果。将制备例1中制备的Stx1蛋白和SNAP-25蛋白分别处理为3μM后，再用buforin-1、buforin-2或parasin进行处理，然后，在4℃的温度下进行2小时的反应后，添加浓度为3μM的syb2，并在4℃的温度下进行一晚的反应。然后，利用SDS-PAGE样品缓冲液使反应终止。通过使用SDS-PAGE和考马斯亮蓝染色法来确认SNARE复合体的形成。通过使用浓度计(GS-900,Bio-Rad公司)对SNARE复合体分子量75kDa的条带的进行定量。
其结果为，当用buforin-1时，随着buforin-1的浓度的增加，对SNARE复合体形成的抑制效果越大。相反，与buforin-1相比，buforin-2对SNARE复合体形成的抑制效果较低，而parasin对SNARE复合体形成的抑制效果微弱(图6和图7)。由此可确认，与buforin-2及parasin相比，buforin-1可有效地抑制SNARE复合体的形成。
[实施例3.2]对神经性SNARE-介导膜融合的抑制作用的比较
通过进行如下所述的实验，以确认使用buforin-1、buforin-2、parasin的酶液泡、及神经性SNARE复合体的膜融合抑制效果。
具体地，在含有264nM的纯化Pbi2p(IB2)、10μM的CoA、1mM的ATP、1mM/mL的肌酸激酶、29mM的肌酸磷酸的反应缓冲液(125mM的KCl，5mM的MgCl2，10mM的Pipes-KOH(pH值6.8))以及200mM的山梨糖醇中，使包含syb2蛋白但缺少pep4p蛋白及prb1p蛋白的BJ-syb2的液泡(酿酒酵母株BJ3505衍生物，3μg)与包含Stx1蛋白和SNAP-25蛋白但缺少Pho8p蛋白的DKY-Stx1/S25液泡(DKY6281菌株的衍生物，3μg)进行神经性SNARE-介导液泡融合反应(30μl)。在27℃的温度下进行90分钟的反应后，通过测量碱性磷酸酶的活性来确定酵母液泡的融合。通过生成的对硝基苯酯(p-nitrophenylate)的浓度(μM)/分钟/pep4p-保留液泡的质量，来计算出融合的酵母液泡。根据结果证实，buforin-1以浓度依赖的方式抑制SNARE-介导膜融合(图8和图9)。
[实施例4]buforin-1、Stx1及SNAP-25在PC-12细胞和HEK-293细胞中的分布位置的比较
将作为神经细胞模型的源自小鼠的PC-12细胞和源自人类的HEK-293细胞中的buforin-1进行处理后，通过免疫荧光法和共聚焦显微镜来比较buforin-1的分布。
首先，在直径为10mm的盖玻片上，对细胞数为2×105的PC-12细胞或HEK-293细胞进行24小时的培养后，用FITC标记的浓度为20μM的buforin-1进行处理，并进行2小时的反应。然后，用4％的多聚甲醛将PC-12细胞或HEK-293细胞固定10分钟，并在含有0.1％的TritonX-100(聚乙二醇辛基苯基醚)的PBS中进行10分钟的培养。然后，在含有5％BSA的PBS中，对PC-12细胞或HEK-293细胞进行1小时的培养。用抗-SNAP-25抗体(sc-376713)和抗-Stx1抗体(sc-12736)对PC-12细胞进行处理，在进行1小时的反应后，与被Alexa Fluor594染料标记的第二抗体(Invitrogen公司)进行1小时的反应。之后，用温度为4℃的PBS将PC-12细胞或HEK-293细胞洗涤3次。通过配备有100x/NA 1.40油浸物镜(oil-immersionobjective lens(Uplan-SApochromat))的共聚焦显微镜进行观察荧光染色，并使用奥林巴斯Fluoview程序进行分析。
结果，在PC-12细胞中，buforin-1的分布与原生质体膜周围的Stx1蛋白及SNAP-25蛋白的分布位置相同，而在缺乏Stx1蛋白和SNAP-25蛋白的HEK-293细胞中未观察到buforin-1(图10和图11)。由此可确认，buforin-1与Stx1蛋白以及SNAP-25蛋白在器内(体外)发生结合。
[实施例5]buforin-1的CgA-EAP释放抑制效果的确认
通过进行如下所述的实验，以确认buforin-1抑制CgA-EAP释放的效果。首先，使用pCMV-CgA-EAP质粒(赛默飞世尔公司)转化PC-12细胞。16小时后，将培养基替换为DMEM培养基，再进行4小时的培养。然后，用溶解于KREB-HEPES缓冲液的buforin-1对PC-12细胞进行处理并进行90分钟的反应后，用刺激缓冲液(10mM的HEPES(pH值7.4)、150mM的NaCl、5mM的KCl、及2mM的BaCl2)进行处理并进行15分钟的反应。
然后，收集培养液，在4℃温度下以1700×g的条件进行3分钟的离心分离，以获得上清液。将附着的细胞沉淀用0.1％的Triton X-100进行再悬浮后，通过实施冷冻/解冻(freeze/thaw)3次使细胞溶解，然后在4℃温度下以12,000×g的条件进行5分钟的离心分离。使用磷-光化学发光分析试剂盒(Phospha-Light chemiluminescence assay kit，AppliedBiosystems公司)确认磷酸酶活性。通过计算使用微孔板式发光检测仪(CentroXS3 LB 960)在培养液和细胞中测得的亮度(luminance)与培养基中的亮度的相对值，从而得出释放的CgA-EAP量。
其结果证实，当用buforin-1进行处理时，CgA-EAP的释放受到抑制。由此可确认，buforin-1可以抑制神经递质的释放(图12)。
[实施例6]buforin-1及人buforin-1样肽(人类组蛋白H2A的氨基酸1-39)的比较
[实施例6.1]PC-12细胞和HEK-293细胞中分布的比较
通过与实施例5相同的方法，在从韩国细胞株银行(首尔，韩国)获得的源自小鼠的PC-12细胞以及源自人类的HEK-293细胞中用人buforin-1样肽处理后，比较它们的分布。
结果，在PC-12细胞中，人buforin-1样肽的分布与原生质体膜周围的Stx1蛋白及SNAP-25蛋白的分布位置相同，而在缺乏Stx1蛋白和SNAP-25蛋白的HEK-293细胞中未观察到H2A(图13)。由此可确认，与buforin-1类似，人buforin-1样肽也与Stx1蛋白以及SNAP-25蛋白在器内(体外)发生结合。
[实施例6.2]是否与SNARE蛋白结合的确认
通过用人buforin-1样肽(SEQ ID NO.115)对实施例1中匀浆化处理后的小鼠脑组织进行处理后，再使用抗-组蛋白H2A抗体进行免疫共沉淀(Co-immunoprecipitation)处理，以鉴定与人-1样肽结合的SNARE蛋白。
具体地，另外再摘取C57/BL6雄性小鼠(12周龄)的脑组织，在含有150mM的NaCl、2mM的CaCl2、1％的Triton X-100(聚乙二醇辛基苯基醚)、蛋白酶抑制剂混合物(Roche)的20mM的Tris(pH7.8)缓冲液中进行匀浆化处理，然后在4℃的温度下以13,000×g的条件进行30分钟的离心分离。收集上清液后，用200μg的人buforin-1样肽进行处理，然后再添加与G-琼脂糖蛋白结合的抗-组蛋白H2A抗体(ab18975，Abcam公司)，并在4℃的温度下进行一晚的反应。此时，将脑组织匀浆与兔血清进行反应，以作为对照组。将珠粒用含有300mM的NaCl、0.1mM的EDTA、1％的Triton X-100、蛋白酶抑制剂的缓冲液洗涤3次。通过与1×SDS样品缓冲液一起加热10分钟获得与抗体结合的蛋白质。然后，通过使用SDS-PAGE及蛋白质印迹对蛋白质进行分离和分析。
其结果证实，Stx1蛋白及SNAP-25蛋白与人buforin-1样肽结合，但未与Syb2结合(图14)。由此可确认，人buforin-1样肽和/或其衍生物可以代替syb2与神经性t-SNARE蛋白发生相互作用。
[实施例6.3]对神经性SNARE复合体的形成以及神经性SNARE-介导膜融合的抑制效果的比较
通过与实施例3及4相同的方法，比较buforin-1及人buforin-1样肽(人类组蛋白H2A的氨基酸1-39)对神经性SNARE复合体的形成的抑制效果以及对神经性SNARE-介导膜融合的抑制作用。
其结果证实，人buforin-1样肽可有效地抑制神经性SNARE复合体的形成，并抑制神经性SNARE-介导的膜融合。由此可确认，与buforin-1类似，人buforin-1样肽也表现出对神经SNARE复合体形成的抑制效果以及对神经性SNARE-介导膜融合的抑制效果(图15和图16)。
[制备例2]buforin衍生物的制备
将编码下表2中所示的buforin-1-1、buforin-1-2、buforin-1-3和buforin-1-4的氨基酸序列的基因分别插入pHis-parallel载体(Addgene公司)。然后，通过与制备例1相同的方法，分离buforin-1-1、buforin-1-2、buforin-1-3和buforin-1-4。
【表2】
通过用Amicon离心超滤装置(Millipore，默克密理博公司)，将分离出的buforin-1-1、buforin-1-2、buforin-1-3和buforin-1-4分别更换到含有150mM的NaCl及0.5％的正辛基-d-吡喃葡萄糖苷的20mM的Tris-HCl缓冲液中。通过使用SDS-PAGE和考马斯亮蓝染色法(Coomassie Brilliant Blue staining)来确认每种SNARE蛋白的纯度为90％以上。
[实施例7]buforin衍生物对神经性SNARE复合体形成的抑制效果的比较
通过进行如下所述的实验，以比较所述制备例2中制备的buforin-1-1、buforin-1-2、buforin-1-3和buforin-1-4对神经性SNARE复合体形成的抑制效果。
通过用制备例1中制备的Stx1蛋白和SNAP-25蛋白分别处理为3μM后，再用parasin、buforin-1、buforin-1-1、buforin-1-2、buforin-1-3、buforin-1-4以及buforin-2分别进行处理，然后，在4℃的温度下进行2小时的反应，再添加浓度为3μM的syb2，并在4℃的温度下进行一晚的反应。然后，通过使用SDS-PAGE样品缓冲液使反应终止。通过使用SDS-PAGE和考马斯亮蓝染色法来确认SNARE复合体的形成。通过使用浓度计(GS-900,Bio-Rad公司)对分子量为75kDa的SNARE复合体的条带的进行定量。
其结果证实，除了parasin以外，buforin-1、buforin-1-1、buforin-1-2、buforin-1-3、buforin-1-4以及buforin-2均抑制SNARE复合体的形成(图17)。
统计学分析
所有数值均表示为平均值±标准偏差。使用西格玛图(Sigma plot)的双尾无配对学生t检验(two-tailed unpaired student's t-test)分析两组之间的比较。统计学显著性将用星号标记(*p<0.05、**p<0.01、***p<0.005)。
<110> 安尼根有限公司
<120> buforin衍生物及其用途
<130> PCB909079ANY
<150> KR 10-2018-0128038
<151> 2018-10-25
<160> 125
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Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly
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Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val
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Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly
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Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg
1 5 10 15
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Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val
1 5 10 15
His Arg Leu Leu Arg Lys Gly
20
<210> 35
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 35
Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
1 5 10 15
Arg Leu Leu Arg Lys Gly
20
<210> 36
<211> 38
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 36
Xaa Gly Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg
20 25 30
Leu Leu Arg Lys Gly Asn
35
<210> 37
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 37
Gly Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg
1 5 10 15
Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu
20 25 30
Leu Arg Lys Gly Asn
35
<210> 38
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 38
Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser
1 5 10 15
Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu
20 25 30
Arg Lys Gly Asn
35
<210> 39
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 39
Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser
1 5 10 15
Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg
20 25 30
Lys Gly Asn
35
<210> 40
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 40
Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg
1 5 10 15
Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys
20 25 30
Gly Asn
<210> 41
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 41
Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala
1 5 10 15
Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly
20 25 30
Asn
<210> 42
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 42
Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly
1 5 10 15
Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25 30
<210> 43
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 43
Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu
1 5 10 15
Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25 30
<210> 44
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 44
Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln
1 5 10 15
Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25 30
<210> 45
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 45
Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe
1 5 10 15
Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25
<210> 46
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 46
Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro
1 5 10 15
Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25
<210> 47
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 47
Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val
1 5 10 15
Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25
<210> 48
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 48
Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly
1 5 10 15
Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25
<210> 49
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 49
Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg
1 5 10 15
Val His Arg Leu Leu Arg Lys Gly Asn
20 25
<210> 50
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 50
Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val
1 5 10 15
His Arg Leu Leu Arg Lys Gly Asn
20
<210> 51
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 51
Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
1 5 10 15
Arg Leu Leu Arg Lys Gly Asn
20
<210> 52
<211> 39
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 52
Xaa Gly Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg
20 25 30
Leu Leu Arg Lys Gly Asn Tyr
35
<210> 53
<211> 38
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 53
Gly Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg
1 5 10 15
Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu
20 25 30
Leu Arg Lys Gly Asn Tyr
35
<210> 54
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 54
Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser
1 5 10 15
Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu
20 25 30
Arg Lys Gly Asn Tyr
35
<210> 55
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 55
Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser
1 5 10 15
Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg
20 25 30
Lys Gly Asn Tyr
35
<210> 56
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 56
Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg
1 5 10 15
Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys
20 25 30
Gly Asn Tyr
35
<210> 57
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 57
Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala
1 5 10 15
Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly
20 25 30
Asn Tyr
<210> 58
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 58
Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly
1 5 10 15
Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25 30
Tyr
<210> 59
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 59
Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu
1 5 10 15
Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25 30
<210> 60
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 60
Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln
1 5 10 15
Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25 30
<210> 61
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 61
Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe
1 5 10 15
Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25 30
<210> 62
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 62
Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro
1 5 10 15
Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25
<210> 63
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 63
Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val
1 5 10 15
Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25
<210> 64
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 64
Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly
1 5 10 15
Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25
<210> 65
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 65
Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg
1 5 10 15
Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25
<210> 66
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 66
Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val
1 5 10 15
His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25
<210> 67
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 67
Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
1 5 10 15
Arg Leu Leu Arg Lys Gly Asn Tyr
20
<210> 68
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Core domain 2
<400> 68
Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg
1 5 10 15
Leu Leu Arg Lys
20
<210> 69
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> C-terminal Domain
<400> 69
Gly Asn Tyr
1
<210> 70
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 70
Xaa Gly Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg
20 25 30
Leu Leu Arg Lys Gly
35
<210> 71
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 71
Gly Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg
1 5 10 15
Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu
20 25 30
Leu Arg Lys Gly
35
<210> 72
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 72
Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser
1 5 10 15
Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu
20 25 30
Arg Lys Gly
35
<210> 73
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 73
Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser
1 5 10 15
Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg
20 25 30
Lys Gly
<210> 74
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 74
Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg
1 5 10 15
Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys
20 25 30
Gly
<210> 75
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 75
Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala
1 5 10 15
Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly
20 25 30
<210> 76
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 76
Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly
1 5 10 15
Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly
20 25 30
<210> 77
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 77
Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu
1 5 10 15
Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly
20 25 30
<210> 78
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 78
Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln
1 5 10 15
Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly
20 25
<210> 79
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 79
Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe
1 5 10 15
Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly
20 25
<210> 80
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 80
Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro
1 5 10 15
Val Gly Arg Val His Arg Leu Leu Arg Lys Gly
20 25
<210> 81
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 81
Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val
1 5 10 15
Gly Arg Val His Arg Leu Leu Arg Lys Gly
20 25
<210> 82
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 82
Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly
1 5 10 15
Arg Val His Arg Leu Leu Arg Lys Gly
20 25
<210> 83
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 83
Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg
1 5 10 15
Val His Arg Leu Leu Arg Lys Gly
20
<210> 84
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 84
Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val
1 5 10 15
His Arg Leu Leu Arg Lys Gly
20
<210> 85
<211> 38
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 85
Xaa Gly Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg
20 25 30
Leu Leu Arg Lys Gly Asn
35
<210> 86
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 86
Gly Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg
1 5 10 15
Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu
20 25 30
Leu Arg Lys Gly Asn
35
<210> 87
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 87
Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser
1 5 10 15
Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu
20 25 30
Arg Lys Gly Asn
35
<210> 88
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 88
Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser
1 5 10 15
Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg
20 25 30
Lys Gly Asn
35
<210> 89
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 89
Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg
1 5 10 15
Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys
20 25 30
Gly Asn
<210> 90
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 90
Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala
1 5 10 15
Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly
20 25 30
Asn
<210> 91
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 91
Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly
1 5 10 15
Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25 30
<210> 92
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 92
Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu
1 5 10 15
Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25 30
<210> 93
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 93
Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln
1 5 10 15
Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25 30
<210> 94
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 94
Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe
1 5 10 15
Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25
<210> 95
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 95
Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro
1 5 10 15
Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25
<210> 96
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 96
Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val
1 5 10 15
Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25
<210> 97
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 97
Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly
1 5 10 15
Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25
<210> 98
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 98
Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg
1 5 10 15
Val His Arg Leu Leu Arg Lys Gly Asn
20 25
<210> 99
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 99
Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val
1 5 10 15
His Arg Leu Leu Arg Lys Gly Asn
20
<210> 100
<211> 39
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 100
Xaa Gly Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg
20 25 30
Leu Leu Arg Lys Gly Asn Tyr
35
<210> 101
<211> 38
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 101
Gly Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg
1 5 10 15
Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu
20 25 30
Leu Arg Lys Gly Asn Tyr
35
<210> 102
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 102
Arg Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser
1 5 10 15
Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu
20 25 30
Arg Lys Gly Asn Tyr
35
<210> 103
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 103
Gly Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser
1 5 10 15
Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg
20 25 30
Lys Gly Asn Tyr
35
<210> 104
<211> 35
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 104
Lys Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg
1 5 10 15
Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys
20 25 30
Gly Asn Tyr
35
<210> 105
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 105
Gln Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala
1 5 10 15
Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly
20 25 30
Asn Tyr
<210> 106
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 106
Gly Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly
1 5 10 15
Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn
20 25 30
Tyr
<210> 107
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 107
Gly Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu
1 5 10 15
Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25 30
<210> 108
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 108
Lys Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln
1 5 10 15
Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25 30
<210> 109
<211> 30
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 109
Xaa Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe
1 5 10 15
Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25 30
<210> 110
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 110
Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro
1 5 10 15
Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25
<210> 111
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 111
Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val
1 5 10 15
Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25
<210> 112
<211> 27
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 112
Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly
1 5 10 15
Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25
<210> 113
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 113
Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg
1 5 10 15
Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25
<210> 114
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin
<400> 114
Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val
1 5 10 15
His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25
<210> 115
<211> 39
<212> PRT
<213> Artificial Sequence
<220>
<223> N-terminal of human histone H2A(1-39)
<400> 115
Ser Gly Arg Gly Lys Gln Gly Gly Lys Ala Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg
20 25 30
Leu Leu Arg Lys Gly Asn Tyr
35
<210> 116
<211> 39
<212> PRT
<213> Artificial Sequence
<220>
<223> buforin-1
<400> 116
Ala Gly Arg Gly Lys Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg
20 25 30
Leu Leu Arg Lys Gly Asn Tyr
35
<210> 117
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> buforin-2
<400> 117
Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
1 5 10 15
Arg Leu Leu Arg Lys
20
<210> 118
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> parasin
<400> 118
Lys Gly Arg Gly Lys Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser Arg
20
<210> 119
<211> 34
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin 1-1
<400> 119
Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala
1 5 10 15
Gly Leu Gln Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys Gly
20 25 30
Asn Tyr
<210> 120
<211> 29
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin 1-2
<400> 120
Arg Ala Lys Ala Lys Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro
1 5 10 15
Val Gly Arg Val His Arg Leu Leu Arg Lys Gly Asn Tyr
20 25
<210> 121
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin 1-3
<400> 121
Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
1 5 10 15
Arg Leu Leu Arg Lys Gly Asn Tyr
20
<210> 122
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Buforin 1-4
<400> 122
Ala Gly Arg Gly Lys Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg
20 25 30
Leu Leu Arg Lys Gly
35
<210> 123
<211> 265
<212> PRT
<213> Artificial Sequence
<220>
<223> Stx1(1-265)
<400> 123
Met Lys Asp Arg Thr Gln Glu Leu Arg Thr Ala Lys Asp Ser Asp Asp
1 5 10 15
Asp Asp Asp Val Ala Val Thr Val Asp Arg Asp Arg Phe Met Asp Glu
20 25 30
Phe Phe Glu Gln Val Glu Glu Ile Arg Gly Phe Ile Asp Lys Ile Ala
35 40 45
Glu Asn Val Glu Glu Val Lys Arg Lys His Ser Ala Ile Leu Ala Ser
50 55 60
Pro Asn Pro Asp Glu Lys Thr Lys Glu Glu Leu Glu Glu Leu Met Ser
65 70 75 80
Asp Ile Lys Lys Thr Ala Asn Lys Val Arg Ser Lys Leu Lys Ser Ile
85 90 95
Glu Gln Ser Ile Glu Gln Glu Glu Gly Leu Asn Arg Ser Ser Ala Asp
100 105 110
Leu Arg Ile Arg Lys Thr Gln His Ser Thr Leu Ser Arg Lys Phe Val
115 120 125
Glu Val Met Ser Glu Tyr Asn Ala Thr Gln Ser Asp Tyr Arg Glu Arg
130 135 140
Cys Lys Gly Arg Ile Gln Arg Gln Leu Glu Ile Thr Gly Arg Thr Thr
145 150 155 160
Thr Ser Glu Glu Leu Glu Asp Met Leu Glu Ser Gly Asn Pro Ala Ile
165 170 175
Phe Ala Ser Gly Ile Ile Met Asp Ser Ser Ile Ser Lys Gln Ala Leu
180 185 190
Ser Glu Ile Glu Thr Arg His Ser Glu Ile Ile Lys Leu Glu Asn Ser
195 200 205
Ile Arg Glu Leu His Asp Met Phe Met Asp Met Ala Met Leu Val Glu
210 215 220
Ser Gln Gly Glu Met Ile Asp Arg Ile Glu Tyr Asn Val Glu His Ala
225 230 235 240
Val Asp Tyr Val Glu Arg Ala Val Ser Asp Thr Lys Lys Ala Val Lys
245 250 255
Tyr Gln Ser Lys Ala Arg Arg Lys Lys
260 265
<210> 124
<211> 206
<212> PRT
<213> Artificial Sequence
<220>
<223> SNAP-25(1-206)
<400> 124
Met Ala Glu Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg
1 5 10 15
Arg Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met
20 25 30
Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val
35 40 45
Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met
50 55 60
Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp
65 70 75 80
Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys
85 90 95
Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val
100 105 110
Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala
115 120 125
Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn
130 135 140
Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu
145 150 155 160
Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg
165 170 175
Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile
180 185 190
Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly
195 200 205
<210> 125
<211> 96
<212> PRT
<213> Artificial Sequence
<220>
<223> syb2(1-96)
<400> 125
Met Ser Ala Thr Ala Ala Thr Ala Pro Pro Ala Ala Pro Ala Gly Glu
1 5 10 15
Gly Gly Pro Pro Ala Pro Pro Pro Asn Leu Thr Ser Asn Arg Arg Leu
20 25 30
Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met Arg Val
35 40 45
Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu Leu Asp
50 55 60
Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu Thr Ser
65 70 75 80
Ala Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn Leu Lys Met Met
85 90 95
Claims (2)
1.buforin衍生物在制备用于预防或治疗SNARE蛋白相关神经性疾病的药物组合物上的用途，所述buforin衍生物的氨基酸序列如SEQ ID NO. 119 至SEQ ID NO. 122中的任何一个所示，所述神经性疾病为选自多汗症、皮肤疼痛、过敏性疾病及其组合中的任意一种疾病。
2. buforin衍生物在制备用于预防或改善皮肤皱纹的化妆品组合物上的用途，所述buforin衍生物的氨基酸序列如SEQ ID NO. 119 至SEQ ID NO. 122中的任何一个所示。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2369369A1 (en) * | 1999-05-14 | 2000-11-23 | Gregory E. Garcia | Buforin 1 as a specific inhibitor and therapeutic agent for botulinum toxin b and tetanus neurotoxins |
| KR100805212B1 (ko) * | 2007-01-05 | 2008-02-21 | 한국과학기술원 | 부포린 유도체 카이신을 포함하는 항암제 |
| CN101570569A (zh) * | 2008-04-29 | 2009-11-04 | 昆山博青生物科技有限公司 | 合成抗菌肽、其制备方法及应用 |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5936063A (en) * | 1996-08-24 | 1999-08-10 | Samyang Genex Co., Ltd. | Antimicrobial peptide isolated from Bufo bufo gargarizans |
| US6713444B1 (en) * | 1999-05-14 | 2004-03-30 | The United States Of America As Represented By The Secretary Of The Army | Buforin I as a specific inhibitor and therapeutic agent for botulinum toxin B and tetanus neurotoxins |
| US20070207209A1 (en) * | 2004-08-27 | 2007-09-06 | Murphy Christopher J | Trophic factor combinations for nervous system treatment |
| US9044438B2 (en) * | 2005-06-17 | 2015-06-02 | Yitzchak Hillman | Disease treatment via antimicrobial peptides or their inhibitors |
| US9259432B1 (en) * | 2011-01-31 | 2016-02-16 | Parminder J. S. Vig | Composition and methods for targeted delivery of a therapeutic compound to the brain or spinal cord of a subject for treatment of neurodegenerative diseases |
| EP3194426A2 (en) | 2014-08-12 | 2017-07-26 | Biomadison, Inc. | Botulinum neurotoxins with modified light chain specifity and methods for producing same |
| KR102049987B1 (ko) * | 2016-11-09 | 2019-11-28 | 아이씨엠 주식회사 | Nkx3.2 단편 및 이를 함유하는 약학 조성물 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2369369A1 (en) * | 1999-05-14 | 2000-11-23 | Gregory E. Garcia | Buforin 1 as a specific inhibitor and therapeutic agent for botulinum toxin b and tetanus neurotoxins |
| KR100805212B1 (ko) * | 2007-01-05 | 2008-02-21 | 한국과학기술원 | 부포린 유도체 카이신을 포함하는 항암제 |
| CN101570569A (zh) * | 2008-04-29 | 2009-11-04 | 昆山博青生物科技有限公司 | 合成抗菌肽、其制备方法及应用 |
Non-Patent Citations (7)
| Title |
|---|
| "Unveiling the Multifaceted Mechanisms of Antibacterial Activity of Buforin II and Frenatin 2.3S Peptides from Skin Micro-Organs of the Orinoco Lime Treefrog (Sphaenorhynchus lacteus)";Carolina Muñoz-Camargo et al.;《Int. J. Mol. Sci.》;第19卷;第3页页 * |
| AltName: Full=Buforin I ; Contains: RecName: Full=Buforin-2 * |
| AltName: Full=Buforin II".《NCBI》.2018,第1-2页. * |
| Contains: RecName: Full=Buforin-1 * |
| ncbi."PREDICTED: histone H2A type 1-H-like, partial [Vicugna pacos]".《NCBI》.2015,第1页. * |
| ncbi."RecName: Full=Histone H2A * |
| 郝刚等."Buforin Ⅱ衍生肽的分子设计、结构分析及活性研究".《药学学报》.2013,第48卷(第48卷),第366-371页. * |
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| KR20200047428A (ko) | 2020-05-07 |
| CN111100195A (zh) | 2020-05-05 |
| KR102336515B1 (ko) | 2021-12-07 |
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