CN111467476A - 解毒药物组合物及其用途 - Google Patents

解毒药物组合物及其用途 Download PDF

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CN111467476A
CN111467476A CN202010148668.6A CN202010148668A CN111467476A CN 111467476 A CN111467476 A CN 111467476A CN 202010148668 A CN202010148668 A CN 202010148668A CN 111467476 A CN111467476 A CN 111467476A
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王宏伟
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Abstract

本发明公开一种解毒药物组合物及其用途。本发明的药物组合物包含去环氧基活性多肽和可选的药物学可接受载体，其中该活性多肽能够使单端孢霉烯族毒素与谷胱甘肽催化反应产生谷胱甘肽化衍生物，从而去除引起毒素毒性的环氧基，实现解毒效果。

Description

解毒药物组合物及其用途
技术领域
本发明涉及医药领域，具体地涉及一种解毒药物组合物及其用途。
背景技术
单端孢霉烯族毒素是主要由镰孢菌、头孢霉、漆斑菌、葡萄穗霉、木霉和其他一些霉菌产生的生物活性和化学结构相似的有毒代谢产物，在自然界中广泛存在。单端孢霉烯族毒素主要存在于由上述细菌污染的食品、饲料及其加工物中，例如大麦、小麦、燕麦、玉米等谷物。
食用受到该类毒素污染的食品或饲料后会导致食欲减退、胃肠炎症，或者出血、呕吐、腹泻、坏死性皮炎、运动失调、血凝不良、贫血和白细胞数量减少、免疫机能降低和流产等，严重威胁人畜健康。过量摄入时则容易导致严重疾病甚至死亡。
自从二十世纪70年代，科研工作者已经探明单端孢霉烯族毒素的环氧基是毒性主要来源基团。目前研究发现在高碱性条件下可体外化学合成去环氧基的单端孢霉烯族毒素衍生物，去环氧基后的衍生物毒素消除。然而目前并没有基于去环氧基解毒的有效药物。分离出可高效对单端孢霉烯族毒素去环氧基的基因或酶，并由此制成药物，有望提供解毒的有效方案。遗憾的是，目前尚未报道有明确的基因或蛋白可通过去环氧基催化单端孢霉烯族毒素进行解毒。
发明内容
针对解决现有技术中的技术问题，本发明提供一种解毒药物组合物，其能够有效去除单端孢霉烯族毒素中的环氧基，从而消除或降低由单端孢霉烯族毒素引发的病况。具体地，本发明包括以下内容。
本发明的第一方面，提供一种解毒药物组合物，其包含去环氧基活性多肽或者编码核酸和可选的药物学可接受载体，所述活性多肽能够使单端孢霉烯族毒素与谷胱甘肽(GSH)催化反应产生GSH化衍生物，从而去除毒素中的环氧基。
在某些实施方案中，本发明的活性多肽具有选自由下述(1)-(5)组成的组中的一种氨基酸序列：
(1)SEQ ID No.:1-35所示的氨基酸序列，其中SEQ ID No.:1表示来源于长穗偃麦草十倍体的氨基酸序列，SEQ ID No.:2表示来源于长穗偃麦草二倍体的氨基酸序列，SEQID No.:3-24表示已验证具有原活性的SEQ ID No.:1的突变体序列，SEQ ID No.:25-35表示来源于香柱菌属
Figure BDA0002401667240000011
的不同物种的氨基酸序列。
(2)与(1)中的氨基酸序列的序列同一性为90％以上，优选95％以上，更优选97％以上，还优选98％以上，进一步优选99％以上，且来源于相同属，优选来源于相同物种的氨基酸序列；进一步优选这些序列组成的多肽仍具有原蛋白酶活性。在某些实施方案中，活性多肽的氨基酸序列与(1)中的氨基酸序列的序列同一性为95％以上，且均来源于香柱菌属。
(3)与(1)或(2)的氨基酸序列相比具有一个或多个氨基酸突变且序列同一性为90％以上，优选95％以上，更优选97％以上，还优选98％以上，进一步优选99％以上的氨基酸序列，此处的氨基酸突变包括氨基酸的插入、缺失或替换。
(4)源自(1)-(3)任一项所述的氨基酸序列的部分连续序列，优选具有该部分连续序列的多肽(或截短多肽)仍具有原多肽的酶催化活性，更优选具有位于(1)-(3)任一项所述的氨基酸序列N端的部分连续序列，例如，具有从N端起前200至前250个氨基酸序列的多肽，例如具有N端起前208个氨基酸序列的多肽，或具有从N端起前242个氨基酸序列的多肽。在某些实施方案中，部分连续序列为第92-110位之间的氨基酸序列或第144-184位之间的氨基酸序列，或者两者的组合。
(5)在(1)-(4)任一项所述的氨基酸序列的N端和/或C端连接其他氨基酸序列的嵌合序列。即，本发明的活性多肽可以为嵌合多肽。在某些实施方案中，其他氨基酸序列为增强多肽表达或分泌的序列，其实例包括但不限于引导肽、信号肽和转动肽。在某些实施方案中，活性多肽为全长蛋白的活性片段与其他氨基酸序列的嵌合多肽，其中其他氨基酸序列为对应于该活性片段之外的来源于同源的其他蛋白的序列，例如结构区或功能区的序列。例如，当来源于某物种中的蛋白酶全长由A+B两部分组成，与其相同属但不同物种的另一同源蛋白酶全长对应的由A’+B’两部分组成，且A与A’为同源对应区域，B与B’为同源对应区域的情况下，嵌合多肽可以由A’+B或A+B’组成。在某些实施方案中，其他氨基酸序列包含非功能序列，例如连接臂或间隔序列。在某些实施方案中，其他氨基酸序列为具有独立功能的多肽，其通过非功能序列，例如连接臂或间隔序列与本发明的活性多肽连接。
在某些实施方案中，本发明的活性多肽具有选自下述至少之一的保守位点：第98位氨基酸A、第99位氨基酸A。
在某些实施方案中，本发明的解毒药物组合物进一步包含谷胱甘肽(GSH)。谷胱甘肽的浓度不特别限定。在某些实施方案中，谷胱甘肽的浓度为0.05mol/L-0.5mol/L，优选0.8-0.2mol/L，更优选0.9-0.15mol/L。
在某些实施方案中，本发明的解毒药物组合物中包含细胞或细胞组分。此处的细胞是指能够施用至人体的体外细胞。所述细胞内包含或能够表达产生本发明的活性多肽。在此情况下，本发明的解毒药物组合物还可包含用于维持细胞活性的成分。此的细胞组分是指将包含本发明的活性多肽的细胞裂解得到的混合物或由此类细胞分泌得到的混合物。
本发明中，药物学可接受载体为在领域内熟知的载体，本领域普通技术人员能够确定其符合临床标准。药物学可接受载体包括但不限于稀释剂和赋形剂。合适的药物学可接受载体的实例包括但不限于：(1)磷酸缓冲盐溶液，pH约7.4，包含或不包含大约1mg/ml到25mg/ml人血清白蛋白；(2)0.9％盐水(0.9％w/v氯化钠)，(3)5％(w/v)葡萄糖；也可以包含抗氧化剂例如色胺和稳定剂例如Tween20。
本发明的解毒药物组合物可以是任何适宜的剂型。例如，注射剂、悬浮剂、乳化剂等。本发明的解毒药物组合物可通过已知的方式施用至体内。例如，通过肌肉注射递送到感兴趣组织中，可选地经由静脉内、经皮、鼻内、经口、粘膜、或其他递送方法进行施用。这样的施用可以经由单剂量或多剂量来进行。本领域技术人员理解的是，本文有待施用的实际剂量可以在很大程度上取决于多种因素而变化，如靶细胞、生物类型或其组织、待治疗受试者的一般状况、给药途径、给药方式等等。
本发明的第二方面，提供一种用于预防细胞中毒或解除细胞毒性的方法，其包括使待处理细胞与去环氧基活性多肽或产生该活性多肽的细胞和可选的谷胱甘肽接触的步骤。
在某些实施方案中，本发明的待处理细胞为体外细胞，例如动物细胞。产生去环氧基活性多肽的细胞包括酵母细胞、大肠杆菌等。
本发明的第三方面，提供去环氧基活性多肽在制备减轻或缓解毒素引发的病况的解毒药物中的用途，其中，所述活性多肽具有选自由下述(1)-(5)组成的组中的一种氨基酸序列：
(1)SEQ ID No.:1-35所示的氨基酸序列；
(2)与(1)中的氨基酸序列的序列同一性为90％以上且来源于相同属的氨基酸序列；
(3)与(1)或(2)的氨基酸序列相比具有一个或多个氨基酸突变且序列同一性为90％以上的氨基酸序列；
(4)源自(1)-(3)任一项所述的氨基酸序列的部分连续序列；
(5)在(1)-(4)任一项所述的氨基酸序列的N端和/或C端连接其他氨基酸序列的嵌合序列。
附图说明
图1A为LC-HRMS(方法1)DON与GSH体外酶促反应的提取离子流谱图EIC。
图1B为LC-HRMS2(方法2)DON与GSH体外酶促反应获得的DON-GSH的高能碰撞解离产生的子离子质谱图。
图2A为LC-HRMS(方法1)3-ADON与GSH体外酶促反应的提取离子流谱图EIC。
图2B为LC-HRMS2(方法2)3-ADON与GSH体外酶促反应获得的3-ADON-GSH通过高能碰撞解离产生的子离子质谱图。
图3A为LC-HRMS(方法1)15-ADON与GSH体外酶促反应提取的提取离子流谱图EIC。
图3B LC-HRMS2(方法2)15-ADON与GSH体外酶促反应获得的15-ADON-GSH的高能碰撞解离产生的子离子质谱图。
图4A为LC-HRMS(方法1)NIV与GSH体外酶促反应的提取离子流谱图EIC。
图4B为LC-HRMS2(方法2)NIV与GSH体外酶促反应获得的NIV-GSH的高能碰撞解离产生的子离子质谱图。
图5A为LC-HRMS(方法1)Fus-X与GSH体外酶促反应的提取离子流谱图EIC。
图5B为LC-HRMS2(方法2)Fus-X与GSH体外酶促反应获得的Fus-X-GSH的高能碰撞解离产生的子离子质谱图。
图6A为LC-HRMS(方法1)DAS与GSH体外酶促反应提取的提取离子流谱图EIC。
图6B为LC-HRMS2(方法2)DAS与GSH体外酶促反应获得的DAS-GSH的高能碰撞解离产生的子离子质谱图。
图7A为LC-HRMS(方法1)HT-2与GSH体外酶促反应提取的提取离子流谱图EIC。
图7B为LC-HRMS2(方法2)HT-2与GSH体外酶促反应获得的HT-GSH加合物的高能碰撞解离产生的子离子质谱图。
图8A为LC-HRMS(方法1)T-2与GSH体外酶促反应提取的提取离子流谱图EIC。
图8B为LC-HRMS2(方法2)T-2与GSH体外酶促反应获得的T2-GSH加合物的高能碰撞解离产生的子离子质谱图。
图9单端孢霉烯族化合物对人体细胞系活力的影响。不同浓度梯度的DON(a)、3ADON(b)、15ADON(c)、FUS-X(d)、NIV(e)、T-2(f)、HT-2(g)、DAS(h)处理细胞48h后，测定OD450nm。
图10FTCD及同源序列进化树。
图11LC-HRMS(方法1)DON处理FTCD同源序列转基因酵母提取的离子色谱图。正离子模式提取到DON-GSH加合物，m/z 604.21730(对应于[M+H]+，Δ±5ppm)。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为具体公开了该范围的上限和下限以及它们之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。除非另有说明，否则“％”为基于重量的百分数。
本文中，术语“多肽”和“蛋白”可互换使用，是指氨基酸残基的聚合物及其变体和合成的和天然存在的类似物。这两个术语适用于其中一个或多个氨基酸残基是合成的非天然存在的氨基酸聚合物，以及适用于天然存在的氨基酸聚合物及其天然存在的化学衍生物。此类化学衍生物包括例如翻译后修饰和降解产物，包括焦谷氨酰、异天冬氨酰、蛋白水解的、磷酸化的、糖基化的、氧化的、异构化的和脱氨基化的变体。
本文中，术语“活性多肽”是指具有去环氧基酶催化活性的多肽，即将环氧基转化为其他基团或去除该基团的活性多肽。本文有时也称作“蛋白酶”。
本文中，术语“序列同一性”是指基因或蛋白质之间分别在核苷酸或氨基酸水平下的序列同一性。“序列同一性”是蛋白质之间的同一性在氨基酸水平的量度和核酸之间的同一性在核苷酸水平下的量度。蛋白质序列同一性可以在比对序列时通过比较每个序列中的给定位置的氨基酸序列来确定。类似地，核酸序列同一性可以在比对序列时通过比较每个序列中的给定位置的核苷酸序列来确定。用于比对用以比较的序列的方法为本领域熟知的，此类方法包括GAP、BESTFIT、BLAST、FASTA和TFASTA。BLAST算法计算序列同一性百分比并且对两个序列之间的相似性执行统计分析。用于执行BLAST分析的软件可通过国家生物技术信息中心(NCBI)网站公开获得。
本文中，术语“第x位氨基酸”或类似表述均以来源于长穗偃麦草十倍体的去环氧酶的氨基酸序列为位置参考，即以SEQ ID No.:1所示的氨基酸序列为位置参考，除非另有明确说明。
本文中，术语“单端孢霉烯族毒素”或“单端孢霉烯族化合物”是指基本化学结构为倍半萜烯，在第12位碳、第13位碳上形成环氧基的一类化合物的统称。优选地，单端孢霉烯族毒素具有下述通式(II)所示的结构：
Figure BDA0002401667240000041
其中，R1、R2和R3各自独立地表示氢原子、羟基或由-OCO-R’表示的酯基，其中R’为直链或支链C1-C5烷基，例如CH3、CH2CH3、CH2CH2CH3或CH2(CH3)2，R4表示氢原子或羟基，R5表示氢原子、＝O、羟基或由-OCO-R”表示的酯基，其中R”为直链或支链C1-C10烷基，优选为CH3、CH2CH3，还优选为直链或支链C3-C8烷基，更优选CH2CH(CH3)2。在某些实施方案中，本发明的单端孢霉烯族毒素包括脱氧雪腐镰刀菌烯醇(DON)、15-乙酰脱氧雪腐镰刀菌烯醇(15-ADON)、3-乙酰脱氧雪腐镰刀菌烯醇(3-ADON)、雪腐镰刀菌烯醇(NIV)、镰刀菌烯酮-X(Fus-X)、二乙酰氧基草镰刀菌烯醇(DAS)、T-2毒素(T-2)、HT-2毒素(HT-2)。
本文中，术语“去环氧基催化活性”是指能够将单端孢霉烯族毒素中的环氧基(优选在第12位碳、第13位碳上形成的环氧基)去除的活性或功能。具体催化过程如下所示：
Figure BDA0002401667240000051
其中，R1-R5的含义与通(I)和(II)中的含义相同。
实施例
一、活性多肽FTCD催化单端孢霉烯族毒素去环氧基反应
1.实验方法：
1.1体外酶促反应：
0.05mol/L磷酸缓冲液(PBS)：0.05mol/L Na2HPO4与0.05mol/L KH2PO4以3:2的比例混合，pH＝7.0；0.1mol/L谷胱甘肽(GSH)：称取谷胱甘肽3.073g，加蒸馏水定容至100ml；0.5mg/ml单端孢霉烯族化合物(DON、3DON、15ADON、FUS-X、NIV、T2、H-T2、DAS)：1mg单端孢霉烯族化合物，加蒸馏水至2ml，过滤灭菌。
将DON、3-DON、15-ADON、NIV、DAS、HT-2、T-2毒素(1mg)分别溶解于新鲜制备的GSH(30.7mg，100μmol)的PBS缓冲液中，并加入活性多肽，于20℃水浴锅保温24h。
1.2LC-HRMS(/MS)分析
体外反应液通过0.22μm滤膜过滤后，转移到进样小瓶中准备进行LC-HRMS检测。使用Thermo ScientificTMQ ExactiveTM组合型四级杆Orbitrap质谱仪。UHPLC系统与配备有电喷雾电离(ESI)源的Orbitrap联用。色谱法在反相XBridge C18，内径150×2.1mm，粒径3.5μm(Waters，Dublin，Ireland)上进行，柱温：35℃。流速为300μLmin-1，进样量3μL。使用U3000液相色谱仪，流动相：A：0.1％乙酸水溶液，B：乙腈；洗脱梯度：0-0.2min，A＝90％；0.2-6min，A递减至10％；6～8min，A＝10％；8.1min，A递增至90％；8.1～10min，A＝90％。
(1)Full scan全扫描模式：该模式在m/z 200～1000的范围内快速交替正负离子扫描。正离子模式的ESI接口采用以下设置进行：鞘气，40；辅助气体，10；毛细电压3.8kV；毛细管温度为350℃。将AGC target设置为2×e5。负离子模式的ESI接口设置为2.9kV；鞘气，4；辅助气体，0。该模式分辨力设置为70,000。
(2)Fullscan+ddms(一级全扫描+自动触发二级)模式的液相方法及色谱条件同上。该方法交替使用全扫描和MS2扫描，归一化碰撞能设置为20eV，产物离子扫描过程中分辨率设置为17500。
(3)PRM模式可用于量化样品中毒素及其衍生物的相对丰度。PRM模式筛选母离子后，在归一化碰撞能量(HCID)下诱导解离，随后在Orbitrap中进行子离子的碎片检测，其分辨力设置为17500。采用归一化碰撞能，所施加的碰撞能量(15、30和45eV)取决于具体分析物。
LC-HRMS(/MS)分析使用Xcalibur 2.1.0分析数据。采用所提出的生物转化产物的色谱峰形状，保留时间(±0.2分钟)和质量(±5ppm)对毒素及其衍生物的提取离子色谱图(EIC)进行了研究。根据二级图谱以及物质基本结构解析中性丢失，推测化学结构。
2.实验结果
2.1FTCD催化DON毒素并转化成谷胱甘肽加合物DON-GSH
图1A为LC-HRMS1(方法1)DON与GSH体外酶促反应的提取离子流谱图EIC。如图1A所示，以负离子模式从LC-HRMS(Full scan模式)中得到DON的提取离子流谱图EIC，m/z355.13984(对应[M+CH3COO]-形式，Δ±5ppm)；正离子模式提取到DON-GSH加合物，m/z604.21707(对应于[M+H]+，Δ±5ppm)。
图1B为LC-HRMS2(方法2)DON与GSH体外酶促反应获得的DON-GSH的高能碰撞解离产生的子离子质谱图，[M+H]+(m/z 604.21707，Δ±5ppm)。通过对带正电([M+H]+)的离子进行靶向HRMS2分析，研究了DON-GSH环氧加合物的MS片段。DON-GSH的离子碎裂产生m/z299.0939的特征离子，对应于C14H19O5S+。该特征离子可归因于在C-6处侧链的裂解和除S以外GSH部分的损失。该片段还可以进一步裂解产生m/z281.08482(C14H17O4S+)，263.07425(C14H15O3S+)和231.10218(C14H15O3 +)。m/z 263.07425处的产物离子是HRMS2质谱图的基峰，该产物离子在m/z 299.0939的基础上脱去两分子H2O。
DON-GSH在甘氨酸损失之后，可得到碎片离子m/z 529.18503(C23H33O10N2S+)，也可得到损失脱水谷氨酸的碎片离子475.17466(C20H31O9N2S+)。损失了C-6处侧链的m/z574.20717(C24H36O11N3S+)离子碎片从其GSH部分损失脱水谷氨酸可得到m/z445.16389的特征离子(C19H29O8N2S+)；也可得到脱去谷氨酰胺的428.13733(C19H26O8NS+)。
产物离子为m/z 308.09108(C10H18O6N3S+，对应GSH的[M+H]+)。该碎片离子损失脱水谷氨酸得到m/z 179.04907(C5H11O3N2S+)；损失谷氨酰胺得到m/z162.02251(C5H9O3NS+)。此外，m/z130.05044(C5H8O3N+)、m/z145.06077(C5H9O3N2 +)的产物离子均与GSH相关。
1.2FTCD催化3-ADON毒素并转化成谷胱甘肽加合物3-ADON-GSH
图2A为LC-HRMS(方法1)3-ADON与GSH体外酶促反应的提取离子流谱图EIC。如图2A所示，以负离子模式从LC-HRMS(Full scan模式)中得到3-ADON的提取离子流谱图EIC，m/z397.15041(对应[M+CH3COO]-形式，Δ±5ppm)；正离子模式提取到3-ADON-GSH加合物，m/z646.22764(对应于[M+H]+，Δ±5ppm)。
图2B为LC-HRMS2(方法2)3-ADON与GSH体外酶促反应获得的3-ADON-GSH通过高能碰撞解离产生的子离子质谱图，[M+H]+(m/z 646.22764，Δ±5ppm)。对带正电([M+H]+)的3-ADON-GSH环氧加合物离子进行靶向HRMS2分析：3-ADON-GSH的离子碎裂会产生m/z323.09539的特征离子，对应于C16H19O5S+。该特征离子可归因于在C-6处连接的侧链的裂解、脱水以及除S以外GSH部分的损失。该片段还可以进一步裂解产生m/z，263.07425(C14H15O3S+)和231.10218(C14H15O3 +)。m/z 263.07425处的子离子是HRMS2质谱图的基峰，该产物离子是在m/z 323.09539离子的基础上于C-3处脱去CH3COOH。
3-ADON-GSH甘氨酸损失之后，可得到碎片离子m/z 571.19560(C25H35O11N2S+)，其C-6处侧链进一步断裂可产生的m/z 541.18503(C24H33O10N2S+)的碎片离子；脱去1分子H2O的碎片离子m/z 628.21707(C27H38O12N3S+)损失甘氨酸可得到m/z 553.18503(C25H33O10N2S+)；损失脱水谷氨酸可得到499.17466(C22H31O9N2S+)。
1.3FTCD催化15-ADON毒素并转化成谷胱甘肽加合物15-ADON-GSH
图3A为LC-HRMS(方法1)15-ADON与GSH体外酶促反应提取的提取离子流谱图EIC。如图3A所示，以负离子模式从LC-HRMS(Full scan模式)中得到15-ADON的提取离子流谱图EIC，m/z 397.15041(对应[M+CH3COO]-形式，Δ±5ppm)；正离子模式提取到15-ADON-GSH加合物，m/z 646.22764(对应于[M+H]+，Δ±5ppm)。
图3B LC-HRMS2(方法2)15-ADON与GSH体外酶促反应获得的15-ADON-GSH的高能碰撞解离产生的子离子质谱图，[M+H]+(m/z 646.22764，Δ±5ppm)。通过对带正电([M+H]+)的离子进行靶向HRMS2分析，研究了15-ADON-GSH环氧加合物的MS片段。15-ADON-GSH的离子碎裂产生m/z 311.09475的特征离子，对应于C15H19O5S+。该特征离子可归因于在C-15处连接的侧链CH3COOH的裂解和除S以外GSH部分的损失。
同3-ADON-GSH一样，15-ADON-GSH甘氨酸损失之后，可得到子离子m/z 571.1956(C25H35O11N2S+)。脱去1分子H2O的m/z 628.21707(C27H38O12N3S+)损失甘氨酸可得到m/z553.18503(C25H33O10N2S+)。损失脱水谷氨酸的可得到m/z 499.17466(C22H31O9N2S+)。
特征离子m/z 440.13736(C20H26O8NS+)损失脱水谷氨酸可得到m/z 311.09475(C15H19O5S+)的碎片离子；特征离子m/z 450.15471(C17H28O9N3S+)损失甘氨酸得到m/z375.12267(C15H23O7N2S+)子离子，损失脱水谷氨酸得到m/z 321.1121(C12H21O6N2S+)，此外该离子还可以脱去两分子H2O，形成子离子m/z 414.13295(C17H24O7N3S+)，该特征离子损失甘氨酸可得到m/z 339.10091(C15H19O5N2S+)，损失脱水谷氨酸得到m/z 285.09035(C12H17O4N2S+)。该片段进一步脱水还可以产生m/z 267.07979(C12H15O3N2S+)。与GSH相关的m/z 145.06077(C5H9O3N2 +，Δ±5ppm)特征离子是质谱图的基峰。
1.4FTCD催化NIV毒素并转化成谷胱甘肽加合物NIV-GSH
如图4A所示，以负离子模式从LC-HRMS(Full scan模式)中得到NIV的提取离子流谱图EIC，m/z 371.13366(对应[M+CH3COO]-形式，Δ±5ppm)；正离子模式提取到NIV-GSH加合物，m/z 620.21199(对应于[M+H]+，Δ±5ppm)。
图4B为LC-HRMS2(方法2)NIV与GSH体外酶促反应获得的NIV-GSH的高能碰撞解离产生的子离子质谱图，[M+H]+(m/z 620.21199，Δ±5ppm)。通过对带正电([M+H]+)的NIV-GSH环氧加合物离子进行靶向HRMS2分析，研究其MS片段。NIV-GSH的离子碎裂产生m/z229.08652的产物离子，对应于C14H13O3 +。该产物离子可归因于在C-6处侧链的裂解、3分子H2O的断裂以及GSH部分的损失，该结构保留NIV的基本骨架。
NIV-GSH在甘氨酸损失之后，可得到子离子m/z545.17995(C23H33O11N2S+)。也可得到损失脱水谷氨酸的子离子491.16938(C20H31O10N2S+)。C-6处侧链断裂后形成m/z 590.20142(C24H36O12N3S+)，该离子的GSH部分损失脱水谷氨酸可得到m/z 461.15881的子离子(C19H29O9N2S+)。
[M+H]+形式的GSH损失谷氨酰胺可得到子离子m/z 162.02251(C5H9O3NS+)；损失脱水谷氨酸得到m/z 179.04907(C5H11O3N2S+)，该离子是HRMS2质谱中最突出的产物离子。此外，子离子m/z 130.05044(C5H8O3N+)和子离子m/z 145.06077(C5H9O3N2 +)均与GSH相关。
1.5FTCD催化Fus-X毒素并转化成谷胱甘肽加合物Fus-X-GSH
图5A为LC-HRMS(方法1)Fus-X与GSH体外酶促反应的提取离子流谱图EIC。如图5A所示，以负离子模式从LC-HRMS(Full scan模式)中得到Fus-X的提取离子流谱图EIC，m/z377.12069(对应[M+Na]+形式，Δ±5ppm)；正离子模式提取到Fus-X-GSH加合物，m/z662.22255(对应于[M+H]+，Δ±5ppm)。
图5B为LC-HRMS2(方法2)Fus-X与GSH体外酶促反应获得的Fus-X-GSH的高能碰撞解离产生的子离子质谱图。通过对带正电([M+H]+)的FusX-GSH环氧加合物离子进行靶向HRMS2分析，研究其MS片段。FusX-GSH的离子碎裂产生m/z 297.07973的产物离子，对应于C14H17O5S+。该产物离子可归因于在C-4处侧链的裂解、C-6处侧链的裂解及除S以外的GSH部分的损失，该结构仅仅保留Fus-X的基本骨架。
FusX-GSH在甘氨酸损失之后，可得到子离子m/z 587.19051(C25H35O12N2S+)。C-6处侧链断裂后形成m/z 632.21198(C26H38O13N3S+)的特征离子损失脱水谷氨酸可得到m/z503.16937(C21H31O10N2S+)的子离子，损失谷氨酰胺可以得到m/z 486.14281(C21H28O10NS+)。其中m/z 503.16937(C24H36O12N3S+)的子离子是HRMS2质谱中最突出的产物离子。
[M+H]+形式的GSH损失谷氨酰胺可得到子离子m/z 162.02251(C5H9O3NS+)；损失脱水谷氨酸得到m/z 179.04907(C5H11O3N2S+)，此外，子离子m/z 130.05044(C5H8O3N+)和子离子m/z 145.06077(C5H9O3N2 +)均与GSH相关。
1.6FTCD催化DAS毒素并转化成谷胱甘肽加合物DAS-GSH
图6A为LC-HRMS(方法1)DAS与GSH体外酶促反应提取的提取离子流谱图EIC。以正离子模式从LC-HRMS(Full scan模式)中得到DAS的提取离子流谱图EIC，m/z 389.15707(对应[M+Na]+形式，Δ±5ppm)；DAS-GSH加合物，m/z 674.25894(对应于[M+H]+，Δ±5ppm)。
图6B为LC-HRMS2(方法2)DAS与GSH体外酶促反应获得的DAS-GSH的高能碰撞解离产生的子离子质谱图，[M+H]+(m/z 674.25894，Δ±5ppm)。通过对带正电([M+H]+)的离子进行靶向HRMS2分析，研究DAS-GSH环氧加合物的MS片段。DAS-GSH的离子碎裂产生m/z229.12231的产物离子，对应于C15H17O2 +。该产物离子可归因于C-4、C-15处连接的侧链CH3COOH的裂解、脱水以及GSH部分的损失。
DAS-GSH在甘氨酸损失之后，可得到子离子m/z 599.22690(C27H39O11N2S+)；损失谷氨酰胺，可得到m/z 528.18977(C24H34O10NS+)的子离子；也可得到损失脱水谷氨酸的子离子545.21633(C24H37O10N2S+)；损失了CH3COOH可得到m/z 614.23781(C27H40O11N3S+)的特征离子。
与GSH相关的m/z 130.05044(C5H8O3N+)、m/z 145.06077(C5H9O3N2 +)、m/z162.02251(C5H9O3NS+)、m/z 179.04907(C5H11O3N2S+)子离子中，失去脱水谷氨酸m/z的特征离子在179.04907处(C5H11O3N2S+)是质谱图的基峰。
1.7FTCD催化HT-2并转化成谷胱甘肽加合物HT-2-GSH
图7A为LC-HRMS(方法1)HT-2与GSH体外酶促反应提取的提取离子流谱图EIC。以正离子模式从LC-HRMS(Full scan模式)中得到HT-2的提取离子流谱图EIC，m/z 447.19894(对应[M+Na]+形式，Δ±5ppm)；HT-GSH的加合物，m/z 732.30080(对应于[M+H]+，Δ±5ppm)。
图7B为LC-HRMS2(方法2)HT-2与GSH体外酶促反应获得的HT-GSH加合物的高能碰撞解离产生的子离子质谱图，[M+H]+(m/z 732.30080，Δ±5ppm)。通过对带正电([M+H]+)的HT-GSH环氧加合物离子进行靶向HRMS2分析，研究其MS片段。HT-GSH碎裂产生m/z295.10048的产物离子，对应于C15H19O4S+。该产物离子可归因于C-8处((CH3)2CHCH2COOH)的断裂，C-15处CH3COOH的裂解、除S以外GSH部分的损失，该结构保留了HT-2的基本骨架。而m/z 274.10335是[M+H]+形式的GSH的—SH键断裂，形成H2S的中性丢失所致。
HT-GSH在C-8、C-15处的侧链断裂之后可得到m/z 570.21226(C25H36O10N3S+)的特征离子，该离子损失甘氨酸可得到m/z 495.18022(C23H31O8N2S+)；损失脱水谷氨酸可得到m/z441.16965(C20H29O7N2S+)；损失谷氨酰胺可得到m/z 424.14309(C20H26O7NS+)的碎片离子。m/z 441.16965处是质谱图的基峰。
此外还检测到与GSH相关的m/z 130.05044(C5H8O3N+)、m/z 145.06077(C5H9O3N2 +)、m/z 162.02251(C5H9O3NS+)、m/z 179.04907(C5H11O3N2S+)离子。
1.8FTCD催化T-2并转化成谷胱甘肽加合物HT-2-GSH
图8A为LC-HRMS(方法1)T-2与GSH体外酶促反应提取的提取离子流谱图EIC。以正离子模式从LC-HRMS(Full scan模式)中得到T-2的提取离子流谱图EIC，m/z489.20950(对应[M+Na]+形式，Δ±5ppm)；T2-GSH的加合物，m/z 774.31136(对应于[M+H]+，Δ±5ppm)。
图8B为LC-HRMS2(方法2)T-2与GSH体外酶促反应获得的T2-GSH加合物的高能碰撞解离产生的子离子质谱图，[M+H]+(m/z 774.31136，Δ±5ppm)。通过对带正电([M+H]+)的T2-GSH环氧加合物离子进行靶向HRMS2分析，研究其MS片段。T2-GSH碎裂产生m/z337.11105的产物离子，对应于C17H21O5S+。该产物离子可归因于C-8处、C-15处所连接侧链的断裂、除S以外GSH部分的损失，该结构保留了T-2的基本骨架。而m/z 274.10335是[M+H]+形式的GSH的-SH键断裂，形成H2S的中性丢失所致。
T2-GSH在C-8、C-15处的侧链断裂之后可得到m/z 612.22283(C27H38O11N3S+)的特征离子，是质谱图的基峰。该离子损失甘氨酸可得到m/z537.19079(C25H33O9N2S+)；损失脱水谷氨酸可得到m/z 483.18022(C22H31O8N2S+)；损失谷氨酰胺得到m/z 466.15366(C22H28O8NS+)的碎片离子。
同HT2-GSH一样，检测到与GSH相关的130.05044(C5H8O3N+)、m/z 145.06077(C5H9O3N2 +)、m/z 162.02251(C5H9O3NS+)、m/z 179.04907(C5H11O 3N2S+)离子。
3.实验结论
本发明的活性多肽在体外可以高效将单端孢霉烯族毒素(包括DON、3DON、15ADON、FUS-X、NIV、T2、H-T2、DAS等)催化成谷胱甘肽加合物，并且由二级谱图可以知该加合物的形成破坏了在单端孢霉烯的毒性中起主要作用的环氧环结构，可以大大降低毒素的毒性。
二、单端孢霉烯族毒素GSH衍生物的细胞毒性试验
使用DMEM基础培养基，添加10％的胎牛血清和500μl的青霉素链霉素双抗，在37℃、5％CO2的恒温培养箱中培养胰腺癌细胞株(PATU8988)、人胚肾细胞293衍生株(293T)以及人正常食管上皮细胞(HEEC)，待细胞长至瓶壁80％～90％，每2～3d传代一次，用胰蛋白酶消化收集细胞并传代，根据细胞生长状态，选取对数生长期细胞进行实验。
结果如图9所示，采用相应浓度的单端孢霉烯族化合物(DON、3-ADON、15-ADON、FUS-X、NIV、T-2、HT-2、DAS)处理48h后，PATU8988、293T以及HEEC活力均急剧下降，说明不同的单端孢霉烯族化合物对细胞均具有较大毒性；而单端孢霉烯族化合物DON、3-ADON、15-ADON、FUS-X、NIV、T-2、HT-2、DAS经反应后产生的相应衍生物，在对应相同的浓度下细胞活力与空白对照基本一致，说明8种单端孢霉烯族化合物相应的谷胱甘肽加合物对细胞基本没有毒性作用。
三、同源序列基因功能分析
在获得来源于长穗偃麦草的去环氧基酶基因的序列(SEQ ID No.:36)的基础上，通过NCBI进行blastn比对，在默认参数的情况下，未搜到任何注释的高度同源基因。但根据
Figure BDA0002401667240000091
菌属存在同源基因的信息，发明人联合搜索了其他实验室的基因组数据库，获得了11条来源于该菌属的序列。如图10所示，这些序列与长穗偃麦草十倍体的去环氧基酶基因具有90％以上的序列同一性。另外，发明人还从长穗偃麦草二倍体中分离得到与十倍体的去环氧基酶基因的序列同一性为98％的基因。这些基因编码的多肽序列如SEQ ID No.:2和25-35所示。
将上述同源基因构建于酵母表达质粒pPICZαA-FTCD中，并转化毕赤酵母，将筛选到的阳性酵母单菌落(X33/pPICZαA-FTCD)与阴性酵母单菌落(X33/pPICZαA)分别接种于25ml的BMGY培养基，28℃～30℃培养至OD600为2～6。室温离心弃去上清，收集细胞，用BMMY液体培养基重悬细胞至OD600＝1左右，转移至500ml的锥形瓶中，28℃～30℃培养，每24h向其中加入甲醇至终浓度为0.5％以保持诱导表达。在诱导48h后，分装菌液5ml到15ml离心管中，并向其中加入多种单端孢霉烯族化合物至终浓度为25μg/ml，继续诱导48h～72h，收集菌体，用于LC-HRMS分析。
结果如图11所示，12个同源序列转入毕赤酵母中，经DON处理。LC-HRMS检测显示均有DON-GSH的生成。RT＝1.68min处存在独立且特异的峰，该峰为C-13处的GSH加合物。在LC-HRMS(方法1)DON处理转基因酵母提取的离子色谱图，正离子模式提取到DON-GSH加合物，m/z 604.21730(对应于[M+H]+，Δ±5ppm)。
在此述分析的基础上，发明人进一步分析这些同源基因产生的蛋白之间的保守性，得到具有第92～110位之间的氨基酸序列的多肽片段和具有第144～184位之间的氨基酸序列的多肽片段。
四、FTCD突变研究
利用诱导突变技术，对来源于长穗偃麦草十倍体的去环氧酶进行随机突变，得到22个氨基酸序列发生改变的突变体。这些突变体的氨基酸序列分别如SEQ ID No.:3-24所示。
经功能分析，在22个突变体中均不同程度地保留了原去环氧基活性。其中存在两个终止突变，分别终止于209位和243位氨基酸，但这两种终止不会导致该酶的功能完全丢失。因此，表明该酶的功能结构域主要是N端。
尽管本发明已经参考示例性实施方案进行了描述，但应理解本发明不限于公开的示例性实施方案。在不背离本发明的范围或精神的情况下，可对本发明说明书的示例性实施方案做多种调整或变化。权利要求的范围应基于最宽的解释以涵盖所有修改和等同结构与功能。
序列表
<110> 山东农之安生物科技有限公司
<120> 解毒药物组合物及其用途
<130> BH2000028-1
<141> 2020-03-05
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Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 12
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 12
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Lys Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 13
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 13
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Phe Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 14
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 14
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Leu Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 15
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 15
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile His Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 16
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 16
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Thr Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 17
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 17
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Phe Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 18
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 18
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Ile Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 19
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 19
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Val Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 20
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 20
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Val Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 21
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 21
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Val Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 22
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 22
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Val Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 23
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 23
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Met Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 24
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 24
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg His Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 25
<211> 280
<212> PRT
<213> Epichloe bromicola
<400> 25
Met Ala Thr Ser Thr Ser Ile Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Gln
115 120 125
Leu Leu Ile Pro Leu Ser Glu Thr Arg Ala Ser Pro Glu Leu Ala Asp
130 135 140
Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val
145 150 155 160
Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val
165 170 175
Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp
180 185 190
Phe Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg
195 200 205
Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly
210 215 220
Pro Phe Leu Leu Gly Gln Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly
225 230 235 240
Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln
245 250 255
Glu Val Arg Ala Cys His Gly Ala Val Phe Gly Gln Leu His Asp Ala
260 265 270
Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 26
<211> 279
<212> PRT
<213> Epichloe amarillans
<400> 26
Met Ala Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile Ala Gln
1 5 10 15
Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn Pro Trp Lys Ser
20 25 30
Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr Trp Val
35 40 45
Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Leu Pro Ala
50 55 60
Cys Arg Lys Phe Ala Asp Gly Thr Asp Phe Asp Thr Leu Pro Ile Ile
65 70 75 80
His Asp Pro Ala Thr Gly Ser Leu Ile Gly Asp Ser Phe Asp Ile Ala
85 90 95
Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp Leu Phe
100 105 110
Pro Pro Gln Lys Leu Asp Tyr Ala Ala Gly Arg Asp Thr Gln Leu Leu
115 120 125
Ile Pro Leu Ser Glu Val Arg Ala Ala Ser Pro Glu Leu Ala Asp Tyr
130 135 140
Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val Gly
145 150 155 160
Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val Thr
165 170 175
Lys Ala Glu Phe Val Arg Arg Ala Ala Val Ser Ser Trp Asp Asp Leu
180 185 190
Asp Met Val Gly Asp Ala Arg Asp Lys Met Met Gln Ser Leu Arg Asn
195 200 205
Thr Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly Pro
210 215 220
Phe Leu Leu Gly Pro Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly Gly
225 230 235 240
Trp Leu Arg Met Met Arg Ala Thr Leu Pro Pro Ser Glu Trp Gln Ala
245 250 255
Ala Arg Ala Trp His Gly Ala Val Phe Gly Gln Leu His Asp Ala Leu
260 265 270
Asp Lys Tyr Ala Glu Val Lys
275
<210> 27
<211> 264
<212> PRT
<213> Epichloe baconii
<400> 27
Met Ala Thr Ser Thr Ser Thr Ser Thr Ser Thr Ser Thr Pro Ile Ile
1 5 10 15
Phe Tyr Asp Ile Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala
20 25 30
Val Asn Pro Trp Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro
35 40 45
Tyr Thr Thr Thr Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala
50 55 60
Ser Leu Asn Leu Pro Ala Cys Arg Lys Phe Ala Asp Gly Thr Asp Phe
65 70 75 80
Asn Thr Leu Pro Ile Ile His Asp Pro Ala Thr Gly Ser Leu Val Gly
85 90 95
Asp Ser Phe Asp Ile Ala Ala Tyr Leu Gln Arg Thr Asp Thr Gln Leu
100 105 110
Leu Ile Pro Leu Ser Glu Val Arg Ala Ala Ser Ser Asp Leu Ala Asp
115 120 125
Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val
130 135 140
Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val
145 150 155 160
Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Asp Asp
165 170 175
Phe Glu Met Ala Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg
180 185 190
Asn Thr Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly
195 200 205
Pro Phe Leu Leu Gly Arg Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly
210 215 220
Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln
225 230 235 240
Ala Ala Arg Ala Trp His Gly Ala Val Phe Gly Gln Leu His Asp Ala
245 250 255
Leu Asp Lys Tyr Ala Glu Val Lys
260
<210> 28
<211> 286
<212> PRT
<213> Epichloe festucae
<400> 28
Met Ala Thr Ser Thr Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr
1 5 10 15
Asp Ile Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn
20 25 30
Pro Trp Lys Thr Arg Leu Ala Leu Asn Phe Lys Ala Val Thr Tyr Thr
35 40 45
Thr Thr Trp Val Lys Met Pro Asp Ile Ser Gly Val Arg Ala Ser Leu
50 55 60
Asn Val Pro Ala Cys Arg Lys Phe Ala Asp Gly Thr Asp Phe Asn Thr
65 70 75 80
Leu Pro Ile Ile His Asp Pro Ala Thr Gly Ser Leu Ile Gly Asp Ser
85 90 95
Phe Asp Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala
100 105 110
Gly His Leu Phe Pro Pro Leu Pro Pro Pro Gln Lys Leu Asp Tyr Ala
115 120 125
Val Gly Arg Asp Met Gln Leu Leu Ile Pro Leu Ser Glu Val Arg Ala
130 135 140
Ser Ser Glu Leu Ala Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala
145 150 155 160
Ala Phe Thr Ala His Val Gly Val Met Val His Gly Leu Pro Leu Asp
165 170 175
Pro Ala Thr Ala Asp Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly
180 185 190
Val Ser Ser Trp Glu Asp Phe Glu Met Val Gly Glu Ala Arg Glu Lys
195 200 205
Met Met Gln Ser Leu Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe
210 215 220
Arg Arg Asp Ala Ser Gly Pro Phe Leu Leu Gly Gln Gln Ala Thr Tyr
225 230 235 240
Ala Asp Leu Ile Val Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu
245 250 255
Pro Ala Ser Glu Trp Gln Glu Val Arg Ala Trp His Gly Ala Val Phe
260 265 270
Gly Arg Leu His Asp Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280 285
<210> 29
<211> 281
<212> PRT
<213> Epichloe gansuensis
<400> 29
Met Ala Thr Ser Thr Ser Thr Ser Ala Ser Thr Pro Ile Ile Phe Tyr
1 5 10 15
Asp Ile Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn
20 25 30
Pro Trp Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr
35 40 45
Thr Thr Trp Val Glu Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu
50 55 60
Asn Leu Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr
65 70 75 80
Leu Pro Ile Ile His Asp Pro Ala Thr Gly Ser Leu Ile Gly Asp Ser
85 90 95
Phe Asp Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala
100 105 110
Asp Asp Leu Phe Pro Pro Gln Lys Leu Asp Tyr Val Val Gly Ser His
115 120 125
Val Gln Pro Phe Ile Pro Leu Ser Asp Ile Arg Ala Ser Glu Phe Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Leu His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu
180 185 190
Asp Phe Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Phe
195 200 205
Arg Thr Met Leu Glu Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Thr
210 215 220
Gly Pro Phe Leu Leu Gly Gln Lys Ala Thr Tyr Ala Asp Leu Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Trp His Gly Ala Val Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 30
<211> 281
<212> PRT
<213> Epichloe typhina
<400> 30
Met Ala Thr Ser Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp
1 5 10 15
Ile Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro
20 25 30
Trp Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr
35 40 45
Thr Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn
50 55 60
Val Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu
65 70 75 80
Pro Ile Met His Asp Pro Ala Thr Asp Ser Leu Ile Gly Asp Ser Phe
85 90 95
Asp Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly
100 105 110
Asp Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Val Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu
180 185 190
Asp Phe Glu Met Val Gly Glu Val Arg Glu Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Leu Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 31
<211> 281
<212> PRT
<213> Epichloe uncinata
<400> 31
Met Ala Thr Ser Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp
1 5 10 15
Ile Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro
20 25 30
Trp Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr
35 40 45
Thr Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn
50 55 60
Val Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu
65 70 75 80
Pro Ile Met His Asp Pro Ala Thr Asp Ser Leu Ile Gly Asp Ser Phe
85 90 95
Asp Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly
100 105 110
Asp Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Val Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu
180 185 190
Asp Phe Glu Met Val Gly Glu Val Arg Glu Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Leu Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 32
<211> 280
<212> PRT
<213> Epichloe sylvatica
<400> 32
Met Thr Thr Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Lys Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Lys Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Met His Asp Pro Ala Thr Asp Ser Leu Leu Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Gln
115 120 125
Leu Leu Ile Pro Leu Ser Glu Val Arg Ala Ser Pro Glu Leu Ala Asp
130 135 140
Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val
145 150 155 160
Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val
165 170 175
Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp
180 185 190
Leu Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg
195 200 205
Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly
210 215 220
Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Leu Ile Val Gly
225 230 235 240
Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln
245 250 255
Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp Ala
260 265 270
Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 33
<211> 280
<212> PRT
<213> Epichloe aotearoae
<400> 33
Met Ala Thr Pro Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Met His Asp Pro Ala Thr Ser Ser Leu Ile Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Ser Gln Lys Leu Asp Tyr Ala Val Ala Arg Asp Thr Gln
115 120 125
Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Ser Glu Leu Ala Asp
130 135 140
Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val
145 150 155 160
Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val
165 170 175
Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp
180 185 190
Phe Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg
195 200 205
Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly
210 215 220
Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Leu Ile Val Gly
225 230 235 240
Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln
245 250 255
Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp Ala
260 265 270
Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 34
<211> 275
<212> PRT
<213> Epichloe glyceriae
<400> 34
Met Ala Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile Ala Gln Arg Pro
1 5 10 15
Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp Lys Ser Arg Leu
20 25 30
Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr Trp Val Ser Met
35 40 45
Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val Pro Ala Cys Arg
50 55 60
Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro Ile Ile His Asp
65 70 75 80
Pro Ala Thr Gly Ser Leu Ile Gly Asp Ser Phe Asp Ile Ala Ala His
85 90 95
Leu Gln Arg Ala Tyr Pro Ala Ser Gly Ala Gly Asp Leu Phe Pro Pro
100 105 110
Gln Glu Leu Asp Tyr Val Val Ala Arg Asp Thr Arg Leu Leu Val Pro
115 120 125
Leu Ser Glu Thr Arg Ala Ser Glu Phe Ala Asp Tyr Ala Arg Phe Asn
130 135 140
Ser Asn Val Asp Ala Ala Phe Thr Ala His Val Gly Leu Met Val His
145 150 155 160
Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val Thr Lys Ala Glu Phe
165 170 175
Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp Phe Glu Leu Val Gly
180 185 190
Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg Asn Val Leu Gly Asp
195 200 205
Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly Pro Phe Leu Leu Gly
210 215 220
Gln Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly Gly Trp Leu Arg Met
225 230 235 240
Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln Glu Ala Arg Ala Trp
245 250 255
His Gly Ala Val Phe Gly Gln Leu His Asp Ala Leu Asp Lys Tyr Ala
260 265 270
Glu Val Lys
275
<210> 35
<211> 280
<212> PRT
<213> Epichloe brachyelytri
<400> 35
Met Ala Thr Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Ile Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Ser Arg Asp Met Gln
115 120 125
Leu Leu Ile Pro Leu Ser Glu Met Arg Ala Ser Ser Glu Leu Ala Asp
130 135 140
Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val
145 150 155 160
Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val
165 170 175
Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp
180 185 190
Phe Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg
195 200 205
Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly
210 215 220
Pro Phe Leu Leu Gly Gln Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly
225 230 235 240
Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln
245 250 255
Glu Val Arg Ala Trp His Gly Ala Ile Phe Gly Gln Leu His Asp Ala
260 265 270
Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 36
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 36
atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60
cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120
aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180
agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240
atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300
ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tcccccccca gaagctcgac 360
tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420
ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480
gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540
gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600
cgcgacaaga tgatgcagtc cctccgaaac atgctggggg acctggctgc cttgtttcgg 660
aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720
ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780
gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840
aagtag 846

Claims (10)

1.一种解毒药物组合物，其特征在于，包含去环氧基活性多肽或其编码核酸和可选的药物学可接受载体，所述活性多肽能够使单端孢霉烯族毒素与谷胱甘肽催化反应产生谷胱甘肽化衍生物，从而去除引起毒素毒性的环氧基。
2.根据权利要求1所述的解毒药物组合物，其特征在于，所述活性多肽具有选自由下述(1)-(5)组成的组中的一种氨基酸序列：
(1)SEQ ID No.:1-35所示的氨基酸序列；
(2)与(1)中的氨基酸序列的序列同一性为90％以上且来源于相同属的氨基酸序列；
(3)与(1)或(2)的氨基酸序列相比具有一个或多个氨基酸突变且序列同一性为90％以上的氨基酸序列；
(4)源自(1)-(3)任一项所述的氨基酸序列的部分连续序列；
(5)在(1)-(4)任一项所述的氨基酸序列的N端和/或C端连接其他氨基酸序列的嵌合序列。
3.根据权利要求2所述的解毒药物组合物，其特征在于，所述活性多肽具有第98位氨基酸A和第99位氨基酸A的保守位点。
4.根据权利要求2所述的解毒药物组合物，其特征在于，所述活性多肽具有源自(1)-(3)任一项所述的氨基酸序列的N端部分连续序列，且仍具有原多肽活性。
5.根据权利要求1-4任一项所述的解毒药物组合物，其特征在于，进一步包含谷胱甘肽。
6.一种用于预防细胞中毒或解除细胞毒性的方法，其特征在于，包括使待处理细胞与去环氧基活性多肽或及编码核酸，或产生该活性多肽的细胞和可选的谷胱甘肽接触的步骤。
7.根据权利要求6所述的方法，其特征在于，所述待处理细胞为体外细胞。
8.根据权利要求6所述的方法，其特征在于，所述待处理细胞为动物细胞，产生去环氧基活性多肽的细胞为酵母细胞。
9.去环氧基活性多肽在制备用于减轻或缓解毒素引发的病况的药物中的用途，其中，所述活性多肽具有选自由下述(1)-(5)组成的组中的一种氨基酸序列：
(1)SEQ ID No.:1-35所示的氨基酸序列；
(2)与(1)中的氨基酸序列的序列同一性为90％以上且来源于相同属的氨基酸序列；
(3)与(1)或(2)的氨基酸序列相比具有一个或多个氨基酸突变且序列同一性为90％以上的氨基酸序列；
(4)源自(1)-(3)任一项所述的氨基酸序列的部分连续序列；
(5)在(1)-(4)任一项所述的氨基酸序列的N端和/或C端连接其他氨基酸序列的嵌合序列。
10.根据权利要求9所述的用途，其特征在于，所述病况包括食欲减退、胃肠炎症、出血、呕吐、腹泻、坏死性皮炎、运动失调、血凝不良、贫血和白细胞数量减少、免疫机能降低和流产。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021174950A1 (zh) * 2020-03-05 2021-09-10 山东维赞生物科技有限公司 具有去环氧基催化活性的多肽及其编码核酸和用途

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105123507A (zh) * 2015-09-08 2015-12-09 山东农业大学 源于长穗偃麦草抗赤霉病基因Fhb7短片段易位系及其应用
WO2016134387A1 (de) * 2015-02-24 2016-09-01 Erber Aktiengesellschaft Fusariumtoxine spaltende polypeptidvarianten, zusatzstoff enthaltend dieselben und verwendung derselben sowie verfahren zur spaltung von fusariumtoxinen
CN106260742A (zh) * 2016-08-08 2017-01-04 广东海洋大学 抗氧化剂在消减动物体内真菌毒素残留中的应用
CN106879250A (zh) * 2014-05-06 2017-06-20 兽医研究所 单端孢霉烯脱毒的方法
CN109452490A (zh) * 2018-11-15 2019-03-12 山西大学 一种防治由霉菌毒素中毒引起的鸡肌胃炎、腺胃炎的饲料添加剂及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106879250A (zh) * 2014-05-06 2017-06-20 兽医研究所 单端孢霉烯脱毒的方法
WO2016134387A1 (de) * 2015-02-24 2016-09-01 Erber Aktiengesellschaft Fusariumtoxine spaltende polypeptidvarianten, zusatzstoff enthaltend dieselben und verwendung derselben sowie verfahren zur spaltung von fusariumtoxinen
CN107208077A (zh) * 2015-02-24 2017-09-26 爱尔伯股份公司 裂解镰孢毒素的多肽变体、包含所述多肽变体的添加剂和所述添加剂的用途，以及用于裂解镰孢毒素的方法
CN105123507A (zh) * 2015-09-08 2015-12-09 山东农业大学 源于长穗偃麦草抗赤霉病基因Fhb7短片段易位系及其应用
CN106260742A (zh) * 2016-08-08 2017-01-04 广东海洋大学 抗氧化剂在消减动物体内真菌毒素残留中的应用
CN109452490A (zh) * 2018-11-15 2019-03-12 山西大学 一种防治由霉菌毒素中毒引起的鸡肌胃炎、腺胃炎的饲料添加剂及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FOSTER等: "A Possible Enzymatic Assay for Trichothecene Mycotoxins in Animal Feedstuffs", 《BIOCHEMICAL SOCIETY TRANSACTIONS》 *
SILVIO UHLIG等: "Glutathione-Conjugates of Deoxynivalenol in Naturally Contaminated Grain Are Primarily Linked via the Epoxide Group", 《TOXINS (BASEL)》 *
STEPHANIE A GARDINER等: "Transcriptome analysis of the barley-deoxynivalenol interaction: evidence for a role of glutathione in deoxynivalenol detoxification", 《MOL PLANT MICROBE INTERACT》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021174950A1 (zh) * 2020-03-05 2021-09-10 山东维赞生物科技有限公司 具有去环氧基催化活性的多肽及其编码核酸和用途

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