CN111647043A - 含有Hyp-Gly序列的一类抗血小板和抗血栓功能的寡肽 - Google Patents

含有Hyp-Gly序列的一类抗血小板和抗血栓功能的寡肽 Download PDF

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CN111647043A
CN111647043A CN201910725022.7A CN201910725022A CN111647043A CN 111647043 A CN111647043 A CN 111647043A CN 201910725022 A CN201910725022 A CN 201910725022A CN 111647043 A CN111647043 A CN 111647043A
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李博
田淇
杨毅杰
王博
宋洪东
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Abstract

本发明公开了含有Hyp‑Gly序列的一类抗血小板和抗血栓功能的寡肽。本发明提供了含有Hyp‑Gly序列寡肽或其盐形式。本发明含OG序列寡肽可以通过人工合成，也可以通过鱼皮酶解制备获得。含OG序列寡肽分子量小于1000Da，该寡肽占总酶切产物的质量比为65％‑95％，对ADP诱导的血小板聚集有特异性抑制作用，可抑制血栓形成，不影响凝血功能，不延长出血时间，出血风险很小，具有耐胃肠道消化酶酶解和易吸收的特点。

Description

含有Hyp-Gly序列的一类抗血小板和抗血栓功能的寡肽
技术领域
本发明属于生物医药领域，也属于功能食品(保健食品)领域，具体涉及含有Hyp-Gly序列的一类抗血小板和抗血栓功能的寡肽。
背景技术
中国水产品产量约占世界总产量的三分之一，但加工率约为30％，与美国等发达国家的差距较大，水产品加工和综合利用技术还需进一步提高。目前，我国水产品加工副产物利用率较低，而国外已经达到根据副产物化学组成和生化特性进行有效分类利用，加工产值甚至超过鱼肉的几倍到几十倍。在鱼类加工生产过程中会产生大量的副产物，如鱼皮、鱼骨、鱼鳞等，含有丰富的胶原蛋白和活性物质，具有良好的应用前景。
血小板是哺乳动物血液中的有形成分之一，一般呈圆盘状，无细胞核，却具有丰富的胞内颗粒。在正常的生理条件下，血小板的主要功能是促进止血和加速凝血，同时还有维护毛细血管壁完整性的功能。但是血小板的过度活化参与许多病理性过程，包括血栓形成、动脉粥样硬化和癌症等(Blood Reviews,2005,19(2),111-123)。当受到激动剂刺激而活化时，血小板一方面被激活发生聚集，参与血栓形成；另一方面，活化的血小板发生释放反应，将贮存在致密颗粒、α-颗粒或溶酶体内的物质排出，释放的多种生物活性物质对血栓形成、动脉粥样硬化和癌症等发生和发展具有非常重要的作用。从致密颗粒中释放的活性成分的功能是募集其他血小板发生聚集。从致密体释放的物质主要有腺苷二磷酸(ADP)、三磷酸腺苷(ATP)、5-羟色胺(5-HT)和Ca2+。其中ADP、5-HT和Ca2+均可促进血小板聚集，从而放大血小板的聚集作用。而α-颗粒含有大量的蛋白，这些蛋白包括粘着蛋白、趋化因子、细胞有丝分裂原和蛋白酶抑制剂。而从α-颗粒中释放的趋化因子和炎症因子，如IL-1β，CD40配体，PF4，MIP-1α和PDGF等可促进动脉粥样硬化的发生和发展(Trends in CardiovascularMedicine,2004,14(1),18-22.)。因此，抑制血小板的聚集和释放功能对预防和治疗血栓形成、动脉粥样硬化等心脑血管疾病的发生具有重要意义。而用于临床预防和治疗心脑血管疾病的药物虽然疗效显著，但存在多种副作用，易引起出血、皮疹等并发症。因此，研发高效、安全、无毒副作用的天然抗血栓活性物质成为热点。
研究表明来自天然食物或动植物的生物活性肽对人体健康有着积极的作用，能够通过调节人体内各种生理过程从而对人体产生有益影响，主要包括抗氧化肽，降血压肽、免疫调剂肽、抗菌肽等。近年来，抗血小板活性肽的研究得到越来越多的关注，相比传统的抗血小板药物，这些抗血小板活性肽具有毒副作用小、易吸收、药效持续时间长等优点。
目前，研究的来源于胶原的寡肽GPR、GPRG、GPRGP、GPRPP和GPRPPP等，虽然在体外对ADP诱导的人和大鼠血小板聚集均有抑制作用，但是，GPR序列多肽的作用靶点为整合素受体αIIbβ3，是血小板活化和聚集的最终通路，抑制整合素受体αIIbβ3的激活导致出血风险较高，增加出血事件的发生。
因此，寻找一种既能够抑制血小板聚集又能够减少出血的多肽或者提取物是非常重要的。
发明内容
为了制备或获得不出血且抗血小板的生物活性肽，本发明提供如下技术方案：
本发明一个目的是提供一种含有Hyp-Gly序列寡肽或其盐形式。
本发明提供的含有Hyp-Gly序列寡肽或其盐形式，所述寡肽包括如下12种氨基酸序列所示寡肽的任1种或任多种组合：Pro-Gly-Glu-Hyp-Gly-Glu(PGEOGE)、Glu-Hyp-Gly-Glu(EOGE)、Hyp-Gly-Arg(OGR)、Hyp-Gly-Ser--Glu(OGSE)、Hyp-Gly-Glu(OGE)、Hyp-Gly-Ser-Ala(OGSA)、Arg-Hyp-Gly-Glu(ROGE)、Hyp-Gly-Gln(OGQ)、Pro-Gly-Glu-Hyp-Gly(PGEOG)、Gly-Glu-Hyp-Gly(GEOG)、Hyp-Gly-Hyp-Met-Gly(OGOMG)、Hyp-Gly-Glu-Phe-Gly(OGEFG)。
上述寡肽序列中的-表示2个氨基酸残基连接。
上述含有Hyp-Gly序列寡肽为上述氨基酸序列所示寡肽的任1种或任多种组合，在本发明的实施例中以四肽EOGE、四肽OGSA、三肽OGE、五肽PGEOG为例证明寡肽具有抗血栓的作用。
任多种组合为任意2种、任意3种、任意4种、任意5种、任意6种、任意7种、任意8种、任意9种、任意10种、任意11种、任意12种。
上述寡肽可以人工合成；也可以自鱼皮明胶；具体源自鱼类鱼皮的明胶(即胶原蛋白)，如鲢鱼胶原蛋白、大西洋鲑鱼胶原蛋白、红鳍东方鲀胶原蛋白、虹鳟鱼胶原蛋白、鲫鱼胶原蛋白或日本鳗鲡胶原蛋白等：12条含有Hyp-Gly序列寡肽在上述几种鱼皮中出现的频次如表3所示。
具体列举一些胶原蛋白的氨基酸序列，鲢鱼鱼皮胶原蛋白氨基酸序列如序列1所示，大西洋鲑鱼鱼皮胶原蛋白氨基酸序列如序列2所示，红鳍东方鲀鱼皮胶原蛋白氨基酸序列如序列3所示，虹鳟鱼鱼皮胶原蛋白氨基酸序列如序列4所示，鲫鱼鱼皮胶原蛋白氨基酸序列如序列5所示，日本鳗鲡鱼皮胶原蛋白氨基酸序列如序列6所示；可以看出，鱼皮胶原蛋白氨基酸序列中五种共同含有的抗血小板寡肽序列所在的位置(注：Hyp在原胶原蛋白中为脯氨酸P，在后期形成胶原纤维过程中羟基化而成)。
本发明另一个目的是提供一种含有Hyp-Gly序列寡肽或其盐形式的酶解产物的制备方法。
本发明提供的方法，包括如下步骤：用碱性蛋白酶和胰蛋白酶酶解鱼类鱼皮的明胶，得到明胶酶解产物，即为含有Hyp-Gly序列寡肽或其盐的酶解产物。
上述方法中，所述鱼类为鲢鱼、大西洋鲑鱼、红鳍东方鲀、虹鳟鱼、鲫鱼或日本鳗鲡。
上述方法中，所述用碱性蛋白酶和胰蛋白酶酶解鱼类鱼皮的明胶包括如下步骤：
1)提取所述鱼类鱼皮明胶；
2)用所述碱性蛋白酶酶解所述明胶，得到第一次酶解产物；
3)用所述胰蛋白酶酶解所述第一次酶解产物，得到明胶酶解产物；
在本发明的实施例中，每种所述蛋白酶的使用量均为：所述蛋白酶与所述明胶的质量比为1:50-200；
或，所述碱性蛋白酶酶解条件为：60℃酶解2-6h；
或，所述胰蛋白酶酶解条件为37℃酶解2-6h。
所述方法还包括如下步骤：再将所述明胶酶解产物层析纯化，收集不同组分；再检测不同组分对抑制血小板聚集抑制，选取抑制率最高的组分作为目的组分，即为含有Hyp-Gly序列寡肽或其盐的酶解产物。
上述碱性蛋白酶，酶活：200U/mg，即每毫克酶蛋白所具有的酶活力为200U；品牌：Solarbio货号：B8360；酶活定义：指酶催化一定化学反应的能力；单位U，在8.0pH值60℃温度下，1min内水解酪蛋白产生1μg酪氨酸所需的酶量为一个活力单位U。
上述胰蛋白酶，酶活：250U/mg，即每毫克酶蛋白所具有的酶活力为250U；品牌：Amresco，货号：0458；酶活定义：指酶催化一定化学反应的能力；单位U，在7.0pH值37℃温度下，1min水解酪蛋白产生1μg酪氨酸所需的酶量为一个活力单位U。
所述层析纯化采用的层析柱为反相C18填料层析色谱柱或离子交换树脂的层析色谱柱。
上述方法中，所述含有Hyp-Gly序列寡肽的酶解产物中的寡肽包括如下12种氨基酸序列所示寡肽的任1种或任多种组合：Pro-Gly-Glu-Hyp-Gly-Glu(PGEOGE)、Glu-Hyp-Gly-Glu(EOGE)、Hyp-Gly-Arg(OGR)、Hyp-Gly-Ser--Glu(OGSE)、Hyp-Gly-Glu(OGE)、Hyp-Gly-Ser-Ala(OGSA)、Arg-Hyp-Gly-Glu(ROGE)、Hyp-Gly-Gln(OGQ)、Pro-Gly-Glu-Hyp-Gly(PGEOG)、Gly-Glu-Hyp-Gly(GEOG)、Hyp-Gly-Hyp-Met-Gly(OGOMG)、Hyp-Gly-Glu-Phe-Gly(OGEFG)。
上述方法中，所述寡肽的分子量均小于1000Da，在本发明的实施例中，寡肽占酶解产物的质量百分比为65％-90％。
由上述方法制备的含有Hyp-Gly序列寡肽或其盐形式的酶解产物也是本发明保护的范围；
或，上述含有Hyp-Gly序列寡肽或其盐的酶解产物或上述含有Hyp-Gly序列寡肽或其盐形式在制备具有如下1)-6)中至少一种产品中的应用也是本发明保护的范围：
1)预防或辅助治疗心血管疾病；
2)预防或抑制血栓形成；
3)抑制动脉粥样硬化；
4)抑制动脉粥样硬化引起的疾病；
5)预防或抑制血小板聚集引起的疾病；
6)抑制血小板聚集。
本发明还提供了一种产品，其包括上述含有Hyp-Gly序列寡肽或其盐形式的酶解产物，或，上述含有Hyp-Gly序列寡肽或其盐形式；
所述产品具有如下1)-6)中至少一种功能：
1)预防或辅助治疗心血管疾病；
2)预防或抑制血栓形成；
3)抑制动脉粥样硬化；
4)抑制动脉粥样硬化引起的疾病；
5)预防或抑制血小板聚集引起的疾病；
6)抑制血小板聚集。
上述产品可以为如下形式：药品、健康辅助食品、保健食品或特殊医疗用途食品。
本发明是在前期对胶原生物活性肽的抗血小板聚集活性评价基础上，筛选具有高血小板聚集抑制率的寡肽，其可以克服现有的肽段GPR出血的问题。本发明含OG序列寡肽可以通过人工合成，也可以通过鱼皮酶解制备获得。含OG序列寡肽分子量小于1000Da，该寡肽占总酶切产物的质量比为65％-95％，对ADP诱导的血小板聚集有特异性抑制作用；可抑制血栓形成，不影响凝血功能，不延长出血时间，出血风险很小，具有耐胃肠道消化酶酶解和易吸收的特点。本发明所涉及的新型寡肽序列结构，迄今尚未见相关报道。相较传统的抗血小板药物，生物活性肽具有毒副作用小、更加安全的优势。因此，本发明的含OG序列寡肽，克服现有的肽段GPR出血的问题，更有利于开发成针对动脉粥样硬化、和有血栓倾向的心脑血管人群的药品、营养健康食品、保健食品或特殊医疗用途食品。
附图说明
图1为含OG序列寡肽化学合成后的质谱图。
图2为12条寡肽在1mM浓度下对ADP诱导的血小板聚集的抑制作用。
图3为鲢鱼皮胶原蛋白的碱性蛋白酶酶解物经反相C18层析柱分离的图谱。
图4为鲢鱼皮胶原蛋白的碱性蛋白酶+胰蛋白酶酶解物各组分对血小板聚集的影响。
图5为鲢鱼皮胶原蛋白的碱性蛋白酶+胰蛋白酶酶解物F1组分的总离子流图和质谱图(+ESI Scan)。
图6为鲢鱼皮胶原蛋白的碱性蛋白酶+胰蛋白酶酶解物F1组分的2条抗血小板活性肽OGE和PGEOG的MS/MS图谱及图谱解析。
图7为大西洋鲑鱼鱼皮胶原蛋白的碱性蛋白酶+胰蛋白酶酶解物经反相C18层析柱分离的图谱。
图8为大西洋鲑鱼鱼皮胶原蛋白的碱性蛋白酶+胰蛋白酶酶解物各组分对血小板聚集的抑制率。其中F2组分和F3组分抗血小板聚集活性较高，在4mg/mL浓度下血小板的抑制率分别为81％和74％。
图9为大西洋鲑鱼鱼皮胶原酶解物(碱性蛋白酶+胰蛋白酶)中F2组分的质谱图。
图10为从实施例2中分离鉴定的抗血小板活性肽OGOMG的MS/MS图谱及图谱解析(m/z＝490.78)。
图11为从实施例3中分离鉴定的抗血小板活性肽OGEFG的MS/MS图谱及图谱解析(m/z＝522.22)。
图12为从实施例3中分离鉴定的抗血小板活性肽OGSA的MS/MS图谱及图谱解析(m/z＝347.15)。
图13为四肽EOGE对SD大鼠颈动脉血栓形成的影响的结果图。
图14为四肽EOGE对SD大鼠胸腺脾脏指数的影响的结果图。
图15四肽OGSA对SD大鼠颈动脉血栓形成的影响的结果图。
图16为四肽OGSA对SD大鼠胸腺脾脏指数的影响的结果图。
图17为三肽OGE对SD大鼠颈动脉血栓形成的影响的结果图。
图18为三肽OGE对SD大鼠胸腺脾脏指数的影响的结果图。
图19五肽PGEOG对SD大鼠颈动脉血栓形成的影响的结果图。
图20为五肽PGEOG对SD大鼠胸腺脾脏指数的影响的结果图。
图21为鲢鱼皮明胶碱性蛋白酶酶解产物对SD大鼠颈动脉血栓形成的影响的结果图。
图22为鲢鱼皮明胶碱性蛋白酶酶解产物对SD大鼠胸腺脾脏指数的影响的结果图。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中w/v为质量比体积，如无特殊说明，单位均为g:ml。
下述实施例中碱性蛋白酶，酶活：200U/mg，即每毫克酶蛋白所具有的酶活力为200U；品牌：Solarbio货号：B8360；酶活定义：指酶催化一定化学反应的能力；单位U，在8.0pH值60℃温度下，1min内水解酪蛋白产生1μg酪氨酸所需的酶量为一个活力单位U。
胰蛋白酶，酶活：250U/mg，即每毫克酶蛋白所具有的酶活力为250U；品牌：Amresco，货号：0458；酶活定义：指酶催化一定化学反应的能力；单位U，在7.0pH值37℃温度下，1min内水解酪蛋白产生1μg酪氨酸所需的酶量为一个活力单位U。
下述实施例中鉴定抗血小板肽采用LC-MS和MS/MS技术如下：
所用毛细管液相色谱的流动相为含0.1％甲酸的2％乙腈(流动相A)和含0.1％甲酸的80％乙腈(流动相B)，洗脱条件为：0min，6％B；5min，9％B；50min，50％B；52min，95％B；56min，95％B。流速为300nL/min。一级质谱扫描范围为100-1500m/z，二级质谱扫描范围为50-1500m/z，分析二级质谱鉴定抗血小板肽序列。
实施例1、含OG序列寡肽的合成与功能验证
一、含OG序列寡肽的合成
合成如下多肽：Pro-Gly-Glu-Hyp-Gly-Glu(PGEOGE)、Glu-Hyp-Gly-Glu(EOGE)、Hyp-Gly-Arg(OGR)、Hyp-Gly-Ser-Glu(OGSE)、Hyp-Gly-Glu(OGE)、Hyp-Gly-Ser-Ala(OGSA)、Arg-Hyp-Gly-Glu(ROGE)、Hyp-Gly-Gln(OGQ)、Pro-Gly-Glu-Hyp-Gly(PGEOG)、Gly-Glu-Hyp-Gly(GEOG)、Hyp-Gly-Hyp-Met-Gly(OGOMG)、Hyp-Gly-Glu-Phe-Gly(OGEFG)。
上述12条由“浙江鸿拓科技有限公司”采用多肽固相合成法合成，通过质谱分析验证了合成物的纯度大于98％。结果由图1所示。
含OG序列的寡肽溶液由含OG序列寡肽和溶剂组成，溶剂为去离子水，该溶液的浓度为10mM(溶液中含OG序列寡肽的浓度)。
二、含OG序列寡肽对ADP诱导的血小板聚集的影响
(1)血小板的制备
雄性健康成年SD大鼠戊巴比妥钠腹腔麻醉，注射剂量为50mg/kg。经大鼠腹腔主动脉取血，并与3.8％的柠檬酸钠溶液(w/v)按照9:1(v/v)的比例混合制成抗凝血。取得的抗凝血加入等体积PBS溶液于23℃下50×g离心10min，取上清后同条件二次离心，以除去残留的红细胞，得到的上清液中富集血小板。将上清于23℃下750×g离心10min，上层即为贫血小板血浆(PPP)，沉淀即为血小板。血小板用PPP调整浓度为2-3×108个/mL，即为富血小板的血浆(PRP)。用含0.5mM EGTA的台式液(Tyrode’s buffer)对血小板进行清洗，于23℃下750×g离心10min，取下层血小板沉淀，加入适量台式液重悬，将血小板数目调为2-3×108个/ml，即得洗涤血小板。血小板制备后均在2h内使用。
(2)含OG序列寡肽的血小板聚集抑制率
血小板聚集率的测定采用普利生LBY-NJ4型血小板聚集仪。
将血小板聚集仪预热30min，将330μL PPP加入比色杯中，并用此调零。
模型组：将270μL实施例1制备的PRP与30μL台式液(台式液配方NaCl 8.0g、10％KCl 2.0ml(0.2g)、10％MgSO4·7H2O 2.6ml(0.26g)、5％NaH2PO4·2H2O 1.3ml(0.065g)、NaHCO3 1.0g、1M CaCL2 1.8ml(0.2g)、葡萄糖1.0g)预先在37℃孵育5min；
样品组：将270μL PRP与30μL的含OG序列寡肽溶液(配制浓度为10mM)预先在37℃孵育5min。
分别向2组中加入30μL ADP(腺苷二磷酸，配制浓度为1.0mM，溶剂为水)诱导血小板聚集，测定血小板在5min时的聚集率，并按照如下公式计算血小板聚集的抑制率。
血小板聚集抑制率(％)＝【(模型组血小板聚集率－样品组血小板聚集率)/模型组血小板聚集率】×100％
上述含OG序列寡肽溶液分别为上述一合成的12条含有OG序列寡肽溶液。
12条含有OG序列寡肽对ADP诱导的血小板聚集抑制率的影响如图2所示，可以看出，含OG序列寡肽可以显著抑制ADP诱导的血小板聚集。
(3)含OG序列寡肽抑制血小板聚集IC50的测定
测定上述一合成的12条含有OG序列寡肽在不同浓度下的血小板聚集抑制率，然后利用Graphpad Prism 6.0软件，对浓度取对数(以10为底的对数)，在浓度(对数)-抑制率的半对数坐标图上进行非线性回归分析，血小板聚集的抑制率为50％时对应的样品浓度即为IC50值。
含OG序列寡肽对ADP诱导的血小板聚集抑制的IC50结果如表1所示。
表1含OG系列寡肽抑制ADP诱导的血小板聚集的IC50值
Figure BDA0002158606220000071
Figure BDA0002158606220000081
由结果可以看到，含OG序列寡肽对ADP诱导的血小板聚集均具有较强的抑制作用，除了OGOMG(IC50＝1.49mM)以外，各条寡肽的半抑制浓度都在1mM以下。因此上述一得到的12条寡肽为抗血小板肽。
检测现有的GPR系列肽的IC50值(检测方法与上述相同)，现有的GPR系列肽的IC50值结果如表2所示。与GPR系列肽的IC50值(表2)进行比较，可以看出，OGEFG和PGEOGE的抑制活性强于3条GPR系列肽，EOGE和OGR强于三肽GPR。总体来说，含OG系列寡肽抑制ADP诱导的血小板聚集活性与GPR系列肽大体上相当。
表2为GPR系列肽的IC50值
Figure BDA0002158606220000082
上表中寡肽序列如下：Gly-Pro-Arg、Gly-Pro-Arg-Gly、Gly-Pro-Arg-Gly-Pro
三、分析不同鱼皮来源的胶原蛋白
这12条抗血小板肽均为天然的胶原寡肽，存在于鱼皮的胶原蛋白酶解物中。通过检索NCBI数据库发现，除了鲢鱼的鱼皮胶原蛋白(序列1)和大西洋鲑鱼(俗称三文鱼)的鱼皮胶原蛋白(序列2)外，OG系列抗血小板肽还存在于其他物种的胶原蛋白中，包括红鳍东方鲀胶原蛋白(序列3)、虹鳟鱼胶原蛋白(序列4)、鲫鱼胶原蛋白(序列5)或日本鳗鲡胶原蛋白(序列6)等，具体的出现频次如表3所示。
表3为六种物种(鱼皮来源)胶原蛋白氨基酸序列12条含有OG序列的抗血小板肽的出现频次
Figure BDA0002158606220000083
Figure BDA0002158606220000091
Figure BDA0002158606220000101
根据活性肽在胶原蛋白序列中出现频次总和，大西洋鲑鱼最高(315次)，其次是鲢鱼和鲫鱼(大于100)，日本鳗鲡和红鳍东方鲀分别出现95和94次，可见活性肽序列来源的丰富性。根据各个活性肽的出现频次发现在各种鱼皮中OGE的含量最高，GEOG其次；除了日本鳗鲡外，含量排在第三和第四位的分别是OGQ和OGR。
实施例2、制备鲢鱼皮抗血小板寡肽
一、利用碱性蛋白酶和胰蛋白酶制备鲢鱼皮抗血小板寡肽的方法
1、含OG序列寡肽的胶原酶解物的制备
(1)鲢鱼皮明胶的提取：
鲢鱼皮解冻后，除去表面的鱼鳞、皮下脂肪及肌肉组织后，用自来水冲洗干净。将鱼皮剪成约0.5cm2大小的小块，用0.05mol/L的NaOH水溶液(料液比1:6，质量w/体积v)浸泡1h，以除去脂肪和杂蛋白，浸泡结束后，用自来水冲洗至中性或偏碱性，得到预处理后的鱼皮。
用0.2％(v/v体积百分含量)的H2SO4溶液(料液比1:6，w/v)浸泡预处理后的鱼皮1h，使其充分溶胀，浸泡结束后，用自来水冲洗至中性或偏酸性，得到溶胀后的鱼皮。
将上述溶胀后的鱼皮放入蒸馏水(料液比1:6，w/v)中，于45℃条件下水浴振荡12h，真空抽滤除杂后，收集抽滤后的溶液，冷冻干燥，即得鲢鱼皮明胶(注：明胶为胶原蛋白的一种形式)。
(2)二步酶解法制备鲢鱼皮胶原酶解物：
第一步酶解：将上述(1)制备的明胶与水按照1:25(g:ml，w/v)混合均匀，将混合液置于90℃水浴中保温10min，进行巴氏杀菌，冷却后，得到灭菌后明胶溶液。
再用1mol/L NaOH水溶液调节灭菌后明胶溶液至pH＝8.0，再按照酶与底物明胶的质量百分比1％(碱性蛋白酶的质量：明胶的质量)加入碱性蛋白酶(酶活：200U/mg，)60℃下酶解6h，得到第一步酶解液。
第二步酶解：将上一步的第一步酶解液用1mM HCl调pH值至7.0，再按照酶/底物(E/S)质量比为1:100(胰蛋白酶的质量：明胶的质量)加入胰蛋白酶(酶活：250U/mg)，置于37℃恒温水浴振荡器中酶解2h。酶解结束后在95-100℃灭酶10min，冷冻干燥，收集产物，得到明胶酶解产物。
(3)分子量分布
采用岛津LC-15C HPLC系统进行分子量分布的分析测定：将上述(2)获得的明胶酶解产物制备成2mg/mL的待分析样品，溶剂为水，用0.22μm的水相滤膜过滤。用手动进样器取10μL样品待测液注入TSK gel G2000SWXL柱，用含0.1％三氟乙酸的45％乙腈溶液进行洗脱，流速为0.5mL/min，检测波长为214nm。用标准品：抑肽酶(6512Da，北京宝如亿生物技术有限公司，产品目录号：P-I1013)、十四肽VYPFPGPIHNSLPQ(1566Da)、九肽LVYPFPGPI(1002Da)、三肽GPR(328Da)、和二肽Gly-Ser(146Da)作为标品，洗脱时间对log分子量做图获得标准曲线：y＝-0.2829x+8.4421(y：logMW，x：洗脱时间)，计算分子量大小。
结果如表4所示，鲢鱼皮胶原酶解物中分子量在1000Da以下的组分占93％，表明酶解物中主要成分是胶原寡肽，适合制备含OG系列寡肽。
表4鲢鱼皮胶原酶解物的分子量分布
Figure BDA0002158606220000121
(4)测定酶解产物的血小板聚集抑制率
实验方法：同实施例1中的二(2)
实验结果：鲢鱼皮明胶碱性蛋白酶与胰蛋白酶的酶解产物(简称鲢鱼皮酶解物)在浓度为4mg/ml时(水溶液)的血小板聚集抑制率为76.4±8.1％。
2、抗血小板肽高活性组分的分离纯化
将上述1的(2)获得的胶原酶解物复溶于水中，使其浓度为100mg/mL，取1mL添加到装有ODSA反相C18填料的层析柱(φ1.0cm×10cm)中，床体积7mL，洗脱液依次为去离子水、体积百分含量10％甲醇水溶液、体积百分含量30％甲醇水溶液和体积百分含量50％甲醇水溶液。整个过程流速为1mL/min，洗脱时间为60min，检测波长为220nm，用色谱处理系统记录分离过程中图谱变化，并收集样品组分(见图3)，在旋转蒸发仪中将各组分中的甲醇挥发干净并浓缩，然后在冻干机中冻干，放在-80℃的冰箱中备用(用于下述2mg/ml和4mg/ml检测溶剂)。
测定图3中各个组分的血小板聚集率，凝血酶和胶原诱导的血小板聚集实验，所用血小板为洗涤血小板，ADP诱导的血小板聚集所用血小板为PRP。实验方法同实施例1中二(2)。实验中所用诱导剂的浓度为：胶原(50μg/mL)，凝血酶(5.0U/mL)，ADP(1.0mM)。
各个组分的血小板聚集率结果如图4所示，(A)胶原诱导，(B)凝血酶诱导，(C)ADP诱导，诱导数值＝平均值±标准差(n＝5)，与模型组(M)相比，*P<0.05，**P<0.01，由于F1组分对三种诱导剂诱导的血小板聚集都有显著的抑制作用，因此选取F1组分作为目标组分。
再将F1组分进行质谱检测，一级质谱扫描范围为100-1500m/z，二级质谱扫描范围为50-1500m/z，结果如图5所示，可以看出，质荷比为188.12、279.10、304.15、318.16、472.20相对丰度较高。
现有的GPR系列肽的质荷比分别是：GPR，329；GPRG，386；GPRGP，483，从图中可以看出F1组分不含GPR系列的3条抗血小板肽。
再将图5中的箭头标识的丰度较高的质荷比472.20和318.16的峰对应的物质经LC-MS和MS/MS进一步解析确定了肽的序列，结果如图6所示(上图为质荷比318.16解析，下图为质荷比472.20解析)，根据a离子、b离子或y离子计算获得氨基酸序列，F1组分中含有OGE(m/z＝318.16)和PGEOG(m/z＝472.20)。其中PGEOG的检测丰度最高，表明其为F1中的主要活性成分。
上述结果表明，从鲢鱼皮胶原蛋白碱性蛋白酶和胰蛋白酶得到的酶解产物的F1组分具有抗血小板聚集的功能，且其中含有抑制血小板聚集的寡肽OGE和PGEOG。
对比例：利用复合蛋白酶和胰蛋白酶制备鲢鱼皮抗血小板寡肽的方法
按照上述一的方法，仅将其中的碱性蛋白酶替换为复合蛋白酶，采用同样的方法测定酶解物的血小板聚集抑制率。实验结果表明，鲢鱼皮胶原蛋白的复合蛋白酶与胰蛋白酶的酶解物(简称鲢鱼皮复合蛋白酶酶解物)在浓度4mg/ml的血小板聚集抑制率为56.7±7.2％。
可以看出，鲢鱼皮复合蛋白酶酶解物的血小板聚集抑制率显著低于鲢鱼皮碱性蛋白酶酶解物，鲢鱼皮碱性蛋白酶酶解物更适合制备含OG系列寡肽的功能配料。
实施例3、制备大西洋鲑鱼鱼皮抗血小板寡肽
一、利用碱性蛋白酶和胰蛋白酶制备大西洋鲑鱼鱼皮抗血小板寡肽的方法
(1)大西洋鲑鱼鱼皮明胶的提取：
与实施例2的一相同。
(2)二步酶解法制备明胶酶解产物：
第一步酶解：将上述(1)制备的明胶与水按照6％(w/v)混合均匀，将混合液置于90℃水浴中保温10min，进行巴氏杀菌，冷却后，得到灭菌后明胶溶液。
再用1mol/L NaOH分别调节明胶溶液至pH＝8.0，再按照酶与底物的质量比2％加入碱性蛋白酶，60℃下酶解4h，得到第一步酶解液。
第二步酶解：将上一步的第一步酶解液用1mol/L HCl调pH值至7.0，再按照酶/底物(E/S)质量比为1:50(胰蛋白酶的质量：明胶的质量)加入胰蛋白酶，置于37℃恒温水浴振荡器中酶解2h。酶解结束后在95-100℃灭酶10min，冷冻干燥，收集产物，得到胶原酶解物。
(3)明胶酶解产物的血小板聚集抑制率
检测方法与实施例2相同，结果表明，大西洋鲑鱼鱼皮胶原蛋白的碱性蛋白酶与胰蛋白酶酶解产物(简称大西洋鲑鱼鱼皮碱性蛋白酶酶解物)的血小板聚集抑制率60.57±13.4％(测试样品浓度4mg/ml)。
2、抗血小板肽高活性组分的分离纯化：
将上述1的(2)获得的明胶酶解产物按照实施例2的一2进行分离纯化。
样品组分分离纯化图谱见图7。
按照实施例2的一、2测定各个组分对ADP诱导血小板聚集的抑制率,结果见图8。从结构可以看出F2和F3组分对ADP诱导的血小板聚集抑制率最高。F2组分作为目标组分，在4mg/mL浓度下的血小板抑制率，F2为81.26±0.15％，F3为74.43±12.35％，差异不显著，可以共同收集起来作为抗血小板功能配料。
首先将F2组分进行质谱检测，结果如图9。从图中质荷比可以看出F2组分不含GPR系列的3条抗血小板肽(质荷比：GPR 329；GPRG 386；GPRGP 483)。F2组分质谱图中质荷比(m/z)为600.80和526.30的序列没有解出来。该组分中仅分析出了质荷比为573.63(肽的序列为VVGOKG)、490.78和522.22的序列。以m/z＝490.78为例进行说明。
将F2组分中m/z＝490.78对应的物质经LC-MS和MS/MS技术进一步解析确定了肽的序列。根据b离子分析出氨基酸序列为OGOMG，结果如图10所示。表明F2组分中含有OGOMG。
再将F2组分中m/z＝522.22的峰对应的物质经LC-MS和MS/MS技术进一步解析确定了肽的序列为OGEFG，结果如图11所示。F2组分中也含有活性肽OGEFG。根据F2质谱图(图9中)肽的丰度可知，OGOMG的丰度远高于OGEFG的丰度，推测F2中OGOMG的含量要高于OGEFG，这与在大西洋鲑鱼胶原蛋白中二者出现的频次相一致。
F3组分的质谱解析过程略。从F3组分中分离出了OGSA(m/z＝347.15)，该活性肽的解谱是根据y离子计算获得氨基酸序列的，结果如图12所示。
对比例：利用复合蛋白酶和胰蛋白酶制备大西洋鲑鱼鱼皮抗血小板寡肽的方法
按照上述一的方法，仅将一中的碱性蛋白酶替换为复合蛋白酶，最终制备的酶解物(复合蛋白酶+胰蛋白酶)对ADP诱导的血小板聚集的抑制率为35.63±9.3％(样品浓度为4mg/ml)，远低于碱性蛋白酶酶解物的抗血小板活性。
实施例4、四肽EOGE的抗血栓效果和凝血级联反应检测
本实施例中的四肽EOGE来自实施例1合成。
一、实验方法
对雄性SD大鼠进行适应性饲养6天，随后将大鼠分为4组，每组5只，分别进行如下处理：
生理盐水组：用生理盐水灌胃大鼠；
EOGE低剂量组(200μM/kg bw.)：灌胃200μM/kg bw.EOGE；
EOGE高剂量组(300μM/kg bw.)：灌胃300μM/kg bw.EOGE；
氯吡格雷(45mg/kg bw.)：灌胃45mg/kg bw.氯吡格雷；
灌胃1小时后，用2％戊巴比妥钠(0.25mL/100g)对SD大鼠麻醉后，沿颈正中线切开，钝性分离右侧1cm长的颈动脉，放入0.6cm宽的封口胶条，浸有10％FeCl3溶液(注：是诱导血小板聚集的诱导剂)的滤纸条(1cm×0.5cm)环裹分离备用的颈动脉段，并用封口胶条封住15min；40min后，结扎滤纸条两段血管(或者拿止血钳夹住两段血管)；然后腹腔主动脉取血；最后摘取大鼠胸腺和脾脏。
血栓的质量检测：精确剪下滤纸条包裹的血管段，用洁净滤纸吸干血管内余血，精确称量含血栓的血管湿重，取出血栓后的血管再称重，两者相减即为该0.5cm长血管段内血栓的质量。
凝血级联反应检测：将腹腔主动脉取血的血液与3.8％的柠檬酸钠混合(9:1，v/v)抗凝，轻轻混合均匀后，1500g条件下离心10min，取上清(血浆，黄色)。取130μL大鼠血浆放入血凝仪中检测，仪器给出PT和APTT值。取200μL大鼠血浆于37℃水浴中孵育5min。孵育结束后，体系中加入200μL的TT试剂，对光观察凝固时间TT(终点判断：以出现混浊的初期凝固为准)。
胸腺指数和脾脏指数检测：将大鼠胸腺和脾脏称重，根据与体重的比值计算得到胸腺指数和脾脏指数。
二、实验结果
1、四肽EOGE对SD大鼠颈动脉血栓形成的影响
血栓的质量检测结果如图13所示，口服EOGE在300μM/kg bw剂量下可以显著降低颈动脉血栓重量，具有良好的抗血栓治疗效果。
2.四肽EOGE对大鼠胸腺脾脏指数的影响
胸腺指数和脾脏指数检测结果如图14所示，口服四肽EOGE不会引起大鼠胸腺脾脏指数的变化，不会引起急性免疫反应。
3.四肽EOGE对SD大鼠凝血级联反应的影响
凝血级联反应检测结果如表5所示，四肽EOGE不会延长凝血三项时间，对凝血功能无影响，无出血风险。
表5口服四肽EOGE后对凝血三项PT，APTT，TT的影响
Figure BDA0002158606220000151
对比例：
检测现有的GPR系列肽的凝血级联反应的影响(检测方法同上)，结果如表6所示。
表6GPR系列肽对凝血级联反应的影响
Figure BDA0002158606220000161
*表示与生理盐水组相比差异显著(P＜0.05)
上表中寡肽序列如下：Gly-Pro-Arg、Gly-Pro-Arg-Gly、Gly-Pro-Arg-Gly-Pro
经过比较，GPR系列肽延长出血时间，有出血风险；而四肽EOGE不会延长凝血三项时间，对凝血功能无影响，无出血风险，克服了GPR系列肽的出血风险。
实施例5、四肽OGSA的抗血栓效果和凝血级联反应检测
本实施例中的四肽OGSA是实施例1合成。
一、实验方法
对雄性SD大鼠进行适应性饲养6天，随后将大鼠分为4组，每组5只，分别如下：
生理盐水组：用生理盐水灌胃大鼠作为阴性对照；
OGSA低剂量组(200μM/kg bw.)：灌胃200μM/kg bw.OGSA；
OGSA高剂量组(300μM/kg bw.)：灌胃300μM/kg bw.OGSA；
氯吡格雷(30mg/kg bw.)：灌胃30mg/kg bw.氯吡格雷；
灌胃1小时后的实验操作与实施例4相同。
二、实验结果
1.四肽OGSA对SD大鼠颈动脉血栓形成的影响
血栓的质量检测结果如图15所示，口服OGSA在高剂量和低剂量下均可以显著降低颈动脉血栓重量，具有良好的抗血栓治疗效果。
2.四肽OGSA对大鼠胸腺脾脏指数的影响
胸腺指数和脾脏指数检测结果如图16所示，口服四肽OGSA不会引起大鼠胸腺脾脏指数的变化，不会引起急性免疫反应。
3.四肽OGSA对SD大鼠凝血级联反应的影响
凝血级联反应检测实验结果如表7所示，四肽OGSA不会延长凝血三项时间，对凝血功能无影响，无出血风险。
表7口服四肽OGSA后对凝血三项PT，APTT，TT的影响
Figure BDA0002158606220000171
实施例6、三肽OGE的抗血栓效果检测
本实施例中的三肽OGE来自实施例1，是采用多肽固相合成法合成的。
一、实验方法
对雄性SD大鼠进行适应性饲养6天，随后将大鼠分为3组，每组5只，分别进行如下处理：
生理盐水组：用生理盐水灌胃大鼠作为阴性对照；
OGE组，灌胃剂量为300μM/kg bw.
阳性对照组(氯吡格雷)，灌胃剂量30mg/kg bw.
灌胃1小时后的实验操作与实施例4相同。
二、实验结果
1.三肽OGE对SD大鼠颈动脉血栓形成的影响
血栓的质量检测结果如图17所示，口服OGE在300μM/kg bw剂量下可以显著降低颈动脉血栓重量，具有良好的抗血栓治疗效果。
2.三肽OGE对大鼠胸腺脾脏指数的影响
胸腺指数和脾脏指数检测结果如图18所示，口服OGE不会引起大鼠胸腺脾脏指数的变化，不会引起急性免疫反应。
3.三肽OGE对SD大鼠凝血级联反应的影响
凝血级联反应检测实验结果如表8所示，OGE不会延长凝血三项时间，对凝血功能无影响，无出血风险。
表8口服三肽OGE后对凝血三项PT，APTT，TT的影响
Figure BDA0002158606220000181
实施例7、五肽PGEOG的抗血栓效果检测
本实施例中的五肽PGEOG来自实施例1，是采用多肽固相合成法合成的。
一、实验方法
对雄性SD大鼠进行适应性饲养6天，随后将大鼠分为3组，每组5只，分组如下：
生理盐水组：用生理盐水灌胃大鼠作为阴性对照；
PGEOG组，灌胃剂量为300μM/kg bw.
阳性对照组(氯吡格雷)，灌胃剂量30mg/kg bw.
实验方法与实施例5完全相同。
二、实验结果
1.五肽PGEOG对SD大鼠颈动脉血栓形成的影响
血栓的质量检测结果五肽PGEOG对血栓形成的影响如图19所示，口服OGE在300μM/kg bw剂量下可以显著降低颈动脉血栓重量，具有良好的抗血栓治疗效果。
2.五肽PGEOG对大鼠胸腺脾脏指数的影响
胸腺指数和脾脏指数检测结果如图20所示，口服OGE不会引起大鼠胸腺脾脏指数的变化，不会引起急性免疫反应。
3.五肽PGEOG对SD大鼠凝血级联反应的影响
凝血级联反应检测实验结果如表9所示，口服五肽PGEOG不会延长凝血三项时间，对凝血功能无影响，无出血风险。
表9口服五肽PGEOG后对凝血三项PT，APTT，TT的影响
Figure BDA0002158606220000182
实施例8、鲢鱼皮明胶碱性蛋白酶酶解产物的抗血栓实验
一、实验方法：对雄性SD大鼠进行适应性饲养6天，随后将大鼠分为4组，每组5只，分别进行如下处理：
生理盐水组：用生理盐水灌胃大鼠作为阴性对照；
胶原肽低剂量组:灌胃剂量为0.07g/kg bw.实施例2中获得的鲢鱼皮明胶碱性蛋白酶酶解产物；
胶原肽中剂量组:灌胃剂量为0.14g/kg bw.实施例2中获得的鲢鱼皮明胶碱性蛋白酶酶解产物；
胶原肽高剂量组:灌胃剂量为0.28g/kg bw.实施例2中获得的鲢鱼皮明胶碱性蛋白酶酶解产物；
阳性对照组(阿司匹林):灌胃剂量12mg/kg bw.阿司匹林。
连续灌胃七天，第七天灌胃1h后的实验操作与实施例4相同。
二、实验结果
1.鲢鱼皮明胶碱性蛋白酶酶解产物对SD大鼠颈动脉血栓形成的影响
鲢鱼皮明胶碱性蛋白酶酶解产物对血栓形成的影响如图21所示，口服鲢鱼皮明胶碱性蛋白酶酶解产物在0.28g/kg bw剂量下可以显著降低颈动脉血栓重量，具有良好的抗血栓治疗效果。
2.鲢鱼皮明胶碱性蛋白酶酶解产物对大鼠胸腺脾脏指数的影响
结果如图22所示，口服鲢鱼皮明胶碱性蛋白酶酶解产物不会引起大鼠胸腺脾脏指数的变化，不会引起急性免疫反应。
SEQUENCE LISTING
<110> 中国农业大学
<120> 含有Hyp-Gly序列的一类抗血小板和抗血栓功能的寡肽
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<170> PatentIn version 3.5
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Met Phe Ser Phe Val Asp Ile Arg Leu Ala Leu Leu Leu Ser Ala Thr
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Val Leu Leu Ala Arg Gly Gln Gly Glu Asp Asp Arg Thr Gly Gly Ser
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Cys Thr Leu Asp Gly Gln Val Tyr Asn Asp Arg Asp Val Trp Lys Pro
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Glu Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Thr Val Met Cys Asp
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Glu Val Ile Cys Glu Asp Thr Thr Asp Cys Pro Asn Pro Val Ile Pro
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His Asp Glu Cys Cys Pro Val Cys Pro Asp Asp Asp Phe Gln Glu Pro
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Pro Pro Gly Pro Pro Gly Asn Asp Gly Ile Pro Gly Gln Pro Gly Leu
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Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe
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Ser Pro Gln Met Ser Gly Gly Phe Asp Glu Lys Ser Gly Gly Ala Met
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Ala Val Pro Gly Pro Met Gly Pro Met Gly Pro Arg Gly Pro Pro Gly
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Pro Pro Gly Thr Pro Gly Pro Gln Gly Phe Thr Gly Pro Pro Gly Glu
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Pro Gly Glu Ala Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Ala
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Gly Pro Pro Gly Lys Asn Gly Glu Asp Gly Glu Ser Gly Lys Pro Gly
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Arg Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Phe
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Pro Gly Thr Pro Gly Leu Pro Gly Ile Lys Gly His Arg Gly Phe Ser
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Gly Leu Asp Gly Ala Lys Gly Asp Thr Gly Pro Ser Gly Pro Lys Gly
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Glu Ala Gly Ala Pro Gly Glu Asn Gly Thr Pro Gly Ala Met Gly Pro
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Arg Gly Leu Pro Gly Glu Arg Gly Arg Ala Gly Pro Pro Gly Ala Ala
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Gly Ala Arg Gly Asn Asp Gly Ala Ala Gly Ala Ala Gly Pro Pro Gly
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Pro Thr Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Gly Pro Gly Ala
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Gly Ala Arg Gly Glu Ala Gly Asn Pro Gly Pro Ala Gly Pro Ala Gly
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Pro Ala Gly Asn Asn Gly Ala Asp Gly Ala Ala Gly Pro Lys Gly Ser
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Pro Gly Thr Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Pro Arg
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Gly Pro Pro Gly Pro Ser Gly Ala Ala Gly Ala Pro Gly Pro Lys Gly
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Asn Thr Gly Glu Val Gly Ala Pro Gly Ala Lys Gly Glu Ala Gly Ala
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Pro Gly Ala Asp Gly Ser Ala Gly Pro Lys Gly Ala Pro Gly Glu Arg
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Gly Gly Pro Gly Val Val Gly Pro Lys Gly Ala Thr Gly Glu Pro Gly
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Arg Asn Gly Glu Pro Gly Met Pro Gly Ser Lys Gly Met Thr Gly Ser
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Pro Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Ser Gly Thr Pro
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Gly Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Val Gly Ala Arg Gly
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Gln Pro Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu
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Gly Ala Pro Gly Lys Asp Gly Asp Val Gly Ala Pro Gly Ala Pro Gly
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Pro Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Pro
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Pro Gly Phe Gln Gly Leu Pro Gly Pro Gln Gly Ala Thr Gly Glu Pro
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Gly Lys Ser Gly Glu Gln Gly Leu Pro Gly Glu Ala Gly Ala Pro Gly
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Pro Ser Gly Ser Arg Gly Asp Arg Gly Phe Pro Gly Glu Arg Gly Ala
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Pro Gly Pro Ala Gly Pro Ala Gly Ala Arg Gly Ser Pro Gly Ser Ala
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Gly Asn Asp Gly Ala Lys Gly Asp Ser Gly Ala Pro Gly Ala Pro Gly
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Ala Gln Gly Pro Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala
705 710 715 720
Ala Gly Leu Pro Gly Leu Lys Gly Asp Arg Gly Asp Gln Gly Ala Lys
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Gly Thr Asp Gly Ala Pro Gly Lys Asp Gly Ile Arg Gly Met Thr Gly
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Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu
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Thr Gly Ala Pro Gly Leu Val Gly Pro Ala Gly Ala Arg Gly Pro Pro
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Gly Glu Arg Gly Glu Thr Gly Ala Pro Gly Pro Ala Gly Phe Ala Gly
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Pro Pro Gly Ala Asp Gly Leu Pro Gly Ala Lys Gly Glu Ala Gly Asp
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Asn Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ser Gly Ala Thr
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Gly Ala Pro Gly Pro Gln Gly Pro Val Gly Ala Thr Gly Pro Lys Gly
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Ala Arg Gly Ala Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala
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Ala Gly Arg Val Gly Pro Pro Gly Pro Ala Gly Asn Ala Gly Pro Pro
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Gly Pro Pro Gly Pro Ala Gly Lys Glu Gly Gln Lys Gly Ser Arg Gly
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Glu Thr Gly Pro Ala Gly Arg Thr Gly Glu Ile Gly Thr Pro Gly Pro
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Pro Gly Ala Pro Gly Glu Lys Gly Thr Pro Gly Ala Glu Gly Pro Thr
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Gly Pro Ser Gly Thr Pro Gly Pro Gln Gly Ile Asn Gly Gln Arg Gly
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Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu
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Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Pro Ser
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Gly Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly
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Pro Pro Gly Glu Pro Gly Arg Glu Gly Thr Pro Gly Asn Glu Gly Ser
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Ala Gly Arg Asp Gly Ala Pro Gly Pro Lys Gly Asp Arg Gly Glu
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Thr Gly Ala Ala Gly Thr Gly Ala Pro Gly Pro Ile Gly Pro Ala
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Gly Lys Thr Gly Asp Arg Gly Glu Ser Gly Pro Ala Gly Pro Ser
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Gly Ala Val Gly Leu Thr Gly Pro Arg Gly Pro Val Gly Pro Ala
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Gly Ala Arg Gly Asp Lys Gly Glu Thr Gly Glu Ala Gly Glu Arg
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Gly Met Lys Gly His Arg Gly Phe Thr Gly Ile Gln Gly Pro Pro
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Gly Pro Pro Gly Pro Ser Gly Glu Pro Gly Pro Ala Gly Ala Ser
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Gly Pro Ala Gly Pro Arg Gly Pro Ala Gly Ser Ser Gly Pro Ala
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Gly Lys Asp Gly Met Ser Gly Leu Pro Gly Pro Ile Gly Pro Pro
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Gly Pro Arg Gly Arg Asn Gly Glu Ile Gly Pro Ala Gly Pro Pro
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Gly Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Ser Gly Gly Gly
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Phe Asp Ile Gly Phe Ile Ala Gln Pro Gln Glu Lys Ala Pro Asp
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Pro Phe Arg His Phe Arg Ala Asp Asp Ala Asn Val Met Arg Asp
1190 1195 1200
Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln
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Ile Glu Ser Ile Met Ser Pro Asp Gly Thr Lys Lys Asn Pro Ala
1220 1225 1230
Arg Thr Cys Arg Asp Leu Lys Met Cys His Pro Asp Trp Lys Ser
1235 1240 1245
Gly Glu Tyr Trp Ile Asp Pro Asp Gln Gly Cys Asn Gln Asp Ala
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Ile Lys Val Tyr Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr
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Pro Thr Glu Ser Thr Ile Pro Lys Lys Asn Trp Tyr Thr Ser Lys
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Asn Ile Lys Glu Lys Lys His Val Trp Phe Gly Glu Ala Met Thr
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Asp Gly Phe Gln Phe Glu Tyr Gly Ser Glu Gly Ser Lys Ala Glu
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Asp Val Asn Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu
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Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Ile Ala Tyr
1340 1345 1350
Met Asp Gln Ala Ser Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln
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Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe
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Thr Tyr Ser Val Thr Glu Asp Gly Cys Thr Ser His Thr Gly Ala
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Trp Gly Lys Thr Val Ile Asp Tyr Lys Thr Thr Lys Thr Ser Arg
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Leu Pro Ile Ile Asp Ile Ala Pro Met Asp Val Gly Ala Pro Asn
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Gln Glu Phe Gly Ile Glu Val Gly Pro Val Cys Phe Leu
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Met Ser Gly Cys Ser Gly Ser Ser Cys Glu Gly Lys Cys Asp Cys Ser
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Gly Val Lys Gly Ala Lys Gly Glu Arg Gly Phe Pro Gly Leu Gln Gly
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Asn Met Gly Phe Pro Gly Met Gln Gly His Glu Gly Pro Ala Gly Pro
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Met Gly Pro Lys Gly Glu Tyr Gly Glu Ser Gly Thr Pro Gly Met Lys
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Gly Thr Arg Gly Pro Asn Gly Leu Pro Gly Phe Pro Gly Asn Pro Gly
65 70 75 80
Leu Pro Gly Ile Pro Gly Gln Asp Gly Pro Pro Gly Ser Pro Gly Ile
85 90 95
Pro Gly Cys Asn Gly Thr Lys Gly Asp Arg Gly Thr Asp Gly Gln Ser
100 105 110
Gly Phe Pro Gly Leu Gln Gly Pro Pro Gly Ile Pro Gly Leu Met Gly
115 120 125
Met Lys Gly Asp Ala Gly Gly Val Ile Gly Val Ile Pro Leu Lys Gly
130 135 140
Asp Lys Gly Phe Pro Gly Thr Pro Gly Leu Leu Gly Pro Asn Gly Pro
145 150 155 160
Ser Gly Pro Glu Gly Pro Pro Gly Asn Gln Gly Ser Asp Gly Pro Arg
165 170 175
Gly Phe Pro Gly Pro Pro Gly Pro Lys Gly Glu Lys Gly Asp Arg Leu
180 185 190
Ser Phe Gln Ser Glu Lys Gly Asp Lys Gly Gln Gln Gly Phe Arg Gly
195 200 205
Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser Gln Glu Ala Thr
210 215 220
Gly Asn Ala Val Ser His Tyr Leu Pro Gly Pro Pro Gly Gln Arg Gly
225 230 235 240
Asp Thr Gly Asp Arg Gly Glu Lys Gly Phe Cys Ile Pro His Leu Asn
245 250 255
Gly Val Lys Gly Glu Gln Gly Pro Pro Gly Pro Arg Gly Lys Pro Gly
260 265 270
Lys Asp Gly Asp Asn Gly Phe Lys Gly Glu Arg Gly Phe Pro Gly Gly
275 280 285
Pro Gly Tyr His Gly Thr Pro Gly Glu Lys Gly Glu Arg Gly Pro Pro
290 295 300
Ala Asn Gly Asp Gly Ala Pro Gly Pro Pro Gly Pro Pro Gly Leu Pro
305 310 315 320
Gly Leu Gln Gly Glu Arg Gly Phe Pro Gly Ile Gln Gly Ala Leu Gly
325 330 335
Leu Pro Gly Arg His Ile Glu Gly Pro Pro Gly Glu Lys Gly Arg Pro
340 345 350
Gly Glu Val Gly Gln Lys Gly Asp Arg Gly Ala Glu Gly Glu Ser Leu
355 360 365
Arg Gly Lys Pro Gly Gln Asp Gly Leu Ile Gly His Pro Gly Pro Pro
370 375 380
Gly Pro Pro Gly Asp Glu Pro Cys Asp Pro Ala Leu Glu Lys Gly Pro
385 390 395 400
Pro Gly Pro Pro Gly Pro Pro Gly Leu Gln Gly Glu Leu Gly Gln Lys
405 410 415
Gly Asp Gln Gly Asp Thr Cys Val Gln Cys Glu Ala Leu Gly Pro Pro
420 425 430
Gly Ile Pro Gly Pro Gln Gly Pro Lys Gly Leu Gln Gly Tyr Pro Gly
435 440 445
Ala Ala Gly Ser Lys Gly Asp Lys Gly Val Pro Gly Pro Ala Gly Leu
450 455 460
Asp Gly Ser Pro Gly Asn Ser Gly Thr Pro Gly Leu Val Gly Ala Pro
465 470 475 480
Gly Ala His Gly Glu Pro Gly Asp Ile Tyr Leu Ala Pro Gly Leu Lys
485 490 495
Gly Asp Lys Gly Leu Pro Gly Phe Val Gly Ala Gln Gly Leu Pro Gly
500 505 510
Val Asp Gly Leu Pro Gly Lys Glu Gly Leu Ala Gly Leu Pro Gly Ser
515 520 525
Lys Gly Glu Pro Ala Arg Glu Gly Ile Lys Gly Asp Arg Gly Leu Asp
530 535 540
Gly Asp Pro Gly Phe Ser Gly Pro Pro Gly Glu Arg Gly Pro Pro Gly
545 550 555 560
Val Pro Gly Phe Gly Arg Pro Gly Glu Pro Gly Glu Lys Gly Thr Ser
565 570 575
Gly Gln Gln Gly Lys Pro Gly Ile Pro Gly Gln Pro Gly Leu Lys Gly
580 585 590
Glu Pro Gly Lys Asp Val Ser Ser Pro Gly Pro Gln Gly Ser Pro Gly
595 600 605
Pro Arg Gly Gln Ser Gly Ile Pro Gly Leu Gln Gly Asp Arg Gly Leu
610 615 620
Gln Gly Asp Pro Gly Met Pro Gly Phe Pro Gly Gln Lys Gly Asp Ser
625 630 635 640
Gly Val Pro Gly Ile Gly Phe Pro Gly Leu Pro Gly Pro Lys Gly Tyr
645 650 655
Ser Gly Ala Pro Gly Ala Pro Gly Leu Pro Gly Glu Pro Gly Arg Ser
660 665 670
Gly Gln Asp Gly Phe Ser Gly Lys Pro Gly Ile Pro Gly Pro Lys Gly
675 680 685
Glu Pro Gly Gln Gly Leu Pro Gly Pro Lys Gly Ser Gln Gly Pro Pro
690 695 700
Gly Glu Thr Gly Phe His Gly Asp Lys Gly Ser Val Gly Pro Thr Gly
705 710 715 720
Ile Pro Gly Gln Glu Gly His Thr Gly Pro Pro Gly Ala Gln Gly Ile
725 730 735
Gln Gly Asp Pro Gly Pro Pro Gly Gln His Gly Gly Thr Gly Pro Pro
740 745 750
Gly Pro Pro Gly Ala Gly Glu Pro Gly Ala Pro Gly Pro Ile Gly Pro
755 760 765
Pro Gly Glu Pro Gly Pro Phe Gly His Asp Gly Val Lys Gly Asp Lys
770 775 780
Gly Phe Pro Gly Ser Pro Gly Leu Asp Met Pro Gly Pro Gln Gly Glu
785 790 795 800
Lys Gly Asp Ser Gly Phe Pro Gly Leu Ser Gly Ser Lys Gly Leu Pro
805 810 815
Gly Arg Pro Gly Pro Ala Gly Arg Asp Gly Phe Pro Gly Asp Pro Gly
820 825 830
Leu Lys Gly Glu Met Gly Val Met Gly Met Pro Gly Thr Pro Gly Tyr
835 840 845
Gln Gly Ser Ala Gly Ser Pro Gly Thr Pro Gly Gln Arg Gly Asn Pro
850 855 860
Gly Val Ser Gly Pro Arg Gly Glu Phe Gly Glu Pro Gly Pro Lys Gly
865 870 875 880
Glu Arg Gly Glu Pro Gly Leu Gln Gly Pro Pro Gly Asn Met Ser Glu
885 890 895
Leu Asn Met Glu His Met Lys Gly Glu Lys Gly Asp Ser Gly Asp Pro
900 905 910
Gly Asp Pro Gly His Thr Gly Glu Lys Gly Tyr Pro Gly Gln Ala Gly
915 920 925
Val Pro Gly Met Pro Gly Lys Asp Gly Glu Pro Gly Thr Pro Gly Gln
930 935 940
Pro Gly Glu Lys Gly Asp Thr Gly Val Pro Gly Glu Pro Gly Ser Thr
945 950 955 960
Gly Tyr Pro Gly Asn Lys Gly Ser Ile Gly Glu Met Gly Tyr Pro Gly
965 970 975
Ser Lys Gly Ser Lys Gly Ala Lys Gly Ile Val Gly Thr Thr Gly His
980 985 990
Pro Gly Phe Arg Gly Thr Glu Gly Val Lys Gly Asp Lys Gly Thr Ala
995 1000 1005
Gly Leu Pro Gly Val Gly Val Pro Gly Pro Pro Gly Glu Lys Gly
1010 1015 1020
Gln Leu Gly Leu Pro Gly Phe Pro Gly Asn Ala Gly Glu Lys Gly
1025 1030 1035
Gln Lys Gly Gly Met Gly Val Pro Gly Met Pro Gly Thr Pro Gly
1040 1045 1050
Thr Lys Gly Asp Thr Gly Tyr Ile Gly His Pro Gly Gln Pro Gly
1055 1060 1065
Arg Pro Gly Glu Lys Gly Val Gly Gly Leu Pro Gly Ser Thr Gly
1070 1075 1080
Glu Pro Gly Gln Thr Gly Arg Pro Gly Glu Pro Gly Leu Gln Gly
1085 1090 1095
Pro Pro Gly Pro Thr Gly Glu Lys Gly Glu Ser Gly Val Asp Gly
1100 1105 1110
Ile Pro Gly Ser Ser Gly Asp Arg Gly Asp Gln Gly Phe Pro Gly
1115 1120 1125
Arg Gly Phe Pro Gly Thr Pro Gly Ser Ser Gly Leu Lys Gly Asp
1130 1135 1140
Lys Gly Ser Pro Gly Phe Pro Gly Ser Pro Gly Ile Pro Gly Ile
1145 1150 1155
Pro Gly Thr Arg Gly Glu Lys Gly Thr Ala Gly Phe Gln Gly Ser
1160 1165 1170
Leu Gly Gln Pro Gly Glu Gln Gly His Pro Gly Pro Ala Met Glu
1175 1180 1185
Gly Pro Lys Gly Asp Gln Gly Val Pro Gly Lys Pro Gly Glu Pro
1190 1195 1200
Gly Thr Ser Gly Val Pro Gly Pro Thr Gly Val Pro Gly Ser Ala
1205 1210 1215
Gly Ala Lys Gly Asp Lys Gly Asp Gln Gly Gly Gln Gly Val Gln
1220 1225 1230
Gly Glu Gln Gly Leu Lys Gly Glu Arg Gly Tyr Ser Gly Leu Pro
1235 1240 1245
Gly Gln Ser Gly Leu Pro Gly Val Asp Gly Leu Lys Gly Glu Met
1250 1255 1260
Gly Leu His Gly Val Pro Gly Phe Pro Gly Thr Lys Gly Glu Leu
1265 1270 1275
Gly Val Phe Gly Leu Lys Gly Glu Leu Gly Asp Arg Gly Phe Pro
1280 1285 1290
Gly Thr Lys Gly Asn Asp Gly Pro Pro Gly Pro Pro Gly Leu His
1295 1300 1305
Thr Phe Ile Lys Gly Glu Ser Gly Phe Pro Gly Gly Gln Gly Pro
1310 1315 1320
Gln Gly Pro Val Gly Pro Ser Gly Phe Pro Gly Leu Lys Gly Gln
1325 1330 1335
Gln Gly Met Thr Gly Ile Gln Gly Ile Lys Gly Asp Glu Gly Asn
1340 1345 1350
Pro Gly Ile Asn Gly Leu Pro Gly Ala Lys Gly Glu Pro Gly Leu
1355 1360 1365
Leu Gly Pro Ser Gly Pro Arg Gly Tyr Pro Gly Pro Pro Gly Pro
1370 1375 1380
Asp Gly Val Pro Gly Gln Val Gly Pro Pro Gly Pro Ser Ser Met
1385 1390 1395
Glu His Gly Phe Leu Val Thr Arg His Ser Gln Ser Val Glu Val
1400 1405 1410
Pro Gln Cys Pro Glu Gly Thr Ser Leu Ile Tyr Asp Gly Tyr Ser
1415 1420 1425
Leu Leu Tyr Ile Gln Gly Asn Glu Arg Ser His Gly Gln Asp Leu
1430 1435 1440
Gly Thr Ala Gly Ser Cys Leu Arg Lys Phe Ser Pro Met Pro Phe
1445 1450 1455
Leu Phe Cys Asn Ile Asn Asn Val Cys Tyr Phe Ala Ser Arg Asn
1460 1465 1470
Asp Tyr Ser Tyr Trp Leu Thr Ser Pro Glu Pro Met Pro Met Asn
1475 1480 1485
Met Asp Pro Ile Thr Gly Gln Gly Ile Arg Pro Phe Ile Ser Arg
1490 1495 1500
Cys Ser Val Cys Glu Ala Pro Ala Met Val Ile Ala Val His Ser
1505 1510 1515
Gln Asn Ile Met Ile Pro Ser Cys Pro Asn Gly Trp Asp Ser Leu
1520 1525 1530
Trp Ile Gly Tyr Ser Phe Val Met His Thr Ser Ala Gly Ala Glu
1535 1540 1545
Gly Ser Gly Gln Ala Leu Ala Ser Pro Gly Ser Cys Leu Glu Glu
1550 1555 1560
Phe Arg Ser Ala Pro Phe Ile Glu Cys His Gly Arg Gly Thr Cys
1565 1570 1575
Asn Tyr Tyr Ala Asn Ser Tyr Ser Phe Trp Leu Ala Ala Ile Glu
1580 1585 1590
Asp Glu Glu Met Phe Thr Lys Pro Val Pro Thr Thr Leu Lys Ala
1595 1600 1605
Gly Ser Leu Arg Thr His Ile Ser Arg Cys Gln Val Cys Met Lys
1610 1615 1620
Arg Thr
1625
<210> 3
<211> 1418
<212> PRT
<213> Artificial sequence
<400> 3
Met Phe Ser Phe Leu Asp Ser Arg Thr Val Leu Leu Leu Val Ala Ser
1 5 10 15
Gln Val Val Leu Leu Ser Val Val Arg Cys Gln Glu Glu Asp Asp His
20 25 30
Val Thr Gly Ala Lys Gly Gln Lys Gly Glu Pro Gly Asp Ile Val Asp
35 40 45
Val Val Gly Pro Lys Gly Pro Pro Gly Pro Met Gly Pro Ser Gly Glu
50 55 60
Gln Gly Pro Arg Gly Glu Val Gly Leu Lys Gly Asp Lys Gly Asn Pro
65 70 75 80
Gly Pro Arg Gly Arg Asp Gly Glu Pro Gly Thr Pro Gly Asn Pro Gly
85 90 95
Pro Ala Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala Ala
100 105 110
Gln Met Ser Ala Gly Phe Asp Glu Lys Ser Gly Gly Ala Gln Met Gly
115 120 125
Val Met Gln Gly Pro Met Gly Pro Met Gly Pro Arg Gly Pro Pro Gly
130 135 140
Pro Ser Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Ala Pro Gly Glu
145 150 155 160
Ala Gly Glu Pro Gly Pro Ala Gly Pro Met Gly Pro Arg Gly Pro Ala
165 170 175
Gly Pro Pro Gly Lys Ala Gly Ser Asp Gly Glu Ala Gly Lys Pro Gly
180 185 190
Lys Ala Gly Glu Arg Gly Pro Ala Gly Pro Gln Gly Ala Arg Gly Phe
195 200 205
Pro Gly Thr Pro Gly Leu Pro Gly Ile Lys Gly His Arg Gly His Pro
210 215 220
Gly Leu Asp Gly Ala Lys Gly Glu Ser Gly Ala Ala Gly Ala Lys Gly
225 230 235 240
Glu Thr Gly Ser Ala Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro
245 250 255
Arg Gly Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Ala Gly Ala Ala
260 265 270
Gly Ala Arg Gly Asn Asp Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly
275 280 285
Pro Val Gly Pro Ala Gly Ala Pro Gly Phe Pro Gly Ser Pro Gly Ala
290 295 300
Lys Gly Glu Ala Gly Pro Thr Gly Asn Arg Gly Ala Glu Gly Gln Gln
305 310 315 320
Gly Pro Arg Gly Glu Ala Gly Thr Pro Gly Ser Pro Gly Pro Ala Gly
325 330 335
Ala Ser Gly Asn Pro Gly Thr Asp Gly Ile Pro Gly Ala Lys Gly Ser
340 345 350
Thr Gly Gly Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Pro Arg
355 360 365
Gly Pro Pro Gly Pro Gln Gly Ala Thr Gly Ser Leu Gly Pro Lys Gly
370 375 380
Gln Ser Gly Asp Pro Gly Leu Pro Gly Leu Lys Gly Glu Thr Gly Pro
385 390 395 400
Lys Gly Glu Leu Gly Pro Leu Gly Pro Gln Gly Ala Pro Gly Pro Ala
405 410 415
Gly Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Ala Ala Gly
420 425 430
Pro Ile Gly Pro Pro Gly Glu Arg Gly Ala Pro Gly Asn Arg Gly Phe
435 440 445
Pro Gly Gln Asp Gly Leu Ala Gly Gly Lys Gly Ala Pro Gly Asp Arg
450 455 460
Gly Val Pro Gly Ala Ala Gly Pro Lys Gly Thr Gly Gly Asp Pro Gly
465 470 475 480
Arg Pro Gly Glu Ser Gly Leu Pro Gly Ala Arg Gly Leu Thr Gly Arg
485 490 495
Pro Gly Asp Ala Gly Pro Gln Gly Lys Val Gly Ala Ser Gly Pro Ala
500 505 510
Gly Asp Asp Gly Arg Pro Gly Pro Pro Gly Pro Leu Gly Ala Arg Gly
515 520 525
Gln Pro Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Asn Gly Glu
530 535 540
Pro Gly Lys Pro Gly Glu Lys Gly Leu Leu Gly Arg Gln Gly Leu Arg
545 550 555 560
Gly Leu Pro Gly Lys Asp Gly Glu Thr Gly Ser Ala Gly Pro Pro Gly
565 570 575
Pro Ala Gly Pro Val Gly Glu Arg Gly Glu Gln Gly Gln Pro Gly Pro
580 585 590
Ser Gly Phe Gln Gly Leu Pro Gly Pro Ser Gly Ser Pro Gly Glu Ala
595 600 605
Gly Lys Pro Gly Asp Gln Gly Leu Pro Gly Glu Gly Gly Val Pro Gly
610 615 620
Ala Ala Gly Pro Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Gly
625 630 635 640
Ala Gly Pro Gln Gly Leu Gln Gly Pro Arg Gly Leu Pro Gly Thr Ala
645 650 655
Gly Ser Asp Gly Pro Lys Gly Ala Ile Gly Pro Ala Gly Ala Ala Gly
660 665 670
Pro Gln Gly Pro Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala
675 680 685
Gly Gly Ile Pro Gly Ala Lys Gly Asp Arg Gly Asp Leu Gly Glu Lys
690 695 700
Gly Pro Glu Gly Ala Pro Gly Lys Asp Gly Ser Arg Gly Leu Thr Gly
705 710 715 720
Pro Ile Gly Pro Pro Gly Pro Ser Gly Pro Asn Gly Ala Lys Gly Glu
725 730 735
Ser Gly Pro Val Gly Pro Asn Gly Ala Pro Gly Ser Arg Gly Thr Pro
740 745 750
Gly Asp Arg Gly Glu Ile Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly
755 760 765
Pro Pro Gly Ala Asp Gly Gln Pro Gly Val Lys Gly Glu Leu Gly Glu
770 775 780
Ser Gly Gln Lys Gly Asp Ser Gly Ser Pro Gly Pro Gln Gly Pro Ser
785 790 795 800
Gly Ala Pro Gly Pro Val Gly Pro Thr Gly Val Ser Gly Pro Lys Gly
805 810 815
Ala Arg Gly Ala Gln Gly Ala Pro Gly Ser Thr Gly Phe Pro Gly Ser
820 825 830
Ala Gly Arg Val Gly Pro Pro Gly Pro Asn Gly Asn Pro Gly Ala Ala
835 840 845
Gly Pro Ala Gly Pro Ala Gly Lys Asp Gly Pro Lys Gly Thr Arg Gly
850 855 860
Asp Ala Gly Pro Pro Gly Arg His Gly Asp Ala Gly Leu Arg Gly Pro
865 870 875 880
Pro Gly Gln Gln Gly Glu Lys Gly Glu Pro Gly Glu Asp Gly Pro Pro
885 890 895
Gly Ser Glu Gly Pro Ser Gly Pro Gln Gly Leu Gly Gly Ser Arg Gly
900 905 910
Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu
915 920 925
Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Ala Ser Gly Ser Ala
930 935 940
Gly Asp Arg Gly Pro Pro Gly Pro Val Gly Pro Pro Gly Leu Thr Gly
945 950 955 960
Pro Ala Gly Asp Pro Gly Arg Glu Gly Ala Pro Gly Ser Asp Gly Pro
965 970 975
Pro Gly Arg Asp Gly Ala Ser Gly Val Lys Gly Glu Arg Gly Asn Ser
980 985 990
Gly Pro Ala Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly
995 1000 1005
Ser Val Gly Pro Leu Gly Lys Gln Gly Asp Arg Gly Glu Ala Gly
1010 1015 1020
Ala Gln Gly Pro Ala Gly Pro Pro Gly Leu Ala Gly Ala Arg Gly
1025 1030 1035
Met Ala Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Ala Gly
1040 1045 1050
Glu Ala Gly Glu Arg Gly Gln Lys Gly His Arg Gly Phe Thr Gly
1055 1060 1065
Leu Gln Gly Leu Pro Gly Pro Pro Gly Pro Ala Gly Asp Ser Gly
1070 1075 1080
Ala Ser Gly Pro Ala Gly Pro Gly Gly Pro Lys Gly Pro Pro Gly
1085 1090 1095
Pro Ala Gly Val Ser Gly Lys Asp Gly Ser Asn Gly Gln Pro Gly
1100 1105 1110
Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Ser Gly Glu Thr Gly
1115 1120 1125
Pro Ser Gly Pro Pro Gly Asn Thr Gly Pro Pro Gly Pro Pro Gly
1130 1135 1140
Pro Pro Gly Pro Gly Ile Asp Ile Ser Ala Phe Ala Gly Leu Gly
1145 1150 1155
Gln Thr Glu Lys Ser Pro Asp Pro Leu Arg Tyr Met Arg Ala Asp
1160 1165 1170
Glu Ala Ser Ser Ser Leu Arg Gln His Asp Val Glu Val Asp Ser
1175 1180 1185
Thr Leu Lys Ser Leu Asn Asn Gln Ile Glu Thr Leu Arg Ser Pro
1190 1195 1200
Asp Gly Thr Gln Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys
1205 1210 1215
Leu Cys His Pro Lys Trp Glu Ser Gly Asn Tyr Trp Val Asp Pro
1220 1225 1230
Asn Leu Gly Cys Thr Ala Asp Ala Met Lys Val Phe Cys Asn Met
1235 1240 1245
Glu Thr Gly Glu Thr Cys Val Tyr Pro Ser Ile Ala Lys Ile Pro
1250 1255 1260
Lys Lys Asn Trp Trp Ser Ser Lys Ser Lys Asp Arg Lys His Val
1265 1270 1275
Trp Phe Gly Glu Thr Met Asn Gly Gly Phe His Phe Ser Tyr Ala
1280 1285 1290
Gln Asp Gly Pro Ala Ala Ala Ala Ala Gly Val Gln Leu Asn Phe
1295 1300 1305
Leu Arg Leu Leu Ser Ala Glu Ala Ser Gln Asn Leu Thr Tyr His
1310 1315 1320
Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Ser Thr Gly Asn Leu
1325 1330 1335
Lys Lys Ala Met Leu Leu Gln Gly Ser Asn Glu Val Glu Ile Arg
1340 1345 1350
Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Leu Glu Asp Gly
1355 1360 1365
Cys Lys Lys His Thr Gly Arg Trp Gly Lys Thr Val Phe Glu Tyr
1370 1375 1380
Lys Thr Gln Lys Thr Ser Arg Leu Pro Ile Val Asp Ile Ala Pro
1385 1390 1395
Met Asp Ile Gly Gly Ala Asp Gln Glu Phe Gly Val Asp Val Gly
1400 1405 1410
Ala Val Cys Phe Leu
1415
<210> 4
<211> 1449
<212> PRT
<213> Artificial sequence
<400> 4
Met Phe Ser Phe Val Asp Ile Arg Leu Ala Leu Leu Leu Ser Ala Thr
1 5 10 15
Val Leu Leu Ala Arg Gly Gln Gly Glu Asp Asp Arg Thr Ala Gly Ser
20 25 30
Cys Thr Leu Asp Gly Gln Phe Tyr Asn Asp Arg Asp Val Trp Lys Pro
35 40 45
Glu Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Thr Val Met Cys Asp
50 55 60
Glu Val Ile Cys Glu Asp Thr Ser Asp Cys Pro Asn Pro Val Ile Pro
65 70 75 80
His Asp Glu Cys Cys Pro Ile Cys Pro Asp Asp Gly Phe Gln Glu Pro
85 90 95
Lys Val Glu Gly Pro Gln Gly Asp Arg Gly Ala Lys Gly Glu Pro Gly
100 105 110
Pro Ala Gly Phe Pro Gly Asn Asp Gly Ile Pro Gly Gln Pro Gly Leu
115 120 125
Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe
130 135 140
Ser Pro Gln Met Ser Gly Gly Phe Asp Glu Lys Ser Gly Gly Gly Met
145 150 155 160
Ser Met Pro Gly Pro Met Gly Pro Met Gly Pro Arg Gly Pro Pro Gly
165 170 175
Pro Pro Gly Ser Ser Gly Pro Gln Gly Phe Thr Gly Pro Pro Gly Glu
180 185 190
Pro Gly Glu Ala Gly Ser Ser Gly Pro Met Gly Pro Arg Gly Pro Ala
195 200 205
Gly Pro Pro Gly Lys Asn Gly Asp Asp Gly Glu Ser Gly Lys Pro Gly
210 215 220
Arg Pro Gly Glu Arg Gly Ala Ser Gly Pro Gln Gly Ala Arg Gly Phe
225 230 235 240
Pro Gly Thr Pro Gly Leu Pro Gly Ile Lys Gly His Arg Gly Phe Ser
245 250 255
Gly Leu Asp Gly Ala Lys Gly Glu Ser Gly Pro Ala Gly Pro Lys Gly
260 265 270
Glu Gly Gly Ala Ser Gly Glu Asn Gly Ala Ala Gly Ala Met Gly Pro
275 280 285
Arg Gly Leu Pro Gly Glu Arg Gly Arg Ala Gly Pro Asn Gly Ala Ala
290 295 300
Gly Ala Arg Gly Asn Asp Gly Ala Ala Gly Ala Ala Gly Pro Pro Gly
305 310 315 320
Pro Thr Gly Pro Ala Gly Ala Pro Gly Phe Pro Gly Gly Pro Gly Ala
325 330 335
Lys Gly Glu Val Gly Ala Gln Gly Ala Arg Gly Gly Glu Gly Pro Gln
340 345 350
Gly Ser Arg Gly Glu Ala Gly Asn Pro Gly Pro Ala Gly Ala Ala Gly
355 360 365
Pro Ala Gly Asn Asn Gly Ala Asp Gly Asn Pro Gly Thr Lys Gly Ala
370 375 380
Pro Gly Ser Ser Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Pro Arg
385 390 395 400
Gly Pro Pro Gly Pro Gln Gly Ala Gly Gly Ala Pro Gly Pro Lys Gly
405 410 415
Asn Thr Gly Glu Val Gly Ala Asn Gly Ala Lys Gly Glu Ala Gly Ala
420 425 430
Lys Gly Glu Ser Gly Pro Ala Gly Val Gln Gly Pro Ala Gly Pro Ala
435 440 445
Gly Glu Glu Gly Lys Arg Gly Gly Arg Gly Glu Pro Gly Gly Ala Gly
450 455 460
Ala Arg Gly Ala Pro Gly Glu Arg Gly Ala Pro Gly Ser Arg Gly Phe
465 470 475 480
Pro Gly Ser Asp Gly Ala Ser Gly Pro Lys Gly Gly Pro Gly Glu Arg
485 490 495
Gly Gly Ala Gly Val Ala Gly Ala Lys Gly Asn Thr Gly Glu Pro Gly
500 505 510
Arg Asn Gly Glu Pro Gly Met Pro Gly Ser Lys Gly Met Thr Gly Ser
515 520 525
Pro Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Ser Gly Ala Gly
530 535 540
Gly Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Val Gly Ala Arg Gly
545 550 555 560
Gln Pro Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu
565 570 575
Gly Gly Lys Pro Gly Glu Arg Gly Val Met Gly Pro Ser Gly Ala Val
580 585 590
Gly Ala Pro Gly Lys Asp Gly Asp Val Gly Ala Pro Gly Ala Pro Gly
595 600 605
Val Ala Gly Pro Ser Gly Glu Arg Gly Glu Gln Gly Ala Gly Gly Pro
610 615 620
Pro Gly Phe Gln Gly Leu Ser Gly Pro Gln Gly Ala Ile Gly Glu Thr
625 630 635 640
Gly Lys Pro Gly Glu Gln Gly Leu Pro Gly Glu Gly Gly Ala Pro Gly
645 650 655
Ser Ala Gly Ser Arg Gly Asp Arg Gly Phe Pro Gly Glu Arg Gly Ala
660 665 670
Pro Gly Pro Ser Gly Pro Ala Gly Ala Arg Gly Ser Pro Gly Ser Ala
675 680 685
Gly Asn Asp Gly Gly Lys Gly Glu Ala Gly Ala Ala Gly Ala Pro Gly
690 695 700
Gly Gln Gly Pro Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala
705 710 715 720
Gly Gly Leu Pro Gly Leu Lys Gly Asp Arg Gly Asp Gln Gly Val Lys
725 730 735
Gly Ala Asp Gly Ala Gly Gly Lys Asp Gly Val Arg Gly Met Thr Gly
740 745 750
Pro Ile Gly Pro Asn Gly Pro Ala Gly Ser Pro Gly Asp Lys Gly Glu
755 760 765
Thr Gly Ala Pro Gly Ala Val Gly Pro Ser Gly Ala Arg Gly Ala Pro
770 775 780
Gly Glu Arg Gly Glu Ser Gly Ala Pro Gly Pro Ala Gly Phe Ala Gly
785 790 795 800
Pro Pro Gly Gly Asp Gly Gln Pro Gly Ala Lys Gly Glu Ala Gly Asp
805 810 815
Asn Gly Ala Lys Gly Asp Gly Gly Ala Gln Gly Pro Ala Gly Pro Thr
820 825 830
Gly Ala Pro Gly Pro Gln Gly Pro Ala Gly Asn Thr Gly Ala Lys Gly
835 840 845
Ala Arg Gly Ala Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala
850 855 860
Ala Gly Arg Phe Gly Pro Pro Gly Pro Ser Gly Asn Asn Gly Pro Pro
865 870 875 880
Gly Thr Pro Gly Pro Gly Gly Lys Glu Gly Gln Lys Gly Asn Arg Gly
885 890 895
Glu Thr Gly Pro Ala Gly Arg Pro Gly Glu Leu Gly Ala Ala Gly Pro
900 905 910
Pro Gly Pro Lys Gly Glu Lys Gly Gln Pro Gly Gly Asp Gly Pro Asn
915 920 925
Gly Pro Ser Gly Thr Pro Gly Pro Gln Gly Ile Gly Gly Gln Arg Gly
930 935 940
Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu
945 950 955 960
Ala Gly Gln Leu Gly Glu Pro Gly Lys Gln Gly Pro Gly Gly Pro Phe
965 970 975
Gly Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly
980 985 990
Ala Pro Gly Glu Pro Gly Arg Glu Gly Thr Pro Gly Asn Glu Gly Ser
995 1000 1005
Ser Gly Arg Asp Gly Ala Ala Gly Pro Lys Gly Glu Arg Gly Glu
1010 1015 1020
Ser Gly Val Ala Gly Ala Ser Gly Ala Pro Gly Pro Pro Gly Ala
1025 1030 1035
Pro Gly Ala Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu
1040 1045 1050
Ser Gly Pro Ala Gly Pro Ala Gly Ile Ala Gly Pro Ala Gly Pro
1055 1060 1065
Arg Gly Pro Ser Gly Pro Ala Gly Ala Arg Gly Asp Lys Gly Glu
1070 1075 1080
Ser Gly Glu Ala Gly Glu Arg Gly Met Lys Gly His Arg Gly Phe
1085 1090 1095
Thr Gly Met Gln Gly Pro Pro Gly Pro Ser Gly Gln Ser Gly Glu
1100 1105 1110
Ser Gly Pro Ala Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro
1115 1120 1125
Ser Gly Ser Ala Gly Ala Ala Gly Lys Asp Gly Met Ser Gly Leu
1130 1135 1140
Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Ser Gly Glu
1145 1150 1155
Met Gly Pro Ser Gly Thr Pro Gly Pro Pro Gly Pro Pro Gly Pro
1160 1165 1170
Pro Gly Pro Pro Gly Gly Gly Phe Asp Met Gly Phe Ile Ala Gln
1175 1180 1185
Pro Ala Gln Glu Lys Ala Pro Asp Pro Phe Arg His Phe Arg Ala
1190 1195 1200
Asp Asp Ala Asn Val Met Arg Asp Arg Asp Leu Glu Val Asp Thr
1205 1210 1215
Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro
1220 1225 1230
Glu Gly Thr Lys Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys
1235 1240 1245
Met Cys His Pro Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro
1250 1255 1260
Asp Gln Gly Cys Thr Gln Asp Ala Ile Lys Val Tyr Cys Asn Met
1265 1270 1275
Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Glu Ala Asp Ile Pro
1280 1285 1290
Lys Lys Ser Trp Tyr Thr Ser Lys Asn Ile Lys Glu Lys Lys His
1295 1300 1305
Val Trp Phe Gly Glu Ala Met Thr Asp Gly Phe Gln Phe Glu Tyr
1310 1315 1320
Gly Ser Glu Gly Ser Asn Ala Lys Asp Val Asn Ile Gln Leu Thr
1325 1330 1335
Phe Leu Arg Leu Met Ala Thr Glu Ala Ser Gln Asn Ile Thr Tyr
1340 1345 1350
His Cys Lys Asn Ser Ile Ala Tyr Met Asp Gln Gln Ser Gly Asn
1355 1360 1365
Leu Lys Lys Ser Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile
1370 1375 1380
Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Glu Asp
1385 1390 1395
Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile Asp
1400 1405 1410
Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Ile Ala
1415 1420 1425
Pro Met Asp Val Gly Ala Pro Asn Gln Glu Phe Gly Ile Glu Val
1430 1435 1440
Gly Pro Val Cys Phe Leu
1445
<210> 5
<211> 1485
<212> PRT
<213> 5
<400> 5
Met Phe Ser Phe Val Asp Ile Arg Leu Ala Leu Leu Leu Ser Ala Thr
1 5 10 15
Val Leu Leu Ala Arg Gly Gln Gly Glu Asp Asp Arg Thr Gly Gly Ser
20 25 30
Cys Thr Leu Asp Gly Gln Val Tyr Asn Asp Arg Asp Val Trp Lys Pro
35 40 45
Glu Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Thr Val Met Cys Asp
50 55 60
Glu Val Ile Cys Glu Asp Thr Thr His Cys Pro Asn Pro Val Ile Pro
65 70 75 80
His Asp Glu Cys Cys Pro Val Cys Pro Asp Asp Glu Phe Leu Glu Pro
85 90 95
Ser Val Glu Gly Pro Ser Gly Pro Ala Gly Glu Lys Gly Asp Arg Gly
100 105 110
Pro Pro Gly Pro Pro Gly Asn Asp Gly Ile Pro Gly Gln Pro Gly Leu
115 120 125
Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe
130 135 140
Ser Pro Gln Met Ser Gly Gly Tyr Asp Glu Lys Ser Gly Gly Ala Met
145 150 155 160
Ala Val Pro Gly Pro Met Gly Ala Met Gly Pro Arg Gly Pro Pro Gly
165 170 175
Pro Pro Gly Thr Pro Gly Pro Gln Gly Phe Thr Gly Pro Pro Gly Glu
180 185 190
Pro Gly Glu Ala Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Ala
195 200 205
Gly Pro Pro Gly Lys Asn Gly Glu Asp Gly Glu Ser Gly Lys Pro Gly
210 215 220
Arg Pro Gly Glu Arg Gly Pro Pro Gly Ala Gln Gly Ala Arg Gly Phe
225 230 235 240
Pro Gly Thr Pro Gly Leu Pro Gly Ile Lys Gly His Arg Gly Phe Ser
245 250 255
Gly Leu Asp Gly Ser Lys Gly Asp Thr Gly Pro Ala Gly Pro Lys Gly
260 265 270
Glu Pro Gly Ala Ala Gly Glu Asn Gly Thr Pro Gly Ala Met Gly Pro
275 280 285
Arg Gly Leu Pro Gly Glu Arg Gly Arg Ala Gly Pro Pro Gly Ala Ala
290 295 300
Gly Ala Arg Gly Asn Asp Gly Ala Ala Gly Ala Ala Gly Pro Pro Gly
305 310 315 320
Pro Thr Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Gly Pro Gly Ala
325 330 335
Lys Gly Glu Val Gly Ala Gln Gly Ala Arg Gly Ala Glu Gly Pro Gln
340 345 350
Gly Ala Arg Gly Glu Pro Gly Asn Pro Gly Pro Ala Gly Ala Ala Gly
355 360 365
Pro Ala Gly Asn Asn Gly Ala Asp Gly Ala Pro Gly Leu Lys Gly Ala
370 375 380
Pro Gly Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Pro Arg
385 390 395 400
Gly Pro Ser Gly Pro Ala Gly Ala Ala Gly Ala Pro Gly Pro Lys Gly
405 410 415
Asn Thr Gly Glu Val Gly Ala Pro Gly Ala Lys Gly Glu Ala Gly Ala
420 425 430
Lys Gly Glu Ala Gly Ala Gln Gly Val Gln Gly Pro Pro Gly Pro Ser
435 440 445
Gly Glu Glu Gly Lys Arg Gly Pro Arg Gly Glu Pro Gly Ser Ala Gly
450 455 460
Ser Arg Gly Pro Pro Gly Glu Arg Gly Ala Pro Gly Ala Arg Gly Phe
465 470 475 480
Pro Gly Ala Asp Gly Ser Ala Gly Pro Lys Gly Ala Thr Gly Glu Arg
485 490 495
Gly Gly Pro Gly Ile Val Gly Pro Lys Gly Ala Thr Gly Glu Pro Gly
500 505 510
Arg Asn Gly Glu Pro Gly Leu Pro Gly Ser Lys Gly Met Thr Gly Ser
515 520 525
Pro Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly Ala Pro Gly Asn Pro
530 535 540
Gly Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Val Gly Ala Arg Gly
545 550 555 560
Gln Pro Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu
565 570 575
Ala Gly Lys Pro Gly Glu Arg Gly Val Met Gly Ala Leu Gly Ala Thr
580 585 590
Gly Ala Pro Gly Lys Asp Gly Asp Val Gly Ala Pro Gly Ala Pro Gly
595 600 605
Pro Ala Gly Pro Thr Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Pro
610 615 620
Pro Gly Phe Gln Gly Leu Thr Gly Pro Gln Gly Ala Thr Gly Glu Pro
625 630 635 640
Gly Lys Ala Gly Glu Gln Gly Val Pro Gly Glu Ala Gly Ala Pro Gly
645 650 655
Pro Ser Gly Ser Arg Gly Asp Arg Gly Phe Pro Gly Glu Arg Gly Ala
660 665 670
Pro Gly Pro Ala Gly Pro Ala Gly Ala Arg Gly Ser Pro Gly Ser Ala
675 680 685
Gly Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly
690 695 700
Ala Gln Gly Pro Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ser
705 710 715 720
Ala Gly Leu Pro Gly Leu Lys Gly Asp Arg Gly Asp Gln Gly Ala Lys
725 730 735
Gly Thr Asp Gly Ala Pro Gly Lys Asp Gly Ile Arg Gly Met Thr Gly
740 745 750
Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu
755 760 765
Thr Gly Ala Pro Gly Leu Val Gly Pro Asn Gly Ala Arg Gly Pro Pro
770 775 780
Gly Glu Arg Gly Glu Thr Gly Ala Pro Gly Pro Ala Gly Phe Ala Gly
785 790 795 800
Pro Pro Gly Ala Asp Gly Leu Pro Gly Ala Lys Gly Glu Pro Gly Asp
805 810 815
Asn Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Pro Ala Gly Ala Thr
820 825 830
Gly Ala Pro Gly Pro Gln Gly Pro Val Gly Ser Thr Gly Pro Lys Gly
835 840 845
Ala Arg Gly Ala Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala
850 855 860
Ala Gly Arg Val Gly Pro Pro Gly Pro Ala Gly Asn Ala Gly Pro Ala
865 870 875 880
Gly Pro Ser Gly Ala Pro Gly Lys Glu Gly Gln Lys Gly Asn Arg Gly
885 890 895
Glu Thr Gly Pro Ala Gly Arg Thr Gly Glu Val Gly Ala Ala Gly Pro
900 905 910
Pro Gly Ala Pro Gly Glu Lys Gly Asn Pro Gly Ala Glu Gly Ala Pro
915 920 925
Gly Ser Ala Gly Thr Pro Gly Pro Ala Gly Ile Ala Gly Gln Arg Gly
930 935 940
Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu
945 950 955 960
Pro Gly Gln Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Pro Ser
965 970 975
Gly Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly
980 985 990
Pro Pro Gly Glu Pro Gly Arg Glu Gly Thr Pro Gly Asn Glu Gly Ser
995 1000 1005
Ala Gly Arg Asp Gly Ala Ala Gly Pro Lys Gly Asp Arg Gly Glu
1010 1015 1020
Thr Gly Ser Ala Gly Thr Pro Gly Ala Pro Gly Pro Pro Gly Ala
1025 1030 1035
Pro Gly Pro Ile Gly Pro Ala Gly Lys Thr Gly Asp Arg Gly Glu
1040 1045 1050
Ser Gly Pro Ala Gly Pro Ala Gly Ala Val Gly Pro Ala Gly Pro
1055 1060 1065
Arg Gly Pro Ala Gly Pro Ala Gly Ala Arg Gly Asp Arg Gly Glu
1070 1075 1080
Thr Gly Glu Ala Gly Glu Arg Gly Met Lys Gly His Arg Gly Phe
1085 1090 1095
Thr Gly Met Gln Gly Pro Pro Gly Pro Pro Gly Pro Ser Gly Glu
1100 1105 1110
Pro Gly Pro Ala Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro
1115 1120 1125
Gly Gly Ser Ala Gly Ala Ala Gly Lys Asp Gly Met Ser Gly Leu
1130 1135 1140
Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Asn Gly Glu
1145 1150 1155
Ile Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Leu Pro Gly Pro
1160 1165 1170
Pro Gly Pro Ser Gly Gly Gly Phe Asp Ile Gly Phe Ile Ala Gln
1175 1180 1185
Pro Met Glu Lys Ala Pro Asp Pro Phe Arg Ser Tyr Arg Ala Asp
1190 1195 1200
Asp Ala Asn Val Met Arg Asp Arg Asp Leu Glu Val Asp Thr Thr
1205 1210 1215
Leu Lys Ser Leu Ser Gln Gln Ile Glu Ser Ile Met Ser Pro Asp
1220 1225 1230
Gly Thr Lys Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met
1235 1240 1245
Cys His Pro Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asp
1250 1255 1260
Gln Gly Cys Asn Gln Asp Ala Ile Lys Val Tyr Cys Asn Met Glu
1265 1270 1275
Thr Gly Glu Thr Cys Val Tyr Pro Ala Glu Ser Ser Ile Pro Lys
1280 1285 1290
Lys Asn Trp Tyr Thr Ser Lys Asn Ile Lys Glu Lys Lys His Val
1295 1300 1305
Trp Phe Gly Glu Ala Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly
1310 1315 1320
Ser Glu Gly Ser Lys Pro Glu Asp Val Asn Ile Gln Leu Thr Phe
1325 1330 1335
Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His
1340 1345 1350
Cys Lys Asn Ser Ile Ala Tyr Met Asp Gln Ala Ser Gly Asn Leu
1355 1360 1365
Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg
1370 1375 1380
Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Glu Asp Gly
1385 1390 1395
Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile Asp His
1400 1405 1410
Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Ile Ala Pro
1415 1420 1425
Met Asp Val Gly Ala Pro Asn Gln Glu Phe Gly Ile Glu Val Gly
1430 1435 1440
Pro Val Cys Phe Leu Asp His Lys Thr Thr Lys Thr Ser Arg Leu
1445 1450 1455
Pro Ile Ile Asp Ile Ala Pro Met Asp Val Gly Ala Pro Asn Gln
1460 1465 1470
Glu Phe Gly Ile Glu Val Gly Pro Val Cys Phe Leu
1475 1480 1485
<210> 6
<211> 1446
<212> PRT
<213> 6
<400> 6
Met Phe Ser Phe Val Asp Ile Arg Leu Val Leu Leu Leu Ala Ala Thr
1 5 10 15
Ala Leu Leu Ala Arg Gly Gln Gly Glu Asp Asp Gln Ile Gly Thr Ser
20 25 30
Cys Thr Leu Asp Gly Gln Leu Tyr Asn Asn Lys Asp Val Trp Lys Pro
35 40 45
Glu Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Thr Val Met Cys Asp
50 55 60
Glu Val Ile Cys Glu Asp Thr Ser Asp Cys Pro Asn Pro Glu Ile Pro
65 70 75 80
Phe Gly Asp Cys Cys Pro Ile Cys Pro Gln Gly Pro Asp Lys Gly Pro
85 90 95
Pro Gly Asp Asp Gly Pro Lys Gly Asp Arg Gly Leu Thr Gly Pro Arg
100 105 110
Gly Asn Asp Gly Ile Pro Gly Gln Pro Gly Leu Pro Gly Pro Pro Gly
115 120 125
Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ser Pro Gln Met Ser
130 135 140
His Gly Tyr Asp Ala Lys Ser Gly Gly Gly Asp Met Ala Met Pro Gly
145 150 155 160
Pro Met Gly Pro Met Gly Pro Arg Gly Pro Pro Gly Pro Pro Gly Leu
165 170 175
Asn Gly Pro Gln Gly Phe Pro Gly Pro Ala Gly Glu Pro Gly Glu Pro
180 185 190
Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Ala Gly Pro Pro Gly
195 200 205
Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg Pro Gly Glu
210 215 220
Arg Gly Pro Ser Gly Pro Gln Gly Gly Arg Gly Phe Pro Gly Thr Pro
225 230 235 240
Gly Leu Pro Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Asp Gly
245 250 255
Ala Lys Gly Asp Ser Gly Pro Ala Gly Pro Lys Gly Glu Ala Gly His
260 265 270
His Gly Glu Asn Gly Ala Ala Gly Ala Met Gly Ala Arg Gly Leu Pro
275 280 285
Gly Glu Arg Gly Arg Pro Gly Pro Pro Gly Pro Ala Gly Ala Arg Gly
290 295 300
Asn Asp Gly Asn Ser Gly Ala Ala Gly Pro Pro Gly Pro Thr Gly Pro
305 310 315 320
Ala Gly Pro Pro Gly Phe Pro Gly Gly Ala Gly Pro Lys Gly Glu Thr
325 330 335
Gly Pro Ala Gly Gly Arg Gly Ser Glu Gly Pro Gln Gly Ser Arg Gly
340 345 350
Glu Pro Gly Asn Pro Gly Pro Ala Gly Pro Ala Gly Pro Ala Gly Asn
355 360 365
Pro Gly Ser Asp Gly Ala Pro Gly Ala Lys Gly Ser Pro Gly Ala Ala
370 375 380
Gly Ile Ala Gly Ala Ser Gly Phe Pro Gly Ser Arg Gly Ala Ala Gly
385 390 395 400
Gly Pro Gly Pro Gly Gly Ala Pro Gly Pro Lys Gly Asn Asn Gly Asp
405 410 415
Ala Gly Thr Pro Gly Pro Lys Gly Glu Pro Gly Thr Lys Gly Glu Pro
420 425 430
Gly Pro Ala Gly Ile Gln Gly Ala Pro Gly Pro Ser Gly Glu Glu Gly
435 440 445
Lys Arg Gly Gly Arg Gly Glu Pro Gly Gly Ala Gly Pro Arg Gly Pro
450 455 460
Pro Gly Glu Arg Gly Ala Pro Gly Asn Arg Gly Phe Pro Gly Ala Asp
465 470 475 480
Gly Ala Gly Gly Pro Lys Gly Ala Pro Gly Glu Arg Gly Pro Ser Gly
485 490 495
Pro Ala Gly Ala Gln Gly Ala Thr Gly Glu Ala Gly Arg Pro Gly Glu
500 505 510
Pro Gly Asn Pro Gly Ser Lys Gly Met Thr Gly Ser Pro Gly Ser Pro
515 520 525
Gly Pro Asp Gly Lys Thr Gly Pro Ser Gly Leu Pro Gly Gln Asp Gly
530 535 540
Arg Pro Gly Ala Pro Gly Pro Ala Gly Ser Arg Gly Ala Pro Gly Val
545 550 555 560
Met Gly Phe Pro Gly Pro Lys Gly Thr Ala Gly Asp Ala Gly Lys Pro
565 570 575
Gly Glu Arg Gly Ala Val Gly Pro Ala Gly Pro Leu Gly Ala Pro Gly
580 585 590
Lys Asp Gly Asp Val Gly Ala Pro Gly Ala Pro Gly Pro Ala Gly Pro
595 600 605
Ala Gly Glu Lys Gly Glu Gln Gly Pro Ala Gly Ala Pro Gly Phe Gln
610 615 620
Gly Leu Pro Gly Pro Gln Gly Ala Thr Gly Glu Ala Gly Lys Pro Gly
625 630 635 640
Glu Gly Gly Pro Ala Gly Glu Thr Gly Gly Pro Gly Pro Ser Gly Pro
645 650 655
Arg Gly Asp Arg Gly Phe Pro Gly Glu Arg Gly Ala Pro Gly Gly Val
660 665 670
Gly Pro Ala Gly His Arg Gly Ser Pro Gly Pro Ala Gly Asn Asp Gly
675 680 685
Pro Lys Gly Glu Pro Gly Ala Ala Gly Ala Pro Gly Ala Leu Gly Ala
690 695 700
Pro Gly Met Gln Gly Met Pro Gly Glu Arg Gly Ala Gly Gly Met Pro
705 710 715 720
Gly Ala Arg Gly Glu Arg Gly Asp Gly Gly Pro Lys Gly Ala Asp Gly
725 730 735
Gly Pro Gly Lys Asp Gly Leu Arg Gly Leu Thr Gly Pro Ile Gly Leu
740 745 750
Pro Gly Pro Pro Gly Gly Ala Gly Glu Lys Gly Glu Gly Gly Pro Val
755 760 765
Gly Pro Ala Gly Pro Thr Gly Gly Arg Gly Ala Pro Gly Glu Arg Gly
770 775 780
Glu Pro Gly Ala Pro Gly Pro Ala Gly Phe Ala Gly Pro Pro Gly Ala
785 790 795 800
Asp Gly Gln Pro Gly Ala Lys Gly Glu Thr Gly Asp Thr Gly Pro Lys
805 810 815
Gly Asp Ala Gly Ala Pro Gly His Ala Gly Pro Ala Gly Ala Ala Gly
820 825 830
Pro Gln Gly Pro Ala Gly Asn Ala Gly Pro Lys Gly Ala Arg Gly Gly
835 840 845
Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Val Gly Arg Val
850 855 860
Gly Ala Pro Gly Pro Ala Gly Val Ala Gly Pro Pro Gly Pro Pro Gly
865 870 875 880
Pro Gly Gly Lys Glu Gly Ala Arg Gly Asn Arg Gly Glu Thr Gly Ile
885 890 895
Ala Gly Arg Pro Gly Glu Pro Gly Pro Ala Gly Pro Ala Gly Pro His
900 905 910
Gly Glu Lys Gly Ser Ala Gly Ser Asp Gly Pro Ala Gly Ala Pro Gly
915 920 925
Ile Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Ile Val Gly Leu
930 935 940
Pro Gly Gln Arg Gly Glu Arg Gly Phe Gly Gly Leu Pro Gly Pro Ser
945 950 955 960
Gly Glu Pro Gly Lys Gln Gly Pro Val Gly Pro Ala Gly Glu Arg Gly
965 970 975
Pro Pro Gly Pro Met Gly Pro Pro Gly Met Ser Gly Ala Pro Gly Glu
980 985 990
Ala Gly Arg Glu Gly Ser Pro Gly His Asp Gly Ala Pro Gly Arg Asp
995 1000 1005
Gly Ala Ala Gly Pro Lys Gly Asp Arg Gly Glu Ser Gly Pro Ala
1010 1015 1020
Gly Ala Pro Gly Ala Pro Gly Pro Pro Gly Pro Pro Gly Ala Ile
1025 1030 1035
Gly Pro Ser Gly Lys Asn Gly Asp Arg Gly Glu Ala Gly Pro Ala
1040 1045 1050
Gly Pro Ser Gly Pro Ala Gly Pro Ala Gly Val Arg Gly Pro Ala
1055 1060 1065
Gly Pro Ala Gly Ala Arg Gly Asp Lys Gly Glu Ala Gly Glu Ala
1070 1075 1080
Gly Asp Arg Gly Met Lys Gly His Arg Gly Phe Ser Gly Leu Gln
1085 1090 1095
Gly Leu Pro Gly Pro Ala Gly Ala His Gly Glu Gln Gly Pro Ala
1100 1105 1110
Gly Pro Ser Gly Ala Pro Gly Pro Arg Gly Pro Ala Gly Ser Ser
1115 1120 1125
Gly Ser His Gly Lys Asp Gly Met Asn Gly Leu Pro Gly Pro Ile
1130 1135 1140
Gly Pro Pro Gly Pro Arg Gly Arg Ala Gly Glu Met Gly Pro Ala
1145 1150 1155
Gly Ala Pro Gly Leu Pro Gly Pro Pro Gly Pro Pro Gly Ala Pro
1160 1165 1170
Gly Gly Gly Phe Asp Phe Gly Phe Ile Ala Gln Pro Ser Gln Glu
1175 1180 1185
Lys Ala Pro Asp Pro Phe Arg Ser Gly Tyr Arg Ala Asp Asp Ala
1190 1195 1200
Asn Ser Val Arg Asn Arg Asp Val Glu Val Asp Thr Thr Leu Lys
1205 1210 1215
Ser Leu Ser Gln Lys Ile Glu Asn Ile Arg Ser Pro Glu Gly Thr
1220 1225 1230
Gln Lys Asn Pro Ala Arg Ala Cys Arg Asp Leu Lys Met Cys His
1235 1240 1245
Pro Glu Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly
1250 1255 1260
Ser Ala Leu Asp Ala Ile Lys Val Tyr Cys Asn Met Glu Thr Gly
1265 1270 1275
Gln Thr Cys Val Ala Pro Ser Gln Ala Glu Ile Ala Lys Lys Asn
1280 1285 1290
Trp Tyr Thr Ser Lys Asn Pro Lys Glu Lys Lys His Val Trp Phe
1295 1300 1305
Gly Glu Ser Met Thr Glu Gly Phe Gln Phe Gln Tyr Gly Ser Glu
1310 1315 1320
Gly Ser Asp Pro Glu Asp Val Asn Ile Gln Leu Thr Phe Leu Arg
1325 1330 1335
Leu Met Ala Asn Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys
1340 1345 1350
Asn Ser Ile Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys Lys
1355 1360 1365
Ala Leu Leu Leu Gln Gly Ser Asn Asp Ile Glu Ile Arg Ala Glu
1370 1375 1380
Gly Asn Ser Arg Phe Thr Tyr Ser Val Ser Glu Asp Gly Cys Thr
1385 1390 1395
Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile Asp Tyr Lys Thr
1400 1405 1410
Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Ile Ala Pro Met Asp
1415 1420 1425
Val Gly Ala Asn Asn Gln Glu Phe Gly Ile Glu Val Gly Pro Val
1430 1435 1440
Cys Phe Leu
1445

Claims (10)

1.含有Hyp-Gly序列寡肽或其盐形式，所述寡肽包括如下如下12种氨基酸序列所示寡肽的任1种或任多种组合：Pro-Gly-Glu-Hyp-Gly-Glu、Glu-Hyp-Gly-Glu、Hyp-Gly-Arg、Hyp-Gly-Ser-Glu、Hyp-Gly-Glu、Hyp-Gly-Ser-Ala、Arg-Hyp-Gly-Glu、Hyp-Gly-Gln、Pro-Gly-Glu-Hyp-Gly、Gly-Glu-Hyp-Gly、Hyp-Gly-Hyp-Met-Gly和Hyp-Gly-Glu-Phe-Gly。
2.根据权利要求1所述的寡肽或其盐形式，其特征在于：所述寡肽为人工合成；
或所述寡肽来自鱼皮明胶。
3.根据权利要求1或2所述的寡肽或其盐形式，其特征在于：所述鱼皮所属的鱼类为鲢鱼、大西洋鲑鱼、红鳍东方鲀、虹鳟鱼、鲫鱼或日本鳗鲡。
4.一种含有Hyp-Gly序列寡肽或其盐形式的酶解产物的制备方法，包括如下步骤：用碱性蛋白酶和胰蛋白酶酶解鱼类鱼皮的明胶，得到明胶酶解产物，即为含有Hyp-Gly序列寡肽或其盐的酶解产物。
5.根据权利要求4所述的方法，其特征在于：所述鱼类为鲢鱼、大西洋鲑鱼、红鳍东方鲀、虹鳟鱼、鲫鱼或日本鳗鲡。
6.根据权利要求4或5所述的方法，其特征在于：所述用碱性蛋白酶和胰蛋白酶酶解鱼类鱼皮的明胶包括如下步骤：
1)提取所述鱼类鱼皮明胶；
2)用所述碱性蛋白酶酶解所述明胶，得到第一次酶解产物；
3)用所述胰蛋白酶酶解所述第一次酶解产物，得到明胶酶解产物；
或，每种所述蛋白酶的使用量均为：所述蛋白酶与所述明胶的质量比为1:50-200；
或，所述碱性蛋白酶酶解条件为60℃酶解2-6h；
或，所述胰蛋白酶酶解条件为37℃酶解2-4h。
7.根据权利要求4-6中任一所述的方法，其特征在于：所述方法在酶解后还包括如下步骤：将所述明胶酶解产物层析纯化，收集不同组分；再检测不同组分中对抑制血小板聚集抑制，选取抑制率最高的组分作为目的组分，即为含有Hyp-Gly序列寡肽或其盐的酶解产物。
8.由权利要求4-6任一所述的方法制备的含有Hyp-Gly序列寡肽或其盐形式的酶解产物。
9.权利要求1所述的含有Hyp-Gly序列寡肽或其盐形式、权利要求8所述的含有Hyp-Gly序列寡肽或其盐形式的酶解产物在制备具有如下1)-6)中至少一种产品中的应用：
1)预防或辅助治疗心血管疾病；
2)预防或抑制血栓形成；
3)抑制动脉粥样硬化；
4)抑制动脉粥样硬化引起的疾病；
5)预防或抑制血小板聚集引起的疾病；
6)抑制血小板聚集。
10.一种产品，其中包含权利要求1所述的含有Hyp-Gly序列寡肽或其盐，或，权利要求8所述的含有Hyp-Gly序列寡肽或其盐形式的酶解产物；
所述产品具有如下1)-6)中至少一种功能：
1)预防或辅助治疗心血管疾病；
2)预防或抑制血栓形成；
3)抑制动脉粥样硬化；
4)抑制动脉粥样硬化引起的疾病；
5)预防或抑制血小板聚集引起的疾病；
6)抑制血小板聚集。
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CN112111003B (zh) * 2019-06-20 2022-02-11 中国农业大学 含有me序列的一类新型抑制血小板聚集和抗血栓形成寡肽
CN114032269A (zh) * 2021-10-19 2022-02-11 华南理工大学 一种富含二肽Hyp-Gly的胶原小分子肽及其制备方法和用途
CN114032269B (zh) * 2021-10-19 2023-08-04 华南理工大学 一种富含二肽Hyp-Gly的胶原小分子肽及其制备方法和用途
CN115353551A (zh) * 2022-06-27 2022-11-18 上海理工大学 一种燕麦源促glp-1分泌寡肽及其制备方法和应用
CN115353551B (zh) * 2022-06-27 2024-01-26 上海理工大学 一种燕麦源促glp-1分泌寡肽及其制备方法和应用
CN116143876A (zh) * 2022-09-08 2023-05-23 浙江大学 一种鲤鱼鳞抗菌肽及其制备方法和应用
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CN117143194A (zh) * 2023-11-01 2023-12-01 北京大学第一医院 抗血小板聚集多肽、其制备方法及应用
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