CN107865962A - 制备改良猪血浆纤连蛋白以增强伤口愈合的应用 - Google Patents

制备改良猪血浆纤连蛋白以增强伤口愈合的应用 Download PDF

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CN107865962A
CN107865962A CN201710874862.0A CN201710874862A CN107865962A CN 107865962 A CN107865962 A CN 107865962A CN 201710874862 A CN201710874862 A CN 201710874862A CN 107865962 A CN107865962 A CN 107865962A
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郭津岑
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Abstract

本发明揭示了可用于临床伤口愈合与组织修复的材料及其应用。为了寻求安全的血浆纤连蛋白来源以使用于伤口敷料的实际应用，本发明从猪血浆中分离并修饰纤连蛋白，经修饰后的猪血浆纤连蛋白适用于人体，且可以作为刺激细胞贴附和定向细胞移动的基础材料。本发明还揭示了一种增强伤口愈合的材料与一种医药组合物。

Description

制备改良猪血浆纤连蛋白以增强伤口愈合的应用
【技术领域】
本发明涉及改良猪血浆纤连蛋白以作为临床伤口愈合和组织修复的安全材料的用途。本发明还揭示了一种增强伤口愈合的材料与一种医药组合物。
【现有技术】
伤口愈合是一种动态过程，其包括止血、发炎、增生和重塑。纤连蛋白是细胞外基质(ECM)糖蛋白，在伤口愈合的不同阶段起重要作用，其主要功能是调节细胞贴附和细胞迁移。纤连蛋白作用以激活细胞表面整合素受体，其诱导一系列细胞蛋白质聚集以连接细胞内的肌动蛋白细胞骨架，以形成整联蛋白的黏着胞器，黏着斑(focal adhesions,FAs)。黏着斑(focal adhesions,FAs)连接细胞内的肌动蛋白细胞骨架(actin cytoskeleton)和细胞外基质(ECM)的纤连蛋白(fibronectin)以动力驱动伤口愈合中的定向细胞迁移。
伤口愈合时，肌动蛋白聚合(actin polymerization)产生细胞突起并连结成为致密的肌动蛋白网，然后往细胞外基质(ECM)纤连蛋白(fibronectin)的方向延伸，进而在接触后形成新生的黏着斑(nascent adhesions,new-born FAs)。进一步，新生的黏着斑经由肌动蛋白丝上肌球蛋白-II(myosin II)产生的收缩力进入成熟作用而增大。成熟的黏着斑提供细胞施力点，传递从肌动蛋白细胞骨架上肌球蛋白II驱动的收缩力到细胞外基质的纤连蛋白，而将细胞体向前拉。最后，伴随着肌球蛋白II驱动的收缩力将细胞后端的黏着斑从细胞外基质(ECM)纤连蛋白拔起以促使细胞后端回缩。细胞外基质(ECM)纤连蛋白在细胞外活化细胞表面整合素受体并促进黏着斑形成，以连接细胞内细胞骨架(actincytoskeleton)，所以在透过黏着斑(FAs)动态调控，控制伤口愈合过程中的细胞黏着和细胞移动。
纤连蛋白有两种形式：血浆纤连蛋白(plasma fibronectin)和细胞纤连蛋白(cellular fibronectin)。血浆纤维连接蛋白由肝细胞合成并分泌到血浆中，而细胞纤连蛋白是由许多细胞类型如纤维母细胞(fibroblasts)、内皮细胞 (endothelial cells)、肌原细胞(myocytes)和软骨细胞(chondrocytes)产生。在伤口愈合过程中，已经有许多研究表示血浆纤连蛋白在伤口产生时会累积在伤口处，这对血小板、纤维母细胞和内皮细胞的各种功能如黏着(adhesion)、迁移(migration)和聚集(aggregation)是至关重要的，显示血浆纤连蛋白适合作为加速伤口修复的物质。在动物实验中，含有血浆纤连蛋白的临时基质在表皮细胞黏着和迁移均有显著效果，这些先前研究显示血浆纤连蛋白在人伤口愈合和组织修复中的临床潜力。
然而，由人类血浆中取得的血浆纤连蛋白是既不安全而且昂贵的来源。尤其人血浆纤连蛋白对人伤口愈合的临床应用尚未得到验证，原因是来自人体的纤维连接蛋白并不适合用于医疗产品。过去的研究证明癌症患者中的纤连蛋白具有特异性异常糖基化修饰，其具有促进癌症转移的作用，因此若高纯度纤维连接蛋白是来自于未知健康状况的个体所提供的血液纯化而来，在使用上会产生医疗风险。另外，在过去的其他文献中，纤连蛋白纯化的方法太粗糙，不但纤连蛋白的纯度太低，且并未阐明其对糖基保护的作用。
回顾前人已发表的文献方法，包括利用基因重组的纤连蛋白重组体，和从人血液中纯化的纤维连接蛋白等方式皆是有缺陷的。由于纤连蛋白上的糖基化在促进伤口愈合中具有重要作用，而由基因重组表达的重组纤维连接蛋白并不具有糖基化修饰，因此其作用是有缺陷的。
【发明内容】
在发明人的研究中，发现人类和猪纤连蛋白有相似的聚糖(glycans)，且功能相似。此外，发明人进一步分析人血和猪血浆纤连蛋白上的糖基化 (glycosylation)位点和聚糖结构的特征，发现血浆纤维连接蛋白上的聚糖藉由调节细胞表面整合素受体介导的黏着讯号在细胞黏着和定向细胞迁移中具有重要影响。因此，猪血浆纤连蛋白的应用于伤口愈合，可以补足人血浆纤连蛋白应用可能产生的缺失，以取代人血浆纤连蛋白的功能。
经发明人研究，猪纤连蛋白上的唾液酸(sialic acids)的结构是N-乙酰神经氨酸(Neu5AC)和N-羟乙酰神经氨酸(Neu5GC)，而人纤连蛋白的结构仅为 Neu5AC，这样的结果显示Neu5GC在临床应用中具有引起人体免疫反应的可能性，但由于发明人研究证实，纤连蛋白的唾液酸在细胞黏附和定向细胞迁移中并不具备关键的作用，故本发明中特别针对猪血浆纤连蛋白中的唾液酸做去除，以修饰为较适合人类伤口愈合和组织修复的安全材料。
鉴于血浆纤连蛋白对伤口愈合的重要性，以及其在医学应用中的潜力，发明人从猪只的血浆中分离猪血浆纤连蛋白，并修饰为适合人类伤口愈合与组织修复的纤连蛋白，可作为替代人血浆纤连蛋白的材料，相较人血浆纤连蛋白具备较佳且安全性和更高的量。
为了解决前述的问题，本发明提供了一种用于制备治疗伤口愈合的纤连蛋白的用途，其中该纤连蛋白包含酵素修饰的糖基化的猪纤连蛋白。
在本发明的一个实施方案中，其中在猪纤连蛋白上被修饰的糖基是指复数个唾液酸分子糖基。
在本发明的一个实施方案中，其中所述唾液酸分子糖基为N-乙酰神经氨酸(Neu5Ac)和/或N-羟乙酰神经氨酸(Neu5GC)。
在本发明的一个实施方案中，其中所述唾液酸分子糖基被除去大于 80％。
在本发明的一个实施方案中，其中所述酵素为α2-3,6,8神经氨酸酶(α 2-3,6,8Neuraminidase)。
在本发明的一个实施方案中，其中所述酵素进一步包含蛋白酶，所述蛋白酶具备将纤连蛋白切割成纤连蛋白胜肽(纤连蛋白多肽)的功效。
在本发明的一个实施方案中，其中所述酵素包含基质金属蛋白酶 3(matrixmetalloproteinase 3)。
在本发明的一个实施方案中，所述糖基化的猪纤连蛋白由唯一的明胶- 琼脂糖凝胶快速流动4B的预柱(gelatin-Sepharose Fast Flow 4B)制备。
为了解决前述的问题，本发明提供了一种用于促进伤口愈合的医药组合物，包含：酵素修饰的糖基化的猪纤连蛋白；胶原蛋白；以及透明质酸；和/或其药学上可接受的盐。
为了解决前述的问题，本发明提供了一种用于促进伤口愈合的材料，包含酵素修饰的糖基化的猪纤连蛋白，其中被修饰的糖基是指猪纤连蛋白上复数个唾液酸分子糖基，其中所述唾液酸分子糖基被除去大于80％，其中所述酵素为α2-3,6,8神经氨酸酶(α2-3,6,8Neuraminidase)。
本发明提供了一种用于纯化修饰的糖基化的猪血浆纤连蛋白的方法，其包括：步骤1：提供干净的猪血浆；步骤2：使用干净的猪血浆通过明胶- 琼脂糖凝胶快速流动4B的预柱；步骤3：通过用含有EDTA(乙二胺四乙酸) 的TBS(tris buffered saline，三乙醇胺缓冲盐水溶液)顺序洗涤去除凝胶中非特异性吸附的蛋白质；步骤4：通过用1M NaCl溶液依次洗涤除去凝胶中非特异性吸附的蛋白质；步骤5：使用小于0.5摩尔浓度的精氨酸(Arg) 溶液顺序洗涤去除凝胶中非特异性吸附的蛋白质；步骤6：使用大于0.5摩尔浓度的精氨酸(Arg)溶液洗脱，制得猪血浆纤连蛋白样品。
本发明提供了修饰糖基化猪纤连蛋白的修饰方法，包括：步骤1：在缓冲液(pH5～7)中配置猪血浆纤维连接蛋白(1毫克)；步骤2：加入5～50 单位α2-3,6,8神经氨酸酶(一个单位定义为从1nmol Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-7-氨基-4-甲基香豆素(AMC)切割大于95％的末端α-Neu5Ac所需的酶量，在37℃下作用5分钟，总反应体积为10μl)；步骤3：在37℃下作用1～24小时。
本发明提供了修饰糖基化猪纤连蛋白的修饰方法，包括：将猪血浆纤连蛋白与基质金属蛋白酶3(matrix metalloproteinase-3,MMP3)以酵素-受质比在1:5到1:30的比例，于37℃环境中隔夜共同反应。
本发明提供猪血浆纤连蛋白经过修饰后，对于细胞黏附和定向细胞迁移的正调节具有正向作用，此外，将纤连蛋白适当地切割成纤连蛋白胜肽，则更有助于达成伤口愈合与组织修复的目的，故经修饰后的猪血浆纤连蛋白，可作为替代人血浆纤连蛋白材料，相较人血浆纤连蛋白具备较佳地安全性和更高的量。
【附图说明】
图1显示本发明的纯化方法。
图2显示从明胶-琼脂糖凝胶快速流动4B的预柱(gelatin-Sepharose Fast Flow4B column)获得的洗涤物质和洗脱物。
图3显示相对于所有检测到的N-糖基化位点的总光谱计数，具有或不具有唾液酸(sialic acids)的N-糖基化残基的光谱计数的百分比。
图4a-c显示使用人类和猪纤连蛋白测试U2OS、HFF1和Hela细胞伤口愈合效果。
图5显示将U2OS细胞接种于指定浓度的纤连蛋白(μg/ml)涂覆的盖玻片上30分钟，然后通过相差显微镜检查图像，比例尺：100微米。
图6显示将U2OS细胞接种于指定浓度的纤连蛋白(μg/ml)涂覆的盖玻片上贴附扩散的细胞面积。
图7显示将U2OS细胞接种于指定浓度的纤连蛋白上30分钟，然后测量其相对于0μg/ml纤连蛋白组别的细胞附着程度的倍数。
图8显示将U2OS细胞接种于指定的纤连蛋白浓度涂覆的盖玻片上1.5 小时，并用黏着斑标记蛋白(Paxillin)免疫荧光染色的TIRFM图像，比例尺： 10微米。
图9显示细胞对应指定浓度纤连蛋白(μg/ml)，其形成黏着斑蛋白(Paxillin)蛋白标记(paxillin-marked)的黏着斑总面积。
图10显示从猪纤连蛋白中去除唾液酸(sialic acid)不影响细胞纤连蛋白和细胞缔合的程度。
图11显示经基质金属蛋白酶3修饰前后的猪纤连蛋白变化。
图12显示经基质金属蛋白酶3修饰后的猪纤连蛋白较修饰前有较好的伤口愈合效果。
【实施方式】
本发明为了在伤口敷料中寻求安全的血浆纤维连接蛋白，发明人从人类 (homo)和猪(procine)的血浆中分离出纤连蛋白，证明猪血浆纤维连接蛋白具有与人血浆纤连蛋白相似的能力，可作为适合的伤口敷料，以刺激细胞黏附和定向细胞迁移。
本发明进一步鉴定猪血浆纤连蛋白上的糖基化位置，猪血浆纤连蛋白的这些糖基化修饰和其蛋白质功能具有协同作用以支持整合素受体所传递的信号，此为调节细胞黏附和定向细胞迁移所必须的。本研究不仅确定了聚糖在人和猪血浆纤连蛋白调解的细胞黏附和定向细胞迁移中的重要功能，而且揭示了猪血浆纤连蛋白可被作为临床伤口愈合和组织修复材料的潜在应用价值。
实施例1.材料和细胞
U2OS(人骨肉瘤细胞系)和Hela(人宫颈腺癌上皮细胞系)是由陈瑞华教授的实验室(中央研究院，台湾，台北市)提供，且在5％CO2环境下，于10％FBS(胎牛血清，美国Invitrogen公司)和1％抗生素溶液(青霉素和链霉素；Invitrogen公司)的DMEM-高葡萄糖(Invitrogen公司)中培养。 HFF1(人包皮成纤维细胞)细胞购自ATCC(American TypeCulture Collection，美国典型培养物保藏中心)，并在5％CO2环境下，于15％FBS 和1％抗生素溶液(青霉素和链霉素)的DMEM-高葡萄糖中培养；人血浆是由自愿者捐献的人血液获得的。所有与人体血液相关的方法，均按照有关指引和规定进行。所有与人体血液相关的实验方案，均经阳明大学人体伦理委员会(IRB)批准。获得所有受试者的知情同意书。猪血浆由台湾嘉一香食品股份有限公司提供。
实施例2.血浆纤连蛋白制备步骤
血浆纤连蛋白纯化:本发明提供了一种纯化修饰的糖基化猪纤连蛋白的方法，其包括：步骤1：将干净的血浆通过明胶-琼脂糖凝胶快速流动4B的预柱、步骤2：通过50毫升含有EDTA(乙二胺四乙酸)的TBS(tris buffered saline，三乙醇胺缓冲盐水溶液)洗涤，去除非特异性吸附的蛋白质、步骤3：通过50毫升1摩尔浓度的NaCl洗涤，去除非特异性吸附的蛋白质、步骤4：通过50毫升小于0.5摩尔浓度精氨酸(Arg)顺序洗涤，去除非特异性吸附的蛋白质、步骤5：通过大于0.5摩尔浓度精氨酸(Arg)洗脱纤连蛋白样品、步骤6：用TBS(trisbuffered saline，三乙醇胺缓冲盐水溶液)(pH 5-8)透析纤连蛋白样品在4℃下24小时、步骤7：使用Vivaspin 20 离心浓缩器浓缩纤连蛋白样品(截留分子量：100kDa)。
参考图1、2，本发明使用血浆纤连蛋白纯化方法从猪血浆中分离出高质量，并且具备糖基化的纤连蛋白，将血浆装入明胶-琼脂糖凝胶快速流动4B的预柱中，将凝胶依次用含有EDTA(乙二胺四乙酸)的TBS(tris buffered saline，三乙醇胺缓冲盐水溶液)，1摩尔浓度的NaCl和<0.5摩尔浓度的精氨酸(Arg)洗涤以除去非特异性结合蛋白，留下结合的纤连蛋白用>0.5 摩尔浓度精氨酸洗脱，合并洗脱的纤维连接蛋白的部分，并在TBS(trisbuffered saline，三乙醇胺缓冲盐水溶液)中在4℃下透析24小时，并使用Vivaspin 20离心浓缩器(截留分子量：100kDa)浓缩。
血浆纤连蛋白修饰:本发明提供了修饰糖基化猪纤连蛋白的修饰方法，包括：步骤1：在缓冲液(pH5～7)中调配猪血浆纤维连接蛋白(1毫克)、步骤2：加入5～50单位α2-3,6,8的神经氨酸酶(一个单位定义为从1nmol Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-7-氨基-4-甲基香豆素(AMC) 切割>95％末端α-Neu5Ac所需的酶量，在37℃下反应5分钟，总反应体积为10μl)、步骤3：于37℃反应1～24小时。
血浆纤连蛋白的糖蛋白鉴定:将沉淀的纤连蛋白(～10微克)用胰蛋白酵素进行蛋白质分解成小的胜肽(多肽)。再将胜肽混合物溶解在0.1％甲酸中，使用Orbitrap FusionTribrid质谱仪(Thermo Scientific)搭配Dionex Ultimate 3000nanoLC(ThermoScientific)液相层析系统和25cm×75μm碳 -18层析管柱(Acclaim PepMap RSLC，ThermoScientific)进行分析，使用 100％移动相A(0.1％甲酸水溶液)在120分钟线性梯度分离至40％移动相B(含0.1％甲酸的乙腈)，流速为300nL/min。使用PicoView(New Objective)纳米喷雾源以正离子模式检测，质谱仪以每3秒钟循环进行数据收集，包括Orbitrap全扫描母离子MS1(m/z:400-2000)，在m/z 400 下分辨率为120,000，自动增益控制(AGC)目标在200,000，之后是以30％的碰撞能量进行高能量碰撞解离(HCD)产生子离子MS2图谱，并在Orbitrap 分析仪中以30000分辨率(AGC目标为100,000)检测HCD MS2子离子图谱。质谱数据分析使用ByonicTM搜索软件(Protein Metrics，v.2.7.4)，蛋白质序列使用Swiss Prot(瑞士生物信息学研究所)数据库包含FN1_人类或 FN1_Sus scrofa，分别进行人和猪血浆纤连蛋白的质谱数据比对，将蛋白质修饰设置为羧甲基酰基甲基(C)(固定)，脱酰胺(N)(可变)，氧化(M) (可变)和N-连接的糖基化修饰(包含182种人类的N-糖糖基化数据库；309 种哺乳动物N-糖基化数据库)，允许两切点不完全消化，误差值限定为母离子MS1质谱图小于5ppm，MS2子离子质谱图误差小于10ppm。糖化胜肽鉴定，选择Byonic分数超过100，错误发现率(FDR)小于1％。
如图3，本发明使用α2-3,6,8神经氨酸酶去除猪纤连蛋白上的N-乙酰神经氨酸(Neu5Ac)和N-羟乙酰神经氨酸(Neu5GC)糖基，结果显示修饰后的猪纤连蛋白可用于伤口愈合，发挥与人纤连蛋白相同的功能。
实施例3.人和猪血浆纤连蛋白在细胞粘附和迁移方面显示出相同的效果
伤口愈合分析:将在组织培养板上生长的U2OS，Hela或HFF1细胞以胰蛋白酶切下，并再次接种在培养基中具有10μg/ml纤连蛋白涂层的6孔板上 16小时，然后置于配有温度和CO2控制，并配有10×NA0.25物镜(Zeiss) 的显微镜(Axio Observer.Z1，Zeiss)，使用由Zen图像分析软件(Zeiss) 操作的AxioCamMR3CCD照相机，在12小时内以15分钟间隔获得延时图像，为了计算伤口闭合的百分比，使用Metamorph图像分析软件(Molecular Device)从延时图像中获得6小时或12小时移植期间的伤口愈合的面积，并计算为创伤后0小时伤口面积与净伤口愈合面积的比值。
如图4a-c，为了比较来自人和猪血浆的分离的纤连蛋白的功能，发明人使用预先涂覆来自人或猪血浆的纤连蛋白涂层的玻片，进行细胞伤口愈合的迁移测定。结果显示，使用人和猪纤连蛋白，在U2OS，HFF1和Hela的细胞伤口愈合分析中，表现出相似的伤口闭合效应，因此人和猪纤连蛋白在调节细胞迁移方面具有相同的能力。
细胞贴附试验:细胞贴附测定使用已经在37℃下用1％变性BSA(牛血清白蛋白，Albumin from bovine serum)预处理1小时的96孔盘，然后用指定浓度的人或猪血浆纤连蛋白涂覆。为了进行实验，将生长在组织培养板上的U2OS细胞进行胰蛋白酶处理，以悬浮于无血清培养基中，然后重新接种在预处理的96孔板上10分钟或过夜(～16h)。培养后，通过用PBS 洗涤两次，以完全除去未附着的细胞，并在室温下用5％戊二醛25分钟以固定附着的细胞，接着于室温下用0.1％结晶紫染色25分钟，并除去所有未结合的结晶紫后，将结晶紫标记的细胞溶解在50μl溶液A(50％乙醇和 0.1％乙酸的水)中，使用Thermo ScientificMultiskan以OD 550nm光谱测量结晶紫的量，结果以图形方式使用Excel软件(Microsoft)显示。
免疫荧光染色和图像分析:首先将黏着斑标的蛋白(Paxillin)以免疫荧光染色方式作染色。在TIRFM成像部分，将细胞安装在含有没食子酸丙酯的 PBS的载玻片上，使用配备有100×1.49NA物镜(Nikon)的TIRF(Roper) /共轭焦显微镜(CSUX1，Yokogawa)显微镜系统，以EMCCD相机 (Photometrics)获得TIRFM图像。为了确定黏附面积，将Paxillin染色的细胞的TIRFM图像阈值化以仅突出黏着斑讯号范围(focal adhesions,FAs)，并且使用Metamorph记录这些区域的面积，记录的黏着斑总面积相加得到细胞黏附总面积，结果以图形方式使用Excel软件(Microsoft)显示。
细胞贴附扩散测定和图像分析:将生长在组织培养盘上的细胞进行胰蛋白酶作用切下，并再次接种在涂有指定浓度的人或猪血浆纤连蛋白的玻片上，以使其贴附并扩散附着(30分钟)。接下来，在室温下用4％多聚甲醛在PBS中固定细胞20分钟，然后使用配备有20×0.45NA物镜(Nikon)的显微镜(Eclipse TS100；Nikon)系统以ISCapture软件操作的WHITE CCD 照相机成像(TUCSEN)。为了计算细胞贴附扩散面积，使用Metamorph图像分析软件(Molecular Device)，在相位图像上手动盘点细胞区域，结果用 Excel软件(Microsoft)以图形方式显示。
为了比较人和猪纤连蛋白在调节细胞黏附强度，以及比较人和猪纤连蛋白对细胞贴附扩散的影响，发明人比较了接种在逐渐增加浓度的人或猪纤连蛋白涂覆的玻片上的U2OS细胞，在30分钟后测量细胞贴附扩散的面积，结果显示，随着纤连蛋白的浓度增加，细胞贴附扩散面积增加(如图5)，细胞黏附能力也增强(如图6)，这也表明了人或猪纤连蛋白随着浓度的增加而促进细胞黏附到纤连蛋白的能力是相同的(如图7)。接下来，发明人比较接种在增加浓度的人或猪纤连蛋白上1.5小时的细胞免疫荧光染色讯号显示黏着斑标的蛋白(Paxillin)细胞模式(如图8)，结果显示，随着纤连蛋白浓度的增加，黏着斑形成面积增加(μm2)；根据黏着斑标的蛋白 (Paxillin)标记的黏着斑面积进行定量(如图9)。
为了进一步确定在黏附增强期间，唾液酸(Neu5Ac与Neu5GC)是否具备关键的作用，发明人首先使用α2-3,6,8神经氨酸酶(唾液酸酶)从猪纤连蛋白切割唾液酸(如图3)。从结果得知，将猪纤连蛋白中去除唾液酸不影响细胞和纤连蛋白结合的能力(如图10)。由于在人体内不存在内源性 Neu5Gc，故直接利用猪纤连蛋白的临床应用可能因为Neu5Gc的存在而引起异常免疫反应，且来自猪血浆纤连蛋白，予以去除唾液酸之后并没有改变纤连蛋白对细胞黏贴附着的能力(如图3、10)，故综合上述结果可知，在生产过程中从猪血浆纤连蛋白中去除唾液酸，以作为具有新颖性的伤口敷料材料，可增强伤口愈合，而不会由于Neu5Gc的存在引起异常免疫反应的可能。
实施例4.猪血浆纤连蛋白经过蛋白酶修饰后在伤口闭合中表现出更好的效果
猪血浆纤连蛋白经MMP3修饰:为了使猪血浆纤连蛋白上的聚糖结构更完整暴露，以提供更好的伤口闭合功能，本发明提供了一种修饰糖基化猪血浆纤连蛋白的方法，包括：将猪血浆纤连蛋白与基质金属蛋白酶3(matrix metalloproteinase-3,MMP3)以酵素-受质比在1:5到1:30的比例，于37℃环境中隔夜共同反应。
伤口愈合分析:将在组织培养盘上生长的U2OS细胞进行胰蛋白酶作用切下，并再次接种在具有10μg/ml纤连蛋白涂覆的6孔盘上16小时，然后置于具温度和CO2控制，并配有10XNA0.25物镜(Zeiss)的显微镜(Axio Observer.Z1，Zeiss)，使用由Zen图像分析软件(Zeiss)操作的AxioCamMR3 CCD照相机，在12小时内以15分钟间隔获得延时图像，为了计算伤口闭合的百分比，使用Metamorph图像分析软件(Molecular Device)从延时图像中获得12小时移植期间的伤口愈合的面积，并计算为创伤后0小时伤口面积与净伤口愈合面积的比值。
本发明使用MMP3以产生猪纤连蛋白胜肽(如图11)。为了比较经由 MMP3修饰前后的猪纤连蛋白胜肽的伤口愈合效果，发明人使用已经接种在经过MMP3修饰的猪纤连蛋白胜肽，或未经过MMP3修饰的猪纤连蛋白涂抹玻片上的细胞进行伤口愈合迁移测定。结果显示经过MMP3修饰的猪纤连蛋白胜肽具有显著的促进伤口闭合效果(如图12)。因此，经MMP3 修饰处理后的猪血浆纤连蛋白，在调节细胞迁移方面具有更好的效果。
上列详细说明是针对本发明之一可行实施例的具体说明，惟该实施例并非用以限制本发明的专利范围，凡未脱离本发明技艺精神所为的等效实施或变更，均应包含于本发明的专利范围中。
序列表
<110> 战国策智权股份有限公司
<120> 制备改良猪血浆纤连蛋白以增强伤口愈合的应用
<130> 2016RDPA002
<150> US 62/399,688
<151> 2016-09-26
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2477
<212> PRT
<213> Homo sapiens
<400> 1
Met Leu Arg Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Gln Cys
1 5 10 15
Leu Gly Thr Ala Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln
20 25 30
Ala Gln Gln Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser
35 40 45
Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln
50 55 60
Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr Cys Tyr Gly
65 70 75 80
Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu Glu Thr
85 90 95
Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr
100 105 110
Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala
115 120 125
Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly
130 135 140
Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr
145 150 155 160
Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu
165 170 175
Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly
180 185 190
Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly Trp
195 200 205
Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr
210 215 220
Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr
225 230 235 240
Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu Leu
245 250 255
Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg
260 265 270
His Thr Ser Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp
275 280 285
Val Arg Ala Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro
290 295 300
Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly Met
305 310 315 320
Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys Leu
325 330 335
Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr Gly
340 345 350
Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn Gly
355 360 365
Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His Leu
370 375 380
Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe
385 390 395 400
Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn
405 410 415
Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr
420 425 430
Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly Thr
435 440 445
Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met Ala
450 455 460
Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile
465 470 475 480
Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys
485 490 495
Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser
500 505 510
Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn
515 520 525
Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr
530 535 540
Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln
545 550 555 560
Cys Gln Asp Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp Ser Trp
565 570 575
Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly Arg
580 585 590
Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Ser Ser
595 600 605
Ser Gly Pro Val Glu Val Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn
610 615 620
Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys
625 630 635 640
Tyr Ile Leu Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu
645 650 655
Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys
660 665 670
Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly
675 680 685
His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr
690 695 700
Pro Val Thr Ser Asn Thr Val Thr Gly Glu Thr Thr Pro Phe Ser Pro
705 710 715 720
Leu Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser Phe
725 730 735
Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg Val
740 745 750
Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu
755 760 765
Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg
770 775 780
Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser
785 790 795 800
Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro Asp
805 810 815
Thr Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp Ser
820 825 830
Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro Ser
835 840 845
Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn Ser
850 855 860
Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile
865 870 875 880
Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln
885 890 895
Glu Thr Thr Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp
900 905 910
Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr
915 920 925
Pro Pro Glu Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile Pro Val
930 935 940
Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn Thr
945 950 955 960
Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr Tyr Phe Lys
965 970 975
Val Phe Ala Val Ser His Gly Arg Glu Ser Lys Pro Leu Thr Ala Gln
980 985 990
Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu
995 1000 1005
Thr Asp Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln Ile
1010 1015 1020
Thr Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Arg Gly Gln Pro Arg
1025 1030 1035 1040
Gln Tyr Asn Val Gly Pro Ser Val Ser Lys Tyr Pro Leu Arg Asn Leu
1045 1050 1055
Gln Pro Ala Ser Glu Tyr Thr Val Ser Leu Val Ala Ile Lys Gly Asn
1060 1065 1070
Gln Glu Ser Pro Lys Ala Thr Gly Val Phe Thr Thr Leu Gln Pro Gly
1075 1080 1085
Ser Ser Ile Pro Pro Tyr Asn Thr Glu Val Thr Glu Thr Thr Ile Val
1090 1095 1100
Ile Thr Trp Thr Pro Ala Pro Arg Ile Gly Phe Lys Leu Gly Val Arg
1105 1110 1115 1120
Pro Ser Gln Gly Gly Glu Ala Pro Arg Glu Val Thr Ser Asp Ser Gly
1125 1130 1135
Ser Ile Val Val Ser Gly Leu Thr Pro Gly Val Glu Tyr Val Tyr Thr
1140 1145 1150
Ile Gln Val Leu Arg Asp Gly Gln Glu Arg Asp Ala Pro Ile Val Asn
1155 1160 1165
Lys Val Val Thr Pro Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala
1170 1175 1180
Asn Pro Asp Thr Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr
1185 1190 1195 1200
Pro Asp Ile Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln
1205 1210 1215
Gln Gly Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys
1220 1225 1230
Thr Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr
1235 1240 1245
Thr Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile Ile
1250 1255 1260
Pro Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val Asp Ile Thr Asp
1265 1270 1275 1280
Ser Ser Ile Gly Leu Arg Trp Thr Pro Leu Asn Ser Ser Thr Ile Ile
1285 1290 1295
Gly Tyr Arg Ile Thr Val Val Ala Ala Gly Glu Gly Ile Pro Ile Phe
1300 1305 1310
Glu Asp Phe Val Asp Ser Ser Val Gly Tyr Tyr Thr Val Thr Gly Leu
1315 1320 1325
Glu Pro Gly Ile Asp Tyr Asp Ile Ser Val Ile Thr Leu Ile Asn Gly
1330 1335 1340
Gly Glu Ser Ala Pro Thr Thr Leu Thr Gln Gln Thr Ala Val Pro Pro
1345 1350 1355 1360
Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg Val
1365 1370 1375
Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe Leu Val Arg
1380 1385 1390
Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu Ser Ile Ser
1395 1400 1405
Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu Pro Gly Thr Glu
1410 1415 1420
Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His Glu Ser Thr Pro
1425 1430 1435 1440
Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp
1445 1450 1455
Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro
1460 1465 1470
Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe
1475 1480 1485
Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn Ser Ile
1490 1495 1500
Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser Ile Val
1505 1510 1515 1520
Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln Gln Ser
1525 1530 1535
Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro
1540 1545 1550
Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr
1555 1560 1565
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
1570 1575 1580
Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys
1585 1590 1595 1600
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly
1605 1610 1615
Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu
1620 1625 1630
Ile Asp Lys Pro Ser Gln Met Gln Val Thr Asp Val Gln Asp Asn Ser
1635 1640 1645
Ile Ser Val Lys Trp Leu Pro Ser Ser Ser Pro Val Thr Gly Tyr Arg
1650 1655 1660
Val Thr Thr Thr Pro Lys Asn Gly Pro Gly Pro Thr Lys Thr Lys Thr
1665 1670 1675 1680
Ala Gly Pro Asp Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro Thr
1685 1690 1695
Val Glu Tyr Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly Glu Ser
1700 1705 1710
Gln Pro Leu Val Gln Thr Ala Val Thr Asn Ile Asp Arg Pro Lys Gly
1715 1720 1725
Leu Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp Glu
1730 1735 1740
Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser Ser Pro
1745 1750 1755 1760
Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly Glu Glu Asp
1765 1770 1775
Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr Thr Val Ser
1780 1785 1790
Val Val Ala Leu His Asp Asp Met Glu Ser Gln Pro Leu Ile Gly Thr
1795 1800 1805
Gln Ser Thr Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe Thr Gln Val
1810 1815 1820
Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn Val Gln Leu
1825 1830 1835 1840
Thr Gly Tyr Arg Val Arg Val Thr Pro Lys Glu Lys Thr Gly Pro Met
1845 1850 1855
Lys Glu Ile Asn Leu Ala Pro Asp Ser Ser Ser Val Val Val Ser Gly
1860 1865 1870
Leu Met Val Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala Leu Lys Asp
1875 1880 1885
Thr Leu Thr Ser Arg Pro Ala Gln Gly Val Val Thr Thr Leu Glu Asn
1890 1895 1900
Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr Thr
1905 1910 1915 1920
Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln
1925 1930 1935
Val Asp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile
1940 1945 1950
Lys Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly Thr
1955 1960 1965
Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser Ser
1970 1975 1980
Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn Leu
1985 1990 1995 2000
Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp Gln Pro
2005 2010 2015
Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu Lys Pro Gly
2020 2025 2030
Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro Gly Val Thr Glu
2035 2040 2045
Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr Ile Tyr Val
2050 2055 2060
Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu Pro Leu Ile Gly Arg Lys
2065 2070 2075 2080
Lys Thr Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn Leu
2085 2090 2095
His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr Val Gln Lys Thr Pro
2100 2105 2110
Phe Val Thr His Pro Gly Tyr Asp Thr Gly Asn Gly Ile Gln Leu Pro
2115 2120 2125
Gly Thr Ser Gly Gln Gln Pro Ser Val Gly Gln Gln Met Ile Phe Glu
2130 2135 2140
Glu His Gly Phe Arg Arg Thr Thr Pro Pro Thr Thr Ala Thr Pro Ile
2145 2150 2155 2160
Arg His Arg Pro Arg Pro Tyr Pro Pro Asn Val Gly Glu Glu Ile Gln
2165 2170 2175
Ile Gly His Ile Pro Arg Glu Asp Val Asp Tyr His Leu Tyr Pro His
2180 2185 2190
Gly Pro Gly Leu Asn Pro Asn Ala Ser Thr Gly Gln Glu Ala Leu Ser
2195 2200 2205
Gln Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr Ser Glu Tyr Ile
2210 2215 2220
Ile Ser Cys His Pro Val Gly Thr Asp Glu Glu Pro Leu Gln Phe Arg
2225 2230 2235 2240
Val Pro Gly Thr Ser Thr Ser Ala Thr Leu Thr Gly Leu Thr Arg Gly
2245 2250 2255
Ala Thr Tyr Asn Val Ile Val Glu Ala Leu Lys Asp Gln Gln Arg His
2260 2265 2270
Lys Val Arg Glu Glu Val Val Thr Val Gly Asn Ser Val Asn Glu Gly
2275 2280 2285
Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe Asp Pro Tyr Thr Val Ser
2290 2295 2300
His Tyr Ala Val Gly Asp Glu Trp Glu Arg Met Ser Glu Ser Gly Phe
2305 2310 2315 2320
Lys Leu Leu Cys Gln Cys Leu Gly Phe Gly Ser Gly His Phe Arg Cys
2325 2330 2335
Asp Ser Ser Arg Trp Cys His Asp Asn Gly Val Asn Tyr Lys Ile Gly
2340 2345 2350
Glu Lys Trp Asp Arg Gln Gly Glu Asn Gly Gln Met Met Ser Cys Thr
2355 2360 2365
Cys Leu Gly Asn Gly Lys Gly Glu Phe Lys Cys Asp Pro His Glu Ala
2370 2375 2380
Thr Cys Tyr Asp Asp Gly Lys Thr Tyr His Val Gly Glu Gln Trp Gln
2385 2390 2395 2400
Lys Glu Tyr Leu Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly Gln
2405 2410 2415
Arg Gly Trp Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly Glu Pro Ser
2420 2425 2430
Pro Glu Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser Gln Arg Tyr
2435 2440 2445
His Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe Met
2450 2455 2460
Pro Leu Asp Val Gln Ala Asp Arg Glu Asp Ser Arg Glu
2465 2470 2475
<210> 2
<211> 2478
<212> PRT
<213> Sus scrofa
<400> 2
Met Leu Gly Gly Pro Gly Pro Gly Leu Leu Leu Leu Ala Val Leu Ser
1 5 10 15
Leu Gly Thr Thr Val Pro Ser Thr Gly Ala Ser Lys Ser Lys Arg Gln
20 25 30
Ala Gln Gln Ile Val Gln Pro Gln Ser Pro Leu Val Asp Ser Gln Arg
35 40 45
Lys Pro Gly Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln Gln
50 55 60
Trp Glu Arg Thr Tyr Leu Gly Ser Ala Leu Val Cys Thr Cys Tyr Gly
65 70 75 80
Gly Ser Arg Gly Phe Asn Cys Glu Ser Lys Pro Glu Pro Glu Glu Thr
85 90 95
Cys Phe Asp Lys Tyr Thr Gly Asn Thr Tyr Arg Val Gly Asp Thr Tyr
100 105 110
Glu Arg Pro Lys Asp Ser Met Ile Trp Asp Cys Thr Cys Ile Gly Ala
115 120 125
Gly Arg Gly Arg Ile Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly
130 135 140
Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr
145 150 155 160
Gly Gly Tyr Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu
165 170 175
Trp Thr Cys Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly
180 185 190
Gly Thr Ser Tyr Val Val Gly Glu Thr Trp Glu Lys Pro Tyr Gln Gly
195 200 205
Trp Met Met Val Asp Cys Thr Cys Leu Gly Glu Gly Ser Gly Arg Ile
210 215 220
Thr Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser
225 230 235 240
Tyr Arg Ile Gly Asp Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn Leu
245 250 255
Leu Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu
260 265 270
Arg His Thr Ser Leu Gln Thr Thr Ser Ala Gly Ser Gly Ser Phe Thr
275 280 285
Asp Val Arg Thr Ala Ile Tyr Gln Pro Gln Pro His Pro Gln Pro Ala
290 295 300
Pro Tyr Gly His Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly
305 310 315 320
Met Gln Trp Leu Lys Thr Gln Gly Asn Lys Gln Met Leu Cys Thr Cys
325 330 335
Leu Gly Asn Gly Val Ser Cys Gln Glu Thr Ala Val Thr Gln Thr Tyr
340 345 350
Gly Gly Asn Ser Asn Gly Glu Pro Cys Val Leu Pro Phe Thr Tyr Asn
355 360 365
Gly Arg Thr Phe Tyr Ser Cys Thr Thr Glu Gly Arg Gln Asp Gly His
370 375 380
Leu Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser
385 390 395 400
Phe Cys Thr Asp His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser
405 410 415
Asn Gly Ala Leu Cys His Phe Pro Phe Leu Tyr Asn Asn Arg Asn Tyr
420 425 430
Thr Asp Cys Thr Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys Gly
435 440 445
Thr Thr Gln Asn Tyr Asp Ala Asp Gln Lys Phe Gly Phe Cys Pro Met
450 455 460
Ala Ala His Glu Glu Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg
465 470 475 480
Ile Gly Asp Gln Trp Asp Lys Gln His Asp Met Gly His Met Met Arg
485 490 495
Cys Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Val Ala Tyr
500 505 510
Ser Gln Leu Arg Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val
515 520 525
Asn Asp Thr Phe His Lys Arg His Glu Glu Gly His Met Leu Asn Cys
530 535 540
Thr Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp
545 550 555 560
Gln Cys Gln Asp Ser Glu Thr Arg Thr Phe Tyr Gln Ile Gly Asp Ser
565 570 575
Trp Glu Lys Tyr Val His Gly Val Arg Tyr Gln Cys Tyr Cys Tyr Gly
580 585 590
Arg Gly Ile Gly Glu Trp His Cys Gln Pro Leu Gln Thr Tyr Pro Gly
595 600 605
Thr Thr Gly Pro Val Gln Val Ile Ile Thr Glu Thr Pro Ser Gln Pro
610 615 620
Asn Ser His Pro Ile Gln Trp Asn Ala Pro Glu Pro Ser His Ile Ser
625 630 635 640
Lys Tyr Ile Leu Arg Trp Lys Pro Lys Asn Ser Pro Asn Arg Trp Lys
645 650 655
Glu Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu
660 665 670
Arg Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Val Gln His Tyr
675 680 685
Gly His Arg Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser
690 695 700
Ser Ala Val Thr Ser Asn Thr Val Val Gly Glu Thr Thr Pro Phe Ser
705 710 715 720
Pro Val Val Ala Thr Ser Glu Ser Val Thr Glu Ile Thr Ala Ser Ser
725 730 735
Phe Val Val Ser Trp Val Ser Ala Ser Asp Thr Val Ser Gly Phe Arg
740 745 750
Val Glu Tyr Glu Leu Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp
755 760 765
Leu Pro Ser Thr Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly
770 775 780
Arg Lys Tyr Ile Val Asn Val Tyr Gln Ile Ser Glu Glu Gly Glu Gln
785 790 795 800
Ser Leu Ile Leu Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro
805 810 815
Asp Pro Thr Val Asp Gln Val Asp Asp Thr Ser Ile Val Val Arg Trp
820 825 830
Ser Arg Pro Gln Ala Pro Ile Thr Gly Tyr Arg Ile Val Tyr Ser Pro
835 840 845
Ser Val Glu Gly Ser Ser Thr Glu Leu Asn Leu Pro Glu Thr Ala Asn
850 855 860
Ser Val Thr Leu Ser Asp Leu Gln Pro Gly Val Gln Tyr Asn Ile Thr
865 870 875 880
Ile Tyr Ala Val Glu Glu Asn Gln Glu Ser Thr Pro Val Phe Ile Gln
885 890 895
Gln Glu Thr Thr Gly Val Pro Arg Pro Asp Lys Val Pro Pro Pro Lys
900 905 910
Asp Leu Gln Phe Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp
915 920 925
Thr Pro Pro Glu Ser Pro Val Thr Gly Tyr Arg Val Asp Val Ile Pro
930 935 940
Val Asn Leu Pro Gly Glu His Gly Gln Arg Leu Pro Ile Ser Arg Asn
945 950 955 960
Thr Phe Ala Glu Val Thr Gly Leu Ser Pro Gly Val Thr Tyr His Phe
965 970 975
Lys Val Phe Ala Val Asn Gln Gly Arg Glu Ser Lys Pro Leu Thr Ala
980 985 990
Gln Gln Thr Thr Lys Leu Asp Ala Pro Thr Asn Leu Gln Phe Ile Asn
995 1000 1005
Glu Thr Asp Ser Thr Val Met Val Thr Trp Thr Pro Pro Arg Ala Arg
1010 1015 1020
Ile Ala Gly Tyr Arg Leu Thr Val Gly Leu Thr Arg Gly Gly Gln Pro
1025 1030 1035 1040
Lys Gln Tyr Asn Val Gly Pro Ser Ala Ser Gln Tyr Leu Leu Arg Asn
1045 1050 1055
Leu Gln Pro Gly Ser Glu Tyr Ala Val Thr Leu Val Ala Val Lys Gly
1060 1065 1070
Asn Gln Gln Ser Pro Arg Ala Thr Gly Val Phe Thr Thr Leu Gln Pro
1075 1080 1085
Val Gly Ser Ile Pro Pro Tyr Asn Thr Glu Val Thr Glu Thr Thr Ile
1090 1095 1100
Val Ile Thr Trp Thr Pro Ala Pro Arg Ile Gly Phe Lys Leu Gly Val
1105 1110 1115 1120
Arg Pro Ser Gln Gly Gly Glu Ala Pro Arg Glu Val Thr Ser Asp Ser
1125 1130 1135
Gly Ser Ile Val Val Ser Gly Leu Thr Pro Gly Val Glu Tyr Val Tyr
1140 1145 1150
Thr Ile Ser Val Leu Arg Asp Gly Gln Glu Arg Asp Thr Pro Ile Val
1155 1160 1165
Lys Lys Val Val Thr Pro Leu Ser Pro Pro Thr Asn Leu His Leu Glu
1170 1175 1180
Ala Asn Pro Asp Thr Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr
1185 1190 1195 1200
Thr Pro Asp Ile Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly
1205 1210 1215
Gln Gln Gly Tyr Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser
1220 1225 1230
Cys Thr Phe Glu Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val
1235 1240 1245
Tyr Thr Val Lys Asn Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile
1250 1255 1260
Ile Pro Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val Asp Ile Thr
1265 1270 1275 1280
Asp Ser Ser Ile Gly Leu Arg Trp Thr Pro Ile Asn Ser Ser Thr Ile
1285 1290 1295
Ile Gly Tyr Arg Ile Thr Val Val Ala Ala Gly Glu Gly Ile Pro Ile
1300 1305 1310
Phe Glu Asp Phe Ala Asp Ser Ser Val Gly Tyr Tyr Thr Val Thr Gly
1315 1320 1325
Leu Glu Pro Gly Ile Asp Tyr Asp Ile Ser Val Ile Thr Leu Ile Asn
1330 1335 1340
Gly Gly Glu Ser Ala Pro Thr Thr Leu Thr Gln Gln Thr Ala Val Pro
1345 1350 1355 1360
Pro Pro Thr Asp Leu Arg Phe Thr Asn Val Gly Pro Asp Thr Ile Arg
1365 1370 1375
Val Thr Trp Ala Pro Pro Pro Ser Ile Glu Leu Thr Asn Phe Leu Val
1380 1385 1390
Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu Ser Ile
1395 1400 1405
Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu Pro Gly Thr
1410 1415 1420
Glu Tyr Leu Val Ser Val Ser Ser Val Tyr Glu Gln His Glu Ser Ile
1425 1430 1435 1440
Pro Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile
1445 1450 1455
Asp Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val Tyr Trp Ile Ala
1460 1465 1470
Pro Arg Ala Thr Ile Thr Gly Tyr Lys Ile Arg His His Pro Glu His
1475 1480 1485
Met Gly Gly Arg Pro Arg Glu Asp Arg Val Pro Pro Ser Arg Asn Ser
1490 1495 1500
Ile Thr Leu Thr Asn Leu Ile Pro Gly Val Glu Tyr Val Val Ser Ile
1505 1510 1515 1520
Val Ala Val Asn Gly Arg Glu Glu Ser Pro Pro Leu Val Gly Gln Gln
1525 1530 1535
Ser Thr Val Ser Asp Val Pro Arg Asp Leu Gln Val Ile Ala Thr Thr
1540 1545 1550
Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg
1555 1560 1565
Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln
1570 1575 1580
Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu
1585 1590 1595 1600
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg
1605 1610 1615
Gly Asp Ser Pro Ala Ser Ser Lys Pro Val Ser Ile Asp Tyr Arg Thr
1620 1625 1630
Glu Ile Asp Lys Pro Ser Gln Met Gln Val Thr Asp Val Gln Asp Asn
1635 1640 1645
Ser Ile Ser Val Arg Trp Leu Pro Ser Ser Ser His Val Thr Gly Tyr
1650 1655 1660
Arg Val Thr Thr Thr Pro Lys Asn Gly Ser Gly Pro Ser Lys Thr Lys
1665 1670 1675 1680
Thr Val Gly Pro Asp Gln Thr Glu Met Thr Ile Glu Gly Leu Gln Pro
1685 1690 1695
Thr Val Glu Tyr Val Val Ser Val Tyr Ala Gln Asn Gln Asn Gly Glu
1700 1705 1710
Ser Gln Pro Leu Val Gln Thr Ala Val Thr Asn Ile Asp Arg Pro Lys
1715 1720 1725
Gly Leu Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys Ile Ala Trp
1730 1735 1740
Glu Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser Ser
1745 1750 1755 1760
Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly Glu Glu
1765 1770 1775
Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr Thr Val
1780 1785 1790
Ser Val Val Ala Leu His Asp Asp Met Glu Ser Gln Pro Leu Ile Gly
1795 1800 1805
Thr Gln Ser Thr Ala Ile Pro Ala Pro Thr Asn Leu Lys Phe Thr Gln
1810 1815 1820
Val Thr Pro Thr Ser Leu Thr Ala Gln Trp Thr Ala Pro Asn Val Gln
1825 1830 1835 1840
Leu Thr Gly Tyr Arg Val Arg Val Thr Pro Lys Glu Lys Thr Gly Pro
1845 1850 1855
Met Lys Glu Ile Asn Leu Ala Pro Asp Ser Ser Ser Val Val Val Ser
1860 1865 1870
Gly Leu Met Val Ala Thr Lys Tyr Glu Val Ser Ile Tyr Ala Leu Lys
1875 1880 1885
Asp Thr Leu Thr Ser Arg Pro Ala Gln Gly Val Val Thr Thr Leu Glu
1890 1895 1900
Asn Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu Thr
1905 1910 1915 1920
Thr Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe
1925 1930 1935
Gln Val Asp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr
1940 1945 1950
Ile Lys Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly
1955 1960 1965
Thr Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser
1970 1975 1980
Ser Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser Asn
1985 1990 1995 2000
Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp Gln
2005 2010 2015
Pro Pro Arg Ala Lys Ile Thr Gly Tyr Ile Ile Lys Tyr Glu Lys Pro
2020 2025 2030
Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro Gly Val Thr
2035 2040 2045
Glu Ala Thr Ile Thr Gly Leu Glu Pro Ala Thr Glu Tyr Thr Ile Gln
2050 2055 2060
Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu Pro Leu Ile Gly Arg
2065 2070 2075 2080
Lys Arg Thr Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His Pro Asn
2085 2090 2095
Leu His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr Val Gln Lys Thr
2100 2105 2110
Pro Phe Val Thr Lys Pro Gly Tyr Asp Thr Gly Asn Gly Ile Gln Leu
2115 2120 2125
Pro Gly Thr Ser Gly Gln Gln Pro Ser Leu Gly Gln Gln Met Ile Phe
2130 2135 2140
Glu Glu His Gly Phe Arg Arg Thr Thr Pro Pro Thr Thr Ala Thr Pro
2145 2150 2155 2160
Val Arg His Arg Pro Gly Pro Tyr Thr Pro Asn Val Asn Glu Glu Ile
2165 2170 2175
Gln Val Gly His Val Pro Arg Gly Asp Val Asp His His Leu Tyr Pro
2180 2185 2190
His Val Leu Gly Leu Asn Pro Asn Ala Ser Thr Gly Gln Glu Ala Leu
2195 2200 2205
Ser Gln Thr Thr Ile Ser Trp Thr Pro Phe Gln Glu Ser Ser Glu Tyr
2210 2215 2220
Ile Ile Ser Cys His Pro Val Gly Ile Asp Glu Glu Pro Leu Gln Phe
2225 2230 2235 2240
Arg Val Pro Gly Thr Ser Ala Ser Ala Thr Leu Thr Gly Leu Thr Arg
2245 2250 2255
Gly Ala Thr Tyr Asn Ile Ile Val Glu Ala Leu Lys Asp Gln Lys Arg
2260 2265 2270
His Lys Ile Arg Glu Glu Val Val Thr Val Gly Asn Ser Val Asp Gln
2275 2280 2285
Gly Leu Ser Gln Pro Thr Asp Asp Ser Cys Phe Asp Pro Tyr Thr Val
2290 2295 2300
Ser His Tyr Ala Ile Gly Glu Glu Trp Glu Arg Leu Ser Glu Ser Gly
2305 2310 2315 2320
Phe Lys Leu Ser Cys Gln Cys Leu Gly Phe Gly Ser Gly His Phe Arg
2325 2330 2335
Cys Asp Ser Ser Lys Trp Cys His Asp Asn Gly Val Asn Tyr Lys Ile
2340 2345 2350
Gly Glu Lys Trp Asp Arg Gln Gly Glu Asn Gly Gln Met Met Ser Cys
2355 2360 2365
Thr Cys Leu Gly Asn Gly Lys Gly Glu Phe Lys Cys Asp Pro His Glu
2370 2375 2380
Ala Thr Cys Tyr Asp Asp Gly Lys Thr Tyr His Val Gly Glu Gln Trp
2385 2390 2395 2400
Gln Lys Glu Tyr Leu Gly Ala Ile Cys Ser Cys Thr Cys Phe Gly Gly
2405 2410 2415
Gln Arg Gly Trp Arg Cys Asp Asn Cys Arg Arg Pro Gly Ala Glu Leu
2420 2425 2430
Gly Pro Glu Gly Ser Thr Gly His Ser Tyr Asn Gln Tyr Ser Gln Arg
2435 2440 2445
Tyr His Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile Glu Cys Phe
2450 2455 2460
Met Pro Leu Asp Val Gln Ala Asp Ile Glu Asp Ser Arg Glu
2465 2470 2475

Claims (19)

1.一种用于制备治疗伤口愈合的纤连蛋白的用途，其特征是，所述纤连蛋白包含酵素修饰的糖基化的猪纤连蛋白。
2.如权利要求1所述的用途，其特征是，所述修饰化的糖基是指猪纤连蛋白上复数个唾液酸分子糖基。
3.如权利要求2所述的用途，其特征是，所述唾液酸分子糖基是N-乙酰神经氨酸和/或N-羟乙酰神经氨酸。
4.如权利要求2的用途，其特征是，所述唾液酸分子糖基被除去大于80％。
5.如权利要求1的用途，其特征是，所述酵素为α2-3,6,8神经氨酸酶。
6.如权利要求5的用途，其特征是，所述酵素为进一步包含蛋白酶，所述蛋白酶具备将纤连蛋白切割成纤连蛋白胜肽的功效。
7.如权利要求6的用途，其特征是，所述酵素为包含基质金属蛋白酶3。
8.如权利要求1的用途，其特征是，所述糖基化的猪纤连蛋白由明胶-琼脂糖凝胶快速流动4B的预装管柱制备。
9.一种用于促进伤口愈合的医药组合物，其特征是，包含：
酵素修饰的糖基化的猪纤连蛋白；
胶原蛋白；以及
透明质酸；
和/或其药学上可接受的盐。
10.如权利要求9所述的组合物，其特征是，所述修饰的糖基是指猪纤连蛋白上复数个唾液酸分子糖基。
11.如权利要求10所述的组合物，其特征是，所述唾液酸分子糖基为N-乙酰神经氨酸和/或N-羟乙酰神经氨酸。
12.如权利要求10所述的组合物，其特征是，所述唾液酸分子糖基被除去大于80％。
13.如权利要求9所述的组合物，其特征是，所述酵素为α2-3,6,8神经氨酸酶。
14.如权利要求13所述的组合物，其特征是，所述酵素为进一步包含蛋白酶，所述蛋白酶具备将纤连蛋白切割成纤连蛋白胜肽的功效。
15.如权利要求14所述的组合物，其特征是，所述酵素为包含基质金属蛋白酶3。
16.如权利要求9所述的组合物，其特征是，所述糖基化的猪纤连蛋白由明胶-琼脂糖凝胶快速流动4B的预装管柱制备。
17.一种用于促进伤口愈合的材料，其特征是，包含酵素修饰的糖基化的猪纤连蛋白，其中所述修饰的糖基是指猪纤连蛋白上复数个唾液酸分子糖基，其中所述唾液酸分子糖基被除去大于80％，其中所述酵素为α2-3,6,8神经氨酸酶。
18.如权利要求17所述的材料，其特征是，所述酵素为进一步包含蛋白酶，所述蛋白酶具备将纤连蛋白切割成纤连蛋白胜肽的功效。
19.如权利要求18所述的材料，其特征是，所述酵素为包含基质金属蛋白酶3。
CN201710874862.0A 2016-09-26 2017-09-25 制备改良猪血浆纤连蛋白以增强伤口愈合的应用 Pending CN107865962A (zh)

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