CN114874339B - 蛋白粘合剂及其制备方法和应用 - Google Patents

蛋白粘合剂及其制备方法和应用 Download PDF

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CN114874339B
CN114874339B CN202210587956.0A CN202210587956A CN114874339B CN 114874339 B CN114874339 B CN 114874339B CN 202210587956 A CN202210587956 A CN 202210587956A CN 114874339 B CN114874339 B CN 114874339B
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刘凯
李铭
柳柏梅
李敬敬
张洪杰
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Tsinghua University
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Abstract

本发明涉及生物材料领域，尤其涉及蛋白粘合剂及其制备方法和应用。本发明提供了蛋白粘合剂包括：融合蛋白和鲑鱼精DNA；上述融合蛋白包括：生长因子和类弹性蛋白。本发明通过基因重组技术合成EGF‑ELP，利用蛋白与DNA之间发生的液液相分离，制备出一种具有良好的细胞相容性、生物可降解性以及粘附性能的蛋白粘合剂(EKD粘合剂)。本发明的蛋白粘合剂在亲疏水表面以及动物组织上均具有优异的粘合性能，能够促进细胞的增殖和迁移，可用于伤口止血并促进伤口愈合。

Description

蛋白粘合剂及其制备方法和应用
技术领域
本发明涉及生物材料领域，尤其涉及蛋白粘合剂及其制备方法和应用。
背景技术
针对外科手术中伤口闭合处理过程容易产生二次损伤、感染、瘢痕等问题，用于伤口闭合、止血和愈合的外科组织粘合剂作为缝合等传统伤口闭合的潜在替代品，具有良好的应用前景。目前临床所使用的第一代组织粘合剂主要有纤维蛋白胶、白蛋白-戊二醛(BioGlues)、氰基丙烯酸酯胶、聚乙二醇(PEG)基对应物等。然而，氰基丙烯酸酯胶生物相容性及生物降解性较差，纤维蛋白胶组织粘附强度(<20kPa)低。此外，包括纤维蛋白胶在内的其他天然来源的粘合剂具有以共价形式交联固化时需要预处理、操作繁琐等缺点，在一定程度上限制了其在临床上的广泛应用。因此，迫切需要开发出一种具有良好的细胞相容性、生物可降解性以及粘附性能的生物粘合剂。
研究表明，海洋类粘合剂中沙塔蠕虫可通过聚阴阳离子复合凝聚发生液液相分离产生粘合剂，类弹性蛋白亦可通过调节pH、温度、第四位氨基酸种类来调控其发生复合凝聚，且具有良好的生物相容性，已被证实在生物材料中具有较大应用前景。
伤口愈合过程中多种生长因子在促进细胞增殖和迁移、细胞外基质沉积、血管生成和组织重塑等方面发挥着至关重要的协同作用。生长因子治疗法已得到人们广泛关注，目前已开发出多种生物相容性材料与生长因子相结合的伤口敷料，如壳聚糖、胶原蛋白、海藻酸盐、聚氨酯等。其中，表皮生长因子(EGF)可与细胞表面EGF受体(EGFR)结合，激活一系列的细胞内信号传导途径，进而促进细胞迁移和增殖。然而，EGF在大肠杆菌中表达产量较低，很难在体外大量制备。此外，由于体内蛋白酶的水解作用，以液体等形式局部应用EGF以及负载EGF的伤口敷料仍存在较大局限性。
因此，开发具有优异粘合性能、良好生物相容性且能促进伤口愈合的蛋白粘合剂对临床上伤口无瘢痕闭合处理具有重大意义。
发明内容
有鉴于此，本发明提供了蛋白粘合剂及其制备方法和应用。本发明通过基因重组技术合成EGF-ELP，利用蛋白与DNA之间发生的液液相分离，制备出一种具有良好的细胞相容性、生物可降解性以及粘附性能的蛋白粘合剂。本发明的蛋白粘合剂在亲疏水表面以及动物组织上均具有优异的粘合性能，能够促进细胞的增殖和迁移，可用于伤口止血并促进伤口愈合。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了蛋白粘合剂包括：融合蛋白和鲑鱼精DNA；
上述融合蛋白包括：生长因子和类弹性蛋白(ELP)。
在本发明的一些实施方案中，上述鲑鱼精DNA片段大小为～2000bp。
在本发明的一些实施方案中，上述生长因子为表皮生长因子(EGF)。
在本发明的一些实施方案中，上述蛋白粘合剂中的所述融合蛋白与所述鲑鱼精DNA的摩尔比为1:(0.5～2)。
在本发明的一些实施方案中，上述蛋白粘合剂中的所述融合蛋白与所述鲑鱼精DNA的摩尔比为1:1。
在本发明的一些实施方案中，上述蛋白粘合剂中的所述类弹性蛋白包含n个重复的五肽序列GKGVP，其中n为≥18的整数。
在本发明的一些实施方案中，上述n为72，即带有72个正电荷。
在本发明的一些实施方案中，上述的蛋白粘合剂中所述融合蛋白具有：
(1)、如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3和/或SEQ ID NO:4所示的氨基酸序列；或
(2)、在如(1)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列；或
(3)、与如(1)所示的氨基酸序列同源性90％以上的序列。
在本发明的一些实施方案中，上述SEQ ID NO:1的序列为：MNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRMGAGP[(GVGVP)(GKGVP)9]8GWPH6。
在本发明的一些实施方案中，上述SEQ ID NO:2的序列为：MNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRMGAGP[(GVGVP)(GKGVP)9]4GWPH6。
在本发明的一些实施方案中，上述SEQ ID NO:3的序列为：MNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRMGAGP[(GVGVP)(GKGVP)9]12GWPH6。
在本发明的一些实施方案中，上述SEQ ID NO:4的序列为：MNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRMGAGP[(GVGVP)(GKGVP)9]16GWPH6。
在本发明的一些实施方案中，上述的蛋白粘合剂中编码所述融合蛋白的核酸分子具有：
(4)、如SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7和/或SEQ ID NO:8所示的核苷酸序列；或
(5)、与(4)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(4)所示的核苷酸序列不同的核苷酸序列；或
(6)、与(4)或(5)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(4)或(5)所示的核苷酸序列功能相同或相似的核苷酸序列。
在本发明的一些实施方案中，上述的蛋白粘合剂中所述生长因子具有：
(7)、如SEQ ID NO:9所示的氨基酸序列；或
(8)、在如(7)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列；或
(9)、与如(7)所示的氨基酸序列同源性90％以上的序列。
在本发明的一些实施方案中，上述SEQ ID NO:9的序列为：MNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR。
本发明提供了上述蛋白粘合剂的融合蛋白的制备方法，包括以下步骤：
S1：构建上述生长因子的引物，PCR扩增，获得基因元件；
S2：取上述基因元件与上述类弹性蛋白的表达载体经酶切后进行同源重组，获得蛋白表达载体；
S3：取上述蛋白表达载体转化，筛选，进而培养、诱导表达，纯化，获得上述融合蛋白。
在本发明的一些实施方案中，上述蛋白粘合剂的制备方法，包括：取上述融合蛋白和/或上述制备方法制得的融合蛋白与上述鲑鱼精DNA混合，离心后收集底部凝聚物。
在本发明的一些实施方案中，上述的制备方法中所述收集底部凝聚物后还包括冻干步骤，所述冻干时间为3～10min，温度为20～30℃。
在本发明的一些实施方案中，上述的制备方法中所述收集底部凝聚物后还包括冻干步骤，所述冻干时间为3～5min，温度为20～30℃。
在本发明的一些实施方案中，上述冻干步骤用于硬底片，上述冻干步骤不用于软组织。
在本发明的一些实施方案中，上述的蛋白粘合剂、上述制备方法制得的蛋白粘合剂在制备促进细胞增殖和/或迁移、伤口和/或组织止血、愈合、粘合的产品中的一种或多种应用。
在本发明的一些实施方案中，上述的蛋白粘合剂在制备不同基材(陶瓷、玻璃、铁等)和/或软组织(肌肉、猪皮等)粘合产品中的一种或多种应用。
在本发明的一些实施方案中，上述的蛋白粘合剂在制备粘合肝脏和/或肾脏的伤口缺损和/或止血产品中的应用。
本发明提供了蛋白粘合剂包括：融合蛋白和鲑鱼精DNA；
上述融合蛋白包括：生长因子和类弹性蛋白。
本发明通过基因重组技术合成EGF-ELP，利用蛋白与DNA之间发生的液液相分离，制备出一种具有良好的细胞相容性、生物可降解性以及粘附性能的蛋白粘合剂(EKD粘合剂)。本发明的蛋白粘合剂在亲疏水表面以及动物组织上均具有优异的粘合性能，能够促进细胞的增殖和迁移，可用于伤口止血并促进伤口愈合。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示本发明实施例2中电荷摩尔比EGF-K72:鲑鱼精DNA＝1:1制备的蛋白粘合剂及粘合效果图；
图2示本发明实施例2中制备不同比例蛋白粘合剂的粘合性能；
图3示本发明实施例2中以电荷摩尔比EGF-K72:鲑鱼精DNA＝1:1制备的蛋白粘合剂的粘合强度；
其中：A示粘合剂在陶瓷、铝、玻璃、铁等上的粘合性能，市售氰基丙烯酸酯在铁上的粘合强度作为对照实验；B示粘合剂在组织上的粘合性能；
图4示本发明实施例1制备的超电融合蛋白及实施例2中以电荷摩尔比EGF-K72:鲑鱼精DNA＝1:1制备的蛋白粘合剂的生物活性评估；
其中：A示蛋白对细胞迁移的影响；B示不同蛋白浓度下的细胞增殖情况；C示蛋白粘合剂下细胞增殖情况；
图5示本发明实施例2中以电荷摩尔比EGF-K72:鲑鱼精DNA＝1:1制备的蛋白粘合剂用于肝脏、肾脏止血效果图；
图6示本发明实施例2中以电荷摩尔比EGF-K72:鲑鱼精DNA＝1:1制备的蛋白粘合剂用于伤口愈合效果图；
其中：A示不同时间点伤口愈合情况；B示组织学评估。
具体实施方式
本发明公开了蛋白粘合剂及其制备方法和应用。
应该理解，表述“……中的一种或多种”单独地包括每个在所述表述后叙述的物体以及所述叙述的物体中的两者或更多者的各种不同组合，除非从上下文和用法中另有理解。与三个或更多个叙述的物体相结合的表述“和/或”应该被理解为具有相同的含义，除非从上下文另有理解。
术语“包括”、“具有”或“含有”，包括其语法同义语的使用，通常应该被理解为开放性和非限制性的，例如不排除其他未叙述的要素或步骤，除非另有具体陈述或从上下文另有理解。
应该理解，只要本发明仍可操作，步骤的顺序或执行某些行动的顺序并不重要。此外，两个或更多个步骤或行动可以同时进行。
本文中的任何和所有实例或示例性语言如“例如”或“包括”的使用，仅仅打算更好地说明本发明，并且除非提出权利要求，否则不对本发明的范围构成限制。本说明书中的任何语言都不应解释为指示任何未要求保护的要素对于本发明的实践是必不可少的。
此外，用以界定本发明的数值范围与参数皆是约略的数值，此处已尽可能精确地呈现具体实施例中的相关数值。然而，任何数值本质上不可避免地含有因个别测试方法所致的标准偏差。因此，除非另有明确的说明，应当理解本公开所用的所有范围、数量、数值与百分比均经过“约”的修饰。在此处，“约”通常是指实际数值在一特定数值或范围的正负10％、5％、1％或0.5％之内。
本发明融合蛋白构建、表达与纯化、蛋白粘合剂的制备及验证测试试验中，所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1融合蛋白构建、表达与纯化
构建EGF蛋白单元的编码序列(如SEQ ID NO:9所示)以及PCR引物1(TTTAAGAAGGAGATATACATATGAACAGTGATTCAGA)(如SEQ ID NO:10所示)、PCR引物2(GCCCGGCCCCGCGCCCATTCGCAGTTCCCACCATTTCA)(如SEQ ID NO:11所示)、酶切位点1(NdeI)，在购自Vazyme公司的重组酶ClonExpressⅡ作用下，与经过NdeI单酶切的类弹性蛋白表达载体(pET-25b-ELP)进行同源重组，获得pET-25b-EGF-ELP蛋白表达载体，所述类弹性蛋白表达载体(pET-25b-ELP)为本实验室之前构建，即将类弹性蛋白通过T4连接酶连接至pET-25b市售载体质粒。
本实施例中的类弹性蛋白(ELP)为包含n个重复的五肽序列(GKGVP)的蛋白，其特征序列为(GKGVP)n。EGF蛋白单元的编码序列分别与不同拷贝单元(GKGVP)的ELP蛋白融合表达，最终获得EGF-K36(如SEQ ID No:2所示)、EGF-K72(如SEQ ID No:1所示)、EGF-K108(如SEQ ID No:3所示)、EGF-K144(如SEQ ID No:4所示)蛋白表达载体；
将上述蛋白表达载体转化进E.coliBLR(DE3)感受态细胞中，挑取阳性单克隆菌落，在LB培养基(100μg/mL氨苄西林)中37℃培养至OD600为3-4，转入TB培养基(100μg/mL氨苄西林)，保证初始OD600<0.1。当OD600为0.6-0.8时，加入终浓度0.1mM的IPTG并在28.5℃条件下进行诱导表达，12小时后离心收集菌体；
裂解缓冲液重悬菌体后加入溶菌酶(1mg/mL，≥40000u/mg)、DNA酶(5μg/mL，2000u/mg)、氯化镁(30mM)，超声破碎，12000rpm离心60min后收集上清液进行亲和层析纯化，获得超电荷融合蛋白，保存在-80℃冰箱中。
实施例2蛋白粘合剂的制备
将实施例1制备的带正电荷的超电荷融合蛋白EGF-K72及鲑鱼精DNA溶液分别以电荷摩尔比1:2、1:1、1:0.5的比例混合，振荡，12000rpm离心3-5min，液氮或冰箱冷冻至固态后冻干，冻干处理3-10min，最终获得所述蛋白粘合剂，所述蛋白粘合剂用于硬底片时需冻干处理，用于软组织时无需冻干。
制备的蛋白粘合剂对猪皮均具有良好的粘附性，以电荷摩尔比1:1制备的粘合剂为例，其在极端外力条件下展现出强有力的粘附行为，见图1，不同电荷摩尔比制备的蛋白粘合剂在钢基材上的粘合强度如图2所示，以下所述蛋白粘合剂均指电荷摩尔比为1:1时所制备。
实施例3蛋白粘合剂的粘合性能
用砂纸将铁、铝基材表面氧化膜打磨后超纯水清洗，干燥后用于粘合性能测试。将实施例2中以电荷摩尔比EGF-K72:鲑鱼精DNA＝1:1制备的蛋白粘合剂涂覆在基材(铁、铝、玻璃、陶瓷)表面，使用夹具将两块板材固定，剪切搭接面积约为5×5mm，室温固化24h，以10mm/min的拉伸速率进行拉伸测试，测试结果如图3A和表1所示。
表1
Glass Al Ceramics Steel Steel(test502)
Stress(MPa) 9.12±0.39 9.03±0.75 16.32±2.09 18.86±0.94 11.52±0.92
将去除脂肪层的猪皮、肝脏、肌肉切成长条状，湿巾覆盖防止组织干燥，将所述蛋白粘合剂涂抹在软组织一端，粘合固定后以50mm/min的拉伸速率测试其粘合强度，测试结果见图3B和表2。该蛋白粘合剂在亲疏水表面以及软组织上均表现出优异的粘合性能。
表2
Adhesionalenergy(J·m-2) Stress(KPa)
Muscle 12.88±3.29 4.04±0.66
Skin 39.96±5.31 28.55±8.01
Liver 74.00±6.30 16.39±2.92
实施例4蛋白及蛋白粘合剂的生物活性
实施例1制备的带正电荷的超电荷融合蛋白EGF-K72蛋白对细胞迁移能力的影响由单层细胞划痕实验评估。NIH/3T3细胞以104个/孔的密度接种到6孔板中，在含有10％FBS的完全培养基中培养至单层细胞汇合，使用10μL移液器吸头刮擦细胞层，PBS洗涤以除去细胞碎片。细胞在溶有1μM本发明所述蛋白的无血清培养基中进一步培养24h，无蛋白无血清培养的细胞作为对照组，Nikon光学显微镜下观察分析细胞再次汇合情况，如图4A所示。
采用CCK8评估蛋白对NIH/3T3细胞增殖的影响。NIH/3T3细胞以5×103个/孔的密度接种在96孔板中，37℃孵箱中培养16-20h。rhEGF(市售)及本发明蛋白在无血清条件下与细胞共培养24h，每孔加入10μL CCK8，孵育0.5-1h后在450nm波长下使用酶标仪读取吸光度，如图4B和表3所示。综上，EGF-K72重组蛋白具有EGF的生物活性，促进细胞的增殖和迁移。
表3
蛋白粘合剂对细胞增殖的影响按以下方法检测，NIH/3T3细胞以4×103个/孔接种到装有100μL的DMEM培养基(含5％FBS和1％青霉素-链霉素)的96孔板中。细胞贴壁后，用本发明中实施例2中以电荷摩尔比EGF-K72:鲑鱼精DNA＝1:1制备的蛋白粘合剂(100μg/mL)在无血清条件下与细胞共培养24h。加入CCK8后按如上所述进行吸光度测定，结果见图4C和表4。所制备的蛋白粘合剂可发挥超电融合蛋白的生物活性，促进细胞增殖。
表4
Control Positive(EGF) KDadhesive EKDadhesive
Relativecellactivity(％) 100.00±1.98 106.96±1.95 104.40±2.04 110.52±0.48
实施例5蛋白粘合剂止血测试
如图5所示，将购自北京生物技术公司的Wistar大鼠麻醉后，用75％乙醇对胸部、腹部进行消毒，手术暴露肝脏与肾脏，采用1mL注射器针头进行穿刺致出血，将实施例2中以电荷摩尔比EGF-K72:鲑鱼精DNA＝1:1制备的蛋白粘合剂(约5mg蛋白所制备的量，以伤口大小为准，覆盖住既可)应用于出血部位，观察伤口止血情况。相较于未处理部位，本发明制备的蛋白粘合剂具有良好的止血效果。
实施例6蛋白粘合剂促进皮肤创面愈合评估
在大鼠背部构建了圆形全层皮肤缺损伤口模型。将实施例2中以电荷摩尔比EGF-K72:鲑鱼精DNA＝1:1获得的超电荷融合蛋白粘合剂(EKD粘合剂)应用于伤口处，空白、医用EGF凝胶及类弹性蛋白粘合剂(KD粘合剂)作为对照组，KD粘合剂为采用相同方法用K72蛋白制备的粘合剂，用量均0.1g，其中EKD粘合剂为10mg蛋白所制备，刚好覆盖伤口。每只老鼠单笼饲养，在第0、3、5、7、10、14天对伤口部位外观进行拍照，观察创面愈合情况。对第10、14天伤口附近的皮肤组织进行组织学评估，如图6所示。
组织切片显示，应用EKD蛋白粘合剂在第10天时，皮下结缔组织愈合完全，毛囊、血管再生情况较好。空白组伤口处仍观察到有大量炎症细胞浸润，KD组及EGF凝胶组中皮下结缔组织未完全愈合且炎症细胞较多。14天时，EKD蛋白粘合剂组胶原纤维丰富且排列紧密。结果证明，本发明实施例2中以电荷摩尔比EGF-K72:鲑鱼精DNA＝1:1制备的蛋白粘合剂具有促进皮肤愈合的优异性能。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
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Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys
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Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly
500 505 510
Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val
515 520 525
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro
530 535 540
Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly
545 550 555 560
Val Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys
565 570 575
Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly
580 585 590
Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val
595 600 605
Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro
610 615 620
Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly
625 630 635 640
Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys
645 650 655
Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly
660 665 670
Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val
675 680 685
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro
690 695 700
Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly
705 710 715 720
Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys
725 730 735
Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly
740 745 750
Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val
755 760 765
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro
770 775 780
Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly
785 790 795 800
Lys Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Lys
805 810 815
Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly
820 825 830
Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Lys Gly Val
835 840 845
Pro Gly Lys Gly Val Pro Gly Lys Gly Val Pro Gly Trp Pro His His
850 855 860
His His His His
865
<210> 5
<211> 1410
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgaacagtg attcagaatg tcctctctca cacgatggat actgcctcca tgacggcgtg 60
tgtatgtata ttgaagcact agacaaatac gcatgcaact gtgtagttgg ctatattggt 120
gaacgatgcc agtaccgaga tctgaaatgg tgggaactgc gaatgggcgc ggggccgggc 180
gtgggtgttc ccggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 240
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 300
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc ccggtaaagg tgttccgggc 360
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 420
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 480
gtgggtgttc ccggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 540
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 600
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc ccggtaaagg tgttccgggc 660
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 720
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 780
gtgggtgttc ccggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 840
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 900
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc ccggtaaagg tgttccgggc 960
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 1020
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 1080
gtgggtgttc ccggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 1140
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 1200
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc ccggtaaagg tgttccgggc 1260
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 1320
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 1380
tggccgcacc accaccacca ccactgataa 1410
<210> 6
<211> 810
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgaacagtg attcagaatg tcctctctca cacgatggat actgcctcca tgacggcgtg 60
tgtatgtata ttgaagcact agacaaatac gcatgcaact gtgtagttgg ctatattggt 120
gaacgatgcc agtaccgaga tctgaaatgg tgggaactgc gaatgggcgc ggggccgggc 180
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 240
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 300
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 360
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 420
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 480
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 540
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 600
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 660
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 720
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 780
tggccgcacc accaccacca ccactgataa 810
<210> 7
<211> 2009
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgaacagtg attcagaatg tcctctctca cacgatggat actgcctcca tgacggcgtg 60
tgtatgtata ttgaagcact agacaaatac gcatgcaact gtgtagttgg ctatattggt 120
gaacgatgcc agtaccgaga tctgaaatgg tgggaactgc gaatgggcgc ggggccgggc 180
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 240
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 300
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 360
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 420
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 480
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 540
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 600
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 660
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 720
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 780
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 840
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 900
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 960
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 1020
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 1080
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 1140
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 1200
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 1260
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 1320
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 1380
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 1440
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 1500
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 1560
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 1620
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 1680
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 1740
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 1800
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 1860
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 1920
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 1980
tggccgcacc accaccacca ccactgata 2009
<210> 8
<211> 2610
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgaacagtg attcagaatg tcctctctca cacgatggat actgcctcca tgacggcgtg 60
tgtatgtata ttgaagcact agacaaatac gcatgcaact gtgtagttgg ctatattggt 120
gaacgatgcc agtaccgaga tctgaaatgg tgggaactgc gaatgggcgc ggggccgggc 180
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 240
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 300
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 360
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 420
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 480
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 540
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 600
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 660
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 720
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 780
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 840
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 900
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 960
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 1020
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 1080
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 1140
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 1200
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 1260
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 1320
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 1380
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 1440
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 1500
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 1560
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 1620
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 1680
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 1740
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 1800
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 1860
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 1920
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 1980
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 2040
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 2100
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 2160
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 2220
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 2280
gtgggtgttc cgggtaaagg tgttccgggc aaaggtgtgc caggcaaagg tgttccgggt 2340
aaaggtgtgc cgggtaaagg cgtaccgggt aaaggcgtac caggcaaagg tgttccgggt 2400
aaaggcgtac caggtaaagg tgtgccgggc gtgggtgttc cgggtaaagg tgttccgggc 2460
aaaggtgtgc caggcaaagg tgttccgggt aaaggtgtgc cgggtaaagg cgtaccgggt 2520
aaaggcgtac caggcaaagg tgttccgggt aaaggcgtac caggtaaagg tgtgccgggc 2580
tggccgcacc accaccacca ccactgataa 2610
<210> 9
<211> 54
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu
1 5 10 15
His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys
20 25 30
Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu
35 40 45
Lys Trp Trp Glu Leu Arg
50
<210> 10
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tttaagaagg agatatacat atgaacagtg attcaga 37
<210> 11
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gcccggcccc gcgcccattc gcagttccca ccatttca 38

Claims (7)

1.蛋白粘合剂，其特征在于，由融合蛋白和鲑鱼精DNA组成；
所述融合蛋白包括：生长因子和类弹性蛋白；
所述融合蛋白与所述鲑鱼精DNA的摩尔比为1:（0.5~2）；
所述类弹性蛋白包含n个重复的五肽序列GKGVP，其中n为≥18的整数。
2.如权利要求1所述的蛋白粘合剂，其特征在于，所述融合蛋白具有：
（1）、如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3和/或SEQ ID NO:4所示的氨基酸序列；或
（2）、在如（1）所示的氨基酸序列的基础上经取代、缺失和/或添加1个或多个氨基酸的序列；或
（3）、与如（1）所示的氨基酸序列同源性90%以上的序列。
3.如权利要求2所述的蛋白粘合剂，其特征在于，编码所述融合蛋白的核酸分子具有：
（4）、如SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7和/或SEQ ID NO:8所示的核苷酸序列；或
（5）、与（4）所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与（4）所示的核苷酸序列不同的核苷酸序列；或
（6）、（4）或（5）所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与（4）或（5）所示的核苷酸序列功能相同或相似的核苷酸序列。
4.如权利要求1所述的蛋白粘合剂，其特征在于，所述生长因子具有：
（7）、如SEQ ID NO:9所示的氨基酸序列；或
（8）、在如（7）所示的氨基酸序列的基础上经取代、缺失和/或添加1个或多个氨基酸的序列；或
（9）、与如（7）所示的氨基酸序列同源性90%以上的序列。
5.如权利要求1至4任一项所述蛋白粘合剂的融合蛋白的制备方法，其特征在于，包括以下步骤：
S1：构建所述生长因子的引物，PCR扩增，获得基因元件；
S2：取所述基因元件与所述类弹性蛋白的表达载体经酶切后进行同源重组，获得蛋白表达载体；
S3：取所述蛋白表达载体转化，筛选，进而培养、诱导表达，纯化，获得所述融合蛋白。
6.如权利要求1至4任一项所述蛋白粘合剂的制备方法，其特征在于，包括：取所述融合蛋白与所述鲑鱼精DNA混合，离心后收集底部凝聚物。
7.如权利要求1至4任一项所述的蛋白粘合剂或如权利要求6所述制备方法制得的蛋白粘合剂在制备促进细胞增殖和/或迁移、伤口和/或组织止血、愈合、粘合的产品中的一种或多种应用。
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