CN113527526B - 一种重组蛋白及其构建方法、用途 - Google Patents
一种重组蛋白及其构建方法、用途 Download PDFInfo
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- CN113527526B CN113527526B CN202111075036.2A CN202111075036A CN113527526B CN 113527526 B CN113527526 B CN 113527526B CN 202111075036 A CN202111075036 A CN 202111075036A CN 113527526 B CN113527526 B CN 113527526B
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Abstract
本发明公开了一种重组透皮纤连蛋白突变体,由透皮肽通过连接肽连接纤连蛋白构成重组透皮纤连蛋白,然后对重组透皮纤连蛋白的氨基酸序列进行E174D和P292G位点突变构建得到的,所述重组透皮纤连蛋白突变体的氨基酸序列如SEQ ID No.1所示。本发明提供的重组蛋白具有较佳的稳定性且功效得以保证,制备简便成本较低,极大地拓展了纤连蛋白和胶原蛋白酶抑制肽在化妆品中的应用。
Description
技术领域
本发明涉及一种重组蛋白,具体地说是一种具有高稳定性的用于化妆品制备或添加的重组蛋白及其构建方法。
背景技术
纤连蛋白(fibronectin,又名Fn)是一种细胞外基质中的高分子量糖蛋白。FN由肝脏及血管内皮细胞生成,广泛存在于动物组织和组织液中,是一种大分子糖蛋白,具有细胞再生修复的生物学功能,目前在医疗临床上已有所应用,包括用于治疗脊柱疾病、烧伤等。经验证明,纤连蛋白对敏感肌肤、红血丝、痤疮、晒后损伤等问题肌肤都有修复作用。
纤连蛋白虽具有优异的护肤功效,但其热稳定性及化学稳定性不佳。热稳定性不佳主要体现在40℃以上的环境下纤连蛋白易变性造成化妆品的析出、沉淀或变色,功效随之受到影响。化学稳定性不佳主要体现在受表面活性剂等常规添加剂影响造成的纤连蛋白变性。上述问题限制了纤连蛋白在化妆品领域的应用。
现有技术对纤连蛋白稳定性提升主要是利用稳定剂,纤连蛋白稳定剂的有效成分为海藻酸钠、胶原水解物或甘油等。其原理是利用稳定剂提高纤连蛋白水溶液在化妆品体系中的聚集稳定性和分散稳定性,有利于纤连蛋白水溶液在较长时期内保持其稳定性。上述稳定剂虽然可以提升纤连蛋白的稳定性,但是会增加化妆品的制备成本,另外上述成分对其他功效成分的效能可能会产生影响,添加比例、制备过程需要严格调试,既要满足纤连蛋白稳定性要求,又要保证其他功效成分功效发挥不受影响,提升了化妆品配方的研发难度。
另外在化妆品配方中,往往会使用多个蛋白肽进行复配,如常用的基质金属蛋白酶抑制肽。基质金属蛋白酶是一类具有降解细胞外基质(ECM)能力的内肽酶家族,包括胶原蛋白酶和弹性蛋白酶等。其中胶原蛋白酶MMP-3,1,8等可以分解皮肤中的胶原蛋白;弹性蛋白酶(MMP-12)能降解弹性蛋白,并且能激活其他基质金属蛋白酶的活性。通过抑制肽抑制胶原蛋白酶和弹性蛋白酶的活性,能够有效减少皮肤中这两类结构蛋白的降解,缓解成纤维细胞的凋亡,从而起到皮肤抗衰老的目的。
现有技术在化妆品配方中一般利用物理混合的方法将上述小分子多肽和纤连蛋白复配,纤连蛋白本身的稳定性欠佳,再添加小分子多肽,稳定性能的挑战更大,制备成本也较高。同时,由于皮肤表面有一层物理屏障,阻止了绝大多数大分子有效成分通过皮肤发挥作用,因为蛋白多肽复合物的透皮性能往往不佳,使得原本具有高活性的多肽无法充分发挥其应有的效果,这也进一步限制了蛋白多肽在化妆品中的应用。
发明内容
本发明所要解决的首要技术问题在于提出一种稳定性能优异的重组透皮纤连蛋白突变体。
本发明所要解决的另一技术问题在于提出一种在上述重组透皮纤连蛋白突变体柔性末端连接胶原蛋白酶抑制肽得到一种重组蛋白。
本发明所要解决的又一技术问题在于提出一种上述重组蛋白的构建方法及用途。
为了实现上述目的,本发明采用下述的技术方案:
一种重组透皮纤连蛋白突变体,由透皮肽通过连接肽连接纤连蛋白构成重组透皮纤连蛋白,然后对重组透皮纤连蛋白的氨基酸序列进行E174D和P292G位点突变构建得到的,所述重组透皮纤连蛋白突变体的氨基酸序列如SEQ ID No.1所示。
编码所述重组蛋白的基因序列如SEQ ID No.2所示。
上述重组透皮纤连蛋白突变体在制备外用护肤品添加剂或护肤制剂中的用途。
一种基于上述重组透皮纤连蛋白突变体制备得到的具有抗衰老功效的重组蛋白,其是在上述重组透皮纤连蛋白突变体的柔性末端通过连接肽连接胶原蛋白酶抑制肽制备得到的。
上述连接肽的氨基酸序列如SEQ ID No.3所示。
上述重组蛋白的氨基酸序列如SEQ ID No.4所示。
编码上述重组蛋白的核苷酸序列如SEQ ID No.5所示。
上述重组蛋白在制备外用化妆品添加剂或化妆品制剂中的用途。
上述外用护肤品添加剂或护肤制剂为抗衰老外用护肤品添加剂或护肤制剂。
上述重组蛋白的制备方法,包括如下的步骤:
(1)在载体上***编码所述重组蛋白核苷酸序列获得重组质粒;
(2)提取上述步骤得到的重组质粒的基因组DNA,并线性化,电转入感受态毕赤酵母;加入预冷的山梨醇,然后将内容物涂于培养基上,孵育,至克隆产生;
(3)筛选可表达目的重组蛋白的菌株,对上述筛选菌株进行表达纯化。
本发明的有益结果如下:
(1) 本发明将透皮肽与纤连蛋白连接,提高了纤连蛋白的透皮性能。本发明还将构建的透皮纤连蛋白的特定区域进行定点突变,提高了透皮纤连蛋白的稳定性,稳定性提升的同时并不影响纤连蛋白功效,更使其功效发挥更充分。
(2) 本发明改进了现有纤连蛋白和胶原蛋白酶抑制肽通过物理混合造成稳定性差、功效下降的问题。本发明将胶原蛋白酶抑制肽通过连接肽连接于上述重组透皮纤连蛋白的柔性末端,进一步提高了重组透皮纤连蛋白的稳定性,同时又提高了抑制肽的稳定性。
(3) 本发明对连接肽进行了优化,避免了由于连接肽和抑制肽形成二级结构进而影响抑制肽功能的问题。
(4) 将纤连蛋白和胶原蛋白酶抑制肽重组后在酵母体系表达,改进了现有技术单独化学合成小分子肽的过程,成本节约了50%以上。
(5) 本发明提供的重组蛋白具有较佳的稳定性且功效得以保证,制备简便成本较低,这极大的拓展了纤连蛋白和胶原蛋白酶抑制肽在化妆品中的应用。
附图说明
图1为本发明实施例中,重组透皮纤连蛋白突变体表达载体质粒图谱;
图2为本发明实施例中,制备的重组蛋白纯化后的表达量图;
图3为本发明实施例中,重组蛋白对成纤维细胞中胶原蛋白的影响;
图4为本发明实施例中,重组蛋白对成纤维细胞存活率的影响;
图5为本发明实施例中,重组蛋白对人成纤维细胞粘附的影响;
图6为本发明实施例中,重组蛋白稳定性测试结果图。
具体实施方式
本实施例所用原料均为本领域公知原料,可通过市售购买获得,符合化妆品领域原料标准。
本发明利用对纤连蛋白的定点突变提升了纤连蛋白的稳定性,同时利用在纤连蛋白柔性末端连接抑制肽实现对其稳定性的进一步提升。本实施例所用胶原蛋白酶抑制肽SEQ ID No.7所示序列的原序列来自于蛋白酶处理深海鳕鱼胶原蛋白后产生的胶原蛋白多肽,该抑制肽效果较佳序列精简。本发明提供的重组透皮纤连蛋白突变体具有极佳的稳定性,胶原蛋白酶抑制肽连接于其柔性末端可进一步增加重组蛋白的稳定性,因此,胶原蛋白酶抑制肽也可以选择本领域其他来源的胶原蛋白酶抑制肽,不影响本发明最终创造性功效的实现。
本发明涉及两个重组蛋白肽,分别为SEQ ID No.1所示的透皮纤连蛋白突变体重组蛋白,以及在SEQ ID No.1的基础上利用连接肽将胶原蛋白酶抑制肽连接于透皮纤连蛋白突变体的柔性末端,最终得到SEQ ID No.4所示的透皮纤连蛋白突变体-胶原蛋白酶抑制肽重组蛋白。上述两个重组蛋白的构建方法相同,下面通过详细的实施例进一步对本发明进行描述。
实施例1透皮纤连蛋白突变体的研发
1、突变体研发思路:
化妆品配方中有表面活性剂,增稠剂,香精,多元醇等成分,它们会影响蛋白肽在化妆品中的稳定性。同时,化妆品在运输和储存中可能遇到高温条件,加速蛋白肽的降解。
本发明将透皮肽与纤连蛋白进行重组后根据重组蛋白的三维结构特征,透皮肽纤连蛋白序列如SEQ ID No.6所示,在其表面进行氨基酸点突变,可显著提高重组蛋白的稳定性,突变点选择有效避开活性位点,对重组纤连蛋白的功能不产生任何影响。
实验设计:从RCSB网站上下载人纤连蛋白的晶体结构文件1fnf.pdb。用PyMOL软件分析其温度因子B-facor,预测热敏感氨基酸位点。B-factor数值越高,模糊度越大,相应部位的构象就越不稳定或者柔性越强。重组透皮纤连蛋白的温度敏感位点为N138、E174、G230、P292,均在蛋白质表面的柔性区。通过PCR对位点进行单点突变或者两个位点同时突变(N138Q,E174D, G230V ,P292G),构造多种突变体,在各突变体溶液中加入表面活性剂SLS,40℃储存1个月来进行热加速老化实验,选择稳定性更高的重组蛋白突变体。重组蛋白突变体的具体构建方法和实施例2一致。
2、重组透皮纤连蛋白突变体稳定性测试方法:
取重组透皮纤连蛋白突变体100毫升300uM的溶液,加入1% SLS溶液,制成重组蛋白+1%SLS溶液。重组蛋白,重组蛋白+1%SLS溶液分到10ml西林瓶中,10ml/瓶, 每个样品准备三瓶。测定各样品各指标初始值,然后在40℃恒温箱中储存,1个月后,每个样品取200μL置于96孔板的样品孔内,每瓶做3个重复,不含蛋白的稀释液作为空白对照,酶标仪630nm下检测样品OD值,OD值越大说明样品越浑浊,越小则越清澈;浑浊度表征蛋白的稳定性,蛋白越不稳定越容易产生沉淀,浑浊程度越高。本研究设计的各突变体及具体实验结果见表1。
表1
编码 | 样品 | SLS含量 | 储存前OD630 | 40℃(1个月)OD630 | OD630变化值 |
C-C | 不含蛋白的空白溶液 | 1% | 0.004 | 0.002 | 0.002 |
C-0 | 重组透皮纤连蛋白 | 1% | 0.029 | 0.064 | 0.035 |
A-1 | 突变体1(N138Q) | 1% | 0.031 | 0.050 | 0.019 |
A-2 | 突变体2(E174D) | 1% | 0.029 | 0.039 | 0.010 |
A-3 | 突变体3 (G230V) | 1% | 0.029 | 0.049 | 0.020 |
A-4 | 突变体4 (P292G) | 1% | 0.030 | 0.046 | 0.011 |
A-5 | 突变体5 (E174D, P292G)(SEQ ID No.1) | 1% | 0.030 | 0.036 | 0.006 |
A-6 | 突变体6(N138Q, G230V) | 1% | 0.031 | 0.039 | 0.018 |
如上表所示,在重组透皮纤连蛋白突变体上同时突变E174D和P292G两个位点得到的重组蛋白A-5在40℃储存1个月后,OD630值的变化最小,表明该突变体增强了蛋白结构的稳定性,并且和化妆品中其他原料的配伍性更好,也更能适应高温的存储条件和运输。
实施例2透皮纤连蛋白突变体重组蛋白质粒的构建
本发明实施例中,重组透皮纤连蛋白突变体(A-5)的氨基酸序列如SEQ ID No.1所示,其中ACSPPHSKSHC为透皮肽序列,可以增强该蛋白的透皮吸收性能,GGGGS为透皮肽和纤连蛋白之间的连接。
构建方法:
(1)根据商用载体pPICZaA相关序列位置设计选择酶切位点EcoR I和SalI;编码透皮纤连蛋白突变体重组蛋白的DNA序列如SEQ ID No.2;
(2)在所合成的重组蛋白DNA序列的5’和3’端都各有一个限制性内切酶位点,分别对应EcoR I和SalI;
(3)将重组蛋白目的片段***载体酶切位点之间,获得编码上述重组蛋白的表达质粒,载体质粒结构图如图1所示。
实施例3透皮纤连蛋白突变体-胶原蛋白酶抑制肽重组蛋白质粒的构建
本发明实施例中,透皮纤连蛋白突变体-胶原蛋白酶抑制肽重组蛋白是在实施例2制备的重组透皮纤连蛋白突变体柔性末端通过连接肽连接胶原蛋白酶抑制肽,该重组蛋白的氨基酸序列如SEQ ID No.4所示。
构建方法:
(1)根据商用载体pPICZaA相关序列位置设计选择酶切位点EcoR I和SalI;编码透皮肽-纤连蛋白突变体-胶原蛋白酶抑制肽重组蛋白的DNA序列如SEQ ID No.5所示;
(2)在所合成的重组蛋白DNA序列的5’和3’端都各有一个限制性内切酶位点,分别对应EcoR I和SalI;
(3)将重组蛋白目的片段***载体酶切位点之间,获得编码上述重组蛋白的表达质粒。
实施例4重组蛋白质粒表达纯化及电泳鉴定方法
实施例2和实施例3所述重组蛋白质粒的表达纯化和电泳鉴定方法相同,具体方法如下述:
(1)酵母菌克隆的制备
提取表达载体重组蛋白基因组DNA,将质粒用限制性内切酶FastDigest SacI酶切(即线性化,Thermo公司),纯化后将线性化质粒与毕赤酵母工程菌(Pichia pastoris)X33(北京百奥莱博科技有限公司)感受态混合,冰浴5min,转入预冷的电击杯中,按照电击仪的预设程序电击(2mm电击杯,电击条件:2000V、25μF、200Ω),电击后立即加入1ml 预冷的1M山梨醇,然后转移到1.5ml离心管中,将离心管置于30°C 培养箱复苏2-3h,分别涂Zeocin浓度为0.1mg/ml、1mg/ml、2mg/ml的YPDS(Yeast Extract Peptone Dextrose Medium)平板,30°C培养3-5天。
(2)重组蛋白的表达纯化
挑取单克隆转入250mL BMGY(Buffered Glycerol-complex Medium)摇瓶培养基中,30℃,200rpm培养至OD600=2.0-6.0,将发酵液于4℃8000rpm离心10min去除上清,收集菌体;取适量菌体重悬于含有0.25mM CuSO4和1%甲醇的BMMY摇瓶培养基至OD600≈0.6,30℃,200rpm。在1L摇瓶中加入上述培养物,加盖两层灭菌纱布或干酪包布,放入摇床继续生长。每隔24h补加1%(w/v,终浓度)甲醇。培养96小时后,将发酵后的菌液3000g离心5分钟,收集上清,弃沉淀。由于重组表达的蛋白带有多聚组氨酸标签(His6·tag),因此使用镍离子亲和层析法分离目标蛋白。
(3)镍离子亲和层析纯化蛋白的步骤:
(a)平衡:用10倍住体积的20mM缓冲液(含5mM的咪唑)平衡HisTrap HP镍离子柱(1mL);
(b)上样:预先处理好的样品以1mL/min的流速上样;
(c)洗脱:用高浓度咪唑进行梯度洗脱,收集洗脱条件下峰型对应的管号,并做SDS-PAGE,跑SDS-PAGE蛋白电泳确认分子量为重组蛋白的管中蛋白液体合并,结合到阴离子交换树脂上,再用150mM NaCl,20mM磷酸盐0.5M尿素溶液进行洗脱。
所获得的蛋白纯度经SDS-PAGE电泳鉴定蛋白条带较单一,纯度在95%以上,结果如图2所示,图中分别为纤连蛋白C-0、重组透皮纤连蛋白突变体A-5及重组透皮纤连蛋白突变体-胶原蛋白酶抑制肽B3。
实施例5连接肽序列的设计及研究及重组透皮纤连蛋白突变体-胶原蛋白酶抑制肽功效实验
本发明实施例中,重组透皮纤连蛋白突变体和胶原蛋白酶抑制肽之间通过连接肽相连。连接肽的长度和序列可能会影响重组透皮纤连蛋白突变体和抑制肽的结构和活力。长度过短,重组透皮纤连蛋白突变体和抑制肽可能对双方造成空间位阻,影响重组透皮纤连蛋白突变体和抑制肽的结合,减弱胶原蛋白酶抑制肽对胶原蛋白酶的抑制作用,同时,纤连蛋白促进细胞黏附的作用也会减弱。长度过长,则突变体、连接肽和抑制肽易形成二级结构,也会影响重组蛋白的功能。选择合适的连接肽对于保证重组蛋白的功能非常重要。样品序列及连接肽设计见表2。
表2
编码 | 样品序列 | 连接肽 |
C-00 | 突变体5(A-5) | 无 |
C-01 | 胶原蛋白酶抑制肽 | 无 |
B-1 | 突变体5+连接肽+胶原蛋白酶抑制肽 | AG |
B-2 | 突变体5+连接肽+胶原蛋白酶抑制肽 | AGG |
B-3 | 突变体5+连接肽+胶原蛋白酶抑制肽 | GGGGS |
B-4 | 突变体5+连接肽+胶原蛋白酶抑制肽 | GGGAAAA |
B-5 | 突变体5+连接肽+胶原蛋白酶抑制肽 | GGGAAAASS |
1、成纤维细胞实验
(1)重组蛋白对光损伤成纤维细胞中胶原蛋白的影响
人皮肤成纤维细胞(HFF,中国科学院典型培养物保藏委员会细胞库);DMEM高糖培养基、胎牛血清、胰蛋白酶、青/链霉素(美国Gibco公司); MTT、DMSO、EDTA(美国Sigma公司)。
取对数生长期的HFF细胞,接种于96孔板培养24h后,弃去旧培养液用PBS洗涤1次,加入少量PBS,使覆盖到培养皿底部,紫外灯下(距紫外灯20厘米处)照射10min,照射后更换新鲜含有300uM重组蛋白的DMEM培养液继续培养24h。
培养细胞后,倒掉含有重组蛋白的DMEM培养基。用 PBS将残留细胞清洗干净,加入0.25%的胰酶消化,轻轻将细胞吹打下来,收集细胞并离心(1000×g,3min)。 之后弃上清液并加入200μLPBS重悬细胞。重复上述离心步骤,弃上清,加入200μL RIPA裂解液处理15min裂解细胞。然后在13000×g,4℃条件下离心5min,收集上清裂解液。使用相关试剂盒,并且根据说明书测定裂解液中胶原蛋白含量。
UV照射后成纤维细胞中的胶原蛋白酶含量明显上升,导致胶原蛋白被分解而含量迅速下降。如图3所示,实验组B-3,即连接肽序列为GGGGS时,胶原蛋白有显著上升,其上升程度和对照组C-01很接近并且相比C-01略有上升,表明连接肽GGGGS没有影响酶抑制肽的功能反而有促进作用。而含有其他连接肽的实验组则上升程度很低,表明其他连接肽降低了抑制肽的活力。
(2)添加重组蛋白后,对光老化成纤维细胞HFF保护作用
细胞培养后,用MTT法测定细胞存活率。UV 具有强穿透作用,会引起成纤维细胞内线粒体上DNA的损伤及MMPs水平的上调等,最终引起成纤维细胞的凋亡。从图4结果中看出,UV照射后,HFF的存活率降低。当受损的成纤维细胞和300uM重组蛋白共同培养24小时后,细胞存活率有不同程度的上升。实验组B-3减少UV诱导的成纤维细胞凋亡的效果更显著,和对照组C-00和C-01很接近且略好于C-01,表明连接肽GGGGS没有影响酶抑制肽的功能反而对其功能有促进作用。而含有其他连接肽的实验组则效果不显著,再次证明其他连接肽降低了抑制肽的活力。
重组蛋白促细胞粘附和生长试验
将各重组蛋白分别配置成20ug/mL后被包被96孔板30分钟,用PBS洗涤两遍。再加1%BSA于37℃封闭30分钟后,加入人成纤维细胞(用无血清培养基培养)。1h后轻轻吸取孔中培养基,用PBS轻轻漂洗未吸附的细胞,用CCD8法检测吸附在孔板底部活细胞的数量,验证重组蛋白的促进细胞粘附的活性。如图5所示,实验组B-3、B-4和B-5随着浓度额升高,显著增强了细胞的粘附,和C-00接近,其中B-3表现较佳。而含有较短连接肽的实验组B-1和B-2则细胞的黏附较差,较短的连接肽可能造成抑制肽对纤连蛋白形成空间位阻,而影响了纤连蛋白促进细胞黏附的功能。
综合上述实验结果,连接肽选择GGGGS时,纤连蛋白突变体和抑制肽的活力及功效均不受影响。
重组蛋白稳定性测定
同突变体A1-A5稳定性筛选的实验方法, 测试样本C-0,A-5,B-1,B-2,B-3,B-4,B-5的热稳定性。取重组纤连蛋白100毫升300uM的溶液,加入1% SLS溶液,制成重组蛋白+1%SLS溶液。重组蛋白,重组蛋白+1%SLS溶液分到10ml西林瓶中,10ml/瓶,每个样品准备三瓶。测定各样品各指标初始值,然后在40℃恒温箱中储存,1个月及3个月后,每个样品取200μL置于96孔板的样品孔内,每瓶做3个重复,不含蛋白的稀释液作为空白对照,酶标仪630nm下检测样品OD值。OD值越大说明样品越浑浊,越小则越清澈;浑浊度表征蛋白的稳定性,蛋白越不稳定越容易产生沉淀,浑浊程度越高。
如图6所示,40℃储存一个月时,各突变体都比C-0的OD630值变化小,且各突变体之间差异不显著;储存3个月后,B1,B2,B3,B4,B5的稳定性都显著比A-5更好。因此,在A-5突变体的基础上,在其C末端连接一段多肽可稳定柔性的C末端, 进一步提高了重组蛋白的稳定性,各连接肽对稳定性的影响差异不显著。
序列表
<110> 美慕(北京)科技有限公司
<120> 一种重组蛋白及其构建方法、用途
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gcgctgaacg gccgcgaaga aagcccgctg ctgattggcc agcagagcac cgtgagcgat 840
gtgccgcgcg atctggaagt ggtggcggcg accggcacca gcctgctgat tagctgggat 900
gcgccggcgg tgaccgtgcg ctattatcgc attacctatg gcgaaaccgg cggcaacagc 960
ccggtgcagg aatttaccgt gccgggcagc aaaagcaccg cgaccattag cggcctgaaa 1020
ccgggcgtgg attataccat taccgtgtat gcggtgaccg gccgcggcga tagcccggcg 1080
agcagcaaac cgattagcat taactatcgc accggcggcg gcggcagcgg cccgccgggc 1140
ccgaccggcg cg 1152
<210> 6
<211> 371
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Ala Cys Ser Pro Pro His Ser Lys Ser His Cys Gly Gly Gly Gly Ser
1 5 10 15
Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys Tyr Ile Leu
20 25 30
Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu Ala Thr Ile
35 40 45
Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys Pro Gly Val
50 55 60
Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly His Gln Glu
65 70 75 80
Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr Gly Gly Ser
85 90 95
Ala Val Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp
100 105 110
Thr Met Arg Val Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn
115 120 125
Phe Leu Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu
130 135 140
Leu Ser Ile Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu
145 150 155 160
Pro Gly Thr Glu Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His
165 170 175
Glu Ser Thr Pro Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro
180 185 190
Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His
195 200 205
Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His
210 215 220
Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser
225 230 235 240
Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val
245 250 255
Val Ser Ile Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile
260 265 270
Gly Gln Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
275 280 285
Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val
290 295 300
Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser
305 310 315 320
Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile
325 330 335
Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val
340 345 350
Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn
355 360 365
Tyr Arg Thr
370
<210> 7
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Gly Pro Pro Gly Pro Thr Gly Ala
1 5
Claims (10)
1.一种重组透皮纤连蛋白突变体,其特征在于,由透皮肽通过连接肽连接纤连蛋白构成重组透皮纤连蛋白,然后对重组透皮纤连蛋白的氨基酸序列进行E174D和P292G位点突变构建得到的,所述重组透皮纤连蛋白突变体的氨基酸序列如SEQ ID No.1所示。
2.如权利要求1所述的重组透皮纤连蛋白突变体,其特征在于,编码所述重组透皮纤连蛋白突变体的基因序列如SEQ ID No.2所示。
3.权利要求1或2所述重组透皮纤连蛋白突变体在制备外用护肤品添加剂或护肤制剂中的用途。
4.一种重组蛋白,其特征在于,在权利要求1或2所述重组透皮纤连蛋白突变体的柔性末端通过连接肽连接胶原蛋白酶抑制肽。
5. 如权利要求4所述的重组蛋白,其特征在于,所述连接肽的氨基酸序列如SEQ IDNo.3所示。
6. 如权利要求5所述的重组蛋白,其特征在于,所述胶原蛋白酶抑制肽的氨基酸序列如SEQ ID No.7所示。
7. 如权利要求6所述的重组蛋白,其特征在于,所述重组蛋白的氨基酸序列如SEQ IDNo.4所示。
8. 如权利要求6所述的重组蛋白,其特征在于,编码所述重组蛋白的核苷酸序列如SEQID No.5所示。
9.权利要求4至8中任意一项所述重组蛋白的制备方法,其特征在于,包括如下步骤:
(1)在载体上***编码所述重组蛋白核苷酸序列获得重组质粒;
(2)提取上述步骤得到的重组质粒的基因组DNA,并线性化,电转入感受态毕赤酵母;加入预冷的山梨醇,然后将内容物涂于培养基上,孵育,至克隆产生;
(3)筛选可表达目的重组蛋白的菌株,对上述筛选菌株进行表达纯化。
10.权利要求4至8中任意一项所述重组蛋白在制备外用化妆品添加剂或化妆品制剂中的用途。
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