CN114805534B - 人成纤维细胞生长因子突变体及其编码基因、制备方法与应用 - Google Patents
人成纤维细胞生长因子突变体及其编码基因、制备方法与应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域，尤其涉及人成纤维细胞生长因子突变体及其编码基因、制备方法与应用。本发明提供了一种高度稳定的碱性成纤维生长因子突变体，其具有SEQ ID NO：2所述的氨基酸序列。该突变体大大提高了在水溶液状态下bFGF的稳定性以及热稳定性，并且增加分子内二硫键的碱性成纤维细胞生长因子具有与天然生长因子具有相似的促细胞增殖能力。在应用中即使在分装和储存期间，也可以生产不失去活性的功能性化妆品和皮肤炎症药物。
Description
技术领域
本发明涉及生物技术领域，尤其涉及人成纤维细胞生长因子突变体及其编码基因，制备方法与应用。
背景技术
碱性成纤维细胞生长因子(Basic fibroblast growth factor, bFGF)最早从牛脑垂体中通过与肝素的高亲和性结合纯化而来，因具有促进成纤维细胞生长的生物活性而命名。
bFGF是一种具有代表性的生长因子，它通过结合和激活成纤维细胞生长因子受体发挥多种功能，对组织的修复和再生具有潜在的作用。目前，已被广泛应用于已被用于受损组织的再生，包括皮肤、血管、肌肉、脂肪、肌腱/韧带、软骨、骨头、牙齿和神经。由于bFGF出色的促增殖能力，目前，已有许多研究报道bFGF被吸附在材料上或封装在材料内直接用于伤口表面修复。
野生型人bFGF自身性质并不稳定，易被伤口表面的金属酶降解失活。有实验表明，室温放置半个小时的bFGF促细胞增殖能力将大大减弱，需不断向伤口处补充有活性的生长因子。因此，进一步研究提高生长因子的稳定性及延长在常温条件下活性保持的时间极其有必要。
发明内容
有鉴于此，本发明要解决的技术问题在于提供高度稳定的碱性成纤维细胞生长因子突变体及其用途。
本发明提供的人碱性成纤维细胞生长因子突变体，其在如SEQ ID NO:1所示野生型人碱性成纤维细胞生长因子的氨基酸序列中，存在如下突变位点中的至少一种：P49C、Y73C、C87S。
本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在P49C突变。
或者本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在Y73C突变。
或者本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在C87S突变。
或者本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在P49C和Y73C突变。
或者本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在Y73C和C87S突变。
或者本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在C87S和P49C突变。
或者本发明所述的bFGF突变体，其在如SEQ ID NO:1所示氨基酸序列中，存在P49C、C87S和P49C突变。该突变体的氨基酸序列如SEQ ID NO:2所示。
现有技术中，大多改造bFGF的方案都是以减少分子间二硫键的形成为出发点。例如，天然bFGF第69位及第87位半胱氨酸暴露在结构表面，而为了提高bFGF的稳定性，有报道将第69位C及第87位C突变为S，以减少分子间二硫键的形成。而本发明将第87位半光氨酸突变为丝氨酸，并在此基础上将第49位P突变为半胱氨酸，将第73位的Y也突变成半胱氨酸从而提高了bFGF的稳定性。经进一步分析，本发明提供的bFGF中，第87位氨基酸的b-factor被降低，第49位氨基酸能够与第69位处半光氨酸形成二硫键，而第73位的Y与第92位处半胱氨酸形成二硫键。通过DSC(差示扫描量热法)检测，与WT-bFGF相比，发明的bFGF突变体Tm值提高约5.1℃。且经体外细胞实验证实，本发明提供的增加二硫键的bFGF 与天然bFGF具有相似的促细胞增殖能力，并且具有较好的稳定性,可延长其保留生物活性的时间。
本发明还提供了编码本发明所述的突变体的核酸。
一些具体实施例中，所述编码人碱性成纤维细胞生长因子突变体的核酸序列如SEQ ID NO:3所示。
本发明还提供了含有所述核酸的质粒载体。
本发明所述的质粒载体能够实现本发明所述核酸在大肠杆菌或其他表达宿主细胞中的保存或表达。一些实施例中，所述质粒的骨架载体为ppGH载体。
本发明还提供了一种宿主细胞，其转化或转染所述的表达载体。所述大肠杆菌为大肠杆菌Origami（DE3）。
本发明所述宿主细胞的构建方法，包括将本发明所述的质粒载体转化入大肠杆菌感受态细胞。
本发明所述突变体的制备方法包括：培养本发明所述的宿主细胞，诱导所述突变体的表达。在本发明所述的制备方法中，所述诱导的诱导剂为IPTG。
本发明所述的突变体、所述的核酸，所述的表达载体，所述的宿主细胞或所述制备方法制得的产物，在制备培养细胞的产品和/或修复皮肤的产品中的应用。
本发明还提供了一种培养细胞的产品，其包括本发明所述的突变体、所述的核酸，所述的表达载体，所述的宿主细胞或所述制备方法制得的产物。
本发明所述培养细胞的产品包括细胞的培养基，或者细胞的培养添加剂。
本发明所述的细胞包括普通体细胞或者干细胞。一些实施例中，所述细胞为成纤维细胞。
一种培养细胞的方法，其以本发明所述培养细胞的产品培养细胞。
本发明所述的培养细胞包括对细胞的增殖培养、分化培养或者细胞的保存。本发明所述的增殖培养包括对体细胞的增殖培养，经过培养后细胞的数量发生扩大。本发明所述的分化培养包括对干细胞的分化培养，经过培养后干细胞分化为目标细胞。本发明中，所述的细胞保存包括在细胞的冷冻保存中保护细胞的活性或在细胞转运过程中保护细胞的活性。
本发明还提供了一种修复皮肤的产品，其包括本发明所述的突变体、所述的核酸，所述的表达载体，所述的宿主细胞或所述制备方法制得的产物。
本发明还提供了一种修复皮肤的方法，其包括给予本发明所述修复皮肤的产品。
本发明中，所述修复皮肤包括促进伤口的愈合，也包括对皮肤屏障的修复，对炎症皮肤的改善等，例如保护皮肤角质层，恢复皮肤的保湿效果、消除皮肤炎症等。
本发明中，所述的修复皮肤的产品中还包括基质成分。一些实施例中，所述修复皮肤的产品包括药品或化妆品。一些实施例中，所述促进伤口愈合的产品为液体制剂、霜膏制剂、喷雾剂或贴敷剂。
本发明提供了一种高度稳定的碱性成纤维生长因子突变体，其具有SEQ ID NO:2所述的氨基酸序列。该突变体大大提高了在水溶液状态下bFGF的稳定性以及热稳定性，并且增加分子内二硫键的碱性成纤维细胞生长因子具有与天然生长因子具有相似的促细胞增殖能力。在应用中即使在分装和储存期间，也可以生产不失去活性的功能性化妆品和皮肤炎症药物。
附图说明
图1示天然bFGF的结构；
图2示天然bFGF的电泳检测图；
图3示发明的bFGF突变体电泳检测图；
图4示不同温度下分子动力学模拟；
图5示天然bFGF与发明的bFGF突变体 DSC曲线；
图6示发明的bFGF突变体二硫键检测图谱；
图7示25℃下放置不同时间天然bFGF与发明bFGF突变体不同浓度对细胞促增殖的能力的检测；
图8示37℃下放置不同时间天然bFGF与发明bFGF突变体不同浓度对细胞促增殖的能力的检测。
具体实施方式
本发明提供了人成纤维细胞生长因子突变体及其编码基因，制备方法与应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in MolecularBiology（Ausubel）。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
本发明中，所述人碱性成纤维细胞生长因子突变体的氨基酸序列为：PALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDCHIKLQLQAEERGVVSIKGVCANRCLAMKEDGRLLASKSVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKCILFLPMSAKS（SEQ ID NO:2）。
编码本发明所述人碱性成纤维细胞生长因子突变体的核酸序列为：gcagcaggtagcattaccaccttaccggcactgccggaagatggtggtagcggtgcatttccgccgggtcattttaaagatccgaaacgtctgtattgcaagaacggcggttttttcctgcgcattcacccggatggtcgtgttgatggtgttcgtgaaaaaagcgattgtcatattaagctgcagctgcaggcagaagaacgtggtgttgttagcattaaaggtgtttgtgcaaatcgttgcctggcaatgaaagaagatggtcgtctgctggcaagcaaaagcgttaccgatgaatgtttctttttcgagcgcctggagagcaataattacaatacctatcgtagccgcaaatacaccagctggtatgttgcactgaaacgtaccggtcagtataaactgggtagcaaaaccggtccgggtcagaaagcaattctgtttctgccgatgagcgcaaaaagc（SEQID NO:3）。
本发明采用的试材皆为普通市售品，皆可于市场购得。下面结合实施例，进一步阐述本发明：
实施例1：天然bFGF的制备
将天然bFGF基因克隆到表达载体pet21b中，酶切位点为酶切位点为：NdEI和XholI。将构建好的质粒转入E.coliBL21（DE3）中，菌生长浓度OD值大于1.0时，加入0.2mMIPTG在22℃下诱导14-16h。在离心条件4000rpm，20min的条件下收集菌体，进行超声破碎，收集上清。目的蛋白用浓度为200mM咪唑从镍柱填料中洗脱下来，得到的蛋白较杂，通过分子筛对蛋白进一步纯化，最终获得纯度较高的目的蛋白以备使用(图2）。
实施例2：bFGF突变体的制备
编码本发明所述突变体（SEQ ID NO:2）的基因（SEQ ID NO:3）克隆到表达载体ppGH载体中，酶切位点为酶切位点为：NdE I和XholI。将构建好的质粒转入有利于二硫键正确形成的感受态Origami（DE3）中，菌生长浓度OD值大于1.0时，加入0.2mM IPTG在22℃下诱导14-16h。在离心条件4000rpm，20min的条件下收集菌体，进行超声破碎，收集上清。将目的蛋白用ppase酶过夜酶切后用浓度为25mM Tris 150mM NaCl的缓冲液从填料中洗脱下来，得到相对较纯的目的蛋白（图3）。
实施例3：不同温度下分子动力学模拟
本研究的全原子分子动力学模拟工作使用AMBER18软件进行。在模拟之前，对体系进行能量优化，包括2500步的最陡下降法和2500步的共轭梯度法。体系能量优化完成后，采用在固定体积和恒定升温速度下，对体系进行200 ps的升温，使体系温度缓慢地从0 K升到298.15 K或310.15K或328.15K或343.15K。在体系维持温度的条件下，进行500ps的NVT(等温等体)系宗模拟，使溶剂分子进一步均匀的分布在溶剂盒子中。最后，NPT(等温等压)的情况下，对整个体系进行500 ps的平衡模拟。最后，两个复合体系在周期边界性条件下，分别进行30ns的NPT(等温等压)系宗模拟。模拟时，非键的截断距离设为10 Å，Particle meshEwald(PME)方法被用于计算长程的静电作用，SHAKE方法用于氢原子键长得限制，Langevin算法用于温控，其中碰撞频率γ设为2ps-1。体系压强为1atm，积分步长为2fs，每隔10ps保存轨迹用于后续分析。
分析工作使用AMBER18中的CPPTRAJ模块进行，主要包括MD后构象提取以及均方波动（RMSF）的计算，结果如图4，结果表明，相对于天然bFGF，本发明所述的突变体在不同的温度下都保持良好的稳定性。
实施例4：DSC曲线
基线用PDS buffer校正，分别在对照池及样品池中加入250pbs及适当浓度蛋白溶液，选择20-80℃温度范围进行进行温度扫描，得到DSC曲线图5。
实施例3：bFGF突变体二硫键的检测
将天然蛋白与bFGF突变体 分别打质谱测定二硫键是否形成以及二硫键的形成位置，图谱如图6。
实施例5：bFGF突变体的生物学活性检测：
将Balb/c 3T3细胞后，以4×103/孔密度种在96孔板中，用完全培养基培养（10%血清）培养24小时，更换维持培养基（0.4%血清）后继续培养24小时，加入不同温度（25℃，37℃）不同时间（1天，2天，3天）不同浓度的天然bFGF和bFGF突变体（100ng/ml，50ng/ml，10ng/ml，5ng/ml，1ng/ml，0.1ng/ml）后，继续培养48h后，用10%浓度CCK在450nm处检测不同检测对象的光吸收值。结果如图7~8，结果显示天然bFGF和bFGF突变体对细胞增殖均有明显促进作用，而在37℃条件下，bFGF突变体对细胞增殖的促进更具有优势。说明本发明提供的bFGF突变体能够形成分子内二硫键，从而提高稳定性，并且依然具有良好的生物活性。
以上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。
序列表
<110> 北京理工大学
<120> 人成纤维细胞生长因子突变体及其编码基因，制备方法与应用
<130> MP22002965
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 146
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His
1 5 10 15
Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu
20 25 30
Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp
35 40 45
Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser
50 55 60
Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly
65 70 75 80
Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu
85 90 95
Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr
100 105 110
Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser
115 120 125
Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala
130 135 140
Lys Ser
145
<210> 2
<211> 146
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His
1 5 10 15
Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu
20 25 30
Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp
35 40 45
Cys His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser
50 55 60
Ile Lys Gly Val Cys Ala Asn Arg Cys Leu Ala Met Lys Glu Asp Gly
65 70 75 80
Arg Leu Leu Ala Ser Lys Ser Val Thr Asp Glu Cys Phe Phe Phe Glu
85 90 95
Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr
100 105 110
Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser
115 120 125
Lys Thr Gly Pro Gly Gln Lys Cys Ile Leu Phe Leu Pro Met Ser Ala
130 135 140
Lys Ser
145
Claims (8)
1.人碱性成纤维细胞生长因子突变体，其特征在于，其氨基酸序列如SEQ ID NO:2所示。
2.编码权利要求1所述的突变体的核酸。
3.含有权利要求2所述核酸的表达载体。
4.宿主细胞，其含有转化或转染的权利要求3所述的表达载体。
5.权利要求1所述的突变体的制备方法，其特征在于，包括以下步骤：培养权利要求4所述的宿主细胞，诱导所述突变体的表达。
6.权利要求1所述的突变体、权利要求2所述的核酸，权利要求3所述的表达载体，权利要求4所述的宿主细胞或权利要求5所述制备方法制得的产物，在制备培养细胞的产品和/或修复皮肤的产品中的应用。
7.一种培养细胞的产品，其特征在于，该产品包括权利要求1所述的突变体。
8.一种修复皮肤的产品，其特征在于，该产品包括权利要求1所述的突变体。
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| WO2016195157A1 (ko) * | 2015-06-04 | 2016-12-08 | (주)피앤피바이오팜 | 안정성이 증가된 인간 섬유아세포 성장인자-2 변이체 및 이의 용도 |
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| WO2016195157A1 (ko) * | 2015-06-04 | 2016-12-08 | (주)피앤피바이오팜 | 안정성이 증가된 인간 섬유아세포 성장인자-2 변이체 및 이의 용도 |
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| Structure-function relationship of basic fibroblast growth factor: site-directed mutagenesis of a putative heparin-binding and receptor-binding region;M Presta等;《Biochem Biophys Res Commun》;185(3);第1098-1107页 * |
| 重组人碱性成纤维细胞生长因子突变体[Ser69,87]的表达、纯化及其稳定性研究;吴晓萍等;《中国药科大学学报》;第36卷(第6期);第168-172页 * |
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