CN111499729B - 一种调控类i型胶原蛋白纤维条纹周期长度的方法 - Google Patents
一种调控类i型胶原蛋白纤维条纹周期长度的方法 Download PDFInfo
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Abstract
本发明公开了一种调控类I型胶原蛋白纤维条纹周期长度的方法，属于基因工程技术领域。本发明以N和C端(PPG)n序列为基础，在中间插入连续的具有不同数量Gly‑Xaa‑Yaa三联体的胶原蛋白序列，形成三段式的嵌合胶原蛋白P‑CL‑P模式，通过N和C端的(PPG)n三股螺旋间的相互作用驱动自组装，形成具有不同暗条纹长度的周期性明暗相间条纹的带状纤维。本发明的方法制备工艺简单，能够大规模生产成本低的胶原蛋白纤维，在生物材料领域有着广阔的应用前景。
Description
技术领域
本发明涉及一种调控类I型胶原蛋白纤维条纹周期长度的方法，属于基因工程技术领域。
背景技术
胶原蛋白是一种生物性的高分子，是由三条链相互缠绕形成的三股螺旋结构。胶原蛋白根据其基因序列和功能作用可分为28种类型，其中最为主要是的I型胶原蛋白。在高等生物细胞中，I型胶原的原蛋白经过翻译后修饰、折叠、切割等一系列成熟化过程后，多个胶原蛋白三股螺旋(简称胶原蛋白)交错排列，形成间距均匀的明暗条纹状的胶原蛋白纤维(图1),其形貌对于细胞的粘附与生长有关键性的作用[1]，也是促进人体组织和器官的修复和再生的生物材料。
胶原蛋白目前来源主要为以下五种：目前最主要的来源是动物皮肤提取，虽然价格低廉，但是容易携带病源；化学合成多肽虽然可控性强、纯度高，但是最明显的缺点就是价格昂贵、长度受限制、不适合批量生产。利用转基因植物和哺乳动物细胞等真核体系表达的优势在于能够指导蛋白质的正确折叠，提供复杂的翻译后加工功能，但是普遍存在的问题是培养成本高、周期长、表达量低和难于规模化生产。微生物表达体系具有成本低和表达量高等明显优势。目前研究表明，越来越多的哺乳动物和细菌胶原被证实能够在细菌和酵母等宿主中高效的异源表达，并且正确折叠成胶原三股螺旋。重组胶原在生物材料生产中具有潜在的应用，但是缺乏形成高级结构的自组装驱动力，不能形成更高级的结构，限制了其在生物材料和组织工程方面的应用。Barbara Brodsky等人通过表达全长和两倍长度的酿脓链球菌胶原蛋白Scl2，切除球状引导折叠域后的胶原区域能够自组装形成纤维，通过加长序列长度能够促进自组装的能力，其胶原序列中没有N和C端(PPG)10，形成的胶原纤维无明暗相间的条纹[2]。
参考文献：
1.Friedrichs,J.,A.Taubenberger,C.M.Franz,and D.J.Muller,CellularRemodelling of Individual Collagen Fibrils Visualized by Time-lapseAFM.Journal of Molecular Biology,2007.372(3):p.594-607.
2.Nelson,D.L.,A.L.Lehninger,and M.M.Cox,Lehninger principles ofbiochemistry.2008:Macmillan.
发明内容
本发明通过调节胶原区域长度控制胶原纤维暗条纹的长度，这为研究天然I型胶原纤维明暗条纹机制及明暗条纹纤维的作用，针对性制备不同长度的胶原蛋白纤维提供了基础。
本发明的目的是提供一种调控类I型胶原蛋白条纹周期长度的方法，是向具有
所示蛋白单链的CL-domain区域引入不同来源，或不同长度的氨基酸序列，以调控暗条纹长度；再将所述蛋白单链通过微生物表达，收集表达后的具有不同暗条纹长度胶原蛋白；其中，V-domain的氨基酸序列如SEQ IDNO.1所示；(GPP)n中的n≥5；所述不同长度的氨基酸序列是如SEQ ID NO.2～6任一所示的氨基酸序列，或SEQ ID NO.2～6中任一序列的多次重复序列，或SEQ ID NO.2～6中两个及以上序列按任意顺序的组合。
在一种实施方式中，所述V-domain和(GPP)n之间通过LVPRGSP连接。
在一种实施方式中，所述V-domain前端还融合6×His标签。
在一种实施方式中，所述调控具体为：在(GPP)n区域引入n个甘氨酸-脯氨酸-脯氨酸(GPP)，使明条纹的长度达n×1纳米；其中n为≥5的整数。
在一种实施方式中，所述胶原蛋白可通过高聚自组装形成胶原蛋白纤维。
在一种实施方式中，所述调控是在CL-domain区域引入如SEQ ID NO.2～6任一所示的氨基酸序列，使胶原蛋白纤维中的暗条纹达到：(CL-domain区域的氨基酸个数÷3×0.9)±1nm。
在一种实施方式中，所述调控是在CL-domain区域引入长度为81，81×2，81×3和108个氨基酸胶原蛋白序列，使胶原蛋白纤维中的暗条纹达到24.0nm,47.4nm,72.3nm和32.6nm的长度。
在一种实施方式中，所述调控是在CL-domain区域引入1个、2个或3个SEQ ID NO.2所示序列，或引入1个、2个或3个SEQ ID NO.3所示序列，或引入1个、2个或3个SEQ ID NO.4所示序列，或引入1个、2个或3个SEQ ID NO.5所示序列，使胶原蛋白纤维中的暗条纹达到24.0nm,47.4nm,72.3nm的长度。
在一种实施方式中，所述调控是在CL-domain区域引入1个或n个SEQ ID NO.6所示序列，使胶原蛋白纤维中的暗条纹达到32.6nm或n×32.6nm的长度；其中，n为大于1的整数。
在一种实施方式中，所述方法的步骤包括：
(1)将编码
所示蛋白单链的基因在微生物中表达；
(2)将微生物表达的产物进行纯化，获得纯化后的蛋白；
(3)向纯化产物中加入胰蛋白酶，25℃温度下反应至少6h，切除球状引导折叠域后脱盐冻干。
(4)将蛋白冻干粉用10mM PB配成终浓度为0.5mM的溶液，4℃-37℃下放置3.5天数后，即可得到类I型胶原蛋白纤维。
在一种实施方式中，所述制备方法包括如下步骤：
1)胶原蛋白重组质粒的构建：分别合成如SEQ ID NO.13～18所示的编码胶原蛋白基因v-P10AP10,v-P10BP10,v-P10CP10,v-P10B2P10,v-P10ABCP10和v-P10HP10，并分别构建到质粒pColdIII-Tu上，；所述pColdIII-Tu是以pCOLD-TU(Nco I)-S:CTCGAGGGATCCGAATTCA(SEQID NO.19所示),pCOLD-TU(Nco I)-A:GAGCTCCATGGGCACTTTG(SEQ ID NO.20所示)为引物，对pColdIII质粒进行突变，以引入Nco I位点；
2)转化：将步骤(1)构建的重组质粒分别转化到E.coli BL21(DE3)中。
3)诱导表达：接种单菌落于LB液体培养基过夜培养，然后按1％的接种量转接TB液体培养基，37℃培养24h，加入IPTG，25℃诱导10h，转15℃诱导14h。
4)纯化：收集发酵菌体。用磷酸盐缓冲液重悬菌体，在冰浴条件下，利用超声波细胞破碎仪破碎细胞，然后在4℃下，10000rpm离心20min去除细胞碎片，再将上清用微孔滤膜过滤去除杂质；将样品注入安装于蛋白纯化仪的His-trap hp亲和层析柱(5mL)中，然后用洗涤液冲洗8个柱体积，洗脱缓冲液中咪唑的含量呈阶梯状(140mM,400mM)增加，用来洗脱蛋白质，收集出峰蛋白，经胰蛋白酶酶切后透析后，冻干，将其进行SDS-PAGE电泳分析和Maldi-tof分子量鉴定；
5)将步骤(4)冻干后的胶原蛋白配制成浓度为0.5mmol/L的溶液，于4℃～37℃静置至少2天。
本发明还要求保护所述胶原蛋白、基因、质粒、细胞、序列设计模式或制备方法在生物、化工、食品、医药领域制备、生物材料、组织工程和化妆品等领域中的应用。
在一种实施方式中，所述应用是用于制备含有胶原蛋白的产品，包括但不限于制备食品、药物、生物医学材料、化妆品等。
有益效果：本发明通过调整胶原区域的序列长度实现了精确控制纤维的明暗条纹的周期长度，并且其胶原区域序列可置换。用大肠杆菌冷休克表达本发明中涉及的胶原蛋白序列，制备得到清洁来源的能自组装形成具有周期性明暗相间条纹的纤维，其结构与I型胶原蛋白类似，制备工艺简单，能够大规模生产成本低的胶原蛋白纤维。本发明为制备类I型胶原蛋白纤维提供了序列设计方法，该序列胶原区可置换、可扩展，为基于明暗条纹周期型胶原纤维的研究和应用提供了平台，在生物材料应用上有着广阔的前景。
附图说明
图1为I型胶原蛋白纤维形貌；
图2为序列设计示意图及胶原蛋白SDS-PAGE鉴定；A为三段式嵌合序列模式图,x为序列A，B，C或H；B为纯化酶切后胶原蛋白SDS-PAGE；
图3为所设计胶原蛋白MALDI-TOF分子量鉴定；A～H为MALDI-TOF分子量鉴定。
图4为所设计胶原蛋白的二级结构测定；A为圆二色谱全波长扫描谱；B为圆二色谱热变曲线。
图5为置换胶原区域序列对自组装纤维形貌的影响；A～C为P10AP10自组装纤维透射电镜图及明暗条纹长度统计；D～F为P10BP10自组装纤维透射电镜图及明暗条纹长度统计；G～I为P10CP10自组装纤维透射电镜图及明暗条纹长度统计；J～L为P10HP10自组装纤维透射电镜图及明暗条纹长度统计。
图6为改变胶原区域长度对自组装纤维形貌的影响；A～C为P10BP10自组装纤维透射电镜图及明暗条纹长度统计；D～F为P10B2P10自组装纤维透射电镜图及明暗条纹长度统计；G～I为P10ABCP10自组装纤维透射电镜图及明暗条纹长度统计。
图7为改变PPG长度对自组装纤维形貌的影响；A为P5BP5和P5B2P5圆二色谱全波长扫描谱和热变曲线；B为P5BP5和P5B2P5自组装纤维透射电镜图。
图8为P10CLP10的细胞毒性试验结果；A为不同胶原蛋白浓度下平滑肌细胞的粘附能力；B为胶原蛋白浓度为0.02mg/mL时，肌肉细胞的粘附图；C为小鼠3T3成纤细胞在胶原基底生长的细胞数量相对百分比；D为小鼠3T3成纤细胞在胶原基底生长后的荧光染色图。
具体实施方式
1、技术术语：
在不做特别说明的情况下，本申请中的“类I型胶原蛋白”是指由三条具有周期性重复(Gly-Xaa-Yaa)n的蛋白单链围绕一个共同的中心轴盘绕而成的三螺旋结构。“类I型胶原蛋白纤维”是指由类I型胶原蛋白交错排列，自发地聚集或组装形成的间距均匀并具有明暗条纹状形貌的生物大分子。
2、本发明中所用到的材料和方法：
1)培养基
LB固体培养基：15g/L琼脂，10g/L胰蛋白胨，5g/L酵母提取粉，10g/L NaCl，pH7.0。
LB液体培养基：10g/L胰蛋白胨，5g/L酵母提取粉，10g/L NaCl，pH 7.0。
TB液体培养基：12g/L胰蛋白胨，24g酵母提取粉，甘油4mL，2.31g KH2PO4
12.54g K2HPO4，pH 7.5，定容到1L。
2)培养方法
E.coli种子培养条件：将平板划线长出的单菌落接到LB液体培养基中，培养基装液量为10％，采用250mL摇瓶进行培养，培养温度为37℃，培养时间为10h，转速为200rpm。
pET28a重组菌株发酵培养条件：采用TB培养基，培养基装液量为20％，接种量为1％，采用500mL摇瓶进行培养，培养温度为25℃，OD600达到时2.5时，采用终浓度为1mM的IPTG诱导，诱导温度为35℃，诱导时间为24h，转速为200rpm。
pCold重组菌株发酵培养条件：采用TB培养基，培养基装液量为20％，接种量为1％，采用500mL摇瓶进行培养，37℃培养24h后，采用终浓度为1mM的IPTG诱导，25℃诱导10h，然后转入15℃诱导14h，转速为200rpm。
实施例1序列设计及样品制备
按照
所示结构进行设计，具体步骤为：
(1)以N和C端的(GPP)10为固定的序列基元，在中间插入可变的胶原区域，得到三段式嵌合序列
(简写为P10CLP10)。本实施例中CL-domain采用来源于酿脓链球菌的胶原蛋白Scl2(Genbank ID:AAL50184.1)或来源于人源I型胶原α1链(UniProt ID:P02452.5)截取的氨基酸序列(简写为H)作为细菌胶原，其中，Scl2胶原区域分为三个等长的区域A、B和C。在以下实施例中，所设计的CL域分别为A、B、C、BB(重复的2个B区域)和ABC(相当于完整的Scl2胶原区域)。
(2)在序列N端插入来源于Scl2的球状结构域(SEQ ID NO.1所示)用于引导胶原三股螺旋正确折叠，在球状结构域和胶原区域的固定序列单元中间插入蛋白酶切位点LVPRGSP，在序列的N端插入6×His用于纯化。
所述氨基酸序列设计如下：
V-P10AP10的氨基酸序列如SEQ ID NO.7所示；V-P10BP10的氨基酸序列如SEQ IDNO.8所示；V-P10CP10的氨基酸序列如SEQ ID NO.9所示；V-P10HP10的氨基酸序列如SEQ IDNO.12所示。
合成编码上述氨基酸序列的基因，其中，编码V-P10AP10的核苷酸序列如SEQ IDNO.13所示；编码V-P10BP10的基因序列如SEQ ID NO.14所示；编码V-P10CP10的基因序列如SEQID NO.15所示；编码V-P10HP10的基因序列如SEQ ID NO.18所示。
以上核苷酸序列分别包含5'NcoI酶切位点、5'侧翼序列GC和3'BamHI酶切位点，并且将合成的上述基因分别插入到pET28a和pCOLD III-Tu质粒的NcoI和BamHI之间，得到相应的重组胶原蛋白质粒，所述pCOLD III-Tu质粒是以SEQ ID NO.19和SEQ ID NO.20所示的引物，对pColdIII质粒进行突变，以引入NcoI位点。将构建的重组胶原蛋白质粒通过CaCl2方法分别将重组质粒转化到E.coli BL21(DE3)感受态细胞中，涂布含有抗生素的LB平板，培养筛选，获得制备杂合胶原蛋白的重组菌株。
将重组菌株诱导发酵后，将发酵液8000rpm离心5min，收集发酵菌体。用磷酸盐缓冲液重悬菌体，在冰浴条件下，利用超声波细胞破碎仪破碎细胞，然后在4℃下，10000rpm离心20min去除细胞碎片，再将上清采用微孔滤膜(0.45μm)过滤去除杂质。将样品注入安装于蛋白纯化仪上的5mL His-trap hp亲和层析柱中，然后用洗涤液冲洗8个柱体积，洗脱缓冲液中咪唑的含量呈阶梯状(140mM,400mM)增加，用来洗脱蛋白质，收集出峰蛋白，将其进行SDS-PAGE电泳分析。然后利用终浓度为0.05mg/mL胰蛋白酶在25℃下酶切6h切除球状引导折叠域，接着用Desalting脱盐柱脱盐，冷冻干燥，取少量冻干粉末溶于水，采用SDS-PAGE和Maldi-tof进行鉴定。
实施例2序列设计及样品制备
具体实施方式同实施例1，区别在于，增加CL域的氨基酸长度，所设计的CL域分别为BB(重复的2个B区域)和ABC(相当于完整的Scl2胶原区域)，对应的完整氨基酸序列及核苷酸序列为：
V-P10B2P10的氨基酸序列如SEQ ID NO.10所示，编码V-P10B2P10的基因序列如SEQID NO.16所示。
V-P10ABCP10的氨基酸序列如SEQ ID NO.11所示，编码V-P10ABCP10的基因序列如SEQID NO.17所示。
对实施例1～2制备的蛋白进行鉴定。图2(B)显示酶切后的蛋白利用SDS-PAGE均检测到单一的条带，由于胶原蛋白为棒状蛋白，所用蛋白Marker为球状分子，其SDS-PAGE所示分子量大于预想分子量位置。如图3(A)-(H)所示，质谱所得分子量与理论分子量相符，得到分子量正确的胶原蛋白。
实施例3二级结构测定
将实施例1～2制备获得的胶原蛋白配成1mg/mL的浓度。然后4℃静置24h以上，用1mm的比色皿，在4℃下进行圆二色谱全波长扫描，波长从190nm到260nm，波长间隔1nm，每个波长下停留5s。热变实验在220nm下测定，温度从4℃到80℃，每个温度平衡8s，温度递增速度为1℃/6min。典型的胶原蛋白三股螺旋结构CD谱图显示为220nm的正吸收峰。
如图4所示，全波长扫描下，实施例1～2所设计蛋白在220nm附近都有特征吸收峰；热变实验结果显示，随着温度的升高，220nm下的特征吸收值在50℃左右都骤变，表现为胶原蛋白二级结构的破坏，及三股螺旋解螺旋。CD全谱和热变试验结果都说明实施例1所设计的三段式嵌合胶原蛋白P-CL-P都能够正确折叠形成胶原三股螺旋结构，并且有很高的热稳定型。
实施例4置换胶原区域序列对纤维结构的影响
将实施例1制备的冻干的胶原蛋白P10AP10，P10BP10，P10CP10，P10HP10用10mM PB配成终浓度为0.5mM的溶液，4℃下放置3.5天数后取少量滴在铜网上，吸附45s后用滤纸吸干，然后用0.75％的磷钨酸负染20s，滤纸吸干，用Hitachi H-7650透射电子显微镜观察，如图5所示的透射电镜结果，所设计的胶原蛋白均能够自组装形成具有周期性明暗相间条纹的带状纤维，序列A、B和C形成的周期性明暗条纹长度一致。通过对负染的P10BP10纤维明纹和暗纹的测量，至少5张不同的TEM图片，200组以上数据统计得到明纹和暗纹长度分别为10.4nm和24.0nm，与(GPP)10和A,B,C序列的理论长度相符，每个Gly-Xaa-Yaa三联体的长度为0.9nm左右，序列A，B和C的长度都为81个氨基酸，即27个三联体，理论长度为24.3nm，来源于人源序列也能在该模式下自组装形成明暗条纹纤维，其明纹与理论(GPP)10长度相符，暗纹长度为32.6nm，与序列H(36个Gly-Xaa-Yaa三联体)理论长度相符，证明该三段式嵌合的设计模式在N和C端(GPP)10的驱动下能形成稳定的周期型纤维且不受胶原区域序列置换的影响。
实施例5通过胶原区域长度控制纤维的周期长度
将冻干的实施例1制备的胶原蛋白P10BP10和实施例2制备P10B2P10,P10ABCP10按照实例4的方法观察其纤维形貌，透射电镜结果显示如图6，纤维的暗条纹随着序列长度的变化而变化，依次为24.0nm,47.4nm,和72.3nm，与胶原区域B,2B和ABC的理论长度相符合,P10B2P10的暗条纹约为P10BP10的2倍，P10ABCP10的暗条纹约为P10BP10的3倍，明纹长度均为10nm左右，试验结果表明在该三段式嵌合序列模式下，能通过调节胶原区域长度控制胶原纤维暗条纹的长度。
对比例1：
具体实施方式同实施例1，区别在于，将(GPP)10替换为(GPP)5，如图7所示，全波长扫描和热变实验都说明本专利中所设计的嵌合胶原蛋白P5BP5和P5B2P5都能够正确折叠形成胶原三股螺旋结构，并且有很高的热稳定型，但是透射电镜结果显示，所设计的胶原蛋白P5BP5和P5BP5能够自组装形成纤维，但是不具有周期性明暗相间条纹。
实施例6胶原蛋白的功能验证
将实施例3中自组装后的纤维稀释至浓度为0.02，0.04，0.08和0.1mg/mL。然后将不同浓度的胶原蛋白纤维溶液和作为阴性对照的5％牛血清蛋白(BSA)，以及作为阳性对照的0.04mg/mL的I型胶原蛋白分别取200μL加入48孔板中，每组三个平行，于4℃下吸附24h。然后吸出溶液，加入200μL DMEM培养基(含5％BSA)室温放置2h。接着PBS缓冲液清洗3次，然后将肌肉细胞以每孔20000个细胞的密度重悬于含有10％FBS的DMEM，接种200μL到细胞培养板上，2h后，吸出细胞悬浮液，用PBS清洗3次，然后用水晶紫染色测量590nm下的吸收并观察细胞粘附情况。
如图8(A)和(B)所示，P10BP10和P10B2P10相比于BSA，能够很好的促进细胞的粘附，不同浓度下其对细胞的粘附能力没有很大的影响，在0.04mg/mL浓度下，其粘附能力分别约为天然I型胶原的0.58倍和0.57倍。以同样的方式将胶原纤维吸附到96孔板上，然后将小鼠成纤维细胞(3T3细胞)以每孔5000个细胞的密度重悬于含有4％FBS的DMEM，接种100μL到细胞培养板上，培养24h后，利用Dapi和鬼笔环肽染色，统计细胞数量并观察细胞形貌。如图8(C)和(D)所示，P10BP10和P10B2P10对3T3细胞的粘附能力能媲美于天然I型胶原，为I型胶原的0.94倍和1.31倍，且暗条纹的长度长的P10B2P10的细胞粘附性优于P10BP10。观察其细胞形貌可知，以P10BP10和P10B2P10为基底的3T3细胞生长状况良好，细胞伸展度较高。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
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罗格斯大学
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Pro Pro Gly Pro Pro Gly Gln Asp Gly Arg Asn Gly Glu Arg Gly Glu
115 120 125
Gln Gly Pro Thr Gly Pro Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln
130 135 140
Gly Leu Gln Gly Leu Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly
145 150 155 160
Pro Ala Gly Pro Arg Gly Leu Gln Gly Glu Arg Gly Glu Gln Gly Pro
165 170 175
Thr Gly Leu Ala Gly Lys Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr
180 185 190
Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly
195 200 205
Leu Pro Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro
210 215 220
Met Gly Pro Ala Gly Glu Arg Gly Glu Lys Gly Phe Pro Gly Glu Arg
225 230 235 240
Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly
245 250 255
Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys Asp Gly Glu Arg Gly Pro
260 265 270
Val Gly Pro Ala Gly Lys Asp Gly Gln Asn Gly Gln Asp Gly Leu Pro
275 280 285
Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly
290 295 300
Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly Lys
305 310 315 320
Asp Gly Lys Asp Gly Gln Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp
325 330 335
Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Pro Gly
340 345 350
Lys Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro
355 360 365
Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro
370 375 380
Gly
385
<210> 12
<211> 256
<212> PRT
<213> 人工序列
<400> 12
His His His His His His Ala Asp Glu Gln Glu Glu Lys Ala Lys Val
1 5 10 15
Arg Thr Glu Leu Ile Gln Glu Leu Ala Gln Gly Leu Gly Gly Ile Glu
20 25 30
Lys Lys Asn Phe Pro Thr Leu Gly Asp Glu Asp Leu Asp His Thr Tyr
35 40 45
Met Thr Lys Leu Leu Thr Tyr Leu Gln Glu Arg Glu Gln Ala Glu Asn
50 55 60
Ser Trp Arg Lys Arg Leu Leu Lys Gly Ile Gln Asp His Ala Leu Asp
65 70 75 80
Leu Val Pro Arg Gly Ser Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro
85 90 95
Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly
100 105 110
Pro Pro Gly Pro Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala
115 120 125
Arg Gly Leu Pro Gly Ala Pro Gly Gln Met Gly Pro Arg Gly Leu Pro
130 135 140
Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly
145 150 155 160
Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys Gly Glu
165 170 175
Pro Gly Pro Val Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Glu Glu
180 185 190
Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Pro Ala Gly
195 200 205
Pro Lys Gly Ser Pro Gly Glu Ala Gly Arg Pro Gly Glu Ala Gly Leu
210 215 220
Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro
225 230 235 240
Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly
245 250 255
<210> 13
<211> 704
<212> DNA
<213> 人工序列
<400> 13
ccatgggcca ccaccatcac caccacgccg atgaacaaga agaaaaggcg aaggtgcgca 60
cggaactgat tcaagaactg gcccaaggtc tgggcggcat tgagaagaag aactttccga 120
cgctgggtga cgaagacctc gatcacacct acatgaccaa gctgctgacg tatctccaag 180
aacgcgaaca agccgagaat agctggcgta aacgtctgct caaaggcatc caagatcacg 240
cgctggatct ggtgccacgt ggtagtccgg gtccaccggg cccaccgggt ccaccgggcc 300
cgccgggccc gccgggcccg ccgggcccac cgggcccgcc gggcccgccg ggcccaccgg 360
gccaagatgg tcgcaatggt gagcgtggtg aacaaggtcc gacgggtccg accggtccag 420
ccggtccgcg tggtctgcaa ggtctgcaag gcttcccggg cgaacgtggc gaacaaggcc 480
cgacgggtcc agccggccca cgtggtctgc aaggtgaacg cggcgaacaa ggtccaaccg 540
gtctggcggg taaagcgggt gaagccggtg cgaaaggtga aacgggccca gcgggtccac 600
aaggcccgcc gggcccaccg ggtccaccgg gtccaccggg cccaccgggc ccgccgggcc 660
cgccgggccc gccgggcccg ccgggcccgc cgggctaagg atcc 704
<210> 14
<211> 704
<212> DNA
<213> 人工序列
<400> 14
ccatgggcca tcaccaccat catcacgccg atgaacaaga agagaaagcc aaagtgcgca 60
ccgaactgat tcaagaactg gcccaaggtc tgggtggcat tgagaagaag aactttccga 120
cgctgggcga cgaagatctg gaccacacgt acatgaccaa gctgctgacc tatctgcaag 180
aacgcgaaca agccgaaaac agttggcgca aacgtctgct gaaaggcatc caagatcacg 240
cgctggatct cgttccacgt ggtagtccgg gtccaccggg cccaccgggt ccaccgggcc 300
caccgggccc accgggccca ccgggcccgc cgggcccgcc gggcccaccg ggcccaccgg 360
gtccacgcgg tgaacaaggc ccgcaaggtc tgccgggcaa agatggtgag gcgggtgcgc 420
aaggtccagc cggtccaatg ggtccagccg gtttcccggg cgaacgcggt gaaaaaggcg 480
aaccgggtac gcaaggcgcc aaaggtgatc gcggtgaaac gggtccagtt ggcccgcgtg 540
gtgaacgtgg tgaagcgggt ccggccggta aagacggtga acgcggccca gttggtccgg 600
ccggcccacc gggcccaccg ggcccaccgg gcccaccggg cccgccgggc ccgccgggcc 660
cgccgggtcc gccgggtcca ccgggcccac cgggctaagg atcc 704
<210> 15
<211> 695
<212> DNA
<213> 人工序列
<400> 15
ccatgggcca ccatcatcac catcacgcgg atgagcaaga agagaaagcg aaagtgcgca 60
cggagctgat ccaagaactg gcgcaaggcc tcggcggtat cgagaagaag aacttcccga 120
cgctgggtga tgaggatctg gaccacacgt acatgaccaa actgctcacc tatctgcaag 180
aacgcgaaca agccgaaaac agctggcgca agcgtctgct gaaaggcatt caagatcacg 240
ccctcgatct ggttccgcgc ggtagtccgg gcccaccggg cccgccgggc ccgccgggcc 300
caccgggccc gccgggccca ccgggtccac cgggcccgcc gggcccaccg ggcccgccgg 360
gcaaagatgg tcagaatggt caagatggtc tcccgggtaa agatggcaaa gacggtcaaa 420
acggtaaaga cggtctgccg ggcaaggatg gtaaggatgg tcagaacggc aaggacggtc 480
tgccgggcaa agatggtaaa gacggccaag atggtaagga cggtctcccg ggtaaggatg 540
gcaaagatgg tctgccgggc aaggacggca aagatggcca accgggcaaa ccgggcccac 600
cgggcccgcc gggtccaccg ggtccgccgg gcccgccggg tccaccgggc ccaccgggcc 660
cgccgggccc accgggtccg ccgggctaag gatcc 695
<210> 16
<211> 947
<212> DNA
<213> 人工序列
<400> 16
ccatgggcca tcaccaccat catcacgccg atgaacaaga agagaaagcc aaagtgcgca 60
ccgaactgat tcaagaactg gcccaaggtc tgggtggcat tgagaagaag aactttccga 120
cgctgggcga cgaagatctg gaccacacgt acatgaccaa gctgctgacc tatctgcaag 180
aacgcgaaca agccgaaaac agttggcgca aacgtctgct gaaaggcatc caagatcacg 240
cgctggatct cgttccacgt ggtagtccgg gtccaccggg cccaccgggt ccaccgggcc 300
caccgggccc accgggccca ccgggcccgc cgggcccgcc gggcccaccg ggcccaccgg 360
gtccacgcgg tgaacaaggc ccgcaaggtc tgccgggcaa agatggtgag gcgggtgcgc 420
aaggtccagc cggtccaatg ggtccagccg gtttcccggg cgaacgcggt gaaaaaggcg 480
aaccgggtac gcaaggcgcc aaaggtgatc gcggtgaaac gggtccagtt ggcccgcgtg 540
gtgaacgtgg tgaagcgggt ccggccggta aagacggtga acgcggccca gttggtccgg 600
ccggcccacg cggtgaacaa ggcccgcaag gtctgccggg caaagatggt gaggcgggtg 660
cgcaaggtcc agccggtcca atgggtccag ccggtttccc gggcgaacgc ggtgaaaaag 720
gcgaaccggg tacgcaaggc gccaaaggtg atcgcggtga aacgggtcca gttggcccgc 780
gtggtgaacg tggtgaagcg ggtccggccg gtaaagacgg tgaacgcggc ccagttggtc 840
cggccggccc accgggccca ccgggcccac cgggcccacc gggcccgccg ggcccgccgg 900
gcccgccggg tccgccgggt ccaccgggcc caccgggcta aggatcc 947
<210> 17
<211> 1172
<212> DNA
<213> 人工序列
<400> 17
ccatgggcca ccaccatcat catcacgcgg acgagcaaga agagaaagcc aaagttcgca 60
ccgagctgat tcaagaactg gcgcaaggcc tcggcggtat cgagaagaag aactttccga 120
cgctgggcga tgaggatctg gaccatacgt acatgacgaa gctgctgacc tatctgcaag 180
aacgcgaaca agcggaaaac agctggcgca agcgcctcct caaaggcatc caagatcatg 240
ccctcgatct ggttccgcgt ggtagcccgg gcccgccggg cccgccgggc ccaccgggcc 300
cgccgggccc accgggtccg ccgggtccgc cgggcccgcc gggcccaccg ggcccgccgg 360
gccaagatgg ccgtaacggc gaacgtggtg agcaaggccc aacgggcccg acgggtccgg 420
cgggtccacg tggtctccaa ggtctccaag gtctgcaagg cgaacgcggt gaacaaggtc 480
cgaccggtcc ggccggtccg cgtggcctcc aaggcgaacg cggcgaacaa ggcccaaccg 540
gtctggcggg caaagcgggc gaggcgggtg cgaaaggtga aaccggccca gcgggtccac 600
aaggtccgcg tggtgaacaa ggcccgcaag gtctgccggg caaggatggc gaagcgggcg 660
cgcaaggtcc ggccggcccg atgggtccag cgggcgagcg cggtgaaaaa ggcttcccgg 720
gcgagcgtgg cgccaaaggc gatcgcggcg aaacgggtcc agttggtcca cgcggtgaac 780
gcggcgaagc cggtccagcc ggtaaagatg gcgaacgtgg tccagttggc ccagccggta 840
aggatggtca gaatggtcaa gatggcctcc cgggcaagga cggtaaggat ggtcagaatg 900
gtaaagacgg tctgccgggc aaagatggca aggatggcca gaacggcaaa gatggtctcc 960
cgggtaagga cggcaaagac ggccaagatg gcaaagacgg cctcccgggc aaggatggca 1020
aggacggtct cccgggtaaa gacggtaagg atggtcagcc gggcaaaccg ggtccaccgg 1080
gcccgccggg tccgccgggt ccaccgggcc caccgggccc gccgggccca ccgggcccac 1140
cgggtccacc gggcccaccg ggctaaggat cc 1172
<210> 18
<211> 785
<212> DNA
<213> 人工序列
<400> 18
ccatgggcca tcatcaccat caccacgccg acgaacaaga agagaaagcc aaggttcgca 60
ccgaactgat tcaagaactg gcgcaaggtc tgggcggcat cgagaaaaaa aacttcccga 120
ccctcggcga tgaggacctc gatcacacgt acatgacgaa actgctgacg tatctgcaag 180
aacgtgaaca agccgaaaac agctggcgca aacgtctgct gaaaggcatc caagatcacg 240
cgctggatct cgtgccacgc ggtagtccgg gcccgccggg cccaccgggc ccaccgggcc 300
caccgggccc gccgggcccg ccgggtccac cgggcccacc gggtccgccg ggcccgccgg 360
gtgagcgtgg tccgccgggc ccacaaggcg cgcgcggtct gccgggcgcg ccgggccaaa 420
tgggtccacg tggtctgccg ggtgaacgtg gccgtccggg cgcgccgggc ccagcgggcg 480
cccgtggtga accgggtgcc ccgggcagca aaggcgatac gggtgccaaa ggcgaaccgg 540
gcccggttgg cgttcaaggc ccaccgggcc cagccggtga agaaggtaaa cgcggcgccc 600
gcggtgaacc gggcccaacg ggtccagcgg gcccaaaagg tagcccgggc gaagcgggtc 660
gtccgggcga agccggtctg ccgggcccgc cgggcccgcc gggtccaccg ggcccgccgg 720
gcccaccggg cccaccgggc ccgccgggcc caccgggccc accgggccca ccgggctaag 780
gatcc 785
<210> 19
<211> 19
<212> DNA
<213> 人工序列
<400> 19
ctcgagggat ccgaattca 19
<210> 20
<211> 19
<212> DNA
<213> 人工序列
<400> 20
gagctccatg ggcactttg 19
Claims (8)
1.一种调控类I型胶原蛋白条纹周期长度的方法，其特征在于，向具有
所示蛋白单链的CL-domain区域引入不同长度的氨基酸序列；其中，V-domain的氨基酸序列如SEQ ID NO.1所示；(GPP)n中的n=10；所述不同长度的氨基酸序列是如SEQ ID NO.2~6任一所示的氨基酸序列；
所述V-domain和(GPP)n之间通过LVPRGSP连接。
2.根据权利要求1所述的方法，其特征在于，所述V-domain前端还融合6×His标签。
3.根据权利要求1或2所述的方法，其特征在于，所述调控是在CL-domain区域引入如SEQ ID NO.2~6所示的氨基酸序列，使胶原蛋白纤维中的暗条纹宽度达到：（CL-domain区域的氨基酸个数×0.3）±1 nm。
4.根据权利要求1或2所述的方法，其特征在于，所述调控是在CL-domain区域引入1个或多个SEQ ID NO.2所示序列，或引入1个或多个SEQ ID NO.3所示序列，或引入1个或多个SEQ ID NO.4所示序列，或引入1个或多个SEQ ID NO.5所示序列，或引入1个或多个SEQ IDNO.6所示序列。
5.根据权利要求3所述的方法，其特征在于，所述调控是在CL-domain区域引入1个或多个SEQ ID NO.2所示序列，或引入1个或多个SEQ ID NO.3所示序列，或引入1个或多个SEQID NO.4所示序列，或引入1个或多个SEQ ID NO.5所示序列，或引入1个或多个SEQ ID NO.6所示序列。
6.根据权利要求1所述的方法，其特征在于，所述方法的步骤包括：
（1）将编码
所示蛋白单链的基因在微生物中表达；
（2）将微生物表达的产物进行纯化，获得纯化后的蛋白；
（3）向纯化产物中加入胰蛋白酶，25℃温度下反应至少6 h，切除球状引导折叠域后脱盐冻干；
（4）将蛋白冻干粉用10 mM PB配制成终浓度为0.5 mM的溶液，4℃-37℃下放置3.5天数后，即可得到类I型胶原蛋白纤维。
7.根据权利要求6所述的方法，其特征在于，所述步骤（4）配制的溶液于4℃-37℃静置反应至少2天。
8.权利要求1~7任一所述的方法在生物、化工、食品、医药领域制备生物材料或化妆品中的应用。
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