CN115521371A - 一种重组人源化iii型胶原蛋白、制备方法及应用 - Google Patents

一种重组人源化iii型胶原蛋白、制备方法及应用 Download PDF

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CN115521371A
CN115521371A CN202210550416.5A CN202210550416A CN115521371A CN 115521371 A CN115521371 A CN 115521371A CN 202210550416 A CN202210550416 A CN 202210550416A CN 115521371 A CN115521371 A CN 115521371A
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肖建喜
付彩虹
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Collagen Wuhan Biotechnology Co ltd
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Abstract

本发明属于基因工程技术领域，具体涉及一种重组人源化III型胶原蛋白、制备方法及应用。本发明所述的重组人源化III型胶原蛋白包括与人III型胶原蛋白100％相同序列的肽段，100％严格遵守Gly‑X‑Y重复序列模式，同源性高，生物安全性好；并且，所述的重组人源化III型胶原蛋白具有多个人III型胶原蛋白的重要功能位点，生物活性高；而且本发明构建了重组人源化III型胶原蛋白的表达体系，表达量高，易于纯化，制备的重组人源化III型胶原蛋白水溶性好，可广泛应用于护肤品、皮肤修复敷料、医学美容、保健食品等领域。

Description

一种重组人源化III型胶原蛋白、制备方法及应用
技术领域
本发明属于基因工程技术领域，具体涉及一种重组人源化III型胶原蛋白、制备方法及应用。
背景技术
胶原蛋白是人体内含量最丰富的一个蛋白质家族，是细胞外基质的主要组成成分。Ⅲ型胶原蛋白是一种成纤维胶原蛋白，它由3条α1(Ⅲ)肽链组成。III型胶原蛋白是血管、子宫和肠等中空器官的主要结构蛋白，它还经常与Ⅰ型胶原蛋白共生，广泛存在于皮肤、筋膜、肌腱等组织中。III型胶原蛋白为许多器官提供抗张强度和结构完整性，并参与调控细胞的黏附、增殖、迁移、分化等基本活动。III型胶原蛋白在伤口愈合、组织修复等过程中发挥关键作用，它被作为一种主要功能成分，被广泛应用于皮肤修复敷料中。
III型胶原蛋白因为与其他类型胶原蛋白共生，含量较低，因此，从动物组织提取III胶原蛋白存在难度大、纯度低、成本高等严峻问题。重组表达制备的胶原蛋白具有分子量单一、纯度高、无病毒传播隐患等优点，因此开始被用来替代动物胶原蛋白用于护肤品、敷料等领域。转基因植物、哺乳动物细胞等表达体系被用来制备重组胶原蛋白，但是它们存在培养条件严苛、生产周期长、成本高、表达量低等缺陷。大肠杆菌表达系统具有培养周期短、成本低等优点，在重组III型胶原蛋白的表达制备方面越来越受关注。
中国专利CN103122027A公开了一种以大肠杆菌为表达载体制备重组人源胶原蛋白的方法，该方法制备的重组胶原蛋白包括8或16次重复的III型胶原蛋白肽段和部分II型胶原蛋白肽段，并不是100％完全的III型胶原蛋白序列，同时该序列缺乏III型胶原蛋白与其他蛋白质相互作用的功能基序；中国专利CN110194795A制备包括8次重复的III型胶原蛋白肽段GERGGPGGPGPQGPPGKNGETGPQGPPGPT，该序列同样缺乏III型胶原蛋白与其他蛋白质相互作用的功能基序。中国专利CN111087463A和CN111087464A制备包括III型胶原蛋白N-端和C-端的肽段，但是该序列破坏了胶原蛋白特征性的Gly-X-Y重复序列；人体内的III型胶原蛋白要求100％严格遵守Gly-X-Y重复序列，即使单个破坏Gly-X-Y重复序列的突变也会导致疾病。
针对上述技术问题，本发明首先获得一种人III型胶原蛋白片段，所述生物活性高；其次，本发明提供了一种重组人源化III型胶原蛋白，所述重组人源化III型胶原蛋白包括人III型胶原蛋白片段，且与人III型胶原蛋白具有100％相同序列的肽段，同源性高；同时，所述重组人源化III型胶原蛋白100％严格遵守Gly-X-Y重复序列，不存在突变导致的疾病风险；而且，所述重组人源化III型胶原蛋白具有多个人III型胶原蛋白的重要功能位点，生物活性高；最后，本发明构建了一种重组人源化III型胶原蛋白的大肠杆菌表达体系，表达量高，易于纯化，制备的重组人源化III型胶原蛋白水溶性好，质量稳定，可广泛应用于护肤品、皮肤修复敷料、医学美容、保健食品等领域。
发明内容
针对上述技术问题，本发明的目的在于提供一种生物活性高的重组人源化III型胶原蛋白，具体包括以下内容：
第一方面，本发明提供了一种重组人源化III型胶原蛋白，所述重组人源化III型胶原蛋白的氨基酸序列包括至少一段如SEQ ID NO.1所示氨基酸序列。
优选地，所述编码SEQ ID NO.1所示氨基酸序列的基因序列如SEQ ID NO.8所示。
优选地，所述重组人源化III型胶原蛋白的氨基酸序列包括1-10段如SEQ ID NO.1所示氨基酸序列重复。
优选地，所述重组人源化III型胶原蛋白的氨基酸序列如SEQ ID NO.2-7任一所示。
优选地，所述重组人源化III型胶原蛋白的氨基酸序列如SEQ ID NO.3所示。
优选地，所述编码SEQ ID NO.3所示氨基酸序列的基因序列如SQE ID NO.9所示。
优选地，所述重组人源化III型胶原蛋白的氨基酸序列如SEQ ID NO.7所示。
优选地，所述编码SEQ ID NO.7所示氨基酸序列的基因序列如SQE ID NO.10所示。
第二方面，本发明提供了编码上述第一方面所述重组人源化III型胶原蛋白的基因序列，所述基因序列如SEQ ID NO.8-10任一所示。
第三方面，本发明提供了一种携带上述第二方面所述基因的重组质粒、重组载体或重组细菌。
优选地，所述质粒包括pColdIII或pET质粒。
优选地，所述细菌为大肠杆菌。
第四方面，本发明提供了上述第一方面所述的重组人源化III型胶原蛋白在制备护肤品、皮肤修复敷料、植入剂、人工皮肤、医疗器械、保健食品中的应用。
第五方面，本发明提供了一种含有上述第一方面所述重组人源化III型胶原蛋白的胶原蛋白敷料，所述胶原蛋白敷料包括以下质量分数的组分：权利要求1-3任一所述重组人源化III型胶原蛋白0.1％～5％；甘油0.1％～10％；透明质酸钠0.1％～1％；防腐剂0.01％-0.4％。
优选地，所述胶原蛋白敷料类型包括胶原蛋白敷料贴、胶原蛋白冻干粉敷料、胶原蛋白凝胶敷料、胶原蛋白喷雾敷料。
第六方面，本发明提供了一种含有上述第一方面所述重组人源化III型胶原蛋白的护肤品，所述护肤品包括以下质量分数的组分：权利要求1所述重组人源化III型胶原蛋白0.1％～5％；甘油0.1％～3％；透明质酸钠0.1％～2％；β-葡聚糖0.01％-2％；尿囊素0.01％-2％；积雪草提取物0.01％-5％；红景天提取物0.01％-2％；野菊花提取物0.01％-2％；马齿苋提取物0.01％-2％；茶树纯露0.01％-2％；赤藓糖醇0.01％-2％；雪莲花提取物0.01％-2％；生物糖胶0.01％-2％；防腐剂0.01％-0.4％；余量为水。
第七方面，本发明提供了上述第一方面所述的重组人源化III型胶原蛋白的制备方法，所述方法包括如下步骤：
(1)合成编码上述第一方面所述重组人源化III型胶原蛋白的基因序列；
(2)将步骤(1)所述基因与载体连接，转化细菌，构建重组基因工程菌；
(3)表达步骤(2)构建的重组基因工程菌，收集菌体沉淀，破碎得上清液，纯化获得重组人源化III型胶原蛋白。
优选地，所述步骤(1)中基因序列如SEQ ID NO.8-10任一所示。
优选地，所述步骤(2)中细菌为大肠杆菌。
优选地，所述步骤(3)中纯化为盐析和离子交换层析。
本发明的有益效果是：①本发明提供了一种重组人源化III型胶原蛋白，与动物来源的胶原蛋白相比，具有分子量均一、纯度高、无病毒传播隐患等优点；②所述重组人源化III型胶原蛋白包括与人III型胶原蛋白100％相同序列的肽段，同源性高；③所述重组人源化III型胶原蛋白100％严格遵守Gly-X-Y重复序列，不存在突变导致的疾病风险，生物安全性好；④所述重组人源化III型胶原蛋白具有多个人III型胶原蛋白的重要功能位点，生物活性高，可促进人成纤维细胞的黏附和增殖，修复皮肤屏障；⑤本发明构建的重组人源化III型胶原蛋白的大肠杆菌表达体系，表达量高，易于纯化；⑥制备的重组人源化III型胶原蛋白水溶性好，质量稳定，可广泛应用于护肤品、皮肤修复敷料、医学美容、保健食品等领域。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解。
图1本发明所述重组人源化III型胶原蛋白的SDS-PAGE分析图；
图2本发明所述重组人源化III型胶原蛋白的细胞毒性柱状图；
图3本发明所述重组人源化III型胶原蛋白的细胞增殖柱状图；
图4本发明所述重组人源化III型胶原蛋白的细胞黏附图；
图5本发明所述重组人源化III型胶原蛋白的细胞迁移图。
具体实施方式
以下具体结合实施例进一步描述本发明的技术方案，但本发明的保护范围不局限于以下所述。
本本发明提供了一种重组人源化III型胶原蛋白，所述重组人源化III型胶原蛋白包括至少一个氨基酸序列如SEQ ID NO.1所示的氨基酸单体；优选地，所述氨基酸单体为1-10个，优选地，所述重组人源化III型胶原蛋白的氨基酸序列如SEQ ID NO.2-7等序列所示，由100％同源的人源III型胶原蛋白片段组成。本发明所述人源III型胶原蛋白片段来自人的天然氨基酸序列，生物相容性高，且每个单体序列中含有3个多个人III型胶原蛋白的重要功能位点，可供细胞表面受体识别，保证了胶原蛋白的良好生物学性能。
同时，依据本领域公知常识可以推论出：本申请所述重组人源化III型胶原蛋白中SEQ ID NO.1所示的氨基酸序列的重复次数越多，则序列中功能序列越多，可被受体识别的功能位点越多，则该序列的生物学活性越好。其中，当所述重组人源化III型胶原蛋白由1段SEQ ID NO.1所示的氨基酸序列组成时，重组人源化III型胶原蛋白的氨基酸序列如SEQ IDNO.2所示；当所述重组人源化III型胶原蛋白由2段SEQ ID NO.1所示的氨基酸序列重复组成时，重组人源化III型胶原蛋白的氨基酸序列如SEQ ID NO.3所示；当所述重组人源化III型胶原蛋白由4段SEQ ID NO.1所示的氨基酸序列重复组成时，重组人源化III型胶原蛋白的氨基酸序列如SEQ ID NO.4所示；当所述重组人源化III型胶原蛋白由6段SEQ ID NO.1所示的氨基酸序列重复组成时，重组人源化III型胶原蛋白的氨基酸序列如SEQ ID NO.5所示；当所述重组人源化III型胶原蛋白由8段SEQ ID NO.1所示的氨基酸序列重复组成时，重组人源化III型胶原蛋白的氨基酸序列如SEQ ID NO.6所示；当所述重组人源化III型胶原蛋白由10段SEQ ID NO.1所示的氨基酸序列重复组成时，重组人源化III型胶原蛋白的氨基酸序列如SEQ ID NO.7所示；以下实施例以2段SEQ ID NO.1所示的氨基酸序列重复组成的重组人源化III型胶原蛋白(SEQ ID NO.3)为例，具体阐述蛋白制备、生物学评价和应用的内容，其他由不同SEQ ID NO.1所示的氨基酸序列重复组成的重组人源化III型胶原蛋白对应的结果可由此推论。
实施例1重组人源化III型胶原蛋白的制备过程
以SEQ ID NO.1所示的氨基酸为单体，确定单体个数n＝2的重组人源化III型胶原蛋白的氨基酸序列如SEQ ID NO.3所示。
构建重组人源化III型胶原蛋白的表达菌株：合成编码重组人源化III型胶原蛋白的核酸序列(SEQ ID NO.9所示)，构建导入上述核酸的质粒，通过DNA测序确认质粒成功合成；将质粒转化至大肠杆菌BL21-DE3菌株，获得重组人源化III型胶原蛋白表达菌株，将转化成功的菌株加入甘油后，保存于-80度冰箱。
重组人源化III型胶原蛋白的制备与纯化：取微量冻存的重组人源化III型胶原蛋白表达菌株，加入50ml LB培养基(含有抗生素)，在37℃培养过夜；将其转移到1L LB培养基(含有抗生素)，在37℃继续培养，待OD值达到0.5-2.0时，加入IPTG诱导,在25℃、20℃、15℃共培养8hrs-36hrs；离心收集菌体，加入缓冲溶液分散菌体，细胞破碎后，收集上清；通过盐析和离子交换层析纯化得到重组人源化III型胶原蛋白。
取少量洗脱液加入二分之一体积的loading buffer，加热后点样，得到重组人源化III型胶原蛋白的SDS-PAGE如图1所示，纯化后的重组人源化III型胶原蛋白为单一条带，利用IMAGE J软件对该SDS-PAGE图的蛋白条带进行分析，不同批次的重组人源化III型胶原蛋白纯度达95％以上；将离子交换层析洗脱后的溶液进行透析去除无机盐等小分子，冷冻干燥后得到重组人源化III型胶原蛋白粉末。
实施例2重组人源化III型胶原蛋白的生物学评价
1.细胞毒性实验
取生长至培养瓶底面积70％-80％的HeLa细胞，0.25％胰酶消化，用完全培养液制成细胞密度为1×105个/mL的细胞悬液。取100μL细胞悬液接种于96孔培养板中并置于37℃，5％CO2饱和湿度的培养箱中培养。细胞培养24hrs后，吸出完全培养液。在实验组中加入高糖DMEM培养基稀释的不同浓度的重组人源化III型胶原蛋白溶液(0.001-0.1mg/ml，每个浓度设置4个复孔)，对照组为DMEM培养基培养的细胞，空白组为不含细胞的DMEM培养基，继续于37℃、5％CO2饱和湿度的培养箱中培养24hrs。向各组加入CCK-8试剂10μL，细胞培养箱内孵育1-4hrs，使用酶联免疫检测仪在450nm波长下测各孔吸光度值(OD值)。根据各组的吸光度均值按照如下公式计算细胞存活率：
Figure BDA0003654823290000051
实验结果如图2所示，各浓度实验组的细胞活力都在95％以上，表明本发明制备的不同浓度的重组人源化III型胶原蛋白均无细胞毒性，安全性良好。
2.细胞增殖实验
取生长至培养皿面积70％-80％的人皮肤成纤维细胞(HFF-1)，0.25％胰酶消化,用完全培养液制成细胞密度为1×105个/mL的细胞悬液。取100μL细胞悬液接种于96孔培养板中并置于37℃，5％CO2饱和湿度的培养箱中培养。细胞培养24h后，吸出完全培养液。在实验组中加入高糖DMEM培养基稀释的0.1mg/ml的重组人源化III型胶原蛋白(设置4个复孔)，对照组为DMEM培养基培养的细胞，空白组为不含细胞的DMEM培养基，继续于37℃，5％CO2饱和湿度的培养箱中分别培养24h、48h、72h和96h。每隔一段时间，向各组加入CCK-8试剂10μL，细胞培养箱内孵育1-4h，使用酶联免疫检测仪在450nm波长下测各孔吸光度值(OD值)。根据各组的吸光度均值按照如下公式计算细胞的相对增殖率(RGR)：
Figure BDA0003654823290000061
实验结果如图3所示，与空白对照组(100％)相比，本发明制备的重组人源化III型胶原蛋白实验组在第2天、第4天的细胞相对增殖率均大于100％，且随着时间的增加而呈增加的趋势，表明本发明制备的重组人源化III型胶原蛋白可以促进人皮肤成纤维细胞的增殖。
3.细胞黏附实验
用PBS将重组人源化III型胶原蛋白稀释至0.1mg/mL，选用未TC处理的24孔细胞培养板，用0.1mg/mL重组人源化III型胶原蛋白处理孔板，每孔500μL样品，并于4℃冰箱中孵育24h。将生长至培养皿面积70％-80％的人成纤维细胞(HFF-1)用高糖DMEM培养基制成细胞密度为3×105个/mL的细胞悬液。将24孔板中的液体吸出，每孔加入500μL细胞悬液。5h后通过倒置显微镜观察细胞黏附效果。
实验结果如图4所示，在牛血清白蛋白(图4中A所示)中生长的成纤维细胞呈圆形、未铺展的状态，而在重组人源化III型胶原蛋白(图4中B所示)中生长的成纤维细胞大多呈铺展、黏附的状态，表明本发明实施例1制备的重组人源化III型胶原蛋白具有良好的细胞黏附性能。
4.细胞迁移实验
取生长至培养皿面积70％-80％的人皮肤成纤维细胞(HFF-1)，0.25％胰酶消化，用完全培养液制成细胞密度为5×105个/mL的细胞悬液。用马克笔比着直尺在孔板底部画3条横线，取500μL细胞悬液接种于6孔培养板中并置于37℃，5％CO2饱和湿度的培养箱中培养。细胞培养24h后，用10μl枪头比着直尺垂直对准孔板，向下轻推纵向形成划痕，吸出完全培养液，用PBS漂洗3次，去掉划掉的细胞。在实验组中加入高糖DMEM培养基稀释的0.5mg/ml的实施例1制备的重组人源化III型胶原蛋白溶液(设置3个复孔)；对照组为向细胞中加入等量未添加重组人源化III型胶原蛋白的高糖DMEM培养基。在37℃，5％CO2饱和湿度的培养箱中分别培养0h、24h，以横向和纵向划线的交点为核心，在40倍显微镜下拍照，获得9个部位的照片，每组合计27个数据。
结果如图5所示，在0h时(图5中A所示)中央划痕区无细胞，在重组人源化III型胶原蛋白溶液中培养24h(图5中B所示)，HFF-1迁移至中央划痕区，表明重组人源化III型胶原蛋白具有促进细胞迁移的能力。
根据上述，本发明表达的重组人源化III型胶原蛋白具有包含多个功能位点的天然氨基酸序列，分子量均一，可溶性好，具有良好的细胞黏附和增殖性能，可以应用于多种领域，例如医用敷料贴、医用喷雾敷料、医用凝胶敷料、医用冻干粉敷料等不同的形式，还有化妆品和导入性美容产品等。
本发明所述重组人源化III型胶原蛋白包含多个功能位点的天然氨基酸序列，分子量均一，可溶性好，具有良好的细胞黏附和增殖性能，因此，采用了该重组人源化III型胶原蛋白的相关产品也具有相应的生物学功能。具体如实施例3-4所示。
实施例3重组人源III型胶原蛋白医用敷料制备
重组人源III型胶原蛋白制备胶原蛋白贴敷料由本发明制备的重组人源化III型胶原蛋白、保湿剂、防腐剂和无纺布基材组成；所述保湿剂为甘油和透明质酸钠；所述防腐剂为苯氧乙醇。具体包括以下配方：
组方一：本发明制备的重组人源化III型胶原蛋白0.1％；甘油0.1％；透明质酸钠0.1％；防腐剂0.02％；其余为纯化水。制备步骤如下所示：
(1)精确称取各组分，溶于纯化水中，充分搅拌、混匀；
(2)按28mL灌装到含无纺布基材的医用铝箔袋中，辐照灭菌处理，封口，即得胶原蛋白贴敷料产品。
组方二：本发明制备的重组人源化III型胶原蛋白1％；甘油1％；透明质酸钠0.5％；防腐剂0.03％；其余为纯化水。制备步骤如下所示：
(1)精确称取各组分，溶于纯化水中，充分搅拌、混匀；
(2)按28mL灌装到含无纺布基材的医用铝箔袋中，辐照灭菌处理，封口，即得胶原蛋白贴敷料产品。
组方三：本发明制备的重组人源化III型胶原蛋白5％；甘油10％；透明质酸钠1％；防腐剂0.2％；其余为纯化水。制备步骤如下所示：
(1)精确称取各组分，溶于纯化水中，充分搅拌、混匀；
(2)按28mL灌装到含无纺布基材的医用铝箔袋中，辐照灭菌处理，封口，即得胶原蛋白贴敷料产品。
本发明所述的重组人源III型胶原蛋白医用敷料适用于轻中度炎症痤疮、痤疮愈后早期色素沉着、痤疮愈后早期表浅性疤痕的治疗；对治疗皮肤过敏，减轻激光、光子治疗术后疤痕的形成有辅助疗效；在创面愈合期有减轻色素沉着的作用。
实施例4重组人源III型胶原蛋白外用护肤品制备
重组人源III型胶原蛋白外用护肤品含有本发明制备的重组人源化III型胶原蛋白。具体包括以下配方：
组方一：本发明制备的重组人源化III型胶原蛋白0.1％；甘油0.1％；透明质酸钠0.1％；β-葡聚糖0.01％；尿囊素0.01％；积雪草提取物0.01％；红景天提取物0.01％；野菊花提取物0.01％；马齿苋提取物0.01％；茶树纯露0.01％；赤藓糖醇0.01％；雪莲花提取物0.01％；生物糖胶0.01％；苯氧乙醇0.02％；其余为纯化水。
组方二：本发明制备的重组人源化III型胶原蛋白0.5％；甘油1％；透明质酸钠1％；β-葡聚糖1％；尿囊素1％；积雪草提取物2％；红景天提取物1％；野菊花提取物1％；马齿苋提取物1％；茶树纯露1％；赤藓糖醇1％；雪莲花提取物1％；生物糖胶1％；苯氧乙醇0.03％；其余为纯化水。
组方三：本发明制备的重组人源化III型胶原蛋白1％；甘油3％；透明质酸钠2％；β-葡聚糖2％；尿囊素2％；积雪草提取物5％；红景天提取物2％；野菊花提取物2％；马齿苋提取物2％；茶树纯露2％；赤藓糖醇2％；雪莲花提取物2％；生物糖胶2％；苯氧乙醇0.04％；其余为纯化水。
本发明所述的重组人源III型胶原蛋白外用护肤品具有保湿舒缓，促进皮肤晒后修复，加速皮肤新陈代谢的效果。
本发明不限于以上实施例所述，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。
序列表
<110> 胶原蛋白（武汉）生物科技有限公司
<120> 一种重组人源化III型胶原蛋白、制备方法及应用
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Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg
180 185 190
Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys
195 200 205
Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly
210 215 220
Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu
225 230 235 240
Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met
245 250 255
Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly
260 265 270
Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg
275 280
<210> 3
<211> 565
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Gly Thr Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln
1 5 10 15
Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly
20 25 30
Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu
35 40 45
Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro
50 55 60
Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly
65 70 75 80
His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala
85 90 95
Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro
100 105 110
Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly
115 120 125
Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr
130 135 140
Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro
145 150 155 160
Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly
165 170 175
Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg
180 185 190
Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys
195 200 205
Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly
210 215 220
Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu
225 230 235 240
Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met
245 250 255
Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly
260 265 270
Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr Ser Gly His
275 280 285
Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro
290 295 300
Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly
305 310 315 320
Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg
325 330 335
Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala
340 345 350
Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly
355 360 365
Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu
370 375 380
Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg
385 390 395 400
Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly
405 410 415
Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr Ser Gly His Pro Gly Ser
420 425 430
Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala
435 440 445
Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly
450 455 460
Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu
465 470 475 480
Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro
485 490 495
Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn Gly
500 505 510
Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu
515 520 525
Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Pro
530 535 540
Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly
545 550 555 560
Asn Asp Gly Ala Arg
565
<210> 4
<211> 847
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Gly Thr Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln
1 5 10 15
Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly
20 25 30
Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu
35 40 45
Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro
50 55 60
Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly
65 70 75 80
His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala
85 90 95
Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro
100 105 110
Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly
115 120 125
Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr
130 135 140
Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro
145 150 155 160
Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly
165 170 175
Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg
180 185 190
Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys
195 200 205
Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly
210 215 220
Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu
225 230 235 240
Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met
245 250 255
Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly
260 265 270
Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr Ser Gly His
275 280 285
Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro
290 295 300
Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly
305 310 315 320
Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg
325 330 335
Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala
340 345 350
Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly
355 360 365
Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu
370 375 380
Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg
385 390 395 400
Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly
405 410 415
Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr Ser Gly His Pro Gly Ser
420 425 430
Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala
435 440 445
Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly
450 455 460
Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu
465 470 475 480
Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro
485 490 495
Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn Gly
500 505 510
Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu
515 520 525
Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Pro
530 535 540
Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly
545 550 555 560
Asn Asp Gly Ala Arg Gly Thr Ser Gly His Pro Gly Ser Pro Gly Ser
565 570 575
Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser
580 585 590
Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly
595 600 605
Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu
610 615 620
Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro
625 630 635 640
Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly
645 650 655
Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu
660 665 670
Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg
675 680 685
Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly
690 695 700
Ala Arg Gly Thr Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr
705 710 715 720
Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro
725 730 735
Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly
740 745 750
Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro
755 760 765
Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys
770 775 780
Gly His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly
785 790 795 800
Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala
805 810 815
Pro Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro
820 825 830
Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg
835 840 845
<210> 5
<211> 1129
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Gly Thr Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln
1 5 10 15
Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly
20 25 30
Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu
35 40 45
Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro
50 55 60
Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly
65 70 75 80
His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala
85 90 95
Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro
100 105 110
Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly
115 120 125
Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr
130 135 140
Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro
145 150 155 160
Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly
165 170 175
Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg
180 185 190
Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys
195 200 205
Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly
210 215 220
Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu
225 230 235 240
Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met
245 250 255
Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly
260 265 270
Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr Ser Gly His
275 280 285
Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro
290 295 300
Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly
305 310 315 320
Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg
325 330 335
Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala
340 345 350
Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly
355 360 365
Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu
370 375 380
Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg
385 390 395 400
Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly
405 410 415
Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr Ser Gly His Pro Gly Ser
420 425 430
Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala
435 440 445
Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly
450 455 460
Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu
465 470 475 480
Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro
485 490 495
Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn Gly
500 505 510
Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu
515 520 525
Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Pro
530 535 540
Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly
545 550 555 560
Asn Asp Gly Ala Arg Gly Thr Ser Gly His Pro Gly Ser Pro Gly Ser
565 570 575
Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser
580 585 590
Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly
595 600 605
Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu
610 615 620
Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro
625 630 635 640
Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly
645 650 655
Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu
660 665 670
Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg
675 680 685
Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly
690 695 700
Ala Arg Gly Thr Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr
705 710 715 720
Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro
725 730 735
Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly
740 745 750
Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro
755 760 765
Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys
770 775 780
Gly His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly
785 790 795 800
Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala
805 810 815
Pro Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro
820 825 830
Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly
835 840 845
Thr Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro
850 855 860
Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro
865 870 875 880
Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly
885 890 895
Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile
900 905 910
Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg
915 920 925
Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly
930 935 940
Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro
945 950 955 960
Met Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro
965 970 975
Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr Ser Gly
980 985 990
His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu
995 1000 1005
Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile
1010 1015 1020
Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly
1025 1030 1035 1040
Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro
1045 1050 1055
Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp
1060 1065 1070
Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly
1075 1080 1085
Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro
1090 1095 1100
Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala
1105 1110 1115 1120
Gly Ala Arg Gly Asn Asp Gly Ala Arg
1125
<210> 6
<211> 1411
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Gly Thr Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln
1 5 10 15
Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly
20 25 30
Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu
35 40 45
Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro
50 55 60
Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly
65 70 75 80
His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala
85 90 95
Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro
100 105 110
Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly
115 120 125
Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr
130 135 140
Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro
145 150 155 160
Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly
165 170 175
Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg
180 185 190
Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys
195 200 205
Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly
210 215 220
Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu
225 230 235 240
Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met
245 250 255
Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly
260 265 270
Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr Ser Gly His
275 280 285
Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro
290 295 300
Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly
305 310 315 320
Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg
325 330 335
Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala
340 345 350
Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly
355 360 365
Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu
370 375 380
Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg
385 390 395 400
Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly
405 410 415
Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr Ser Gly His Pro Gly Ser
420 425 430
Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala
435 440 445
Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly
450 455 460
Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu
465 470 475 480
Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro
485 490 495
Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn Gly
500 505 510
Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu
515 520 525
Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Pro
530 535 540
Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly
545 550 555 560
Asn Asp Gly Ala Arg Gly Thr Ser Gly His Pro Gly Ser Pro Gly Ser
565 570 575
Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser
580 585 590
Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly
595 600 605
Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu
610 615 620
Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro
625 630 635 640
Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly
645 650 655
Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu
660 665 670
Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg
675 680 685
Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly
690 695 700
Ala Arg Gly Thr Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr
705 710 715 720
Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro
725 730 735
Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly
740 745 750
Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro
755 760 765
Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys
770 775 780
Gly His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly
785 790 795 800
Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala
805 810 815
Pro Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro
820 825 830
Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly
835 840 845
Thr Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro
850 855 860
Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro
865 870 875 880
Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly
885 890 895
Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile
900 905 910
Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg
915 920 925
Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly
930 935 940
Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro
945 950 955 960
Met Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro
965 970 975
Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr Ser Gly
980 985 990
His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu
995 1000 1005
Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile
1010 1015 1020
Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly
1025 1030 1035 1040
Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro
1045 1050 1055
Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp
1060 1065 1070
Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly
1075 1080 1085
Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro
1090 1095 1100
Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala
1105 1110 1115 1120
Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Thr Ser Gly His Pro Gly
1125 1130 1135
Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln
1140 1145 1150
Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser
1155 1160 1165
Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly
1170 1175 1180
Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile
1185 1190 1195 1200
Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn
1205 1210 1215
Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly
1220 1225 1230
Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala
1235 1240 1245
Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg
1250 1255 1260
Gly Asn Asp Gly Ala Arg Gly Thr Ser Gly His Pro Gly Ser Pro Gly
1265 1270 1275 1280
Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro
1285 1290 1295
Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala
1300 1305 1310
Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly
1315 1320 1325
Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe
1330 1335 1340
Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys
1345 1350 1355 1360
Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly
1365 1370 1375
Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu
1380 1385 1390
Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp
1395 1400 1405
Gly Ala Arg
1410
<210> 7
<211> 426
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgggtacat cagggcaccc cggaagtcca ggcagcccgg gttaccaagg tccgccgggc 60
gaaccgggcc aggcaggccc gtccggtcca ccgggcccgc cgggtgcgat tggtccgtct 120
ggtccggcag gcaaggacgg cgaaagcggt cgtcctggca gaccaggcga gcgcggtttg 180
ccgggtccgc caggcatcaa aggtccggcg ggtatcccgg gcttcccggg tatgaaaggc 240
caccgcggtt ttgacggtcg taacggtgag aagggagaaa ccggtgcgcc gggcctgaaa 300
ggtgaaaacg gcctgccggg tgagaacggt gcccctggtc cgatgggtcc gcgtggcgct 360
ccaggcgagc gcggccgtcc gggcctgccg ggcgcggctg gcgcccgtgg taatgatggt 420
gcgcgt 426
<210> 8
<211> 849
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgggtacat cagggcaccc cggaagtcca ggtagcccgg gttatcaggg tccgccgggc 60
gaaccgggcc aagcaggtcc atctggccct ccgggtccgc caggtgccat cggtccgtcc 120
ggtccggctg gcaaggacgg cgaaagcggt cgtccgggtc gtccggggga gcgcggatta 180
ccgggtccgc cgggcattaa aggcccagcg ggcatcccgg gttttccggg tatgaagggc 240
caccgcggtt tcgatggtcg caacggtgag aagggtgaaa ccggtgcgcc tggcctgaaa 300
ggcgagaacg gtctgccggg agagaacggc gcgccgggtc cgatgggccc gcgtggtgcg 360
cctggcgaac gtggtcgtcc gggcttgccg ggcgccgcag gcgctcgtgg taatgatggc 420
gcgcgtggca ccagcggtca tccgggaagc ccgggctccc cgggctacca aggtccgccg 480
ggtgagccgg gccaggcagg tccgtctggt ccaccgggcc caccgggcgc gattggtccg 540
tcgggtcctg ccggcaaaga cggcgagagc ggtcgtccgg gccgcccggg cgaacgtggt 600
ctgcctggcc caccgggtat caaaggcccg gcaggtattc cgggcttccc gggcatgaaa 660
ggtcaccgtg gttttgatgg tcgcaatggt gagaagggtg aaacgggtgc tccgggtctg 720
aagggtgaaa acggtttgcc gggtgaaaat ggtgcgccag gtccgatggg tccgcgtggt 780
gctccgggcg agcgcggacg cccagggctg ccgggcgcgg caggcgcgcg tggcaacgac 840
ggcgcgaga 849
<210> 9
<211> 4233
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atgggtacat cagggcaccc cggaagtcca ggtagcccgg gataccaggg tcccccgggg 60
gagccgggtc aggccggccc gagcggccca ccgggtccgc cgggggcgat tggtccgagt 120
gggccggcgg gcaaggacgg agaatctggc cgtccgggac gtccaggcga gcgtggtctg 180
ccgggtcccc cgggcattaa gggcccggct ggcatcccgg gcttcccggg tatgaaaggt 240
catcgtgggt ttgacggtcg gaacggtgag aaaggcgaaa ccggtgcacc gggactgaaa 300
ggagagaacg gtctgccggg ggaaaatggc gcgccgggtc cgatgggccc acgtggtgct 360
cctggcgaac gcggtcgccc tggtttgccc ggggcagcgg gtgcacgtgg taatgacggc 420
gcacgtggta cgagcggcca cccgggttcc cctggctccc ccggctacca gggtccgcct 480
ggcgagccgg gccaagcggg tccgagcggt ccgccgggtc cgccaggtgc catcggtcca 540
agcggcccgg ccggtaagga cggtgaaagc ggtcgtccag gtcgcccggg cgagcgcgga 600
ctgcctggtc cgcctggcat taaaggcccg gcgggtattc cgggttttcc gggaatgaaa 660
ggtcaccggg gtttcgatgg tcgcaatggt gaaaagggcg aaaccggtgc gccaggcttg 720
aagggtgaga acggattacc gggggagaac ggcgcgccgg ggcctatggg tccgcgtggt 780
gcgcctggtg aacgtggacg tccaggctta ccgggcgccg ccggcgctcg cggcaacgat 840
ggcgcgcgtg gcacctctgg tcacccggga agccctggct cgccgggcta ccagggtcct 900
ccgggagagc cggggcaagc cggaccgagt ggtccgcctg gtcctccggg cgcgattggt 960
cctagtggcc cagcaggcaa ggacggcgaa tcaggacggc cgggcagacc aggggagcgc 1020
ggtttgccgg gaccgccagg tatcaagggt ccggctggta ttccgggttt tccggggatg 1080
aaaggtcatc gtgggttcga tggtaggaat ggtgagaaag gcgaaaccgg tgctcctggg 1140
cttaagggtg agaacggcct gccgggcgag aacggcgcgc ccggtccgat gggtcctcgc 1200
ggtgcgccag gagagcgcgg taggccgggt ctgccgggtg ctgctggcgc ccgtggtaat 1260
gatggcgccc gtggaaccag cggccacccg gggagccccg ggtccccggg ttaccagggt 1320
ccaccgggcg aaccgggtca ggcaggtccg agcggtccac ctgggccgcc gggtgcgatc 1380
ggaccctctg gacctgctgg taaagacggc gaaagcggtc gaccgggtcg tccgggcgaa 1440
agggggctgc cgggcccgcc aggtatcaag ggcccagcag gcatcccggg atttccgggg 1500
atgaaaggcc atcgtggttt cgatggtaga aacggtgaaa aaggtgaaac cggtgcgcca 1560
ggcctgaaag gagagaatgg tttgccgggc gaaaacggtg ctccgggccc gatgggtcca 1620
cgtggagccc cgggagaacg tgggcgtccg ggtttaccgg gcgccgcagg tgctcgcggt 1680
aatgatggtg ctcgcggtac ttccggacat ccgggcagcc cgggtagccc gggttatcaa 1740
ggtccacctg gtgaacccgg tcaggcgggc cctagcggtc cgcctggacc acccggcgcg 1800
attggcccga gcggcccggc cgggaaggac ggtgagtctg gtcgtccggg cagaccggga 1860
gaacgtgggc tgcctggccc tccaggaatt aaagggccag ccggtattcc gggttttccg 1920
ggtatgaagg gtcatcgcgg gttcgacgga cggaacggtg aaaaaggtga aacgggcgct 1980
ccaggactga agggggaaaa cgggctccct ggtgagaacg gtgccccggg tcccatgggc 2040
ccgcgtggtg caccgggcga gcgtggacgt ccgggcctgc cgggcgctgc aggagcgagg 2100
ggtaacgatg gtgcacgtgg caccagcggt caccccggtt cacctggttc gccgggctac 2160
cagggtcctc cgggtgaacc tggacaagcc ggcccatctg gccctccggg tccaccgggg 2220
gcgattggtc cttccggtcc cgcaggtaaa gatggtgaaa gcggtcgacc tggtcgccct 2280
ggagagcgcg gtttaccggg tccaccaggc atcaaaggtc cggcgggcat ccctggattc 2340
ccggggatga agggtcaccg tggcttcgac ggccgtaatg gtgaaaaagg ggaaaccggc 2400
gcgcctggcc ttaagggtga gaacggcctg ccgggcgaaa acggtgcgcc gggcccgatg 2460
ggtccgagag gtgccccggg agaacgtggt aggccgggtc tgccgggcgc tgcgggcgct 2520
cgcggcaacg acggtgcgcg tggtacctct ggtcacccgg gctcgccggg gtctccgggt 2580
tatcagggtc cgcctggcga gccgggtcaa gcgggtccca gcggaccgcc cggtccgccg 2640
ggcgcgattg gtccgagcgg tccggcagga aaggacggtg agagcggccg cccgggccgt 2700
ccaggggaac gtggactgcc gggcccgccg ggtattaaag gccctgcggg tatcccgggt 2760
tttccgggca tgaagggcca cagggggttc gatgggcgta atggtgagaa gggcgaaacc 2820
ggtgccccgg gcctgaaggg cgagaatggc ctgccgggcg aaaatggcgc acctgggcct 2880
atgggtccgc gtggcgcgcc gggcgagcgc ggtagaccgg gtctgccggg tgccgcaggt 2940
gcgcgtggca acgatggtgc tcgtggtacg tcgggtcacc cggggagccc gggtagcccg 3000
ggttaccagg gtccgccggg cgagccggga caagcaggtc cgtcgggccc gccgggcccg 3060
ccgggagcga tcggcccaag tggtccagct ggtaaggacg gtgagtccgg acgtccaggg 3120
cgtccgggcg aacgcggtct gccgggtcct ccgggcatca aaggtccagc gggcatccca 3180
ggcttcccgg gcatgaaagg tcaccgcggt ttcgacggtc gcaacggtga aaagggcgaa 3240
accggggcgc ctggcctcaa gggtgaaaat ggcctgccgg gcgagaacgg cgctccgggg 3300
cccatgggcc cgcgtggagc tcctggcgaa cgtggccgac cgggcctgcc gggtgccgca 3360
ggtgcgcgtg gaaacgatgg cgcgcgtggg accagcggtc atccggggag cccgggtagc 3420
ccgggttatc aaggtccacc aggagagccg ggacaggcag gtccgagcgg tccgccgggc 3480
ccacccggtg cgattggtcc gagcggaccg gcgggcaaag atggcgagag cggtcgtccg 3540
ggacgtccgg gtgaacgcgg gttgccgggt ccgccgggca tcaaaggccc agcaggtatc 3600
ccgggatttc cgggcatgaa aggacatcgt ggttttgacg gtcgtaatgg ggagaaaggc 3660
gaaaccggtg cacccggttt gaaaggcgag aacggtttgc cgggggagaa tggtgctcct 3720
ggccctatgg gtccgcgtgg ggcaccgggt gagcgcggta ggccggggct gccgggggca 3780
gctggtgcac gaggcaacga cggtgcgaga ggcacgtccg gacacccggg aagcccgggt 3840
tccccgggat atcaaggtcc tccgggcgag ccgggtcagg caggtccttc cggtccaccg 3900
ggccccccgg gtgcgatcgg cccgtctggg cctgcgggta aagatggcga gagcggtcgt 3960
ccaggtcgtc ctggcgagcg cggcctgccg ggtccgcccg gcatcaaggg cccggcgggg 4020
attccgggat tccctggtat gaagggccac cgtggttttg atggtcgcaa cggtgagaag 4080
ggcgaaactg gtgcgccggg cctgaaggga gagaacggtt tgcccggcga aaacggcgcg 4140
ccgggtccga tgggtccgag aggcgcacca ggagagcgcg gtagacccgg tctgccgggt 4200
gctgcaggtg cccgtggaaa cgacggcgca aga 4233

Claims (10)

1.一种重组人源化III型胶原蛋白，其特征在于，所述重组人源化III型胶原蛋白的氨基酸序列包括至少一段如SEQ ID NO.1所示氨基酸序列。
2.如权利要求1所述的重组人源化III型胶原蛋白，其特征在于，所述重组人源化III型胶原蛋白的氨基酸序列包括1-10段如SEQ ID NO.1所示氨基酸序列重复。
3.如权利要求2所述的重组人源化III型胶原蛋白，其特征在于，所述重组人源化III型胶原蛋白的氨基酸序列如SEQ ID NO.2-7任一所示。
4.一种编码权利要求1所述的重组人源化III型胶原蛋白的基因序列，其特征在于，所述基因序列如SEQ ID NO.8-10任一所示。
5.一种携带权利要求4所述基因的重组质粒、重组载体或重组细菌。
6.如权利要求1-3任一所述的重组人源化III型胶原蛋白在制备护肤品、皮肤修复敷料、植入剂、人工皮肤、医疗器械、保健食品中的应用。
7.一种含有权利要求1-3任一所述重组人源化III型胶原蛋白的胶原蛋白敷料，其特征在于，所述胶原蛋白敷料包括以下质量分数的组分：权利要求1-3任一所述重组人源化III型胶原蛋白0.1％～5％；甘油0.1％～10％；透明质酸钠0.1％～1％；防腐剂0.01％-0.4％。
8.一种含有权利要求1-3任一所述重组人源化III型胶原蛋白的护肤品，其特征在于，所述护肤品包括以下质量分数的组分：权利要求1所述重组人源化III型胶原蛋白0.1％～5％；甘油0.1％～3％；透明质酸钠0.1％～2％；β-葡聚糖0.01％-2％；尿囊素0.01％-2％；积雪草提取物0.01％-5％；红景天提取物0.01％-2％；野菊花提取物0.01％-2％；马齿苋提取物0.01％-2％；茶树纯露0.01％-2％；赤藓糖醇0.01％-2％；雪莲花提取物0.01％-2％；生物糖胶0.01％-2％；防腐剂0.01％-0.4％；余量为水。
9.如权利要求1-3任一所述的重组人源化III型胶原蛋白的制备方法，其特征在于，所述方法包括如下步骤：
(1)合成编码权利要求1-3任一所述重组人源化III型胶原蛋白的基因序列；
(2)将步骤(1)所述基因与载体连接，转化细菌，构建重组基因工程菌；
(3)表达步骤(2)构建的重组基因工程菌，收集菌体沉淀，破碎得上清液，纯化获得重组人源化III型胶原蛋白。
10.如权利要求9所述的制备方法，其特征在于，所述步骤(1)中的基因序列如SEQ IDNO.8-10任一所示。
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