CN114957449A - 一种重组人源iii型胶原蛋白的制备方法 - Google Patents
一种重组人源iii型胶原蛋白的制备方法 Download PDFInfo
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Images
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C12Y—ENZYMES
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- C12Y114/11002—Procollagen-proline dioxygenase (1.14.11.2), i.e. proline-hydroxylase
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/00—Nucleic acids vectors
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Peptides Or Proteins (AREA)
Abstract
本发明适用于基因工程技术领域，提供了一种重组人源III型胶原蛋白，包含重组人源III型胶原蛋白的核苷酸序列，所述重组人源III型胶原蛋白的核苷酸序列如SEQ ID NO.2所示。其制备方法，包括以下步骤：步骤1：基因设计；步骤2：基因合成及表达载体pPIC9K和pPICZA的构建；步骤3：含有脯氨酸羟化酶基因的毕赤酵母表达系统的制备；步骤4：重组人源III型胶原蛋白的诱导表达步骤。利用本方法所产生的人源III型胶原蛋白，其安全性好、质量稳定，消除了传统提取方法存在的安全隐患。在改善了胶原蛋白的亲水性及免疫排异性的同时，也提高了表达量和有效活性。
Description
技术领域
本发明属于基因工程技术领域，尤其涉及一种重组人源III型胶原蛋白的制 备方法。
背景技术
Ⅲ型胶原蛋白在日常中有着非常广泛的应用，形成了以海绵敷料、膜敷料、 凝胶敷料等具有治疗伤口感染、促进组织再生及伤口闭合功能为主的医用敷料。 目前，市场上的胶原主要源于动物组织提取和基因工程重组技术。动物组织中 提取Ⅲ型胶原蛋白的方法历史悠久，其主要通过用酸、碱水解法从动物结缔组 织(猪皮、牛皮、驴皮、鱼等)中提取，但其工艺复杂、得到的产品成分复杂 且产量较低、存在安全隐患等。基因工程生产人源Ⅲ型胶原蛋白的方法其工艺 简单、安全性高、产量高、成本低、与人体契合度高、不易发生排异反应等优 势，因此，利用分子生物学手段构建基因工程菌株对制备高品质的重组人源III 型胶原蛋白具有重要意义。但因胶原蛋白在体内的合成和修饰复杂，正确折叠 成具有生物活性的胶原蛋白除了胶原链之外，还需要特定的结构，因此按原始 基因序列制备的胶原蛋白不可能在体外自发组织形成正确的空间结构，这使目 前III型类人胶原蛋白种类较少，价格昂贵，且其生物活性等还有待进一步提高。
目前工业上制备胶原蛋白的方法主要为传统提取法，即利用动物组织如猪 皮、牛皮、驴皮等进行酸、碱、盐、酶处理来获取胶原蛋白，虽然该种方法操 作简单、回收率高，但得到的胶原蛋白为水不溶性胶原蛋白，可加工性弱，提 取过程中会造成胶原蛋白的部分活性丧失，且提取的产品还存在携带动物源病 毒的风险隐患，应用于人体会产生免疫排斥反应，这极大地限制了其在生物医 学领域的应用范围。
通过基因工程技术生产重组人源III型胶原蛋白为近年来的一种新型方法， 其通过各种宿主细胞如转基因动物、细菌、酵母等来生产重组人源III型胶原蛋 白，该法解决了传统提取方法存在的各种缺陷。目前重组人源III型胶原蛋白多 采用小片段表达，因其全长表达需要脯氨酸羟化酶的羟基化作用，但常用的细 菌表达系统和酵母表达体系中都不含有脯氨酸羟化酶，其重组表达得到的产物 稳定性非常低，极易降解，此外细菌表达系统还普遍存在以下缺点：目的蛋白 常以包涵体形式表达，使产物纯化困难；原核表达系统的翻译后加工修饰体系 不完善，表达产物生物活性较低；细菌产生的致热源会使表达产物难以应用于 临床等。
发明内容
本发明实施例的目的在于提供一种重组人源III型胶原蛋白的制备方法，旨 在解决在胶原蛋白产品的工业化生产中，针对高品质的重组人源III型胶原蛋白 的生产问题。
本发明实施例是这样实现的，一种重组人源III型胶原蛋白，包含重组人源 III型胶原蛋白的核苷酸序列，所述重组人源III型胶原蛋白的核苷酸序列如SEQ ID NO.2所示。
进一步的技术方案，所述的重组人源III型胶原蛋白的制备方法，包括以下 步骤：
步骤1：基因设计；
步骤2：基因合成及表达载体pPIC9K和pPICZA的构建；
步骤3：含有脯氨酸羟化酶基因的毕赤酵母表达系统的制备；
步骤4：重组人源III型胶原蛋白的诱导表达；
步骤5：SDS-PAGE蛋白胶检测；
步骤6：Western Blot验证；
步骤7：蛋白纯化。
进一步的技术方案，所述步骤1包含以下具体步骤：
步骤1.1：根据GEO数据库中人源III型胶原蛋白的基因序列，选用易于提 取纯化的去掉N端和C端的重组人源III型胶原蛋白核心功能区的cDNA序列， 对应的原始人源III型胶原蛋白基因序列如SEQ ID NO.1所示；
步骤1.2：利用密码子在线翻译工具得到人源III型胶原蛋白氨基酸序列， 所述人源III型胶原蛋白氨基酸序列如SEQ ID NO.3所示；
步骤1.3：针对宿主毕赤酵母表达所需的优选密码子，在设计过程中在N端 和C端分别加入α-MF信号肽序列和六个组氨酸编码序列。
进一步的技术方案，所述步骤2包含以下具体步骤：
步骤2.1：根据与人源P4H的α亚基序列相似的病毒A085R蛋白基因序列 作为脯氨酸羟化酶的基因序列，其基因序列如SEQ ID NO.5所示；
步骤2.2：根据SEQ ID NO.2和SEQ ID NO.5所示的基因序列，分别进行全 基因合成，并构建表达载体。
进一步的技术方案，还包括一种可表达含有脯氨酸羟化酶修饰的重组人源 III型胶原蛋白的酵母，所述的可表达含有脯氨酸羟化酶修饰的重组人源III型胶 原蛋白的酵母是将步骤2.2得到的重组载体转入酵母中得到。
进一步的技术方案，所述的可表达含有脯氨酸羟化酶修饰的重组人源III 型胶原蛋白的酵母的菌种保藏编号是CGMCC NO.24378，分类命名为巴斯德 毕赤酵母(Phchiapastoris)，于2022年1月27日保藏于中国微生物菌种保 藏管理委员会普通微生物中心(CGMCC)，地址：北京市朝阳区北辰西路1号 院3号。
进一步的技术方案，所述的酵母为毕赤酵母
进一步的技术方案，所述步骤3包含以下具体步骤：
步骤3.1：重组质粒pPIC9K-COL3A1、pPICZA-P4H转化至毕赤酵母GS115 感受态细胞，利用限制性内切酶sacI对重组质粒pPIC9K-COL3A1、pPICZA-P4H 线性化，电转法转化至毕赤酵母GS115感受态细胞；
步骤3.2：重组酵母菌的筛选，提取转化后菌落的基因组，利用酵母通用引 物，PCR扩增筛选阳性菌株，反应条件为：95℃5min；95℃30s，60℃60 s，72℃3min，30个循环；72℃10min，于4℃保存；pPIC9K空载体作对 照，毕赤酵母细胞GS115作为空白对照，筛选成功重组到酵母细胞基因组的阳 性重组子。
进一步的技术方案，所述步骤3.2中的酵母通用引物为：
5'测序引物及序列:5’AOX1:5’-GACTGGTTCCAATTGACAAGC-3’；
alpha-Factor-F:5’-TACTATTGCCAGCATTGCTGC-3’；
3'测序引物及序列:3’AOX1:5’-GCAAATGGCATTCTGACATCC-3’。
进一步的技术方案，所述步骤4包含以下具体步骤：
步骤4.1：取保藏于-80℃的菌种GS115/pPIC9K-COL3A1-pPICZA-P4H进行 活化，用玻璃三角涂布棒蘸取菌液在YPD固体平板上均匀涂开；30℃培养2～3 天，挑取单菌落于YPD液体培养基中，30℃，200rpm培养约24h，至OD600达 到0.6～2.0范围；
步骤4.2：取种子液以10％的接种量接种到甘油培养基中，装液量为 50ml/250ml，30℃，200rpm培养24h，后将菌液4℃，5000rpm离心20min得到 大量菌沉淀，弃上清，将菌沉淀转移入提前配制好的200mL甲醇诱导培养基培 养，24h后，向培养基中补加甲醇，使培养基中甲醇浓度为1％，每隔24h补 加一次甲醇，使甲醇浓度维持在1％，培养至120h，收取全部上清(上清需4℃ 低温保存)。
进一步的技术方案，所述步骤5包含以下具体步骤：样品处理：收集步骤 4.2中菌液的上清蛋白样品，按蛋白样品：5×loading buffer＝4:1的比例加入 loading buffer，混合均匀，沸水浴5min，自然冷却备用。选用8％分离凝胶上样， 80V电压下电泳30min，使样品浓缩至一条水平线，后改为120V电压下电泳 40-60min直至溴酚蓝条带跑到凝胶底部。
考马斯亮蓝G250染色：
(1)固定液配制：50ml乙醇，10ml冰醋酸，40ml蒸馏水；
(2)染色液配制：0.25g考马斯亮蓝250加入45ml甲醇、10ml冰醋酸和 45ml蒸馏水；
(3)脱色液配制：25ml乙醇，8ml冰醋酸，蒸馏水定容至100ml；
(4)电泳完成后，撬开胶板取出凝胶，随后放入装有100ml固定液的容器 中，在水平摇床上常温固定30min；
(5)固定完成后，将凝胶转移入装有100ml染色液的染色容器中，在水平 摇床上常温染色30min；
(6)将凝胶从染色液中取出，放入装有100ml脱色液的容器中在水平摇床 上常温脱色，每隔2h更换一次脱色液直至凝胶背景清晰。
进一步的技术方案，所述步骤6的具体步骤为：采用与步骤5相同的方法 进行SDS-PAGE凝胶电泳，并利用所述SDS-PAGE凝胶进行Western Blot验证。
Western Blot步骤如下：
(1)采用湿转方法来进行蛋白转膜：准备2张与海绵大小相同的滤纸，用 剪刀剪出与凝胶大小相同的1张0.45μm的PVDF膜；甲醇激活PVDF膜，并 将滤纸、海绵、激活的PVDF膜浸泡在转膜液中；组装转膜夹，按黑色极板→ 海绵→滤纸→凝胶→PVDF膜→滤纸→海绵→红色极板的顺序叠加铺好，并充分 排除每一层气泡；将转膜夹夹紧，以“黑对黑，红对红”方式放入转膜固定装 置，将其放入转印槽，并在转印槽中注满转膜液，转印槽放置于冰水中；接通 电源，恒压90V-100V，转印1h。
(2)封闭及抗体孵育：关闭电源，取出PVDF膜放入封闭液中，摇床上室 温封闭30min-1h；封闭结束后回收封闭液，并使用TBST短暂洗涤PVDF膜(约 2s)；一抗4℃过夜孵育；回收一抗孵育液，TBST洗涤PVDF膜3次，每次5min； 二抗室温于摇床上孵育2h；回收二抗孵育液，于摇床上使用TBST室温洗涤 PVDF膜3次，每次5min。
(3)显影曝光：以A液：B液＝1:1配制显影液400ml并避光存放，将PVDF 膜放入显影仪中平板上并平铺，用微量移液器将显影液加至膜表面，驱赶气泡 同时使显影液均匀覆盖于膜上，关上显影仪门进行显影，曝光时间控制在 10s-25s。
进一步的技术方案，所述步骤7包含以下具体步骤，使用Ni-NTA 6FF His 标签蛋白纯化试剂盒对重组蛋白进行纯化。
(1)样品处理：取收集的菌液4℃离心，5000rpm 20min，弃沉淀，收集 上清，并使用0.45μm滤器过滤上清液；
(2)镍柱预处理：将镍柱竖直放置，加入5倍填料体积的去离子水冲洗， 加入10倍体积的Binding Buffer平衡柱子(Binding Buffer需置于冰上，上清样 品也需置于冰上，尽可能减少蛋白降解损失)；
(3)目的蛋白与镍柱结合：将菌液上清加入到柱子中，控制流速在3-5s/ 滴，收集流穿液，将其再过一遍镍柱；
(4)镍柱洗涤：加入15倍柱体积的Binding Buffer洗涤柱子，速度可稍快 (此步为洗脱杂蛋白)；
(5)目的蛋白洗脱：加入10倍柱体积的Elution Buffer(洗脱液)洗脱蛋 白，速度要慢3-5s/滴，收集流出的蛋白，10ml即可；
(6)镍柱保存：加入3倍柱体积的Binding Buffer洗涤柱子；加入5倍柱 体积的去离子水洗涤柱子，速度可稍快；加入3倍柱体积的20％乙醇保存柱子。
本发明实施例提供的一种重组人源III型胶原蛋白的制备方法，不仅解决了 动物来源胶原蛋白产品带来的高风险和不确定性的影响，而且还解决了细菌表 达系统产物纯化困难、生物活性较低等问题，并且可实现高表达和高有效活性 的目标。本发明是对编码人III型胶原蛋白基因进行密码子优化并与病毒脯氨酸 羟化酶在酵母系统中共表达，诱导蛋白表达结果显示，通过真核表达系统所产 生的人源III型胶原蛋白，其安全性好、质量稳定，消除了传统提取方法存在的 安全隐患。在改善了胶原蛋白的亲水性及免疫排异性的同时，也提高了表达量 和有效活性.
附图说明
图1是PIC9K质粒的物理图谱，酶切位点为sacI；
图2是PICZA质粒的物理图谱，酶切位点为sacI；
图3是PCR鉴定结果图；
图4是SDS-PAGE结果图；
图5是Western Blot孵育C3抗体结果图；
图6是Western Blot孵育His抗体结果图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实 施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅 仅用以解释本发明，并不用于限定本发明。
以下结合具体实施例对本发明的具体实现进行详细描述。
所述的重组人源III型胶原蛋白的制备方法，包括以下步骤：
步骤1：基因设计，包含以下具体步骤：
步骤1.1：根据GEO数据库中人源III型胶原蛋白的基因序列，选用易于提 取纯化的去掉N端和C端的重组人源III型胶原蛋白核心功能区的cDNA序列， 对应的原始人源III型胶原蛋白基因序列如SEQ ID NO.1所示；
步骤1.2：利用密码子在线翻译工具得到人源III型胶原蛋白氨基酸序列， 所述人源III型胶原蛋白氨基酸序列如SEQ ID NO.3所示；
步骤1.3：针对宿主毕赤酵母表达所需的优选密码子，在设计过程中在N端 和C端分别加入α-MF信号肽序列和六个组氨酸编码序列，有利于后期蛋白纯 化。优化后的基因序列为SEQ ID NO.2所示，该序列与原始序列相比：重组人 源III型胶原蛋白密码子为毕赤酵母所偏爱，有利于宿主菌毕赤酵母高效表达该 基因。
步骤2：基因合成及表达载体pPIC9K和pPICZA的构建，包含以下具体步 骤：
步骤2.1：根据与人源P4H的α亚基序列相似的病毒A085R蛋白基因序列 作为脯氨酸羟化酶的基因序列，其基因序列如SEQ ID NO.5所示；
步骤2.2：根据SEQ ID NO.2和SEQ ID NO.5所示的基因序列，分别进行全 基因合成，并构建表达载体，所构建质粒载体理论图谱为图1、图2所示。
步骤3：含有脯氨酸羟化酶基因的毕赤酵母表达系统的制备，包含以下具体 步骤：
步骤3.1：重组质粒pPIC9K-COL3A1、pPICZA-P4H转化至毕赤酵母GS115 感受态细胞，利用限制性内切酶sacI对重组质粒pPIC9K-COL3A1、pPICZA-P4H 线性化，电转法转化至毕赤酵母GS115感受态细胞，制备感受态细胞步骤如下：
用接菌环蘸取GS115甘油菌平板划线至无抗性的YPD平板，培养至出现单 一菌落，挑取单克隆菌落接种至5ml YPD液体培养基中，30℃220r/min过夜培 养，取500μl菌液接种至50mlYPD液体培养基扩大培养，30℃220r/min扩大 到OD600＝0.6～2.0，将菌液转移至50ml无菌离心管，室温2500rpm离心5min， 弃上清，加入去离子水(预冷)重悬菌体，室温2500rpm离心5min，弃上清， 根据菌体量加入1～2ml去离子水重悬菌体，置于冰上备用。在无菌条件下向预 冷的电转杯中加入20μl线性化质粒和80μl感受态，冰浴15min，1500V，25mF， 200Ω，6ms/次，电击一次，转化后质粒涂布于含博来霉素抗性的YPD培养基 上，倒扣于30℃培养箱中，2～3d直至出现白色菌落。
步骤3.2：重组酵母菌的筛选，提取转化后菌落的基因组，利用酵母通用引 物(如下所示)，PCR扩增筛选阳性菌株，反应条件为：95℃5min；95℃30 s，60℃60s，72℃3min，30个循环；72℃10min，于4℃保存；pPIC9K 空载体作对照，毕赤酵母细胞GS115作为空白对照，筛选成功重组到酵母细胞 基因组的阳性重组子。
酵母通用引物为：
5'测序引物及序列:5’AOX1:5’-GACTGGTTCCAATTGACAAGC-3’；
alpha-Factor-F:5’-TACTATTGCCAGCATTGCTGC-3’；
3'测序引物及序列:3’AOX1:5’-GCAAATGGCATTCTGACATCC-3’。
PCR鉴定结果如图3所示，分析条带大小，实际值与理论值相符合，即证 明我们成功构建了重组人源III型胶原蛋白表达系统。
步骤4：重组人源III型胶原蛋白的诱导表达，包含以下具体步骤：
步骤4.1：取保藏于-80℃的菌种GS115/pPIC9K-COL3A1-pPICZA-P4H进行 活化，用玻璃三角涂布棒蘸取菌液在YPD固体平板上均匀涂开；30℃培养2～3 天，挑取单菌落于YPD液体培养基中，30℃，200rpm培养约24h，至OD600达 到0.6～2.0范围；
步骤4.2：取种子液以10％的接种量接种到甘油培养基中，装液量为 50ml/250ml，30℃，200rpm培养24h，后将菌液4℃，5000rpm离心20min得到 大量菌沉淀，弃上清，将菌沉淀转移入提前配制好的200mL甲醇诱导培养基培 养(此步骤中离心、与转移过程需无菌操作)。24h后，向培养基中补加甲醇， 使培养基中甲醇浓度为1％(培养基总体积为200mL时，则加入2mL甲醇)， 每隔24h补加一次甲醇，使甲醇浓度维持在1％，培养至120h，收取全部上清 (上清需4℃低温保存)。
步骤5：SDS-PAGE蛋白胶检测，包含以下具体步骤：
样品处理：收集步骤4.2中菌液的上清蛋白样品，按蛋白样品：5×loading buffer＝4:1的比例加入loading buffer，混合均匀，沸水浴5min，自然冷却备用。 选用8％分离凝胶上样，80V电压下电泳30min，使样品浓缩至一条水平线，后 改为120V电压下电泳40-60min直至溴酚蓝条带跑到凝胶底部。
考马斯亮蓝G250染色：
(1)固定液配制：50ml乙醇，10ml冰醋酸，40ml蒸馏水；
(2)染色液配制：0.25g考马斯亮蓝250加入45ml甲醇、10ml冰醋酸和 45ml蒸馏水；
(3)脱色液配制：25ml乙醇，8ml冰醋酸，蒸馏水定容至100ml；
(4)电泳完成后，撬开胶板取出凝胶，随后放入装有100ml固定液的容器 中，在水平摇床上常温固定30min；
(5)固定完成后，将凝胶转移入装有100ml染色液的染色容器中，在水平 摇床上常温染色30min；
(6)将凝胶从染色液中取出，放入装有100ml脱色液的容器中在水平摇床 上常温脱色，每隔2h更换一次脱色液直至凝胶背景清晰。
实验结果如图4所示，目的条带大小与理论一致。
步骤6：Western Blot验证，具体步骤为：采用与步骤5相同的方法进行 SDS-PAGE凝胶电泳，并利用所述SDS-PAGE凝胶进行Western Blot验证。
Western Blot步骤如下：
(1)采用湿转方法来进行蛋白转膜：准备2张与海绵大小相同的滤纸，用 剪刀剪出与凝胶大小相同的1张0.45μm的PVDF膜；甲醇激活PVDF膜，并 将滤纸、海绵、激活的PVDF膜浸泡在转膜液中；组装转膜夹，按黑色极板→ 海绵→滤纸→凝胶→PVDF膜→滤纸→海绵→红色极板的顺序叠加铺好，并充分 排除每一层气泡；将转膜夹夹紧，以“黑对黑，红对红”方式放入转膜固定装 置，将其放入转印槽，并在转印槽中注满转膜液，转印槽放置于冰水中；接通 电源，恒压90V-100V，转印1h。
(2)封闭及抗体孵育：关闭电源，取出PVDF膜放入封闭液中，摇床上室 温封闭30min-1h；封闭结束后回收封闭液，并使用TBST短暂洗涤PVDF膜(约 2s)；一抗4℃过夜孵育；回收一抗孵育液，TBST洗涤PVDF膜3次，每次5min； 二抗室温于摇床上孵育2h；回收二抗孵育液，于摇床上使用TBST室温洗涤 PVDF膜3次，每次5min。
(3)显影曝光：以A液：B液＝1:1配制显影液400ml并避光存放，将PVDF 膜放入显影仪中平板上并平铺，用微量移液器将显影液加至膜表面，驱赶气泡 同时使显影液均匀覆盖于膜上，关上显影仪门进行显影，曝光时间控制在10s-25s。
Western Blot结果图如图5和图6所示。
步骤7：蛋白纯化，包含以下具体步骤，使用Ni-NTA 6FF His标签蛋白纯 化试剂盒对重组蛋白进行纯化。
(1)样品处理：取收集的菌液4℃离心，5000rpm 20min，弃沉淀，收集 上清，并使用0.45μm滤器过滤上清液；
(2)镍柱预处理：将镍柱竖直放置，加入5倍填料体积的去离子水冲洗， 加入10倍体积的Binding Buffer平衡柱子(Binding Buffer需置于冰上，上清样 品也需置于冰上，尽可能减少蛋白降解损失)；
(3)目的蛋白与镍柱结合：将菌液上清加入到柱子中，控制流速在3-5s/ 滴，收集流穿液，将其再过一遍镍柱；
(4)镍柱洗涤：加入15倍柱体积的Binding Buffer洗涤柱子，速度可稍快 (此步为洗脱杂蛋白)；
(5)目的蛋白洗脱：加入10倍柱体积的Elution Buffer(洗脱液)洗脱蛋 白，速度要慢3-5s/滴，收集流出的蛋白，10ml即可；
(6)镍柱保存：加入3倍柱体积的Binding Buffer洗涤柱子；加入5倍柱 体积的去离子水洗涤柱子，速度可稍快；加入3倍柱体积的20％乙醇保存柱子 (使乙醇流出一些完全浸没填料为宜)。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发 明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明 的保护范围之内。
序列表
<110> 吉林省国大生物工程有限公司
<120> 一种重组人源III型胶原蛋白的制备方法
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3204
<212> PRT
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Thr Gly Cys Cys Gly Gly Ala Thr Thr Cys Cys Cys Thr Gly Gly Ala
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Gly Thr Cys Cys Cys Gly Cys Thr Gly Gly Cys Ala Thr Thr Cys Cys
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Thr Gly Gly Ala Gly Cys Thr Cys Cys Thr Gly Gly Ala Cys Thr Gly
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Cys Thr Cys Cys Ala Gly Gly Ala Cys Cys Ala Gly Cys Cys Gly Gly
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Gly Ala Gly Cys Cys Cys Gly Gly Ala Cys Cys Ala Cys Gly Thr Gly
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Gly Thr Gly Ala Ala Cys Gly Cys Gly Gly Thr Gly Ala Gly Gly Cys
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Cys Ala Gly Gly Ala Gly Cys Thr Gly Cys Ala Gly Gly Ala Gly Ala
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Thr Gly Gly Gly Ala Ala Ala Cys Cys Ala Gly Gly Gly Cys Cys Thr
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Ala Ala Ala Gly Thr Gly Gly Thr Cys Gly Ala Cys Cys Ala Gly Gly
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Thr Cys Cys Thr Cys Cys Thr Gly Gly Gly Cys Cys Ala Thr Cys Thr
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Ala Gly Gly Thr Gly Gly Cys Cys Cys Thr Gly Gly Ala Gly Gly Ala
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Thr Cys Cys Ala Ala Ala Gly Gly Gly Thr Gly Ala Thr Gly Cys Cys
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Gly Gly Thr Gly Cys Ala Cys Cys Thr Gly Gly Ala Gly Cys Thr Cys
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Gly Cys Thr Gly Gly Thr Cys Cys Cys Cys Cys Thr Gly Gly Thr Cys
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Cys Cys Gly Ala Ala Gly Gly Ala Gly Gly Ala Ala Ala Gly Gly Gly
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Thr Gly Cys Thr Gly Cys Thr Gly Gly Thr Cys Cys Thr Cys Cys Thr
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Gly Gly Gly Cys Cys Ala Cys Cys Thr Gly Gly Thr Gly Cys Thr Gly
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Gly Cys Ala Ala Gly Gly Ala Ala Thr Gly Cys Cys Thr Gly Gly Ala
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Gly Ala Ala Ala Gly Ala Gly Gly Ala Gly Gly Thr Cys Thr Thr Gly
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Gly Ala Ala Gly Thr Cys Cys Thr Gly Gly Thr Cys Cys Ala Ala Ala
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Gly Gly Gly Thr Gly Ala Cys Ala Ala Gly Gly Gly Thr Gly Ala Ala
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Cys Thr Gly Ala Thr Gly Gly Thr Gly Thr Cys Cys Cys Ala Gly Gly
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Gly Ala Ala Ala Gly Ala Thr Gly Gly Cys Cys Cys Ala Ala Gly Gly
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Gly Gly Thr Cys Cys Thr Ala Cys Thr Gly Gly Thr Cys Cys Thr Ala
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Thr Thr Gly Gly Thr Cys Cys Thr Cys Cys Thr Gly Gly Cys Cys Cys
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Gly Ala Thr Ala Ala Gly Gly Gly Thr Gly Ala Ala Gly Gly Thr Gly
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Gly Thr Gly Cys Cys Cys Cys Cys Gly Gly Ala Cys Thr Thr Cys Cys
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Cys Cys Cys Thr Cys Cys Ala Gly Gly Ala Cys Cys Thr Gly Cys Thr
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Cys Thr Gly Gly Ala Cys Ala Gly Ala Ala Thr Gly Gly Thr Gly Ala
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Gly Cys Ala Gly Gly Ala Cys Cys Cys Cys Cys Thr Gly Gly Ala Gly
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Thr Cys Cys Thr Cys Cys Thr Gly Gly Thr Cys Cys Cys Cys Ala Ala
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Gly Gly Thr Gly Thr Cys Ala Ala Ala Gly Gly Thr Gly Ala Ala Cys
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Gly Thr Gly Gly Cys Ala Gly Thr Cys Cys Thr Gly Gly Thr Gly Gly
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Ala Cys Cys Thr Gly Gly Thr Gly Cys Thr Gly Cys Thr Gly Gly Cys
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Thr Thr Cys Cys Cys Thr Gly Gly Thr Gly Cys Thr Cys Gly Thr Gly
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Gly Thr Cys Thr Thr Cys Cys Thr Gly Gly Thr Cys Cys Thr Cys Cys
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Thr Gly Gly Thr Ala Gly Thr Ala Ala Thr Gly Gly Thr Ala Ala Cys
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Cys Cys Ala Gly Gly Ala Cys Cys Cys Cys Cys Ala Gly Gly Thr Cys
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Cys Cys Ala Gly Cys Gly Gly Thr Thr Cys Thr Cys Cys Ala Gly Gly
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Cys Ala Ala Gly Gly Ala Thr Gly Gly Gly Cys Cys Cys Cys Cys Ala
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Cys Cys Cys Thr Gly Gly Ala Gly Thr Gly Thr Cys Thr Gly Gly Ala
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Cys Cys Ala Ala Ala Ala Gly Gly Thr Gly Ala Thr Gly Cys Thr Gly
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Gly Cys Cys Ala Ala Cys Cys Ala Gly Gly Ala Gly Ala Gly Ala Ala
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Gly Gly Gly Ala Thr Cys Gly Cys Cys Thr Gly Gly Thr Gly Cys Cys
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Cys Ala Gly Gly Gly Cys Cys Cys Ala Cys Cys Ala Gly Gly Ala Gly
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Gly Gly Ala Gly Cys Ala Cys Gly Gly Gly Gly Thr Cys Thr Thr Gly
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Cys Ala Gly Gly Ala Cys Cys Ala Cys Cys Ala Gly Gly Cys Ala Thr
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Gly Cys Cys Ala Gly Gly Thr Cys Cys Thr Ala Gly Gly Gly Gly Ala
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Ala Gly Cys Cys Cys Thr Gly Gly Cys Cys Cys Thr Cys Ala Gly Gly
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Gly Thr Gly Thr Cys Ala Ala Gly Gly Gly Thr Gly Ala Ala Ala Gly
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Thr Gly Gly Gly Ala Ala Ala Cys Cys Ala Gly Gly Ala Gly Cys Thr
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Ala Ala Cys Gly Gly Thr Cys Thr Cys Ala Gly Thr Gly Gly Ala Gly
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Ala Cys Cys Cys Cys Ala Gly Gly Gly Thr Cys Thr Thr Cys Cys Thr
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Gly Gly Thr Cys Thr Gly Gly Cys Thr Gly Gly Thr Ala Cys Ala Gly
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Cys Thr Gly Gly Thr Gly Ala Ala Cys Cys Thr Gly Gly Ala Ala Gly
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Ala Gly Ala Thr Gly Gly Ala Ala Ala Cys Cys Cys Thr Gly Gly Ala
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Thr Cys Ala Gly Ala Thr Gly Gly Thr Cys Thr Thr Cys Cys Ala Gly
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Gly Cys Cys Gly Ala Gly Ala Thr Gly Gly Ala Thr Cys Thr Cys Cys
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Thr Gly Gly Thr Gly Gly Cys Ala Ala Gly Gly Gly Thr Gly Ala Thr
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Cys Gly Thr Gly Gly Thr Gly Ala Ala Ala Ala Thr Gly Gly Cys Thr
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Ala Thr Cys Gly Gly Cys Ala Gly Thr Cys Cys Ala Gly Gly Ala Cys
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Ala Cys Cys Thr Gly Thr Thr Gly Gly Ala Cys Cys Cys Ala Gly Thr
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Gly Gly Ala Cys Cys Thr Cys Cys Thr Gly Gly Cys Ala Ala Ala Gly
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Thr Cys Cys Ala Gly Gly Thr Cys Cys Cys Ala Thr Thr Gly Gly Ala
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Cys Cys Ala Cys Cys Ala Gly Gly Gly Cys Cys Thr Cys Gly Ala Gly
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Thr Gly Gly Ala
<210> 2
<211> 3243
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Ala Ala Ala Ala Gly Ala Gly Ala Gly Gly Cys Thr Gly Ala Ala Gly
1 5 10 15
Cys Thr Cys Ala Gly Thr Ala Cys Gly Ala Thr Thr Cys Thr Thr Ala
20 25 30
Cys Gly Ala Thr Gly Thr Gly Ala Ala Gly Ala Gly Thr Gly Gly Thr
35 40 45
Gly Thr Cys Gly Cys Thr Gly Thr Cys Gly Gly Thr Gly Gly Ala Cys
50 55 60
Thr Ala Gly Cys Ala Gly Gly Thr Thr Ala Thr Cys Cys Thr Gly Gly
65 70 75 80
Ala Cys Cys Thr Gly Cys Ala Gly Gly Ala Cys Cys Thr Cys Cys Ala
85 90 95
Gly Gly Thr Cys Cys Thr Cys Cys Thr Gly Gly Ala Cys Cys Thr Cys
100 105 110
Cys Thr Gly Gly Thr Ala Cys Thr Thr Cys Ala Gly Gly Ala Cys Ala
115 120 125
Thr Cys Cys Ala Gly Gly Thr Thr Cys Thr Cys Cys Ala Gly Gly Thr
130 135 140
Thr Cys Thr Cys Cys Ala Gly Gly Thr Thr Ala Thr Cys Ala Ala Gly
145 150 155 160
Gly Ala Cys Cys Ala Cys Cys Thr Gly Gly Thr Gly Ala Ala Cys Cys
165 170 175
Ala Gly Gly Ala Cys Ala Ala Gly Cys Thr Gly Gly Ala Cys Cys Thr
180 185 190
Thr Cys Cys Gly Gly Thr Cys Cys Ala Cys Cys Ala Gly Gly Thr Cys
195 200 205
Cys Ala Cys Cys Ala Gly Gly Ala Gly Cys Thr Ala Thr Thr Gly Gly
210 215 220
Ala Cys Cys Thr Thr Cys Cys Gly Gly Cys Cys Cys Thr Gly Cys Ala
225 230 235 240
Gly Gly Thr Ala Ala Ala Gly Ala Thr Gly Gly Ala Gly Ala Ala Thr
245 250 255
Cys Thr Gly Gly Cys Ala Gly Ala Cys Cys Thr Gly Gly Ala Ala Gly
260 265 270
Ala Cys Cys Thr Gly Gly Ala Gly Ala Ala Cys Gly Thr Gly Gly Thr
275 280 285
Thr Thr Gly Cys Cys Thr Gly Gly Thr Cys Cys Ala Cys Cys Cys Gly
290 295 300
Gly Cys Ala Thr Thr Ala Ala Gly Gly Gly Thr Cys Cys Thr Gly Cys
305 310 315 320
Thr Gly Gly Ala Ala Thr Cys Cys Cys Thr Gly Gly Thr Thr Thr Cys
325 330 335
Cys Cys Thr Gly Gly Ala Ala Thr Gly Ala Ala Gly Gly Gly Thr Cys
340 345 350
Ala Cys Ala Gly Ala Gly Gly Thr Thr Thr Thr Gly Ala Cys Gly Gly
355 360 365
Ala Ala Gly Ala Ala Ala Cys Gly Gly Ala Gly Ala Gly Ala Ala Ala
370 375 380
Gly Gly Thr Gly Ala Gly Ala Cys Cys Gly Gly Thr Gly Cys Ala Cys
385 390 395 400
Cys Ala Gly Gly Cys Thr Thr Gly Ala Ala Ala Gly Gly Ala Gly Ala
405 410 415
Gly Ala Ala Thr Gly Gly Thr Thr Thr Ala Cys Cys Cys Gly Gly Ala
420 425 430
Gly Ala Ala Ala Ala Thr Gly Gly Ala Gly Cys Thr Cys Cys Ala Gly
435 440 445
Gly Cys Cys Cys Cys Ala Thr Gly Gly Gly Thr Cys Cys Thr Ala Gly
450 455 460
Ala Gly Gly Thr Gly Cys Thr Cys Cys Thr Gly Gly Thr Gly Ala Ala
465 470 475 480
Ala Gly Gly Gly Gly Thr Cys Gly Thr Cys Cys Ala Gly Gly Thr Thr
485 490 495
Thr Gly Cys Cys Cys Gly Gly Ala Gly Cys Ala Gly Cys Thr Gly Gly
500 505 510
Thr Gly Cys Thr Ala Gly Ala Gly Gly Ala Ala Ala Cys Gly Ala Cys
515 520 525
Gly Gly Thr Gly Cys Cys Cys Gly Thr Gly Gly Ala Thr Cys Ala Gly
530 535 540
Ala Thr Gly Gly Ala Cys Ala Ala Cys Cys Thr Gly Gly Cys Cys Cys
545 550 555 560
Ala Cys Cys Thr Gly Gly Thr Cys Cys Ala Cys Cys Ala Gly Gly Thr
565 570 575
Ala Cys Thr Gly Cys Ala Gly Gly Thr Thr Thr Cys Cys Cys Ala Gly
580 585 590
Gly Ala Thr Cys Cys Cys Cys Cys Gly Gly Thr Gly Cys Thr Ala Ala
595 600 605
Ala Gly Gly Thr Gly Ala Gly Gly Thr Thr Gly Gly Ala Cys Cys Ala
610 615 620
Gly Cys Thr Gly Gly Thr Thr Cys Ala Cys Cys Thr Gly Gly Ala Thr
625 630 635 640
Cys Thr Ala Ala Cys Gly Gly Thr Gly Cys Cys Cys Cys Cys Gly Gly
645 650 655
Ala Cys Ala Ala Ala Gly Ala Gly Gly Ala Gly Ala Ala Cys Cys Ala
660 665 670
Gly Gly Thr Cys Cys Ala Cys Ala Ala Gly Gly Ala Cys Ala Thr Gly
675 680 685
Cys Thr Gly Gly Thr Gly Cys Thr Cys Ala Ala Gly Gly Ala Cys Cys
690 695 700
Cys Cys Cys Thr Gly Gly Thr Cys Cys Ala Cys Cys Ala Gly Gly Ala
705 710 715 720
Ala Thr Ala Ala Ala Cys Gly Gly Thr Ala Gly Thr Cys Cys Ala Gly
725 730 735
Gly Ala Gly Gly Cys Ala Ala Gly Gly Gly Ala Gly Ala Ala Ala Thr
740 745 750
Gly Gly Gly Thr Cys Cys Ala Gly Cys Thr Gly Gly Ala Ala Thr Thr
755 760 765
Cys Cys Thr Gly Gly Thr Gly Cys Ala Cys Cys Thr Gly Gly Thr Thr
770 775 780
Thr Ala Ala Thr Gly Gly Gly Ala Gly Cys Thr Ala Gly Ala Gly Gly
785 790 795 800
Thr Cys Cys Thr Cys Cys Ala Gly Gly Thr Cys Cys Ala Gly Cys Cys
805 810 815
Gly Gly Ala Gly Cys Thr Ala Ala Cys Gly Gly Thr Gly Cys Thr Cys
820 825 830
Cys Ala Gly Gly Thr Cys Thr Ala Cys Gly Thr Gly Gly Thr Gly Gly
835 840 845
Ala Gly Cys Cys Gly Gly Ala Gly Ala Ala Cys Cys Thr Gly Gly Cys
850 855 860
Ala Ala Ala Ala Ala Thr Gly Gly Ala Gly Cys Thr Ala Ala Ala Gly
865 870 875 880
Gly Thr Gly Ala Ala Cys Cys Thr Gly Gly Thr Cys Cys Cys Cys Gly
885 890 895
Thr Gly Gly Cys Gly Ala Ala Ala Gly Ala Gly Gly Ala Gly Ala Ala
900 905 910
Gly Cys Thr Gly Gly Ala Ala Thr Cys Cys Cys Thr Gly Gly Ala Gly
915 920 925
Thr Thr Cys Cys Ala Gly Gly Cys Gly Cys Thr Ala Ala Ala Gly Gly
930 935 940
Thr Gly Ala Gly Gly Ala Cys Gly Gly Ala Ala Ala Gly Gly Ala Thr
945 950 955 960
Gly Gly Thr Thr Cys Ala Cys Cys Cys Gly Gly Thr Gly Ala Ala Cys
965 970 975
Cys Cys Gly Gly Ala Gly Cys Ala Ala Ala Thr Gly Gly Ala Cys Thr
980 985 990
Thr Cys Cys Ala Gly Gly Ala Gly Cys Thr Gly Cys Thr Gly Gly Cys
995 1000 1005
Gly Ala Ala Ala Gly Ala Gly Gly Cys Gly Cys Ala Cys Cys Ala Gly
1010 1015 1020
Gly Ala Thr Thr Thr Ala Gly Ala Gly Gly Thr Cys Cys Ala Gly Cys
1025 1030 1035 1040
Ala Gly Gly Thr Cys Cys Ala Ala Ala Thr Gly Gly Ala Ala Thr Ala
1045 1050 1055
Cys Cys Thr Gly Gly Thr Gly Ala Gly Ala Ala Ala Gly Gly Ala Cys
1060 1065 1070
Cys Thr Gly Cys Thr Gly Gly Thr Gly Ala Ala Ala Gly Ala Gly Gly
1075 1080 1085
Ala Gly Cys Ala Cys Cys Ala Gly Gly Ala Cys Cys Ala Gly Cys Thr
1090 1095 1100
Gly Gly Ala Cys Cys Thr Cys Gly Ala Gly Gly Thr Gly Cys Thr Gly
1105 1110 1115 1120
Cys Ala Gly Gly Thr Gly Ala Ala Cys Cys Ala Gly Gly Ala Ala Gly
1125 1130 1135
Ala Gly Ala Thr Gly Gly Ala Gly Thr Thr Cys Cys Ala Gly Gly Thr
1140 1145 1150
Gly Gly Ala Cys Cys Thr Gly Gly Thr Ala Thr Gly Ala Gly Ala Gly
1155 1160 1165
Gly Cys Ala Thr Gly Cys Cys Thr Gly Gly Thr Ala Gly Thr Cys Cys
1170 1175 1180
Cys Gly Gly Thr Gly Gly Thr Cys Cys Ala Gly Gly Ala Thr Cys Cys
1185 1190 1195 1200
Gly Ala Cys Gly Gly Thr Ala Ala Ala Cys Cys Thr Gly Gly Thr Cys
1205 1210 1215
Cys Ala Cys Cys Cys Gly Gly Thr Thr Cys Ala Cys Ala Ala Gly Gly
1220 1225 1230
Ala Gly Ala Gly Ala Gly Thr Gly Gly Cys Ala Gly Ala Cys Cys Ala
1235 1240 1245
Gly Gly Ala Cys Cys Thr Cys Cys Thr Gly Gly Thr Cys Cys Thr Thr
1250 1255 1260
Cys Ala Gly Gly Thr Cys Cys Thr Ala Gly Gly Gly Gly Thr Cys Ala
1265 1270 1275 1280
Gly Cys Cys Cys Gly Gly Thr Gly Thr Thr Ala Thr Gly Gly Gly Cys
1285 1290 1295
Thr Thr Thr Cys Cys Ala Gly Gly Ala Cys Cys Thr Ala Ala Gly Gly
1300 1305 1310
Gly Thr Ala Ala Cys Gly Ala Cys Gly Gly Ala Gly Cys Thr Cys Cys
1315 1320 1325
Ala Gly Gly Cys Ala Ala Gly Ala Ala Cys Gly Gly Thr Gly Ala Ala
1330 1335 1340
Cys Gly Thr Gly Gly Thr Gly Gly Ala Cys Cys Cys Gly Gly Ala Gly
1345 1350 1355 1360
Gly Thr Cys Cys Ala Gly Gly Ala Cys Cys Thr Cys Ala Ala Gly Gly
1365 1370 1375
Ala Cys Cys Thr Cys Cys Ala Gly Gly Thr Ala Ala Gly Ala Ala Cys
1380 1385 1390
Gly Gly Thr Gly Ala Ala Ala Cys Thr Gly Gly Ala Cys Cys Ala Cys
1395 1400 1405
Ala Ala Gly Gly Ala Cys Cys Ala Cys Cys Ala Gly Gly Thr Cys Cys
1410 1415 1420
Ala Ala Cys Thr Gly Gly Thr Cys Cys Cys Gly Gly Ala Gly Gly Thr
1425 1430 1435 1440
Gly Ala Cys Ala Ala Ala Gly Gly Thr Gly Ala Thr Ala Cys Thr Gly
1445 1450 1455
Gly Thr Cys Cys Thr Cys Cys Ala Gly Gly Ala Cys Cys Thr Cys Ala
1460 1465 1470
Ala Gly Gly Ala Cys Thr Thr Cys Ala Ala Gly Gly Thr Thr Thr Gly
1475 1480 1485
Cys Cys Thr Gly Gly Thr Ala Cys Cys Gly Gly Thr Gly Gly Thr Cys
1490 1495 1500
Cys Ala Cys Cys Thr Gly Gly Thr Gly Ala Gly Ala Ala Cys Gly Gly
1505 1510 1515 1520
Thr Ala Ala Ala Cys Cys Ala Gly Gly Thr Gly Ala Ala Cys Cys Thr
1525 1530 1535
Gly Gly Thr Cys Cys Thr Ala Ala Gly Gly Gly Thr Gly Ala Cys Gly
1540 1545 1550
Cys Thr Gly Gly Ala Gly Cys Ala Cys Cys Thr Gly Gly Thr Gly Cys
1555 1560 1565
Thr Cys Cys Thr Gly Gly Ala Gly Gly Thr Ala Ala Gly Gly Gly Ala
1570 1575 1580
Gly Ala Cys Gly Cys Cys Gly Gly Thr Gly Cys Thr Cys Cys Ala Gly
1585 1590 1595 1600
Gly Ala Gly Ala Ala Cys Gly Thr Gly Gly Ala Cys Cys Ala Cys Cys
1605 1610 1615
Ala Gly Gly Ala Thr Thr Ala Gly Cys Ala Gly Gly Cys Gly Cys Thr
1620 1625 1630
Cys Cys Ala Gly Gly Thr Thr Thr Ala Ala Gly Ala Gly Gly Ala Gly
1635 1640 1645
Gly Thr Gly Cys Thr Gly Gly Ala Cys Cys Thr Cys Cys Ala Gly Gly
1650 1655 1660
Ala Cys Cys Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Ala Ala Ala
1665 1670 1675 1680
Gly Gly Ala Gly Cys Ala Gly Cys Ala Gly Gly Ala Cys Cys Ala Cys
1685 1690 1695
Cys Ala Gly Gly Ala Cys Cys Ala Cys Cys Thr Gly Gly Ala Gly Cys
1700 1705 1710
Ala Gly Cys Thr Gly Gly Thr Ala Cys Thr Cys Cys Ala Gly Gly Thr
1715 1720 1725
Cys Thr Thr Cys Ala Gly Gly Gly Ala Ala Thr Gly Cys Cys Ala Gly
1730 1735 1740
Gly Ala Gly Ala Gly Ala Gly Gly Gly Gly Ala Gly Gly Cys Cys Thr
1745 1750 1755 1760
Thr Gly Gly Thr Thr Cys Thr Cys Cys Thr Gly Gly Ala Cys Cys Ala
1765 1770 1775
Ala Ala Ala Gly Gly Thr Gly Ala Cys Ala Ala Ala Gly Gly Ala Gly
1780 1785 1790
Ala Ala Cys Cys Ala Gly Gly Thr Gly Gly Thr Cys Cys Ala Gly Gly
1795 1800 1805
Thr Gly Cys Thr Gly Ala Thr Gly Gly Thr Gly Thr Gly Cys Cys Thr
1810 1815 1820
Gly Gly Cys Ala Ala Ala Gly Ala Thr Gly Gly Thr Cys Cys Ala Ala
1825 1830 1835 1840
Gly Ala Gly Gly Ala Cys Cys Ala Ala Cys Thr Gly Gly Ala Cys Cys
1845 1850 1855
Cys Ala Thr Thr Gly Gly Thr Cys Cys Thr Cys Cys Thr Gly Gly Thr
1860 1865 1870
Cys Cys Ala Gly Cys Thr Gly Gly Ala Cys Ala Ala Cys Cys Cys Gly
1875 1880 1885
Gly Thr Gly Ala Thr Ala Ala Gly Gly Gly Ala Gly Ala Gly Gly Gly
1890 1895 1900
Thr Gly Gly Thr Gly Cys Ala Cys Cys Cys Gly Gly Thr Cys Thr Thr
1905 1910 1915 1920
Cys Cys Ala Gly Gly Ala Ala Thr Cys Gly Cys Thr Gly Gly Cys Cys
1925 1930 1935
Cys Ala Ala Gly Ala Gly Gly Ala Ala Gly Thr Cys Cys Ala Gly Gly
1940 1945 1950
Thr Gly Ala Gly Ala Gly Ala Gly Gly Thr Gly Ala Ala Ala Cys Thr
1955 1960 1965
Gly Gly Ala Cys Cys Ala Cys Cys Thr Gly Gly Thr Cys Cys Cys Gly
1970 1975 1980
Cys Ala Gly Gly Thr Thr Thr Thr Cys Cys Ala Gly Gly Thr Gly Cys
1985 1990 1995 2000
Thr Cys Cys Thr Gly Gly Ala Cys Ala Ala Ala Ala Thr Gly Gly Cys
2005 2010 2015
Gly Ala Ala Cys Cys Ala Gly Gly Thr Gly Gly Ala Ala Ala Ala Gly
2020 2025 2030
Gly Thr Gly Ala Ala Ala Gly Ala Gly Gly Ala Gly Cys Thr Cys Cys
2035 2040 2045
Cys Gly Gly Thr Gly Ala Ala Ala Ala Ala Gly Gly Ala Gly Ala Ala
2050 2055 2060
Gly Gly Thr Gly Gly Ala Cys Cys Ala Cys Cys Cys Gly Gly Thr Gly
2065 2070 2075 2080
Thr Thr Gly Cys Thr Gly Gly Ala Cys Cys Ala Cys Cys Thr Gly Gly
2085 2090 2095
Thr Gly Gly Ala Thr Cys Thr Gly Gly Ala Cys Cys Thr Gly Cys Thr
2100 2105 2110
Gly Gly Ala Cys Cys Thr Cys Cys Thr Gly Gly Thr Cys Cys Thr Cys
2115 2120 2125
Ala Ala Gly Gly Thr Gly Thr Thr Ala Ala Gly Gly Gly Thr Gly Ala
2130 2135 2140
Ala Ala Gly Ala Gly Gly Thr Thr Cys Thr Cys Cys Ala Gly Gly Cys
2145 2150 2155 2160
Gly Gly Thr Cys Cys Thr Gly Gly Ala Gly Cys Thr Gly Cys Thr Gly
2165 2170 2175
Gly Thr Thr Thr Thr Cys Cys Ala Gly Gly Thr Gly Cys Thr Ala Gly
2180 2185 2190
Gly Gly Gly Ala Cys Thr Thr Cys Cys Ala Gly Gly Ala Cys Cys Ala
2195 2200 2205
Cys Cys Thr Gly Gly Thr Ala Gly Thr Ala Ala Cys Gly Gly Ala Ala
2210 2215 2220
Ala Cys Cys Cys Ala Gly Gly Ala Cys Cys Ala Cys Cys Thr Gly Gly
2225 2230 2235 2240
Ala Cys Cys Thr Thr Cys Thr Gly Gly Ala Thr Cys Cys Cys Cys Ala
2245 2250 2255
Gly Gly Thr Ala Ala Ala Gly Ala Thr Gly Gly Thr Cys Cys Ala Cys
2260 2265 2270
Cys Ala Gly Gly Thr Cys Cys Ala Gly Cys Ala Gly Gly Thr Ala Ala
2275 2280 2285
Thr Ala Cys Thr Gly Gly Thr Gly Cys Cys Cys Cys Ala Gly Gly Thr
2290 2295 2300
Thr Cys Thr Cys Cys Ala Gly Gly Ala Gly Thr Thr Ala Gly Thr Gly
2305 2310 2315 2320
Gly Thr Cys Cys Thr Ala Ala Ala Gly Gly Thr Gly Ala Thr Gly Cys
2325 2330 2335
Thr Gly Gly Thr Cys Ala Gly Cys Cys Cys Gly Gly Ala Gly Ala Ala
2340 2345 2350
Ala Ala Gly Gly Gly Cys Thr Cys Thr Cys Cys Ala Gly Gly Ala Gly
2355 2360 2365
Cys Thr Cys Ala Ala Gly Gly Thr Cys Cys Thr Cys Cys Thr Gly Gly
2370 2375 2380
Thr Gly Cys Thr Cys Cys Ala Gly Gly Ala Cys Cys Thr Thr Thr Ala
2385 2390 2395 2400
Gly Gly Ala Ala Thr Cys Gly Cys Cys Gly Gly Thr Ala Thr Cys Ala
2405 2410 2415
Cys Ala Gly Gly Thr Gly Cys Thr Cys Gly Thr Gly Gly Thr Cys Thr
2420 2425 2430
Gly Gly Cys Thr Gly Gly Ala Cys Cys Ala Cys Cys Thr Gly Gly Ala
2435 2440 2445
Ala Thr Gly Cys Cys Ala Gly Gly Thr Cys Cys Thr Ala Gly Gly Gly
2450 2455 2460
Gly Thr Thr Cys Cys Cys Cys Ala Gly Gly Ala Cys Cys Thr Cys Ala
2465 2470 2475 2480
Gly Gly Gly Ala Gly Thr Ala Ala Ala Gly Gly Gly Ala Gly Ala Gly
2485 2490 2495
Thr Cys Ala Gly Gly Ala Ala Ala Gly Cys Cys Ala Gly Gly Cys Gly
2500 2505 2510
Cys Thr Ala Ala Cys Gly Gly Thr Cys Thr Thr Ala Gly Thr Gly Gly
2515 2520 2525
Ala Gly Ala Gly Ala Gly Ala Gly Gly Ala Cys Cys Ala Cys Cys Ala
2530 2535 2540
Gly Gly Ala Cys Cys Cys Cys Ala Ala Gly Gly Thr Cys Thr Gly Cys
2545 2550 2555 2560
Cys Ala Gly Gly Thr Thr Thr Gly Gly Cys Ala Gly Gly Thr Ala Cys
2565 2570 2575
Cys Gly Cys Thr Gly Gly Thr Gly Ala Ala Cys Cys Thr Gly Gly Cys
2580 2585 2590
Cys Gly Thr Gly Ala Cys Gly Gly Thr Ala Ala Cys Cys Cys Ala Gly
2595 2600 2605
Gly Thr Ala Gly Thr Gly Ala Thr Gly Gly Thr Cys Thr Thr Cys Cys
2610 2615 2620
Cys Gly Gly Thr Ala Gly Ala Gly Ala Thr Gly Gly Thr Thr Cys Thr
2625 2630 2635 2640
Cys Cys Thr Gly Gly Ala Gly Gly Thr Ala Ala Gly Gly Gly Thr Gly
2645 2650 2655
Ala Thr Ala Gly Ala Gly Gly Thr Gly Ala Gly Ala Ala Cys Gly Gly
2660 2665 2670
Thr Thr Cys Ala Cys Cys Thr Gly Gly Ala Gly Cys Thr Cys Cys Thr
2675 2680 2685
Gly Gly Thr Gly Cys Thr Cys Cys Thr Gly Gly Thr Cys Ala Thr Cys
2690 2695 2700
Cys Ala Gly Gly Thr Cys Cys Thr Cys Cys Ala Gly Gly Ala Cys Cys
2705 2710 2715 2720
Thr Gly Thr Ala Gly Gly Thr Cys Cys Ala Gly Cys Thr Gly Gly Thr
2725 2730 2735
Ala Ala Ala Thr Cys Ala Gly Gly Ala Gly Ala Thr Ala Gly Ala Gly
2740 2745 2750
Gly Thr Gly Ala Ala Thr Cys Ala Gly Gly Thr Cys Cys Ala Gly Cys
2755 2760 2765
Ala Gly Gly Thr Cys Cys Thr Gly Cys Thr Gly Gly Thr Gly Cys Thr
2770 2775 2780
Cys Cys Thr Gly Gly Ala Cys Cys Thr Gly Cys Thr Gly Gly Thr Thr
2785 2790 2795 2800
Cys Ala Cys Gly Thr Gly Gly Cys Gly Cys Ala Cys Cys Thr Gly Gly
2805 2810 2815
Thr Cys Cys Ala Cys Ala Ala Gly Gly Thr Cys Cys Ala Ala Gly Gly
2820 2825 2830
Gly Gly Ala Gly Ala Thr Ala Ala Ala Gly Gly Cys Gly Ala Gly Ala
2835 2840 2845
Cys Thr Gly Gly Thr Gly Ala Ala Ala Gly Gly Gly Gly Thr Gly Cys
2850 2855 2860
Thr Gly Cys Thr Gly Gly Thr Ala Thr Thr Ala Ala Ala Gly Gly Thr
2865 2870 2875 2880
Cys Ala Thr Ala Gly Ala Gly Gly Ala Thr Thr Cys Cys Cys Thr Gly
2885 2890 2895
Gly Thr Ala Ala Cys Cys Cys Thr Gly Gly Ala Gly Cys Ala Cys Cys
2900 2905 2910
Thr Gly Gly Thr Thr Cys Thr Cys Cys Cys Gly Gly Thr Cys Cys Thr
2915 2920 2925
Gly Cys Thr Gly Gly Ala Cys Ala Ala Cys Ala Ala Gly Gly Thr Gly
2930 2935 2940
Cys Cys Ala Thr Cys Gly Gly Ala Thr Cys Ala Cys Cys Thr Gly Gly
2945 2950 2955 2960
Ala Cys Cys Ala Gly Cys Cys Gly Gly Ala Cys Cys Thr Ala Gly Ala
2965 2970 2975
Gly Gly Thr Cys Cys Thr Gly Thr Thr Gly Gly Ala Cys Cys Ala Thr
2980 2985 2990
Cys Thr Gly Gly Thr Cys Cys Thr Cys Cys Thr Gly Gly Ala Ala Ala
2995 3000 3005
Gly Gly Ala Thr Gly Gly Thr Ala Cys Ala Thr Cys Thr Gly Gly Thr
3010 3015 3020
Cys Ala Thr Cys Cys Ala Gly Gly Thr Cys Cys Thr Ala Thr Thr Gly
3025 3030 3035 3040
Gly Ala Cys Cys Thr Cys Cys Thr Gly Gly Thr Cys Cys Ala Ala Gly
3045 3050 3055
Ala Gly Gly Thr Ala Ala Cys Ala Gly Ala Gly Gly Ala Gly Ala Ala
3060 3065 3070
Cys Gly Thr Gly Gly Ala Thr Cys Thr Gly Ala Gly Gly Gly Ala Thr
3075 3080 3085
Cys Ala Cys Cys Cys Gly Gly Thr Cys Ala Thr Cys Cys Thr Gly Gly
3090 3095 3100
Ala Cys Ala Ala Cys Cys Thr Gly Gly Thr Cys Cys Ala Cys Cys Thr
3105 3110 3115 3120
Gly Gly Thr Cys Cys Ala Cys Cys Thr Gly Gly Thr Gly Cys Thr Cys
3125 3130 3135
Cys Ala Gly Gly Ala Cys Cys Ala Thr Gly Cys Thr Gly Thr Gly Gly
3140 3145 3150
Ala Gly Gly Thr Gly Thr Thr Gly Gly Ala Gly Cys Thr Gly Cys Thr
3155 3160 3165
Gly Cys Thr Ala Thr Ala Gly Cys Ala Gly Gly Ala Ala Thr Cys Gly
3170 3175 3180
Gly Ala Gly Gly Ala Gly Ala Ala Ala Ala Gly Gly Cys Cys Gly Gly
3185 3190 3195 3200
Thr Gly Gly Thr Thr Thr Cys Gly Cys Thr Cys Cys Thr Thr Ala Cys
3205 3210 3215
Thr Ala Cys Gly Gly Cys Cys Ala Cys Cys Ala Thr Cys Ala Cys Cys
3220 3225 3230
Ala Cys Cys Ala Thr Cys Ala Thr Thr Ala Gly
3235 3240
<210> 3
<211> 1068
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gln Tyr Asp Ser Tyr Asp Val Lys Ser Gly Val Ala Val Gly Gly Leu
1 5 10 15
Ala Gly Tyr Pro Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Pro Pro
20 25 30
Gly Thr Ser Gly His Pro Gly Ser Pro Gly Ser Pro Gly Tyr Gln Gly
35 40 45
Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro Ser Gly Pro Pro Gly Pro
50 55 60
Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Glu Ser
65 70 75 80
Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly Leu Pro Gly Pro Pro Gly
85 90 95
Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe Pro Gly Met Lys Gly His
100 105 110
Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys Gly Glu Thr Gly Ala Pro
115 120 125
Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly Glu Asn Gly Ala Pro Gly
130 135 140
Pro Met Gly Pro Arg Gly Ala Pro Gly Glu Arg Gly Arg Pro Gly Leu
145 150 155 160
Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Ala Arg Gly Ser Asp
165 170 175
Gly Gln Pro Gly Pro Pro Gly Pro Pro Gly Thr Ala Gly Phe Pro Gly
180 185 190
Ser Pro Gly Ala Lys Gly Glu Val Gly Pro Ala Gly Ser Pro Gly Ser
195 200 205
Asn Gly Ala Pro Gly Gln Arg Gly Glu Pro Gly Pro Gln Gly His Ala
210 215 220
Gly Ala Gln Gly Pro Pro Gly Pro Pro Gly Ile Asn Gly Ser Pro Gly
225 230 235 240
Gly Lys Gly Glu Met Gly Pro Ala Gly Ile Pro Gly Ala Pro Gly Leu
245 250 255
Met Gly Ala Arg Gly Pro Pro Gly Pro Ala Gly Ala Asn Gly Ala Pro
260 265 270
Gly Leu Arg Gly Gly Ala Gly Glu Pro Gly Lys Asn Gly Ala Lys Gly
275 280 285
Glu Pro Gly Pro Arg Gly Glu Arg Gly Glu Ala Gly Ile Pro Gly Val
290 295 300
Pro Gly Ala Lys Gly Glu Asp Gly Lys Asp Gly Ser Pro Gly Glu Pro
305 310 315 320
Gly Ala Asn Gly Leu Pro Gly Ala Ala Gly Glu Arg Gly Ala Pro Gly
325 330 335
Phe Arg Gly Pro Ala Gly Pro Asn Gly Ile Pro Gly Glu Lys Gly Pro
340 345 350
Ala Gly Glu Arg Gly Ala Pro Gly Pro Ala Gly Pro Arg Gly Ala Ala
355 360 365
Gly Glu Pro Gly Arg Asp Gly Val Pro Gly Gly Pro Gly Met Arg Gly
370 375 380
Met Pro Gly Ser Pro Gly Gly Pro Gly Ser Asp Gly Lys Pro Gly Pro
385 390 395 400
Pro Gly Ser Gln Gly Glu Ser Gly Arg Pro Gly Pro Pro Gly Pro Ser
405 410 415
Gly Pro Arg Gly Gln Pro Gly Val Met Gly Phe Pro Gly Pro Lys Gly
420 425 430
Asn Asp Gly Ala Pro Gly Lys Asn Gly Glu Arg Gly Gly Pro Gly Gly
435 440 445
Pro Gly Pro Gln Gly Pro Pro Gly Lys Asn Gly Glu Thr Gly Pro Gln
450 455 460
Gly Pro Pro Gly Pro Thr Gly Pro Gly Gly Asp Lys Gly Asp Thr Gly
465 470 475 480
Pro Pro Gly Pro Gln Gly Leu Gln Gly Leu Pro Gly Thr Gly Gly Pro
485 490 495
Pro Gly Glu Asn Gly Lys Pro Gly Glu Pro Gly Pro Lys Gly Asp Ala
500 505 510
Gly Ala Pro Gly Ala Pro Gly Gly Lys Gly Asp Ala Gly Ala Pro Gly
515 520 525
Glu Arg Gly Pro Pro Gly Leu Ala Gly Ala Pro Gly Leu Arg Gly Gly
530 535 540
Ala Gly Pro Pro Gly Pro Glu Gly Gly Lys Gly Ala Ala Gly Pro Pro
545 550 555 560
Gly Pro Pro Gly Ala Ala Gly Thr Pro Gly Leu Gln Gly Met Pro Gly
565 570 575
Glu Arg Gly Gly Leu Gly Ser Pro Gly Pro Lys Gly Asp Lys Gly Glu
580 585 590
Pro Gly Gly Pro Gly Ala Asp Gly Val Pro Gly Lys Asp Gly Pro Arg
595 600 605
Gly Pro Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly Gln Pro Gly
610 615 620
Asp Lys Gly Glu Gly Gly Ala Pro Gly Leu Pro Gly Ile Ala Gly Pro
625 630 635 640
Arg Gly Ser Pro Gly Glu Arg Gly Glu Thr Gly Pro Pro Gly Pro Ala
645 650 655
Gly Phe Pro Gly Ala Pro Gly Gln Asn Gly Glu Pro Gly Gly Lys Gly
660 665 670
Glu Arg Gly Ala Pro Gly Glu Lys Gly Glu Gly Gly Pro Pro Gly Val
675 680 685
Ala Gly Pro Pro Gly Gly Ser Gly Pro Ala Gly Pro Pro Gly Pro Gln
690 695 700
Gly Val Lys Gly Glu Arg Gly Ser Pro Gly Gly Pro Gly Ala Ala Gly
705 710 715 720
Phe Pro Gly Ala Arg Gly Leu Pro Gly Pro Pro Gly Ser Asn Gly Asn
725 730 735
Pro Gly Pro Pro Gly Pro Ser Gly Ser Pro Gly Lys Asp Gly Pro Pro
740 745 750
Gly Pro Ala Gly Asn Thr Gly Ala Pro Gly Ser Pro Gly Val Ser Gly
755 760 765
Pro Lys Gly Asp Ala Gly Gln Pro Gly Glu Lys Gly Ser Pro Gly Ala
770 775 780
Gln Gly Pro Pro Gly Ala Pro Gly Pro Leu Gly Ile Ala Gly Ile Thr
785 790 795 800
Gly Ala Arg Gly Leu Ala Gly Pro Pro Gly Met Pro Gly Pro Arg Gly
805 810 815
Ser Pro Gly Pro Gln Gly Val Lys Gly Glu Ser Gly Lys Pro Gly Ala
820 825 830
Asn Gly Leu Ser Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Leu Pro
835 840 845
Gly Leu Ala Gly Thr Ala Gly Glu Pro Gly Arg Asp Gly Asn Pro Gly
850 855 860
Ser Asp Gly Leu Pro Gly Arg Asp Gly Ser Pro Gly Gly Lys Gly Asp
865 870 875 880
Arg Gly Glu Asn Gly Ser Pro Gly Ala Pro Gly Ala Pro Gly His Pro
885 890 895
Gly Pro Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly
900 905 910
Glu Ser Gly Pro Ala Gly Pro Ala Gly Ala Pro Gly Pro Ala Gly Ser
915 920 925
Arg Gly Ala Pro Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr
930 935 940
Gly Glu Arg Gly Ala Ala Gly Ile Lys Gly His Arg Gly Phe Pro Gly
945 950 955 960
Asn Pro Gly Ala Pro Gly Ser Pro Gly Pro Ala Gly Gln Gln Gly Ala
965 970 975
Ile Gly Ser Pro Gly Pro Ala Gly Pro Arg Gly Pro Val Gly Pro Ser
980 985 990
Gly Pro Pro Gly Lys Asp Gly Thr Ser Gly His Pro Gly Pro Ile Gly
995 1000 1005
Pro Pro Gly Pro Arg Gly Asn Arg Gly Glu Arg Gly Ser Glu Gly Ser
1010 1015 1020
Pro Gly His Pro Gly Gln Pro Gly Pro Pro Gly Pro Pro Gly Ala Pro
1025 1030 1035 1040
Gly Pro Cys Cys Gly Gly Val Gly Ala Ala Ala Ile Ala Gly Ile Gly
1045 1050 1055
Gly Glu Lys Ala Gly Gly Phe Ala Pro Tyr Tyr Gly
1060 1065
<210> 4
<211> 1080
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Lys Arg Glu Ala Glu Ala Gln Tyr Asp Ser Tyr Asp Val Lys Ser Gly
1 5 10 15
Val Ala Val Gly Gly Leu Ala Gly Tyr Pro Gly Pro Ala Gly Pro Pro
20 25 30
Gly Pro Pro Gly Pro Pro Gly Thr Ser Gly His Pro Gly Ser Pro Gly
35 40 45
Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly Pro
50 55 60
Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro Ala
65 70 75 80
Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly Glu Arg Gly
85 90 95
Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile Pro Gly Phe
100 105 110
Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn Gly Glu Lys
115 120 125
Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly Leu Pro Gly
130 135 140
Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala Pro Gly Glu
145 150 155 160
Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg Gly Asn Asp
165 170 175
Gly Ala Arg Gly Ser Asp Gly Gln Pro Gly Pro Pro Gly Pro Pro Gly
180 185 190
Thr Ala Gly Phe Pro Gly Ser Pro Gly Ala Lys Gly Glu Val Gly Pro
195 200 205
Ala Gly Ser Pro Gly Ser Asn Gly Ala Pro Gly Gln Arg Gly Glu Pro
210 215 220
Gly Pro Gln Gly His Ala Gly Ala Gln Gly Pro Pro Gly Pro Pro Gly
225 230 235 240
Ile Asn Gly Ser Pro Gly Gly Lys Gly Glu Met Gly Pro Ala Gly Ile
245 250 255
Pro Gly Ala Pro Gly Leu Met Gly Ala Arg Gly Pro Pro Gly Pro Ala
260 265 270
Gly Ala Asn Gly Ala Pro Gly Leu Arg Gly Gly Ala Gly Glu Pro Gly
275 280 285
Lys Asn Gly Ala Lys Gly Glu Pro Gly Pro Arg Gly Glu Arg Gly Glu
290 295 300
Ala Gly Ile Pro Gly Val Pro Gly Ala Lys Gly Glu Asp Gly Lys Asp
305 310 315 320
Gly Ser Pro Gly Glu Pro Gly Ala Asn Gly Leu Pro Gly Ala Ala Gly
325 330 335
Glu Arg Gly Ala Pro Gly Phe Arg Gly Pro Ala Gly Pro Asn Gly Ile
340 345 350
Pro Gly Glu Lys Gly Pro Ala Gly Glu Arg Gly Ala Pro Gly Pro Ala
355 360 365
Gly Pro Arg Gly Ala Ala Gly Glu Pro Gly Arg Asp Gly Val Pro Gly
370 375 380
Gly Pro Gly Met Arg Gly Met Pro Gly Ser Pro Gly Gly Pro Gly Ser
385 390 395 400
Asp Gly Lys Pro Gly Pro Pro Gly Ser Gln Gly Glu Ser Gly Arg Pro
405 410 415
Gly Pro Pro Gly Pro Ser Gly Pro Arg Gly Gln Pro Gly Val Met Gly
420 425 430
Phe Pro Gly Pro Lys Gly Asn Asp Gly Ala Pro Gly Lys Asn Gly Glu
435 440 445
Arg Gly Gly Pro Gly Gly Pro Gly Pro Gln Gly Pro Pro Gly Lys Asn
450 455 460
Gly Glu Thr Gly Pro Gln Gly Pro Pro Gly Pro Thr Gly Pro Gly Gly
465 470 475 480
Asp Lys Gly Asp Thr Gly Pro Pro Gly Pro Gln Gly Leu Gln Gly Leu
485 490 495
Pro Gly Thr Gly Gly Pro Pro Gly Glu Asn Gly Lys Pro Gly Glu Pro
500 505 510
Gly Pro Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Gly Lys Gly
515 520 525
Asp Ala Gly Ala Pro Gly Glu Arg Gly Pro Pro Gly Leu Ala Gly Ala
530 535 540
Pro Gly Leu Arg Gly Gly Ala Gly Pro Pro Gly Pro Glu Gly Gly Lys
545 550 555 560
Gly Ala Ala Gly Pro Pro Gly Pro Pro Gly Ala Ala Gly Thr Pro Gly
565 570 575
Leu Gln Gly Met Pro Gly Glu Arg Gly Gly Leu Gly Ser Pro Gly Pro
580 585 590
Lys Gly Asp Lys Gly Glu Pro Gly Gly Pro Gly Ala Asp Gly Val Pro
595 600 605
Gly Lys Asp Gly Pro Arg Gly Pro Thr Gly Pro Ile Gly Pro Pro Gly
610 615 620
Pro Ala Gly Gln Pro Gly Asp Lys Gly Glu Gly Gly Ala Pro Gly Leu
625 630 635 640
Pro Gly Ile Ala Gly Pro Arg Gly Ser Pro Gly Glu Arg Gly Glu Thr
645 650 655
Gly Pro Pro Gly Pro Ala Gly Phe Pro Gly Ala Pro Gly Gln Asn Gly
660 665 670
Glu Pro Gly Gly Lys Gly Glu Arg Gly Ala Pro Gly Glu Lys Gly Glu
675 680 685
Gly Gly Pro Pro Gly Val Ala Gly Pro Pro Gly Gly Ser Gly Pro Ala
690 695 700
Gly Pro Pro Gly Pro Gln Gly Val Lys Gly Glu Arg Gly Ser Pro Gly
705 710 715 720
Gly Pro Gly Ala Ala Gly Phe Pro Gly Ala Arg Gly Leu Pro Gly Pro
725 730 735
Pro Gly Ser Asn Gly Asn Pro Gly Pro Pro Gly Pro Ser Gly Ser Pro
740 745 750
Gly Lys Asp Gly Pro Pro Gly Pro Ala Gly Asn Thr Gly Ala Pro Gly
755 760 765
Ser Pro Gly Val Ser Gly Pro Lys Gly Asp Ala Gly Gln Pro Gly Glu
770 775 780
Lys Gly Ser Pro Gly Ala Gln Gly Pro Pro Gly Ala Pro Gly Pro Leu
785 790 795 800
Gly Ile Ala Gly Ile Thr Gly Ala Arg Gly Leu Ala Gly Pro Pro Gly
805 810 815
Met Pro Gly Pro Arg Gly Ser Pro Gly Pro Gln Gly Val Lys Gly Glu
820 825 830
Ser Gly Lys Pro Gly Ala Asn Gly Leu Ser Gly Glu Arg Gly Pro Pro
835 840 845
Gly Pro Gln Gly Leu Pro Gly Leu Ala Gly Thr Ala Gly Glu Pro Gly
850 855 860
Arg Asp Gly Asn Pro Gly Ser Asp Gly Leu Pro Gly Arg Asp Gly Ser
865 870 875 880
Pro Gly Gly Lys Gly Asp Arg Gly Glu Asn Gly Ser Pro Gly Ala Pro
885 890 895
Gly Ala Pro Gly His Pro Gly Pro Pro Gly Pro Val Gly Pro Ala Gly
900 905 910
Lys Ser Gly Asp Arg Gly Glu Ser Gly Pro Ala Gly Pro Ala Gly Ala
915 920 925
Pro Gly Pro Ala Gly Ser Arg Gly Ala Pro Gly Pro Gln Gly Pro Arg
930 935 940
Gly Asp Lys Gly Glu Thr Gly Glu Arg Gly Ala Ala Gly Ile Lys Gly
945 950 955 960
His Arg Gly Phe Pro Gly Asn Pro Gly Ala Pro Gly Ser Pro Gly Pro
965 970 975
Ala Gly Gln Gln Gly Ala Ile Gly Ser Pro Gly Pro Ala Gly Pro Arg
980 985 990
Gly Pro Val Gly Pro Ser Gly Pro Pro Gly Lys Asp Gly Thr Ser Gly
995 1000 1005
His Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Asn Arg Gly Glu
1010 1015 1020
Arg Gly Ser Glu Gly Ser Pro Gly His Pro Gly Gln Pro Gly Pro Pro
1025 1030 1035 1040
Gly Pro Pro Gly Ala Pro Gly Pro Cys Cys Gly Gly Val Gly Ala Ala
1045 1050 1055
Ala Ile Ala Gly Ile Gly Gly Glu Lys Ala Gly Gly Phe Ala Pro Tyr
1060 1065 1070
Tyr Gly His His His His His His
1075 1080
<210> 5
<211> 729
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ala Thr Gly Ala Cys Ala Ala Ala Thr Ala Ala Ala Thr Thr Thr Ala
1 5 10 15
Thr Thr Thr Cys Gly Thr Ala Cys Ala Ala Thr Ala Ala Ala Ala Thr
20 25 30
Gly Gly Ala Ala Ala Cys Cys Cys Gly Ala Gly Ala Ala Thr Ala Cys
35 40 45
Thr Thr Gly Thr Thr Gly Ala Cys Gly Ala Thr Ala Thr Thr Gly Thr
50 55 60
Thr Cys Gly Thr Gly Ala Thr Ala Gly Cys Thr Thr Gly Thr Thr Thr
65 70 75 80
Cys Ala Thr Gly Gly Thr Ala Thr Thr Ala Ala Ala Thr Cys Thr Cys
85 90 95
Gly Ala Ala Cys Gly Gly Cys Gly Ala Gly Ala Gly Gly Gly Gly Thr
100 105 110
Thr Thr Gly Ala Ala Ala Cys Cys Ala Gly Cys Gly Ala Thr Cys Gly
115 120 125
Thr Cys Cys Gly Gly Gly Thr Gly Thr Thr Thr Gly Thr Gly Ala Thr
130 135 140
Gly Gly Ala Ala Ala Ala Thr Ala Thr Thr Ala Cys Gly Ala Ala Ala
145 150 155 160
Ala Gly Ala Thr Thr Gly Ala Thr Gly Gly Ala Thr Thr Thr Thr Thr
165 170 175
Ala Thr Cys Thr Gly Ala Thr Ala Thr Thr Gly Ala Ala Thr Gly Thr
180 185 190
Gly Ala Thr Gly Thr Ala Thr Thr Ala Ala Thr Cys Ala Ala Thr Gly
195 200 205
Cys Ala Gly Cys Ala Ala Thr Thr Ala Ala Gly Ala Ala Ala Gly Gly
210 215 220
Gly Thr Thr Ala Ala Thr Ala Ala Ala Ala Thr Cys Ala Gly Ala Gly
225 230 235 240
Gly Thr Ala Gly Gly Cys Gly Gly Ala Gly Cys Ala Ala Cys Thr Gly
245 250 255
Ala Ala Ala Ala Thr Gly Ala Thr Cys Cys Thr Ala Thr Ala Ala Ala
260 265 270
Ala Cys Thr Cys Gly Ala Thr Cys Cys Gly Ala Ala Gly Ala Gly Thr
275 280 285
Cys Gly Thr Ala Ala Cys Thr Cys Cys Gly Ala Ala Cys Ala Ala Ala
290 295 300
Cys Ala Thr Gly Gly Thr Thr Thr Ala Thr Gly Cys Cys Cys Gly Gly
305 310 315 320
Cys Gly Ala Ala Cys Ala Cys Gly Ala Ala Gly Thr Thr Ala Thr Thr
325 330 335
Gly Ala Thr Ala Ala Ala Ala Thr Ala Cys Ala Gly Ala Ala Ala Ala
340 345 350
Ala Gly Ala Cys Ala Ala Gly Gly Gly Ala Ala Thr Thr Thr Thr Thr
355 360 365
Ala Ala Ala Cys Ala Gly Thr Ala Ala Ala Ala Ala Ala Cys Ala Thr
370 375 380
Thr Gly Thr Ala Thr Thr Gly Ala Cys Ala Ala Ala Thr Ala Thr Ala
385 390 395 400
Ala Thr Thr Thr Cys Gly Ala Ala Gly Ala Thr Gly Thr Thr Cys Ala
405 410 415
Ala Gly Thr Ala Gly Cys Thr Ala Gly Gly Thr Ala Thr Ala Ala Ala
420 425 430
Cys Cys Cys Gly Gly Gly Cys Ala Ala Thr Ala Cys Thr Ala Thr Thr
435 440 445
Ala Thr Cys Ala Thr Cys Ala Thr Thr Ala Cys Gly Ala Cys Gly Gly
450 455 460
Ala Gly Ala Thr Gly Ala Cys Thr Gly Cys Gly Ala Cys Gly Ala Thr
465 470 475 480
Gly Cys Ala Thr Gly Cys Cys Cys Gly Ala Ala Gly Gly Ala Cys Cys
485 490 495
Ala Ala Ala Gly Ala Thr Thr Ala Gly Cys Thr Ala Cys Gly Thr Thr
500 505 510
Gly Ala Thr Gly Gly Thr Gly Thr Ala Thr Cys Thr Thr Ala Ala Ala
515 520 525
Gly Cys Thr Cys Cys Thr Gly Ala Ala Gly Ala Ala Gly Gly Thr Gly
530 535 540
Gly Thr Gly Gly Thr Gly Gly Thr Gly Ala Gly Ala Cys Thr Gly Ala
545 550 555 560
Thr Thr Thr Thr Cys Cys Gly Ala Cys Ala Cys Thr Cys Ala Ala Ala
565 570 575
Ala Cys Ala Ala Ala Ala Ala Thr Ala Ala Ala Ala Cys Cys Ala Ala
580 585 590
Ala Gly Ala Ala Ala Gly Gly Ala Ala Cys Thr Thr Cys Ala Ala Thr
595 600 605
Cys Thr Thr Thr Thr Thr Thr Thr Gly Gly Gly Thr Thr Gly Cys Gly
610 615 620
Gly Ala Thr Cys Cys Thr Gly Thr Ala Ala Cys Gly Ala Gly Gly Ala
625 630 635 640
Ala Ala Thr Thr Gly Thr Ala Cys Ala Ala Ala Gly Ala Ala Ala Cys
645 650 655
Thr Thr Thr Gly Cys Ala Cys Gly Cys Ala Gly Gly Ala Cys Thr Thr
660 665 670
Cys Cys Cys Gly Thr Gly Ala Ala Ala Ala Gly Thr Gly Gly Ala Gly
675 680 685
Ala Ala Ala Ala Ala Ala Thr Cys Ala Thr Cys Gly Cys Gly Ala Ala
690 695 700
Cys Cys Ala Ala Thr Gly Gly Ala Thr Cys Cys Gly Thr Gly Cys Thr
705 710 715 720
Gly Thr Thr Ala Ala Ala Thr Gly Ala
725
Claims (9)
1.一种重组人源III型胶原蛋白，其特征在于，包含重组人源III型胶原蛋白的核苷酸序列，所述重组人源III型胶原蛋白的核苷酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的重组人源III型胶原蛋白的制备方法，其特征在于，包括以下步骤：
步骤1：基因设计；
步骤2：基因合成及表达载体pPIC9K和pPICZA的构建；
步骤3：含有脯氨酸羟化酶基因的毕赤酵母表达系统的制备；
步骤4：重组人源III型胶原蛋白的诱导表达；
步骤5：SDS-PAGE蛋白胶检测；
步骤6：Western Blot验证；
步骤7：蛋白纯化。
3.根据权利要求2所述的重组人源III型胶原蛋白的制备方法，其特征在于，所述步骤1包含以下具体步骤：
步骤1.1：根据GEO数据库中人源III型胶原蛋白的基因序列，选用易于提取纯化的去掉N端和C端的重组人源III型胶原蛋白核心功能区的cDNA序列，对应的原始人源III型胶原蛋白基因序列如SEQ ID NO.1所示；
步骤1.2：利用密码子在线翻译工具得到人源III型胶原蛋白氨基酸序列，所述人源III型胶原蛋白氨基酸序列如SEQ ID NO.3所示；
步骤1.3：针对宿主毕赤酵母表达所需的优选密码子，在设计过程中在N端和C端分别加入α-MF信号肽序列和六个组氨酸编码序列。
4.根据权利要求2所述的重组人源III型胶原蛋白的制备方法，其特征在于，所述步骤2包含以下具体步骤：
步骤2.1：根据与人源P4H的α亚基序列相似的病毒A085R蛋白基因序列作为脯氨酸羟化酶的基因序列，其基因序列如SEQ ID NO.5所示；
步骤2.2：根据SEQ ID NO.2和SEQ ID NO.5所示的基因序列，分别进行全基因合成，并构建表达载体。
5.根据权利要求4所述的重组人源III型胶原蛋白的制备方法，其特征在于，还包括一种可表达含有脯氨酸羟化酶修饰的重组人源III型胶原蛋白的酵母，所述的可表达含有脯氨酸羟化酶修饰的重组人源III型胶原蛋白的酵母是将步骤2.2得到的重组载体转入酵母中得到。
6.根据权利要求5所述重组人源III型胶原蛋白的制备方法，其特征在于，所述的酵母为毕赤酵母。
7.根据权利要求2所述的重组人源III型胶原蛋白的制备方法，其特征在于，所述步骤3包含以下具体步骤：
步骤3.1：重组质粒pPIC9K-COL3A1、pPICZA-P4H转化至毕赤酵母GS115感受态细胞，利用限制性内切酶sacI对重组质粒pPIC9K-COL3A1、pPICZA-P4H线性化，电转法转化至毕赤酵母GS115感受态细胞；
步骤3.2：重组酵母菌的筛选，提取转化后菌落的基因组，利用酵母通用引物，PCR扩增筛选阳性菌株，反应条件为：95℃5min；95℃30s，60℃60s，72℃3min，30个循环；72℃10min，于4℃保存；pPIC9K空载体作对照，毕赤酵母细胞GS115作为空白对照，筛选成功重组到酵母细胞基因组的阳性重组子。
8.根据权利要求7所述的重组人源III型胶原蛋白的制备方法，其特征在于，所述步骤3.2中的酵母通用引物为：
5'测序引物及序列:5’AOX1:5’-GACTGGTTCCAATTGACAAGC-3’；
alpha-Factor-F:5’-TACTATTGCCAGCATTGCTGC-3’；
3'测序引物及序列:3’AOX1:5’-GCAAATGGCATTCTGACATCC-3’。
9.根据权利要求2所述的重组人源III型胶原蛋白的制备方法，其特征在于，所述步骤4包含以下具体步骤：
步骤4.1：取保藏于-80℃的菌种GS115/pPIC9K-COL3A1-pPICZA-P4H进行活化，用玻璃三角涂布棒蘸取菌液在YPD固体平板上均匀涂开；30℃培养2～3天，挑取单菌落于YPD液体培养基中，30℃，200rpm培养约24h，至OD600达到0.6～2.0范围；
步骤4.2：取种子液以10％的接种量接种到甘油培养基中，装液量为50ml/250ml，30℃，200rpm培养24h，后将菌液4℃，5000rpm离心20min得到大量菌沉淀，弃上清，将菌沉淀转移入提前配制好的200mL甲醇诱导培养基培养，24h后，向培养基中补加甲醇，使培养基中甲醇浓度为1％，每隔24h补加一次甲醇，使甲醇浓度维持在1％，培养至120h，收取全部上清。
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