CN110964099A - 酵母重组人源I型胶原α1链蛋白、合成方法及其应用 - Google Patents
酵母重组人源I型胶原α1链蛋白、合成方法及其应用 Download PDFInfo
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- CN110964099A CN110964099A CN201911135958.0A CN201911135958A CN110964099A CN 110964099 A CN110964099 A CN 110964099A CN 201911135958 A CN201911135958 A CN 201911135958A CN 110964099 A CN110964099 A CN 110964099A
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Abstract
本发明公开了一种酵母重组人源I型胶原α1链蛋白、合成方法及其应用，本发明的重组人源I型胶原α1链蛋白，由N端亲和纯化标记、人源I型胶原蛋白α1链成熟肽序列及C端亲和纯化标记组成，在其两端设计的双特异性亲和纯化标记，有利于该重组蛋白的纯化，也有利于其检测及全长鉴定。本发明首先在基因水平上利用毕赤酵母惯用密码子优化胶原基因序列并人工全基因合成，然后通过PCR、酶切、连接等构建成重组载体，并整合进酵母染色体中，构建成分泌表达重组人源I型胶原α1链蛋白的重组毕赤酵母工程菌。本发明的重组人源I型胶原α1链蛋白携带的双特异性亲和纯化标记，可进行两步特异性纯化，易于得到高纯度的产品。
Description
技术领域
本发明涉及生物工程技术领域，具体是一种酵母重组人源I 型胶原α1链蛋白、合成方法及其应用。
背景技术
胶原蛋白是动物体内含量最丰富的一类蛋白质家族，约占动物机体总蛋白的30％左右。其中I型胶原蛋白在目前已发现的29中胶原蛋白中含量最高，约占90％。它在新组织的形态发生和细胞代谢中，赋予新组织机械强度和生化特性；其主要分布于皮肤、角膜、肌腱等部位，其对维护细胞、组织等的正常生理功能和损伤修复起着重要作用。
I型胶原蛋白具有良好的生物相容性、生物可降解性、无细胞毒性、抗氧化性等功能，并能促进细胞粘附和细胞增殖等功效，可广泛应用在医学、美容、化妆品、保健品等领域中。其在皮肤损伤修复、整形美容、增强机体免疫力等方面的作用也已获得共识。
目前国内虽已有重组人I型胶原蛋白的相关研究，如西北大学和华南理工大学均有有关I型胶原蛋白短肽的研究报道，但其主要研究胶原蛋白短肽片段，尚无有关重组人源I型胶原α1 链全长肽的报道。根据目前研究成果已知，重组天然胶原蛋白短肽片段易降解更短的短肽，且因其结构相似性，难以检测其纯度。而重组人源I型胶原α1链全长肽，因肽链更长，更容易发生降解，更难保证产品中的胶原是否为全长肽，限制了其广泛应用。
目前市场上已流通的重组I型胶原蛋白肽段，虽已表现出较优的功效，但因其片段较小，分子量低，也限制了其功能的发挥。I型胶原蛋白全长肽因其具有更高的分子量，拥有独特的生理生化特性，如其易形成一层透气的薄膜、抗降解时间长、交联后交联度更高，性能更优异；且其随时间推移发生的缓慢降解，可以更持久的补充胶原蛋白短肽，甚至与某些药物结合可以起到缓释作用。因此，为了充分开发其潜在价值，亟需解决其全长肽的生产与全长肽链的检测问题。
目前胶原蛋白的分离纯化，多采用分子筛和离子交换树脂进行分离，而胶原蛋白因其独特的G-X-Y结构使其很难分离开，尤其与同种胶原的降解片段，且该提纯工艺操作复杂及不同批次间提纯工艺难以稳定，导致市场上胶原蛋白短肽产品多为短肽及其降解物的混合物；而亲和纯化技术因其特异性识别，可得到纯度更高的产品，且操作简便，有望解决其纯度难以提高及提纯工艺难以稳定的问题。
发明内容
本发明的目的在于提供一种酵母重组人源I型胶原α1链蛋白、合成方法及其应用，以解决现有技术中的问题。
为实现上述目的，本发明提供如下技术方案：
一种酵母重组人源I型胶原α1链蛋白，所述重组人源I型胶原α1链蛋白的氨基酸序列如SEQ ID NO.1所示；所述重组人源I型胶原α1链蛋白包括N端的Strep-Tag II序列、人源I型胶原蛋白α1链成熟肽序列和C端的6×His序列，使其含有双特异性亲和纯化标记；全长为1071个氨基酸。
较优化地，所述重组人源I型胶原α1链蛋白的基因表达序列如SEQ ID NO.2所示。
较优化地，所述人源I型胶原蛋白α1链成熟肽的基因序列是经利用宿主惯用密码子将天然胶原蛋白基因序列优化后得到的，基因序列如SEQ ID NO.3所示。
较优化地，一种酵母重组人源I型胶原α1链蛋白的合成方法，包括以下步骤：
1)合成人源I型胶原蛋白α1链成熟肽的基因序列；
2)制备含有Strep-Tag II编码序列的改造质粒pPIC9ks；
3)双酶切改造质粒pPIC9ks，构建含有重组人源I型胶原α1 链蛋白基因的重组质粒pPIC9k-colIα1；
4)将重组质粒pPIC9k-colIα1用限制性内切酶SalⅠ线性化，并将其电转化入毕赤酵母SMD1168感受态细胞中，以营养缺陷型标记和G418抗性标记筛选高拷贝阳性重组子，得到毕赤酵母基因工程菌；
5)毕赤酵母基因工程菌的发酵培养；
6)发酵结束后，离心纯化，得到所述重组人源I型胶原α1 链蛋白。
较优化地，包括以下步骤：
1)合成人源I型胶原蛋白α1链成熟肽的基因序列：在人源 I型胶原蛋白α1链成熟肽的氨基酸序列不变条件下，将人I型胶原α1链基因序列中对应的毕赤酵母不常用的密码子优化为毕赤酵母偏爱性密码子；再通过全基因合成人源I型胶原蛋白α1链成熟肽的基因序列；
2)制备含有Strep-Tag II编码序列的改造质粒pPIC9ks；
a)首先以pPIC9k质粒为模板，利用引物P1、P2进行 PCR扩增，产物长度为394bp；其中P1的基因序列如SEQ ID NO.4所示，P2的基因序列如SEQ ID NO.5 所示；
b)对获得的PCR产物和pPIC9k质粒分别做EcoRI和 BamHI双酶切，并经T4 DNA连接酶作用下，16℃连接过夜，得到改造质粒pPIC9ks；
3)构建含有重组人源I型胶原α1链蛋白基因的重组质粒 pPIC9k-colIα1：
a)以步骤1)中合成的人源I型胶原蛋白α1链成熟肽的基因序列为模板，利用引物P3、P4进行PCR扩增，得到长度为3194bp的基因片段；其中P3的基因序列如SEQ ID NO.6所示，P4的基因序列如SEQ ID NO.7 所示；
b)对步骤2)中得到的改造质粒pPIC9ks做ApaI和Not I双酶切，获得载体片段；
c)以P5、P6为引物，以自身为模板，通过PCR相互扩增，得到含有6×His编码序列的基因片段，长度为 65bp；其中P5的基因序列如SEQ ID NO.8所示，P6 的基因序列如SEQ IDNO.9所示；
d)将步骤a)、步骤b)、步骤c)中的各片段混合，利用即用型无缝克隆试剂盒连接，50℃连接1h，得到含有Strep-Tag II编码序列、人源I型胶原蛋白α1链成熟肽基因序列、6×His编码序列的重组质粒 pPIC9k-colIα1；
4)将重组质粒pPIC9k-colIα1用限制性内切酶SalⅠ于37℃线性化，并将其电转化入毕赤酵母SMD1168感受态细胞中，以组氨酸缺陷型标记和G418抗性标记筛选高拷贝阳性重组子，得到毕赤酵母基因工程菌；
5)毕赤酵母基因工程菌的发酵培养：将步骤4)中制备的毕赤酵母基因工程菌接种于YPD液体培养基中，30℃、 220rpm过夜活化培养，而后转接于BMGY培养基，于 30℃、220rpm培养该工程菌16-18h，至OD600＝2.0-6.0；室温下1500g离心5min，收集菌体；再用10mL的BMMY 培养基重悬菌体，使OD600＝1.0左右，将所得的菌液置于100mL无菌三角瓶中，于28℃、220rpm下继续振荡培养；每24小时向培养基中添加甲醇至终浓度为1％进行诱导培养；
6)发酵结束后，取发酵液离心除去酵母细胞，得发酵上清；在非变性条件下，用Ni-NTA His Bands树脂吸附重组胶原，用洗涤液洗去杂质，最后再洗脱重组胶原，收集洗脱液；洗脱液经超滤系统脱盐、浓缩；再将浓缩液用 Strep-Tactin树脂吸附重组胶原，用洗涤液洗去杂质，最后再洗脱重组胶原，收集洗脱液；洗脱液经超滤系统脱盐、浓缩，所制得的浓缩液经真空冷冻干燥制得所述重组人源I型胶原α1链蛋白。
较优化地，酵母重组人源I型胶原α1链蛋白制备的胶原蛋白凝胶敷料，所述胶原蛋白凝胶敷料以纯化水为溶剂，其组分还包括重组人源I型胶原α1链蛋白、凝胶剂、保湿剂；所述凝胶剂为卡波姆，所述保湿剂为甘油和三乙醇胺；其中各组分的质量配比为：重组人源I型胶原α1链蛋白0.1-0.5％、甘油1-10％、卡波姆0.1-4％、三乙醇胺0.1-10％，其余为纯化水；本发明中制备的凝胶敷料可以增加皮肤含水量、调节皮肤pH值及油脂，尤其其中的重组人源I型胶原α1链蛋白可在皮肤表面形成一层透气的薄膜，阻隔外部细菌，防止创面发炎，并促进创面愈合，重建皮肤屏障功能。适用于皮肤屏障功能损伤引起的皮肤干燥脱屑及其他皮肤损伤的修复及激光创面修复等。
较优化地，酵母重组人源I型胶原α1链蛋白制备的胶原蛋白贴敷料，所述胶原蛋白贴敷料由重组人源I型胶原α1链蛋白、保湿剂、医用增稠剂和无纺布基材组成；所述保湿剂为甘油，所述医用增稠剂为黄原胶；其中各组分的质量配比为：重组人源I型胶原α1链蛋白0.1-0.5％、甘油1-10％、黄原胶0.1-2％，其余为纯化水；本发明中制备的胶原蛋白贴敷料能有效地改善皮肤微循环，营造相对密闭湿润环境，增强皮肤的新陈代谢及再生功能。适用于创面愈后早期色素沉着、早期浅表性疤痕修复等。
较优化地，酵母重组人源I型胶原α1链蛋白制备的用于美容的导入性产品，所述导入性产品各原料组分如下：以质量百分比计，重组人源I型胶原α1链蛋白0.1-0.5％，甘油1-10％、透明质酸钠0.1-0.5％、小分子透明质酸钠0.01-0.05％、氯化钠 0.9％、五胜肽0.005-0.02‰，其余为纯化水；本发明中制备的导入性产品能够向皮肤深层补充胶原蛋白，可促进皮肤新陈代谢，及充盈皮肤减轻皱纹，使皮肤水嫩亮白富有弹性。适用于爱美、注重肌肤保养的人群。
较优化地，酵母重组人源I型胶原α1链蛋白制备的外用护肤精华液，所述护肤精华液各原料组分如下：以质量百分比计，甘油1-3％、丁二醇1-3％、透明质酸钠0.01-0.1％、重组人源I 型胶原α1链蛋白0.01-0.1％、维生素C乙基醚0.01-0.1％、维生素C磷酸酯钠0.01-0.1％、甘草酸二钾0.1-0.5％、EDTA·Na2 0.01-0.1％、丝肽0.01-0.1％、β-葡聚糖0.1-0.5％、芦芭油0.1-0.5％、水溶性氮酮0.1-1％，其余为纯化水；本发明中制备的外用护肤精华液可补充皮肤营养，改善皮肤微循环，具有使皮肤光滑湿润及美白等功效，并可阻隔外来细菌、灰尘、紫外线的侵入，保护皮肤免受侵害；适用于肌肤的日常修复保养。
本发明在优化人源I型胶原蛋白α1链成熟肽的基因序列过程中，根据Genbank登录的氨基酸序列和基因序列，在不改变人I型胶原蛋白原始氨基酸序列的前提下优化其对应的基因序列，其优化处理是根据毕赤酵母偏爱性密码子进行，将人I型胶原α1链成熟肽对应的基因序列(colIα1)中毕赤酵母不常用的密码子agg、cgt、ggg、ggc、gca等优化为其偏爱密码子aga、 ggt、gct等，利用DNASTAR软件分析后得到的，经过这一处理，使胶原蛋白更利于表达；同时在合成过程中，将优化的基因序列剔除了ApaI、Bgl II、BamH I、EcoR I、Not I、Sac I、 Sal I等限制性内切酶位点。
本发明中制备得到的改造质粒pPIC9ks中，Strep-Tag II编码序列的紧接于质粒上的KEX2蛋白酶识别位点的基因序列之后，提高了信号肽的切割效率，保证了Strep-TagII不被包埋和在纯化中的高效率识别。
本发明中制备的重组毕赤酵母基因工程菌，已保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号CGMCC NO.17150，保藏日期：2019年1月10日，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，分类命名：巴斯德毕赤酵母Pichiapastoris。
本发明中制备得到的重组人源I型胶原α1链蛋白，其化学结构和性能优于动物源胶原蛋白和原核微生物源重组胶原蛋白及市场上的重组胶原片段和明胶。
本发明利用毕赤酵母构建真核表达系统，通过甲醇诱导表达重组人源I型胶原α1链蛋白，诱导型启动子启动效率高，表达量高；发酵周期短，发酵过程简单，适于规模化生产和高密度发酵生产；胶原蛋白被分泌到细胞外，易于分离纯化；产品无内毒素，更安全。
本发明中制备了一种酵母重组人源I型胶原α1链蛋白，全长为1071个氨基酸，由N端的Strep-Tag II序列、人源I型胶原蛋白α1链成熟肽序列及C端的6×His序列组成，使其含有双特异性亲和纯化标记；该蛋白为单链结构；利用其带有的双特异性亲和纯化标记，易于获取高纯度产品；利用其带有的双特异性亲和纯化标记，便于检测和鉴定其在产品是否为全长胶原蛋白。
本发明使用基因重组工程菌生产的酵母重组人源I型胶原α1链蛋白，进行了相对应的纯化操作；在生产过程中，通过 Ni-NTA His·Bind树脂和Strep-Tactin树脂吸附的方式从工程菌的发酵液中提取重组人源I型胶原α1链蛋白并使其得到纯化。
本发明首次根据毕赤酵母所偏爱的密码子，人工合成了优化的人源I型胶原蛋白α1链成熟肽基因序列，通过PCR扩增、酶切、连接等方法，构建出一个全新的、分泌型的毕赤酵母高效表达载体，在将外源基因导入毕赤酵母细胞后，通过营养缺陷型标记筛选、抗性筛选、表达筛选等方法，获得了能够高效表达重组人源I型胶原α1链蛋白的毕赤酵母工程菌株；经特定条件的发酵培养、亲和层析纯化等操作，得到无毒、高纯度的产品，可用于敷料、化妆品、生物医用原料、整形美容等领域。
与现有技术相比，本发明的有益效果是：
1、本发明的重组人源I型胶原α1链蛋白在其两段携带有双特异性标记，既可以通过亲和纯化获得全长胶原蛋白，亦可以通过夹心法ELISA检验鉴定其在产品中的状态和含量。
2、本发明的重组人源I型胶原α1链蛋白的基因序列依据毕赤酵母惯用密码子对其基因序列进行了密码子优化，消除了不常用密码子及发夹结构，通过同义转换，优化mRNA的二级结构，避免了密码子利用效率限制导致的翻译效率低下，使其更适于在毕赤酵母中表达。
3、与常规的重组胶原片段和明胶相比，重组人源I型胶原α1链蛋白为全长的胶原蛋白，因而具有很高的分子量，作为生物大分子，拥有更优的化学结构和性能，可作为生物医用材料。
4、与传统胶原蛋白生产方法相比，重组人源I型胶原α1 链蛋白为真核表达系统表达的人源化的重组胶原蛋白，且能被分泌到细胞外，其无病毒、内毒素隐患，并避免了免疫排斥反应，其生物相容性和生物安全性更高，工艺过程对环境更友好，产品更安全。
5、本发明的重组人源I型胶原α1链蛋白的亲水性和稳定性好，其氨基酸组成与天然胶原蛋白氨基酸序列相应部分100％相同，其结构和生物活性更接近于天然胶原蛋白。
6、本发明的重组人源I型胶原α1链蛋白携带有N端的 Strep-Tag II标签和C端的6×his标签，利用其双特异性亲和纯化标签，纯化步骤简单，产物纯度高，可满足微创、整形美容、生物医用材料等产品使用。
附图说明
为了使本发明的内容更容易被清楚地理解，下面根据具体实施例并结合附图，对本发明作进一步详细的说明。
图1为构建的重组人源I型胶原α1链蛋白的分泌表达重组质粒pPIC9k-colIα1的图谱；
图2为重组质粒pPIC9k-colIα1的PCR鉴定电泳图，其中泳道M为分子量Marker，1-2为不同重组阳性克隆；
图3为筛选到的重组毕赤酵母工程菌表达重组人源I型胶原α1链蛋白的SDS-PAGE图，其中泳道M为分子量Marker， 1-4为不同重组菌株诱导表达后24h取上清液16μL的电泳图；
图4为筛选到的重组工程菌表达重组人源I型胶原α1链蛋白的免疫印迹实验，其中M为分子量Marker，1、2为同一重组菌株上清液不同上样量的免疫印迹；3、4为同一重组菌株上清液不同上样量的免疫印迹；
图5为筛选到的重组菌表达重组人源I型胶原α1链蛋白的质谱比对结果；图中使用ProtTech’s ProtQuest software suite软件，依据UniProt protein database和NCBI数据库进行比对；
图6为夹心法ELISA对筛选到的重组菌表达重组人源I型胶原α1链蛋白的全长性检验测定，其中降解物为纯化的含有C 端标记的降解蛋白；目标蛋白为纯化的目标蛋白。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
对于本领域技术人员而言，显然本发明不限于以下示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
实施例1：
实施例1中进行重组人源I型胶原α1链蛋白的制备，实验步骤如下所示：
1、合成人源I型胶原蛋白α1链成熟肽的基因序列：在不改变人源I型胶原蛋白α1链成熟肽的氨基酸序列条件下，将其对应的基因序列中对应的毕赤酵母不常用的密码子优化为毕赤酵母偏爱性密码子，利用DNASTAR软件优化基因序列，并剔除了ApaI、Bgl II、BamHI、EcoR I、Not I、Sac I、Sal I等限制性内切酶位点，合成得到人源I型胶原蛋白α1链成熟肽的基因序列，见SEQ ID NO.3；
2、制备含有Strep-Tag II编码序列的改造质粒pPIC9ks：先以pPIC9k质粒为模板，利用引物P1、P2进行PCR扩增，产物长度为394bp；其中P1的基因序列如SEQ ID NO.4所示，P2的基因序列如SEQ ID NO.5所示；再对获得的PCR产物和 pPIC9k质粒分别做EcoRI和BamHI双酶切，并经T4 DNA连接酶作用下，16℃连接过夜；再将连接产物转化进入感受态大肠杆菌DH5α，在含有Kan的LB抗性平板筛选阳性克隆；挑取经 PCR检测正确的阳性克隆，培养后提取质粒，得到改造质粒 pPIC9ks；
3、构建含有重组人源I型胶原α1链蛋白基因的重组质粒 pPIC9k-colIα1：首先以合成的人源I型胶原蛋白α1链成熟肽的基因序列为模板，利用引物P3、P4进行PCR扩增，得到长度为3194bp的基因片段；其中P3的基因序列如SEQ ID NO.6所示，P4的基因序列如SEQ IDNO.7所示；再对得到的改造质粒 pPIC9ks做ApaI和Not I双酶切，获得载体片段；接着以P5、P6为引物，以自身为模板，通过PCR相互扩增，得到含有6×His 编码序列的基因片段，长度为65bp；其中P5的基因序列如SEQ ID NO.8所示，P6的基因序列如SEQ ID NO.9所示；最后将制备的各片段混合，利用即用型无缝克隆试剂盒连接，50℃连接 1h；再将连接产物转化进入感受态大肠杆菌DH5α，在含有Kan 的LB抗性平板筛选阳性克隆；利用引物P1、P7对阳性克隆进行PCR检测，其中P1的基因序列如SEQ ID NO.4所示，P7的基因序列如SEQ ID NO.10所示，挑取经PCR检测正确的阳性克隆，培养后提取质粒，得到含有Strep-Tag II编码序列、人源 I型胶原蛋白α1链成熟肽基因序列、6×His编码序列的重组质粒pPIC9k-colIα1；
4、毕赤酵母基因工程菌的制备：
(1)重组表达质粒pPIC9k-colIα1的线性化：提取重组质粒pPIC9k-colIα1，用限制性内切酶SalⅠ于37℃进行酶切消化，然后以1％琼脂糖凝胶电泳检测是否完全切开，待完全切开后，用胶回收试剂盒对酶切液进行处理，回收线性化质粒，并脱盐处理。
(2)毕赤酵母SMD1168感受态细胞的制备：
①挑取酵母SMD1168单菌落，接种至含有5mL的YPD液体培养基的三角瓶中，30℃、250rpm振荡培养过夜；
②取50μL的过夜培养物接种至含有50mL新鲜YPD液体培养基的300mL三角瓶中，30℃、250rpm振荡培养过夜，至 OD600值达到1.1-1.3；
③将培养物于4℃，1500×g离心5min，用50mL冰预冷的无菌双蒸水重悬菌体；
④按步骤③离心，用25mL冰预冷的无菌双蒸水重悬菌体；
⑤按步骤③离心，用20mL冰预冷的1M的山梨醇溶液重悬菌体；
⑥按步骤③离心，用0.3mL冰预冷的1M的山梨醇溶液重悬菌体，其终体积约为0.5mL；
⑦分装成每80μL一份，于-70℃保存备用。
(3)毕赤酵母的电转化：
①用无水乙醇冲洗电转杯三遍，于灭过菌的超净台中晾干残余乙醇；
②将电转杯密封，转至冰中预冷10min；
③将毕赤酵母感受态细胞于冰预中解冻，加入约10μg的线性化pPIC9k-colIα1质粒，轻轻混匀，转至0.2cm冰预冷的电转杯中，继续于冰上预冷5min；
④电击，电压1.5kV；电容25μF；电阻200Ω；电击时间为 5-10mSec；
⑤电击结束，迅速加入1mL冰预冷的1M的山梨醇溶液，轻轻吹打混匀，并转至1.5mL离心管中；
⑥将菌悬液涂布于MD平板上，每100μL-200μL涂布一块平板，室温静置10min，于30℃倒置培养2-5天，直至有单菌落出现。
(4)多拷贝插入重组子的筛选：
①在生长有转化子的MD平板表面加入2mL无菌双蒸水，然后用无菌三角涂布器轻轻刮下平板表面的His+转化子，并转移到50mL离心管中；
②加入20mL无菌双蒸水稀释，混匀后测定其OD600值(1 OD600＝5×107cells/mL)；
③取105个细胞涂布于含有0.5mg/mL G418的YPD平板上，倒置，30℃培养3-4d；
④在无菌96孔板中每孔加入200μLYPD液体培养基；
⑤用无菌牙签往步骤④分别接入在含有0.5mg/mL G418 的YPD平板上获得的转化子，混匀，于30℃培养48h；
⑥48h后，取一块新的无菌96孔板，每孔加入190μL YPD 液体培养基。在相应孔中加入10μL第一块96孔板所得的培养物，于30℃培养24h；
⑦24h后，再取一块新的无菌96孔板，每孔加入190μL YPD液体培养基；在相应孔中加入10μL第二块96孔板所得的培养物，于30℃培养24h；
⑧24h后，分别从第三块96孔板中取出1μL分别点在含有1.0mg/mL和4mg/mL G418的YPD平板上，于30℃继续培养96h-120h。
5、毕赤酵母基因工程菌的发酵培养：将制备的毕赤酵母基因工程菌接种于YPD液体培养基中，30℃、220rpm过夜活化培养，而后转接于BMGY培养基，于30℃、220rpm培养该工程菌16-18h，至OD600＝2.0-6.0；室温下1500g离心5min，收集菌体；再用10mL的BMMY培养基重悬菌体，使OD600＝1.0左右，将所得的菌液置于100mL无菌三角瓶中，于28℃、220rpm下继续振荡培养；每24小时向培养基中添加甲醇至终浓度为1％进行诱导培养；
6、发酵结束后，分离纯化，得到重组胶原蛋白：
(1)用YPD培养基于30℃、220rpm过夜活化培养毕赤酵母工程菌；
(2)取上述菌液转接于BMGY培养基，于30℃、220rpm 培养该工程菌16-18h，至OD600＝2.0-6.0；
(3)室温下1500g离心5min，收集菌体，用10mL的BMMY 培养基重悬菌体，使OD600＝1.0左右，将所得的菌液置于100mL 无菌三角瓶中，于28℃、220rpm下继续振荡培养；
(4)每24小时向培养基中添加甲醇至终浓度为1％进行诱导培养；
(5)发酵培养结束后，4℃下3000×g离心20min，收集上清；
(6)上清中加入5-7倍体积的纯水，再经超滤浓缩至初始体积的20％；
(7)取1mL浓缩液中加入100μL 1×Ni-NTA结合缓冲液， 4℃充分混合；
(8)加入20μL 50％Ni-NTA His·Bands树脂悬液轻柔混匀，结合30min；
(9)15000×g离心10秒沉淀树脂，弃上清；
(10)用100μL 1×Ni-NTA漂洗缓冲液漂洗树脂，15000×g 离心10秒，小心吸去上清，再重复一次；
(11)用200μL 1×Ni-NTA洗脱缓冲液洗脱目的蛋白， 15000×g离心10秒，小心将上清转移至干净小管中，再重复2 次；
(12)收集上清，使用Strep结合缓冲液4℃透析过夜；
(13)取上述透析液加入100μL Strep-Tactin树脂悬液轻柔混匀，结合30min；
(14)15000×g离心10秒沉淀树脂，弃上清；
(15)用200μL 1×Strep-Tactin漂洗缓冲液漂洗树脂， 15000×g离心10秒，小心吸去上清，再重复一次；
(16)用200μL 1×Strep-Tactin洗脱缓冲液洗脱目的蛋白， 15000×g离心10秒，小心将上清转移至干净小管中，再重复2 次；
(17)将得到的洗脱液经脱盐、浓缩，所制得的浓缩液经真空冷冻干燥制得酵母重组胶原蛋白；
(18)取浓缩液倒入玻璃培养皿中，放入-20℃冰箱中冷冻过夜，然后转入预冷至-45℃的冷冻干燥机中，开启真空泵，维持48h；
(14)冻干结束后，小心打开放气阀，直至内外气压平衡；取出培养皿，得到白色酵母重组人源I型胶原α1链蛋白固体。
实验1：
在上述步骤3中，对筛选得到的阳性克隆以P1/P7为引物进行PCR检测，分析电泳所得条带大小是否与理论值3685bp 一致；实验结果如图2所示。
实验2：
在上述步骤6的步骤(5)中，取少量上清液进行SDS-PAGE 分析检测，实验结果如图3所示。
实验3：
对制备得到的重组人源I型胶原α1链蛋白进行鉴定和序列测定：
1、蛋白免疫印迹(Western blotting)，其步骤如下：取发酵上清液进行SDS-PAGE电泳，电泳直至溴酚蓝完全从分离胶下缘跑出；从玻璃板中取出凝胶，将其浸泡于转膜缓冲液中；取与凝胶大小一致的PVDF膜，于无水甲醇中浸泡10秒后，转移到转膜缓冲液中继续浸泡待用；按顺序在转移夹内放置预先经转膜缓冲液浸泡的海绵、滤纸、PVDF膜、凝胶、滤纸、海绵，保证每层之间没有气泡；将转移夹放入电转槽中，膜靠近正极，胶靠近负极，并将电转槽置于冰水浴中，450mA转移2h；取出 PVDF膜，将其浸泡在用TBS溶液配制的5％的脱脂奶粉中，室温震荡2h；弃去奶粉液，加入用5％奶粉液配制的一抗，室温震荡2h；倒出一抗溶液，以TBS溶液洗膜，洗三次，每次5min；将膜浸泡入用5％奶粉液配制的辣根过氧化物酶(HRP)标记的山羊抗鼠IgG抗体二抗溶液中，室温震荡2h；倒出二抗溶液，以TBS溶液洗膜，洗三次，每次5min；用滤纸吸干膜上残余水分，向膜上均匀涂上TMB显色液(Beyotime)，置于暗光处静置片刻；用滤纸吸除膜上残余水分，拍照保存；检测结果如图4 所示。
2、质谱分析鉴定，其步骤如下：先取蛋白样品进行 SDS-PAGE电泳分析，用考马斯亮蓝R-250染色，脱色后，用洁净的刀片沿目标蛋白条带走向以1mm宽度切下目标条带，送样由普泰生物进行鉴定；检测结果如图5所示。
实验4：
对制备得到的重组人源I型胶原α1链蛋白进行全长性检验测定：利用酵母重组人源I型胶原α1链蛋白两端携带的双亲和纯化标记，可以利用Strep tag II单克隆抗体和偶联HRP的6xHis 单克隆抗体进行夹心法ELISA检测，以定性或/和定量分析样品中的全长重组人源I型胶原α1链蛋白。
实验方法如下：
1)用结合缓冲液(1×PBS缓冲液)将小鼠抗Strep tag II 单克隆抗体稀释1000倍，向ELISA板中加入100μL/孔的抗体稀释液，抗体浓度为0.5μg/mL；
2)用塑料封口膜覆盖板子，并于37℃孵育2h，或者4℃孵育过夜；
3)倒置酶标板，甩出抗体溶液，每孔加入200μL的洗涤液 (添加0.05％吐温-20的PBS缓冲液)漂洗3次，每次震荡5min；
4)每孔加入200μL的封闭液(添加1％BSA的洗涤液)，用以封闭酶标板上的非特异性结合位点；
5)用塑料封口膜覆盖板子，并于37℃孵育2h，或者4℃孵育过夜；
6)弃封闭液，每孔加入200μL的洗涤液漂洗3次，每次震荡5min；
7)每孔加入100μL的经封闭液适当稀释的胶原蛋白样品；
8)用塑料封口膜覆盖板子，并于37℃孵育2h，或者4℃孵育过夜；
9)弃样品溶液，每孔加入200μL的洗涤液漂洗3次，每次震荡5min；
10)用封闭液将偶联HRP的6xHis单克隆抗体稀释1000 倍，每孔加入100μL的抗体稀释液；
11)用塑料封口膜覆盖板子，并于37℃孵育30min；
12)弃HRP-抗体溶液，每孔加入200μL的洗涤液漂洗3次，每次震荡5min；
13)用多通道移液器，向酶标板中加入100μL/孔的TMB 试剂，使其呈现足够深的颜色，约需10-15min；
14)每孔加入100μL的终止液(1mol/L的盐酸)，终止显色反应；
15)于10min内通过酶标仪测定450nm下各孔的吸光值，来检验样品中的全长酵母重组人源I性胶原α1链蛋白。
检测结果如图6所示，其对应实验数据如下。
实施例2：
现给出酵母重组人源I型胶原α1链蛋白用于外用美容护肤产品的实施例：
按以下质量配比，以纯化水为溶剂溶解各原料，并充分搅拌均匀即成一款无色无味透明的保湿护肤精华液；
其中甘油2％、丁二醇2％、透明质酸钠0.5％、重组人源I 型胶原α1链蛋白0.5％、维生素C乙基醚0.5％、维生素C磷酸酯钠0.5％、甘草酸二钾0.3％、EDTA·Na20.5％、丝肽0.5％、β- 葡聚糖0.3％、芦芭油0.3％、水溶性氮酮0.5％，其余为纯化水。
使用方法：早晚洁面后，直接涂抹于面部，轻轻拍打至完全吸收。
实施例3：
现给出重组人源I型胶原α1链蛋白用于外用美容护肤产品的实施例：
按以下质量配比，以纯化水为溶剂溶解各原料，并充分搅拌均匀即成一款无色无味透明的保湿护肤精华液；
其中甘油1％、丁二醇1％、透明质酸钠0.01％、重组人源I 型胶原α1链蛋白0.01％、维生素C乙基醚0.01％、维生素C磷酸酯钠0.01％、甘草酸二钾0.1％、EDTA·Na20.01％、丝肽0.01％、β-葡聚糖0.1％、芦芭油0.1％、水溶性氮酮0.1％，其余为纯化水。
使用方法：早晚洁面后，直接涂抹于面部，轻轻拍打至完全吸收。
实施例4：
现给出重组人源I型胶原α1链蛋白用于外用美容护肤产品的实施例：
按以下质量配比，以纯化水为溶剂溶解各原料，并充分搅拌均匀即成一款无色无味透明的保湿护肤精华液；
其中甘油3％、丁二醇3％、透明质酸钠0.1％、重组人源I 型胶原α1链蛋白0.1％、维生素C乙基醚0.1％、维生素C磷酸酯钠0.1％、甘草酸二钾0.5％、EDTA·Na20.1％、丝肽0.1％、β- 葡聚糖0.5％、芦芭油0.5％、水溶性氮酮1％，其余为纯化水。
使用方法：早晚洁面后，直接涂抹于面部，轻轻拍打至完全吸收。
实施例5：
现给出酵母重组人源I型胶原α1链蛋白在高档微创美容化妆品中的应用实施例：
用于高档微创美容的导入性产品美光针，其溶液由以纯化水为溶剂溶解各原料通过无菌方法制备而成，其步骤为：
①按照以下质量配比，精确称取各组分，并充分搅拌混匀、定容；其中酵母重组人源I型胶原α1链蛋白0.3％、甘油5％、透明质酸钠0.3％、小分子透明质酸钠0.03％、氯化钠0.9％，五胜肽0.01‰。
②用0.22μm微孔滤膜过滤除菌；
③无菌条件下，无菌分装，每支灌装3mL。
④使用时，直接取出配合导入仪器使用。
实施例6：
现给出酵母重组人源I型胶原α1链蛋白制备胶原蛋白凝胶敷料产品的应用实施例：
所述胶原蛋白凝胶以纯化水为溶剂，其组分还包括酵母重组人源I型胶原α1链蛋白、凝胶剂、保湿剂；所述凝胶剂为卡波姆；所述保湿剂为甘油和三乙醇胺；
各组分的质量配比为：酵母重组人源I型胶原α1链蛋白 0.1％、甘油1％、卡波姆0.1％、三乙醇胺0.1％，其余为纯化水。
制备步骤如下所示：
①准确称取甘油和卡波姆，搅拌1h，充分溶解；
②加入纯化水，并准确称取酵母重组人源I型胶原α1链蛋白，搅拌1h，充分溶解；
③加入三乙醇胺，继续搅拌1h，充分混匀，即得胶原蛋白凝胶敷料产品。
实施例7：
现给出酵母重组人源I型胶原α1链蛋白制备胶原蛋白凝胶敷料产品的应用实施例：
所述胶原蛋白凝胶以纯化水为溶剂，其组分还包括酵母重组人源I型胶原α1链蛋白、凝胶剂、保湿剂；所述凝胶剂为卡波姆；所述保湿剂为甘油和三乙醇胺；
各组分的质量配比为：酵母重组人源I型胶原α1链蛋白 0.5％、甘油10％、卡波姆4％、三乙醇胺10％，其余为纯化水。
制备步骤如下所示：
①准确称取甘油和卡波姆，搅拌1h，充分溶解；
②加入纯化水，并准确称取酵母重组人源I型胶原α1链蛋白，搅拌1h，充分溶解；
③加入三乙醇胺，继续搅拌1h，充分混匀，即得胶原蛋白凝胶敷料产品。
实施例8：
现给出酵母重组人源I型胶原α1链蛋白制备胶原蛋白凝胶敷料产品的应用实施例：
所述胶原蛋白凝胶以纯化水为溶剂，其组分还包括酵母重组人源I型胶原α1链蛋白、凝胶剂、保湿剂；所述凝胶剂为卡波姆；所述保湿剂为甘油和三乙醇胺；
各组分的质量配比为：酵母重组人源I型胶原α1链蛋白 0.3％、甘油5％，卡波姆2％、三乙醇胺5％，其余为纯化水。
制备步骤如下所示：
①准确称取甘油和卡波姆，搅拌1h，充分溶解；
②加入纯化水，并准确称取酵母重组人源I型胶原α1链蛋白，搅拌1h，充分溶解；
③加入三乙醇胺，继续搅拌1h，充分混匀，即得胶原蛋白凝胶敷料产品。
实施例9：
现给出酵母重组人源I型胶原α1链蛋白制备胶原蛋白贴敷料产品的应用实施例：
所述胶原蛋白贴敷料由酵母重组人源I型胶原α1链蛋白、保湿剂、医用增稠剂和无纺布基材组成；所述保湿剂为甘油；所述医用增稠剂为黄原胶；
其中各组分的质量配比为：酵母重组人源I型胶原α1链蛋白0.1％、甘油1％、黄原胶0.1％，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定量灌装到含无纺布基材的医用铝箔袋中，封口处理，辐照灭菌，即得胶原蛋白贴敷料产品。
实施例10：
现给出酵母重组人源I型胶原α1链蛋白制备皮肤黏膜保护剂敷料产品的应用实施例：
皮肤黏膜保护剂由以下质量配比的原料组成：酵母重组人源I型胶原α1链蛋白0.5％、透明质酸钠0.5％，甘油20％，其余为纯化水；所制得的制备液灭菌后，无菌灌装。
制备步骤如下所示：
①准确称取各组分，加入纯化水溶解，搅拌1h；
②准确称取酵母重组人源I型胶原α1链蛋白，补足纯化水，继续搅拌，充分溶解混匀；
③无菌条件下灌装，密封包装，辐照灭菌，即得胶原蛋白基皮肤黏膜保护剂敷料，应用于微整形后的皮肤黏膜保护等。
序列表：
SEQUENCE LISTING
〈110〉江苏悦智生物医药有限公司
〈120〉酵母重组人源I型胶原α1链蛋白
〈130〉UCSI1H
〈160〉10
〈170〉PatentIn version 3.5
SEQ ID NO.1酵母重组人源I型胶原α1链蛋白的氨基酸序列
Trp Ser His Pro Gln Phe Glu Lys Gln Leu Ser Tyr Gly Tyr Asp Glu
Lys Ser Thr Gly Gly Ile Ser Val Pro Gly Pro Met Gly Pro Ser Gly
Pro Arg Gly Leu Pro Gly Pro Pro Gly Ala Pro Gly Pro Gln Gly Phe
Gln Gly Pro Pro Gly Glu Pro Gly Glu Pro Gly Ala Ser Gly Pro Met
Gly Pro Arg Gly Pro Pro Gly Pro Pro Gly Lys Asn Gly Asp Asp Gly
Glu Ala Gly Lys Pro Gly Arg Pro Gly Glu Arg Gly Pro Pro Gly Pro
Gln Gly Ala Arg Gly Leu Pro Gly Thr Ala Gly Leu Pro Gly Met Lys
Gly His Arg Gly Phe Ser Gly Leu Asp Gly Ala Lys Gly Asp Ala Gly
Pro Ala Gly Pro Lys Gly Glu Pro Gly Ser Pro Gly Glu Asn Gly Ala
Pro Gly Gln Met Gly Pro Arg Gly Leu Pro Gly Glu Arg Gly Arg Pro
Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Asn Asp Gly Ala Thr Gly
Ala Ala Gly Pro Pro Gly Pro Thr Gly Pro Ala Gly Pro Pro Gly Phe
Pro Gly Ala Val Gly Ala Lys Gly Glu Ala Gly Pro Gln Gly Pro Arg
Gly Ser Glu Gly Pro Gln Gly Val Arg Gly Glu Pro Gly Pro Pro Gly
Pro Ala Gly Ala Ala Gly Pro Ala Gly Asn Pro Gly Ala Asp Gly Gln
Pro Gly Ala Lys Gly Ala Asn Gly Ala Pro Gly Ile Ala Gly Ala Pro
Gly Phe Pro Gly Ala Arg Gly Pro Ser Gly Pro Gln Gly Pro Gly Gly
Pro Pro Gly Pro Lys Gly Asn Ser Gly Glu Pro Gly Ala Pro Gly Ser
Lys Gly Asp Thr Gly Ala Lys Gly Glu Pro Gly Pro Val Gly Val Gln
Gly Pro Pro Gly Pro Ala Gly Glu Glu Gly Lys Arg Gly Ala Arg Gly
Glu Pro Gly Pro Thr Gly Leu Pro Gly Pro Pro Gly Glu Arg Gly Gly
Pro Gly Ser Arg Gly Phe Pro Gly Ala Asp Gly Val Ala Gly Pro Lys
Gly Pro Ala Gly Glu Arg Gly Ser Pro Gly Pro Ala Gly Pro Lys Gly
Ser Pro Gly Glu Ala Gly Arg Pro Gly Glu Ala Gly Leu Pro Gly Ala
Lys Gly Leu Thr Gly Ser Pro Gly Ser Pro Gly Pro Asp Gly Lys Thr
Gly Pro Pro Gly Pro Ala Gly Gln Asp Gly Arg Pro Gly Pro Pro Gly
Pro Pro Gly Ala Arg Gly Gln Ala Gly Val Met Gly Phe Pro Gly Pro
Lys Gly Ala Ala Gly Glu Pro Gly Lys Ala Gly Glu Arg Gly Val Pro
Gly Pro Pro Gly Ala Val Gly Pro Ala Gly Lys Asp Gly Glu Ala Gly
Ala Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu Arg Gly Glu
Gln Gly Pro Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro Gly Pro Ala
Gly Pro Pro Gly Glu Ala Gly Lys Pro Gly Glu Gln Gly Val Pro Gly
Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu Arg Gly Phe
Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Pro Arg
Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly Asp Ala Gly
Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln Gly Met
Pro Gly Glu Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly Asp Arg
Gly Asp Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Lys Asp Gly
Val Arg Gly Leu Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala
Pro Gly Asp Lys Gly Glu Ser Gly Pro Ser Gly Pro Ala Gly Pro Thr
Gly Ala Arg Gly Ala Pro Gly Asp Arg Gly Glu Pro Gly Pro Pro Gly
Pro Ala Gly Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro Gly Ala
Lys Gly Glu Pro Gly Asp Ala Gly Ala Lys Gly Asp Ala Gly Pro Pro
Gly Pro Ala Gly Pro Ala Gly Pro Pro Gly Pro Ile Gly Asn Val Gly
Ala Pro Gly Ala Lys Gly Ala Arg Gly Ser Ala Gly Pro Pro Gly Ala
Thr Gly Phe Pro Gly Ala Ala Gly Arg Val Gly Pro Pro Gly Pro Ser
Gly Asn Ala Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys Glu Gly
Gly Lys Gly Pro Arg Gly Glu Thr Gly Pro Ala Gly Arg Pro Gly Glu
Val Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Glu Lys Gly Ser Pro
Gly Ala Asp Gly Pro Ala Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly
Ile Ala Gly Gln Arg Gly Val Val Gly Leu Pro Gly Gln Arg Gly Glu
Arg Gly Phe Pro Gly Leu Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln
Gly Pro Ser Gly Ala Ser Gly Glu Arg Gly Pro Pro Gly Pro Met Gly
Pro Pro Gly Leu Ala Gly Pro Pro Gly Glu Ser Gly Arg Glu Gly Ala
Pro Gly Ala Glu Gly Ser Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys
Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly Pro Pro Gly Ala Pro Gly
Ala Pro Gly Ala Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp
Arg Gly Glu Thr Gly Pro Ala Gly Pro Ala Gly Pro Val Gly Pro Val
Gly Ala Arg Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly
Glu Thr Gly Glu Gln Gly Asp Arg Gly Ile Lys Gly His Arg Gly Phe
Ser Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Ser Pro Gly Glu Gln
Gly Pro Ser Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro Pro Gly
Ser Ala Gly Ala Pro Gly Lys Asp Gly Leu Asn Gly Leu Pro Gly Pro
Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val
Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser
Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala
His Asp Gly Gly Arg Tyr Tyr Arg Ala His His His His His His
SEQ ID NO.2酵母重组人源I型胶原α1链蛋白的基因DNA序列
TGGTCTCATCCACAATTTGAAAAGCAACTTAGTTATGGATACGATGAAAAAT CCACAGGTGGAATCAGTGTTCCTGGACCTATGGGTCCATCAGGTCCAAGAG GTTTACCAGGACCTCCAGGTGCCCCAGGTCCCCAGGGATTTCAAGGTCCAC CAGGAGAGCCTGGTGAGCCAGGAGCTTCTGGTCCAATGGGTCCCAGAGGA CCACCTGGTCCTCCAGGAAAGAATGGAGATGATGGTGAAGCTGGAAAACC TGGAAGACCTGGAGAAAGAGGACCACCAGGACCCCAGGGTGCCAGAGGA CTGCCAGGTACCGCAGGTCTGCCTGGAATGAAAGGTCATAGAGGATTTTCA GGATTAGACGGTGCAAAGGGAGACGCTGGACCTGCAGGACCAAAGGGTG AGCCAGGAAGTCCAGGAGAGAATGGTGCACCAGGACAGATGGGTCCAAG AGGACTGCCCGGTGAAAGAGGTAGACCCGGAGCACCAGGACCAGCAGGT GCAAGAGGAAATGATGGAGCTACAGGTGCTGCAGGACCCCCAGGTCCAAC AGGACCAGCCGGTCCTCCCGGTTTCCCAGGTGCCGTTGGAGCAAAAGGTG AAGCTGGTCCACAGGGTCCAAGAGGTTCTGAAGGTCCACAGGGAGTTAGA GGAGAACCAGGACCCCCTGGACCAGCTGGTGCAGCAGGACCAGCTGGTA ACCCTGGTGCTGACGGTCAGCCAGGTGCTAAGGGAGCAAATGGAGCACCA GGAATAGCTGGTGCCCCAGGATTTCCCGGTGCTAGAGGTCCAAGTGGTCCA CAAGGACCAGGAGGTCCACCCGGTCCCAAAGGAAACAGTGGAGAACCAG GTGCACCCGGTTCAAAGGGAGATACAGGAGCTAAAGGAGAGCCCGGTCCA GTGGGTGTTCAGGGACCACCCGGACCTGCTGGAGAGGAAGGTAAAAGAG GTGCAAGAGGTGAGCCAGGACCAACAGGTCTGCCTGGTCCCCCTGGTGAA AGAGGTGGTCCAGGTAGTAGAGGATTTCCAGGAGCTGATGGTGTTGCAGG ACCAAAGGGACCCGCAGGTGAGAGAGGATCACCCGGTCCAGCCGGACCA AAAGGATCACCAGGAGAAGCTGGTAGACCAGGAGAAGCTGGTCTGCCAG GTGCTAAAGGATTGACAGGATCACCCGGTTCACCTGGTCCTGATGGAAAGA CAGGACCTCCAGGTCCCGCTGGTCAGGACGGTAGACCAGGACCCCCAGGA CCCCCAGGTGCAAGAGGTCAGGCAGGTGTAATGGGTTTCCCCGGACCTAA AGGAGCAGCTGGAGAACCTGGTAAAGCTGGAGAGAGAGGAGTGCCTGGA CCCCCTGGAGCTGTTGGTCCAGCAGGAAAGGATGGTGAGGCAGGTGCACA AGGTCCACCTGGACCCGCTGGACCTGCAGGTGAGAGAGGAGAGCAAGGT CCCGCAGGTTCTCCAGGTTTTCAGGGTTTGCCAGGTCCAGCCGGTCCTCCT GGAGAGGCAGGAAAGCCAGGAGAACAAGGAGTTCCAGGAGACCTGGGTG CACCAGGACCCTCTGGTGCAAGAGGAGAGAGAGGATTTCCTGGAGAAAG AGGTGTGCAGGGACCACCAGGTCCCGCCGGTCCAAGAGGAGCAAATGGA GCCCCTGGAAATGACGGAGCTAAGGGTGACGCTGGTGCACCAGGAGCACC AGGTTCTCAAGGTGCTCCCGGATTGCAGGGTATGCCTGGAGAGAGAGGTG CAGCTGGACTGCCAGGTCCAAAAGGTGACAGAGGAGACGCCGGTCCTAA GGGAGCTGACGGTTCTCCTGGAAAGGACGGTGTGAGAGGTTTGACAGGAC CAATAGGTCCACCCGGTCCTGCTGGAGCCCCTGGAGACAAAGGTGAATCA GGTCCTTCCGGTCCAGCCGGACCAACAGGAGCAAGAGGAGCACCTGGAG ACAGAGGAGAGCCAGGTCCTCCAGGACCTGCAGGTTTCGCTGGTCCTCCC GGAGCAGATGGACAGCCAGGAGCTAAGGGAGAACCCGGTGACGCTGGTG CTAAGGGAGATGCAGGTCCACCAGGTCCTGCTGGTCCTGCTGGACCTCCCG GACCAATAGGTAATGTTGGAGCACCCGGAGCAAAAGGTGCCAGAGGTTCC GCAGGTCCTCCCGGAGCAACTGGTTTTCCAGGAGCTGCCGGAAGAGTGGG TCCACCTGGTCCTTCTGGAAATGCAGGACCACCAGGTCCTCCTGGTCCAGC CGGAAAGGAAGGTGGAAAGGGACCTAGAGGAGAAACAGGTCCCGCAGGT AGACCCGGTGAGGTGGGTCCACCTGGTCCACCCGGTCCAGCTGGTGAGAA AGGAAGTCCTGGAGCAGACGGACCAGCTGGTGCCCCTGGTACACCAGGAC CCCAAGGAATAGCTGGTCAAAGAGGTGTTGTTGGTTTACCAGGTCAGAGA GGAGAAAGAGGTTTTCCAGGATTACCAGGTCCCTCAGGTGAGCCCGGAAA ACAGGGTCCCTCAGGAGCAAGTGGTGAAAGAGGACCACCAGGACCAATG GGACCTCCAGGATTAGCTGGTCCACCAGGAGAATCAGGAAGAGAGGGTGC TCCTGGAGCAGAAGGTTCACCAGGAAGAGACGGTTCACCCGGAGCCAAG GGAGACAGAGGTGAAACAGGTCCCGCAGGTCCACCAGGAGCACCCGGAG CCCCTGGTGCTCCAGGACCTGTCGGACCAGCAGGAAAATCCGGTGACAGA GGTGAGACTGGACCCGCAGGTCCTGCTGGTCCTGTTGGACCAGTGGGTGC AAGAGGACCAGCAGGTCCACAAGGTCCAAGAGGTGACAAAGGTGAGACA GGTGAGCAGGGTGACAGAGGAATTAAAGGTCACAGAGGATTTTCAGGACT GCAGGGACCACCCGGTCCTCCCGGTTCCCCAGGAGAGCAAGGTCCATCCG GTGCATCCGGTCCAGCTGGACCCAGAGGACCACCTGGTTCTGCTGGTGCA CCAGGTAAAGATGGATTGAACGGTTTGCCTGGTCCAATAGGACCTCCTGGT CCAAGAGGAAGAACTGGTGACGCCGGTCCCGTCGGACCACCCGGTCCACC AGGTCCCCCAGGTCCACCCGGACCACCATCCGCAGGATTTGATTTCTCATT CCTTCCTCAACCTCCTCAAGAGAAAGCACATGATGGAGGTAGATACTATAGAGCCCATCACCACCATCATCATTAA
SEQ ID NO.3人源I型胶原蛋白α1链成熟肽的DNA序列
CAACTTAGTTATGGATACGATGAAAAATCCACAGGTGGAATCAGTGTTCCT GGACCTATGGGTCCATCAGGTCCAAGAGGTTTACCAGGACCTCCAGGTGCC CCAGGTCCCCAGGGATTTCAAGGTCCACCAGGAGAGCCTGGTGAGCCAGG AGCTTCTGGTCCAATGGGTCCCAGAGGACCACCTGGTCCTCCAGGAAAGA ATGGAGATGATGGTGAAGCTGGAAAACCTGGAAGACCTGGAGAAAGAGG ACCACCAGGACCCCAGGGTGCCAGAGGACTGCCAGGTACCGCAGGTCTGC CTGGAATGAAAGGTCATAGAGGATTTTCAGGATTAGACGGTGCAAAGGGA GACGCTGGACCTGCAGGACCAAAGGGTGAGCCAGGAAGTCCAGGAGAGA ATGGTGCACCAGGACAGATGGGTCCAAGAGGACTGCCCGGTGAAAGAGGT AGACCCGGAGCACCAGGACCAGCAGGTGCAAGAGGAAATGATGGAGCTA CAGGTGCTGCAGGACCCCCAGGTCCAACAGGACCAGCCGGTCCTCCCGGT TTCCCAGGTGCCGTTGGAGCAAAAGGTGAAGCTGGTCCACAGGGTCCAAG AGGTTCTGAAGGTCCACAGGGAGTTAGAGGAGAACCAGGACCCCCTGGAC CAGCTGGTGCAGCAGGACCAGCTGGTAACCCTGGTGCTGACGGTCAGCCA GGTGCTAAGGGAGCAAATGGAGCACCAGGAATAGCTGGTGCCCCAGGATT TCCCGGTGCTAGAGGTCCAAGTGGTCCACAAGGACCAGGAGGTCCACCCG GTCCCAAAGGAAACAGTGGAGAACCAGGTGCACCCGGTTCAAAGGGAGA TACAGGAGCTAAAGGAGAGCCCGGTCCAGTGGGTGTTCAGGGACCACCCG GACCTGCTGGAGAGGAAGGTAAAAGAGGTGCAAGAGGTGAGCCAGGACC AACAGGTCTGCCTGGTCCCCCTGGTGAAAGAGGTGGTCCAGGTAGTAGAG GATTTCCAGGAGCTGATGGTGTTGCAGGACCAAAGGGACCCGCAGGTGAG AGAGGATCACCCGGTCCAGCCGGACCAAAAGGATCACCAGGAGAAGCTG GTAGACCAGGAGAAGCTGGTCTGCCAGGTGCTAAAGGATTGACAGGATCA CCCGGTTCACCTGGTCCTGATGGAAAGACAGGACCTCCAGGTCCCGCTGG TCAGGACGGTAGACCAGGACCCCCAGGACCCCCAGGTGCAAGAGGTCAG GCAGGTGTAATGGGTTTCCCCGGACCTAAAGGAGCAGCTGGAGAACCTGG TAAAGCTGGAGAGAGAGGAGTGCCTGGACCCCCTGGAGCTGTTGGTCCAG CAGGAAAGGATGGTGAGGCAGGTGCACAAGGTCCACCTGGACCCGCTGG ACCTGCAGGTGAGAGAGGAGAGCAAGGTCCCGCAGGTTCTCCAGGTTTTC AGGGTTTGCCAGGTCCAGCCGGTCCTCCTGGAGAGGCAGGAAAGCCAGG AGAACAAGGAGTTCCAGGAGACCTGGGTGCACCAGGACCCTCTGGTGCA AGAGGAGAGAGAGGATTTCCTGGAGAAAGAGGTGTGCAGGGACCACCAG GTCCCGCCGGTCCAAGAGGAGCAAATGGAGCCCCTGGAAATGACGGAGCT AAGGGTGACGCTGGTGCACCAGGAGCACCAGGTTCTCAAGGTGCTCCCGG ATTGCAGGGTATGCCTGGAGAGAGAGGTGCAGCTGGACTGCCAGGTCCAA AAGGTGACAGAGGAGACGCCGGTCCTAAGGGAGCTGACGGTTCTCCTGGA AAGGACGGTGTGAGAGGTTTGACAGGACCAATAGGTCCACCCGGTCCTGC TGGAGCCCCTGGAGACAAAGGTGAATCAGGTCCTTCCGGTCCAGCCGGAC CAACAGGAGCAAGAGGAGCACCTGGAGACAGAGGAGAGCCAGGTCCTCC AGGACCTGCAGGTTTCGCTGGTCCTCCCGGAGCAGATGGACAGCCAGGAG CTAAGGGAGAACCCGGTGACGCTGGTGCTAAGGGAGATGCAGGTCCACCA GGTCCTGCTGGTCCTGCTGGACCTCCCGGACCAATAGGTAATGTTGGAGCA CCCGGAGCAAAAGGTGCCAGAGGTTCCGCAGGTCCTCCCGGAGCAACTGG TTTTCCAGGAGCTGCCGGAAGAGTGGGTCCACCTGGTCCTTCTGGAAATGC AGGACCACCAGGTCCTCCTGGTCCAGCCGGAAAGGAAGGTGGAAAGGGA CCTAGAGGAGAAACAGGTCCCGCAGGTAGACCCGGTGAGGTGGGTCCACC TGGTCCACCCGGTCCAGCTGGTGAGAAAGGAAGTCCTGGAGCAGACGGA CCAGCTGGTGCCCCTGGTACACCAGGACCCCAAGGAATAGCTGGTCAAAG AGGTGTTGTTGGTTTACCAGGTCAGAGAGGAGAAAGAGGTTTTCCAGGAT TACCAGGTCCCTCAGGTGAGCCCGGAAAACAGGGTCCCTCAGGAGCAAGT GGTGAAAGAGGACCACCAGGACCAATGGGACCTCCAGGATTAGCTGGTCC ACCAGGAGAATCAGGAAGAGAGGGTGCTCCTGGAGCAGAAGGTTCACCA GGAAGAGACGGTTCACCCGGAGCCAAGGGAGACAGAGGTGAAACAGGTC CCGCAGGTCCACCAGGAGCACCCGGAGCCCCTGGTGCTCCAGGACCTGTC GGACCAGCAGGAAAATCCGGTGACAGAGGTGAGACTGGACCCGCAGGTC CTGCTGGTCCTGTTGGACCAGTGGGTGCAAGAGGACCAGCAGGTCCACAA GGTCCAAGAGGTGACAAAGGTGAGACAGGTGAGCAGGGTGACAGAGGAA TTAAAGGTCACAGAGGATTTTCAGGACTGCAGGGACCACCCGGTCCTCCC GGTTCCCCAGGAGAGCAAGGTCCATCCGGTGCATCCGGTCCAGCTGGACC CAGAGGACCACCTGGTTCTGCTGGTGCACCAGGTAAAGATGGATTGAACG GTTTGCCTGGTCCAATAGGACCTCCTGGTCCAAGAGGAAGAACTGGTGAC GCCGGTCCCGTCGGACCACCCGGTCCACCAGGTCCCCCAGGTCCACCCGG ACCACCATCCGCAGGATTTGATTTCTCATTCCTTCCTCAACCTCCTCAAGAG AAAGCACATGATGGAGGTAGATACTATAGAGCC
SEQ ID NO.4引物DNA序列P1
GACTGGTTCCAATTGACAAGC
SEQ ID NO.5引物DNA序列P2
AGGGAATTCTACGTAGGGCCCCTTTTCAAATTGTGGATGAGACCATCTTTTC TCGAGAGATACCCC
SEQ ID NO.6引物DNA序列P3
GGTCTCATCCACAATTTGAAAAGCAACTTAGTTATGGATACGATGA
SEQ ID NO.7引物DNA序列P4
GGCTCTATAGTATCTACCTC
SEQ ID NO.8引物DNA序列P5
GGAGGTAGATACTATAGAGCCCATCACCACCATCATCATTAAC
SEQ ID NO.9引物DNA序列P6
AATTAATTCGCGGCCGCCCTAGGTTAATGATGATGGTG
SEQ ID NO.10引物DNA序列P7
GCAAATGGCATTCTGACATCC。
序列表
<110> 江苏悦智生物医药有限公司
<120> 酵母重组人源I型胶原α1链蛋白、合成方法及其应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1071
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Trp Ser His Pro Gln Phe Glu Lys Gln Leu Ser Tyr Gly Tyr Asp Glu
1 5 10 15
Lys Ser Thr Gly Gly Ile Ser Val Pro Gly Pro Met Gly Pro Ser Gly
20 25 30
Pro Arg Gly Leu Pro Gly Pro Pro Gly Ala Pro Gly Pro Gln Gly Phe
35 40 45
Gln Gly Pro Pro Gly Glu Pro Gly Glu Pro Gly Ala Ser Gly Pro Met
50 55 60
Gly Pro Arg Gly Pro Pro Gly Pro Pro Gly Lys Asn Gly Asp Asp Gly
65 70 75 80
Glu Ala Gly Lys Pro Gly Arg Pro Gly Glu Arg Gly Pro Pro Gly Pro
85 90 95
Gln Gly Ala Arg Gly Leu Pro Gly Thr Ala Gly Leu Pro Gly Met Lys
100 105 110
Gly His Arg Gly Phe Ser Gly Leu Asp Gly Ala Lys Gly Asp Ala Gly
115 120 125
Pro Ala Gly Pro Lys Gly Glu Pro Gly Ser Pro Gly Glu Asn Gly Ala
130 135 140
Pro Gly Gln Met Gly Pro Arg Gly Leu Pro Gly Glu Arg Gly Arg Pro
145 150 155 160
Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Asn Asp Gly Ala Thr Gly
165 170 175
Ala Ala Gly Pro Pro Gly Pro Thr Gly Pro Ala Gly Pro Pro Gly Phe
180 185 190
Pro Gly Ala Val Gly Ala Lys Gly Glu Ala Gly Pro Gln Gly Pro Arg
195 200 205
Gly Ser Glu Gly Pro Gln Gly Val Arg Gly Glu Pro Gly Pro Pro Gly
210 215 220
Pro Ala Gly Ala Ala Gly Pro Ala Gly Asn Pro Gly Ala Asp Gly Gln
225 230 235 240
Pro Gly Ala Lys Gly Ala Asn Gly Ala Pro Gly Ile Ala Gly Ala Pro
245 250 255
Gly Phe Pro Gly Ala Arg Gly Pro Ser Gly Pro Gln Gly Pro Gly Gly
260 265 270
Pro Pro Gly Pro Lys Gly Asn Ser Gly Glu Pro Gly Ala Pro Gly Ser
275 280 285
Lys Gly Asp Thr Gly Ala Lys Gly Glu Pro Gly Pro Val Gly Val Gln
290 295 300
Gly Pro Pro Gly Pro Ala Gly Glu Glu Gly Lys Arg Gly Ala Arg Gly
305 310 315 320
Glu Pro Gly Pro Thr Gly Leu Pro Gly Pro Pro Gly Glu Arg Gly Gly
325 330 335
Pro Gly Ser Arg Gly Phe Pro Gly Ala Asp Gly Val Ala Gly Pro Lys
340 345 350
Gly Pro Ala Gly Glu Arg Gly Ser Pro Gly Pro Ala Gly Pro Lys Gly
355 360 365
Ser Pro Gly Glu Ala Gly Arg Pro Gly Glu Ala Gly Leu Pro Gly Ala
370 375 380
Lys Gly Leu Thr Gly Ser Pro Gly Ser Pro Gly Pro Asp Gly Lys Thr
385 390 395 400
Gly Pro Pro Gly Pro Ala Gly Gln Asp Gly Arg Pro Gly Pro Pro Gly
405 410 415
Pro Pro Gly Ala Arg Gly Gln Ala Gly Val Met Gly Phe Pro Gly Pro
420 425 430
Lys Gly Ala Ala Gly Glu Pro Gly Lys Ala Gly Glu Arg Gly Val Pro
435 440 445
Gly Pro Pro Gly Ala Val Gly Pro Ala Gly Lys Asp Gly Glu Ala Gly
450 455 460
Ala Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu Arg Gly Glu
465 470 475 480
Gln Gly Pro Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro Gly Pro Ala
485 490 495
Gly Pro Pro Gly Glu Ala Gly Lys Pro Gly Glu Gln Gly Val Pro Gly
500 505 510
Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu Arg Gly Phe
515 520 525
Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Pro Arg
530 535 540
Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly Asp Ala Gly
545 550 555 560
Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln Gly Met
565 570 575
Pro Gly Glu Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly Asp Arg
580 585 590
Gly Asp Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Lys Asp Gly
595 600 605
Val Arg Gly Leu Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly Ala
610 615 620
Pro Gly Asp Lys Gly Glu Ser Gly Pro Ser Gly Pro Ala Gly Pro Thr
625 630 635 640
Gly Ala Arg Gly Ala Pro Gly Asp Arg Gly Glu Pro Gly Pro Pro Gly
645 650 655
Pro Ala Gly Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro Gly Ala
660 665 670
Lys Gly Glu Pro Gly Asp Ala Gly Ala Lys Gly Asp Ala Gly Pro Pro
675 680 685
Gly Pro Ala Gly Pro Ala Gly Pro Pro Gly Pro Ile Gly Asn Val Gly
690 695 700
Ala Pro Gly Ala Lys Gly Ala Arg Gly Ser Ala Gly Pro Pro Gly Ala
705 710 715 720
Thr Gly Phe Pro Gly Ala Ala Gly Arg Val Gly Pro Pro Gly Pro Ser
725 730 735
Gly Asn Ala Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys Glu Gly
740 745 750
Gly Lys Gly Pro Arg Gly Glu Thr Gly Pro Ala Gly Arg Pro Gly Glu
755 760 765
Val Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Glu Lys Gly Ser Pro
770 775 780
Gly Ala Asp Gly Pro Ala Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly
785 790 795 800
Ile Ala Gly Gln Arg Gly Val Val Gly Leu Pro Gly Gln Arg Gly Glu
805 810 815
Arg Gly Phe Pro Gly Leu Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln
820 825 830
Gly Pro Ser Gly Ala Ser Gly Glu Arg Gly Pro Pro Gly Pro Met Gly
835 840 845
Pro Pro Gly Leu Ala Gly Pro Pro Gly Glu Ser Gly Arg Glu Gly Ala
850 855 860
Pro Gly Ala Glu Gly Ser Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys
865 870 875 880
Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly Pro Pro Gly Ala Pro Gly
885 890 895
Ala Pro Gly Ala Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp
900 905 910
Arg Gly Glu Thr Gly Pro Ala Gly Pro Ala Gly Pro Val Gly Pro Val
915 920 925
Gly Ala Arg Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly
930 935 940
Glu Thr Gly Glu Gln Gly Asp Arg Gly Ile Lys Gly His Arg Gly Phe
945 950 955 960
Ser Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Ser Pro Gly Glu Gln
965 970 975
Gly Pro Ser Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro Pro Gly
980 985 990
Ser Ala Gly Ala Pro Gly Lys Asp Gly Leu Asn Gly Leu Pro Gly Pro
995 1000 1005
Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val
1010 1015 1020
Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser
1025 1030 1035 1040
Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala
1045 1050 1055
His Asp Gly Gly Arg Tyr Tyr Arg Ala His His His His His His
1060 1065 1070
<210> 2
<211> 3216
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tggtctcatc cacaatttga aaagcaactt agttatggat acgatgaaaa atccacaggt 60
ggaatcagtg ttcctggacc tatgggtcca tcaggtccaa gaggtttacc aggacctcca 120
ggtgccccag gtccccaggg atttcaaggt ccaccaggag agcctggtga gccaggagct 180
tctggtccaa tgggtcccag aggaccacct ggtcctccag gaaagaatgg agatgatggt 240
gaagctggaa aacctggaag acctggagaa agaggaccac caggacccca gggtgccaga 300
ggactgccag gtaccgcagg tctgcctgga atgaaaggtc atagaggatt ttcaggatta 360
gacggtgcaa agggagacgc tggacctgca ggaccaaagg gtgagccagg aagtccagga 420
gagaatggtg caccaggaca gatgggtcca agaggactgc ccggtgaaag aggtagaccc 480
ggagcaccag gaccagcagg tgcaagagga aatgatggag ctacaggtgc tgcaggaccc 540
ccaggtccaa caggaccagc cggtcctccc ggtttcccag gtgccgttgg agcaaaaggt 600
gaagctggtc cacagggtcc aagaggttct gaaggtccac agggagttag aggagaacca 660
ggaccccctg gaccagctgg tgcagcagga ccagctggta accctggtgc tgacggtcag 720
ccaggtgcta agggagcaaa tggagcacca ggaatagctg gtgccccagg atttcccggt 780
gctagaggtc caagtggtcc acaaggacca ggaggtccac ccggtcccaa aggaaacagt 840
ggagaaccag gtgcacccgg ttcaaaggga gatacaggag ctaaaggaga gcccggtcca 900
gtgggtgttc agggaccacc cggacctgct ggagaggaag gtaaaagagg tgcaagaggt 960
gagccaggac caacaggtct gcctggtccc cctggtgaaa gaggtggtcc aggtagtaga 1020
ggatttccag gagctgatgg tgttgcagga ccaaagggac ccgcaggtga gagaggatca 1080
cccggtccag ccggaccaaa aggatcacca ggagaagctg gtagaccagg agaagctggt 1140
ctgccaggtg ctaaaggatt gacaggatca cccggttcac ctggtcctga tggaaagaca 1200
ggacctccag gtcccgctgg tcaggacggt agaccaggac ccccaggacc cccaggtgca 1260
agaggtcagg caggtgtaat gggtttcccc ggacctaaag gagcagctgg agaacctggt 1320
aaagctggag agagaggagt gcctggaccc cctggagctg ttggtccagc aggaaaggat 1380
ggtgaggcag gtgcacaagg tccacctgga cccgctggac ctgcaggtga gagaggagag 1440
caaggtcccg caggttctcc aggttttcag ggtttgccag gtccagccgg tcctcctgga 1500
gaggcaggaa agccaggaga acaaggagtt ccaggagacc tgggtgcacc aggaccctct 1560
ggtgcaagag gagagagagg atttcctgga gaaagaggtg tgcagggacc accaggtccc 1620
gccggtccaa gaggagcaaa tggagcccct ggaaatgacg gagctaaggg tgacgctggt 1680
gcaccaggag caccaggttc tcaaggtgct cccggattgc agggtatgcc tggagagaga 1740
ggtgcagctg gactgccagg tccaaaaggt gacagaggag acgccggtcc taagggagct 1800
gacggttctc ctggaaagga cggtgtgaga ggtttgacag gaccaatagg tccacccggt 1860
cctgctggag cccctggaga caaaggtgaa tcaggtcctt ccggtccagc cggaccaaca 1920
ggagcaagag gagcacctgg agacagagga gagccaggtc ctccaggacc tgcaggtttc 1980
gctggtcctc ccggagcaga tggacagcca ggagctaagg gagaacccgg tgacgctggt 2040
gctaagggag atgcaggtcc accaggtcct gctggtcctg ctggacctcc cggaccaata 2100
ggtaatgttg gagcacccgg agcaaaaggt gccagaggtt ccgcaggtcc tcccggagca 2160
actggttttc caggagctgc cggaagagtg ggtccacctg gtccttctgg aaatgcagga 2220
ccaccaggtc ctcctggtcc agccggaaag gaaggtggaa agggacctag aggagaaaca 2280
ggtcccgcag gtagacccgg tgaggtgggt ccacctggtc cacccggtcc agctggtgag 2340
aaaggaagtc ctggagcaga cggaccagct ggtgcccctg gtacaccagg accccaagga 2400
atagctggtc aaagaggtgt tgttggttta ccaggtcaga gaggagaaag aggttttcca 2460
ggattaccag gtccctcagg tgagcccgga aaacagggtc cctcaggagc aagtggtgaa 2520
agaggaccac caggaccaat gggacctcca ggattagctg gtccaccagg agaatcagga 2580
agagagggtg ctcctggagc agaaggttca ccaggaagag acggttcacc cggagccaag 2640
ggagacagag gtgaaacagg tcccgcaggt ccaccaggag cacccggagc ccctggtgct 2700
ccaggacctg tcggaccagc aggaaaatcc ggtgacagag gtgagactgg acccgcaggt 2760
cctgctggtc ctgttggacc agtgggtgca agaggaccag caggtccaca aggtccaaga 2820
ggtgacaaag gtgagacagg tgagcagggt gacagaggaa ttaaaggtca cagaggattt 2880
tcaggactgc agggaccacc cggtcctccc ggttccccag gagagcaagg tccatccggt 2940
gcatccggtc cagctggacc cagaggacca cctggttctg ctggtgcacc aggtaaagat 3000
ggattgaacg gtttgcctgg tccaatagga cctcctggtc caagaggaag aactggtgac 3060
gccggtcccg tcggaccacc cggtccacca ggtcccccag gtccacccgg accaccatcc 3120
gcaggatttg atttctcatt ccttcctcaa cctcctcaag agaaagcaca tgatggaggt 3180
agatactata gagcccatca ccaccatcat cattaa 3216
<210> 3
<211> 3171
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
caacttagtt atggatacga tgaaaaatcc acaggtggaa tcagtgttcc tggacctatg 60
ggtccatcag gtccaagagg tttaccagga cctccaggtg ccccaggtcc ccagggattt 120
caaggtccac caggagagcc tggtgagcca ggagcttctg gtccaatggg tcccagagga 180
ccacctggtc ctccaggaaa gaatggagat gatggtgaag ctggaaaacc tggaagacct 240
ggagaaagag gaccaccagg accccagggt gccagaggac tgccaggtac cgcaggtctg 300
cctggaatga aaggtcatag aggattttca ggattagacg gtgcaaaggg agacgctgga 360
cctgcaggac caaagggtga gccaggaagt ccaggagaga atggtgcacc aggacagatg 420
ggtccaagag gactgcccgg tgaaagaggt agacccggag caccaggacc agcaggtgca 480
agaggaaatg atggagctac aggtgctgca ggacccccag gtccaacagg accagccggt 540
cctcccggtt tcccaggtgc cgttggagca aaaggtgaag ctggtccaca gggtccaaga 600
ggttctgaag gtccacaggg agttagagga gaaccaggac cccctggacc agctggtgca 660
gcaggaccag ctggtaaccc tggtgctgac ggtcagccag gtgctaaggg agcaaatgga 720
gcaccaggaa tagctggtgc cccaggattt cccggtgcta gaggtccaag tggtccacaa 780
ggaccaggag gtccacccgg tcccaaagga aacagtggag aaccaggtgc acccggttca 840
aagggagata caggagctaa aggagagccc ggtccagtgg gtgttcaggg accacccgga 900
cctgctggag aggaaggtaa aagaggtgca agaggtgagc caggaccaac aggtctgcct 960
ggtccccctg gtgaaagagg tggtccaggt agtagaggat ttccaggagc tgatggtgtt 1020
gcaggaccaa agggacccgc aggtgagaga ggatcacccg gtccagccgg accaaaagga 1080
tcaccaggag aagctggtag accaggagaa gctggtctgc caggtgctaa aggattgaca 1140
ggatcacccg gttcacctgg tcctgatgga aagacaggac ctccaggtcc cgctggtcag 1200
gacggtagac caggaccccc aggaccccca ggtgcaagag gtcaggcagg tgtaatgggt 1260
ttccccggac ctaaaggagc agctggagaa cctggtaaag ctggagagag aggagtgcct 1320
ggaccccctg gagctgttgg tccagcagga aaggatggtg aggcaggtgc acaaggtcca 1380
cctggacccg ctggacctgc aggtgagaga ggagagcaag gtcccgcagg ttctccaggt 1440
tttcagggtt tgccaggtcc agccggtcct cctggagagg caggaaagcc aggagaacaa 1500
ggagttccag gagacctggg tgcaccagga ccctctggtg caagaggaga gagaggattt 1560
cctggagaaa gaggtgtgca gggaccacca ggtcccgccg gtccaagagg agcaaatgga 1620
gcccctggaa atgacggagc taagggtgac gctggtgcac caggagcacc aggttctcaa 1680
ggtgctcccg gattgcaggg tatgcctgga gagagaggtg cagctggact gccaggtcca 1740
aaaggtgaca gaggagacgc cggtcctaag ggagctgacg gttctcctgg aaaggacggt 1800
gtgagaggtt tgacaggacc aataggtcca cccggtcctg ctggagcccc tggagacaaa 1860
ggtgaatcag gtccttccgg tccagccgga ccaacaggag caagaggagc acctggagac 1920
agaggagagc caggtcctcc aggacctgca ggtttcgctg gtcctcccgg agcagatgga 1980
cagccaggag ctaagggaga acccggtgac gctggtgcta agggagatgc aggtccacca 2040
ggtcctgctg gtcctgctgg acctcccgga ccaataggta atgttggagc acccggagca 2100
aaaggtgcca gaggttccgc aggtcctccc ggagcaactg gttttccagg agctgccgga 2160
agagtgggtc cacctggtcc ttctggaaat gcaggaccac caggtcctcc tggtccagcc 2220
ggaaaggaag gtggaaaggg acctagagga gaaacaggtc ccgcaggtag acccggtgag 2280
gtgggtccac ctggtccacc cggtccagct ggtgagaaag gaagtcctgg agcagacgga 2340
ccagctggtg cccctggtac accaggaccc caaggaatag ctggtcaaag aggtgttgtt 2400
ggtttaccag gtcagagagg agaaagaggt tttccaggat taccaggtcc ctcaggtgag 2460
cccggaaaac agggtccctc aggagcaagt ggtgaaagag gaccaccagg accaatggga 2520
cctccaggat tagctggtcc accaggagaa tcaggaagag agggtgctcc tggagcagaa 2580
ggttcaccag gaagagacgg ttcacccgga gccaagggag acagaggtga aacaggtccc 2640
gcaggtccac caggagcacc cggagcccct ggtgctccag gacctgtcgg accagcagga 2700
aaatccggtg acagaggtga gactggaccc gcaggtcctg ctggtcctgt tggaccagtg 2760
ggtgcaagag gaccagcagg tccacaaggt ccaagaggtg acaaaggtga gacaggtgag 2820
cagggtgaca gaggaattaa aggtcacaga ggattttcag gactgcaggg accacccggt 2880
cctcccggtt ccccaggaga gcaaggtcca tccggtgcat ccggtccagc tggacccaga 2940
ggaccacctg gttctgctgg tgcaccaggt aaagatggat tgaacggttt gcctggtcca 3000
ataggacctc ctggtccaag aggaagaact ggtgacgccg gtcccgtcgg accacccggt 3060
ccaccaggtc ccccaggtcc acccggacca ccatccgcag gatttgattt ctcattcctt 3120
cctcaacctc ctcaagagaa agcacatgat ggaggtagat actatagagc c 3171
<210> 4
<211> 21
<212> DNA
<213> Pichia pastoris
<400> 4
gactggttcc aattgacaag c 21
<210> 5
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
agggaattct acgtagggcc ccttttcaaa ttgtggatga gaccatcttt tctcgagaga 60
tacccc 66
<210> 6
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggtctcatcc acaatttgaa aagcaactta gttatggata cgatga 46
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggctctatag tatctacctc 20
<210> 8
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggaggtagat actatagagc ccatcaccac catcatcatt aac 43
<210> 9
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aattaattcg cggccgccct aggttaatga tgatggtg 38
<210> 10
<211> 21
<212> DNA
<213> Pichia pastoris
<400> 10
gcaaatggca ttctgacatc c 21
Claims (9)
1.一种酵母重组人源I型胶原α1链蛋白，其特征在于：所述重组人源I型胶原α1链蛋白的氨基酸序列如SEQ ID NO.1所示；所述重组人源I型胶原α1链蛋白包括N端的Strep-TagII序列、人源I型胶原蛋白α1链成熟肽序列和C端的6×His序列，使其含有双特异性亲和纯化标记；全长为1071个氨基酸。
2.根据权利要求1所述的酵母重组人源I型胶原α1链蛋白，其特征在于：所述重组人源I型胶原α1链蛋白的基因表达序列如SEQ ID NO.2所示。
3.根据权利要求2所述的酵母重组人源I型胶原α1链蛋白，其特征在于：所述人源I型胶原蛋白α1链成熟肽的基因序列是经利用宿主惯用密码子将天然胶原蛋白基因序列优化后得到的，基因序列如SEQ ID NO.3所示。
4.一种酵母重组人源I型胶原α1链蛋白的合成方法，其特征在于：包括以下步骤：
1)合成人源I型胶原蛋白α1链成熟肽的基因序列；
2)制备含有Strep-TagII编码序列的改造质粒pPIC9ks；
3)双酶切改造质粒pPIC9ks，构建含有重组人源I型胶原α1链蛋白基因的重组质粒pPIC9k-colIα1；
4)将重组质粒pPIC9k-colIα1用限制性内切酶SalⅠ线性化，并将其电转化入毕赤酵母SMD1168感受态细胞中，以营养缺陷型标记和G418抗性标记筛选高拷贝阳性重组子，得到毕赤酵母基因工程菌；
5)毕赤酵母基因工程菌的发酵培养；
6)发酵结束后，离心纯化，得到所述重组人源I型胶原α1链蛋白。
5.根据权利要求4所述的酵母重组人源I型胶原α1链蛋白的合成方法，其特征在于：包括以下步骤：
1)合成人源I型胶原蛋白α1链成熟肽的基因序列：在人源I型胶原蛋白α1链成熟肽的氨基酸序列不变条件下，将人I型胶原α1链基因序列中对应的毕赤酵母不常用的密码子优化为毕赤酵母偏爱性密码子；再通过全基因合成人源I型胶原蛋白α1链成熟肽的基因序列；
2)制备含有Strep-TagII编码序列的改造质粒pPIC9ks；
a)首先以pPIC9k质粒为模板，利用引物P1、P2进行PCR扩增，产物长度为394bp；其中P1的基因序列如SEQ ID NO.4所示，P2的基因序列如SEQ ID NO.5所示；
b)对获得的PCR产物和pPIC9k质粒分别做EcoRI和BamHI双酶切，并经T4 DNA连接酶作用下，16℃连接过夜，得到改造质粒pPIC9ks；
3)构建含有重组人源I型胶原α1链蛋白基因的重组质粒pPIC9k-colIα1：
a)以步骤1)中合成的人源I型胶原蛋白α1链成熟肽的基因序列为模板，利用引物P3、P4进行PCR扩增，得到长度为3194bp的基因片段；其中P3的基因序列如SEQ ID NO.6所示，P4的基因序列如SEQ ID NO.7所示；
b)对步骤2)中得到的改造质粒pPIC9ks做ApaI和NotI双酶切，获得载体片段；
c)以P5、P6为引物，以自身为模板，通过PCR相互扩增，得到含有6×His编码序列的基因片段，长度为65bp；其中P5的基因序列如SEQ ID NO.8所示，P6的基因序列如SEQ ID NO.9所示；
d)将步骤a)、步骤b)、步骤c)中的各片段混合，利用即用型无缝克隆试剂盒连接，50℃连接1h，得到含有Strep-TagII编码序列、人源I型胶原蛋白α1链成熟肽基因序列、6×His编码序列的重组质粒pPIC9k-colIα1；
4)将重组质粒pPIC9k-colIα1用限制性内切酶SalⅠ于37℃线性化，并将其电转化入毕赤酵母SMD1168感受态细胞中，以组氨酸缺陷型标记和G418抗性标记筛选高拷贝阳性重组子，得到毕赤酵母基因工程菌；
5)毕赤酵母基因工程菌的发酵培养：将步骤4)中制备的毕赤酵母基因工程菌接种于YPD液体培养基中，30℃、220rpm过夜活化培养，而后转接于BMGY培养基，于30℃、220rpm培养该工程菌16-18h，至OD600＝2.0-6.0；室温下1500g离心5min，收集菌体；再用10mL的BMMY培养基重悬菌体，使OD600＝1.0左右，将所得的菌液置于100mL无菌三角瓶中，于28℃、220rpm下继续振荡培养；每24小时向培养基中添加甲醇至终浓度为1％进行诱导培养；
6)发酵结束后，取发酵液离心除去酵母细胞，得发酵上清；在非变性条件下，用Ni-NTAHis Bands树脂吸附重组胶原，用洗涤液洗去杂质，最后再洗脱重组胶原，收集洗脱液；洗脱液经超滤系统脱盐、浓缩；再将浓缩液用Strep-Tactin树脂吸附重组胶原，用洗涤液洗去杂质，最后再洗脱重组胶原，收集洗脱液；洗脱液经超滤系统脱盐、浓缩，所制得的浓缩液经真空冷冻干燥制得所述重组人源I型胶原α1链蛋白。
6.酵母重组人源I型胶原α1链蛋白制备的胶原蛋白凝胶敷料，其特征在于：所述胶原蛋白凝胶敷料以纯化水为溶剂，其组分还包括重组人源I型胶原α1链蛋白、凝胶剂、保湿剂；所述凝胶剂为卡波姆，所述保湿剂为甘油和三乙醇胺；其中各组分的质量配比为：重组人源I型胶原α1链蛋白0.1-0.5％、甘油1-10％、卡波姆0.1-4％、三乙醇胺0.1-10％，其余为纯化水。
7.酵母重组人源I型胶原α1链蛋白制备的胶原蛋白贴敷料，其特征在于：所述胶原蛋白贴敷料由重组人源I型胶原α1链蛋白、保湿剂、医用增稠剂和无纺布基材组成；所述保湿剂为甘油，所述医用增稠剂为黄原胶；其中各组分的质量配比为：重组人源I型胶原α1链蛋白0.1-0.5％、甘油1-10％、黄原胶0.1-2％，其余为纯化水。
8.酵母重组人源I型胶原α1链蛋白制备的用于美容的导入性产品，其特征在于：所述导入性产品各原料组分如下：以质量百分比计，重组人源I型胶原α1链蛋白0.1-0.5％，甘油1-10％、透明质酸钠0.1-0.5％、小分子透明质酸钠0.01-0.05％、氯化钠0.9％、五胜肽0.005-0.02‰，其余为纯化水。
9.酵母重组人源I型胶原α1链蛋白制备的外用护肤精华液，其特征在于：所述护肤精华液各原料组分如下：以质量百分比计，甘油1-3％、丁二醇1-3％、透明质酸钠0.01-0.1％、重组人源I型胶原α1链蛋白0.01-0.1％、维生素C乙基醚0.01-0.1％、维生素C磷酸酯钠0.01-0.1％、甘草酸二钾0.1-0.5％、EDTA·Na20.01-0.1％、丝肽0.01-0.1％、β-葡聚糖0.1-0.5％、芦芭油0.1-0.5％、水溶性氮酮0.1-1％，其余为纯化水。
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| CN116333094B (zh) * | 2023-02-04 | 2024-03-29 | 山东多美康生物医药有限公司 | 一种重组人源化I型胶原蛋白α1及表达载体和应用 |
| CN117064788A (zh) * | 2023-09-25 | 2023-11-17 | 广东医保药业有限公司 | 一种口腔护理组合物及其应用 |
| CN117064788B (zh) * | 2023-09-25 | 2024-03-22 | 广东医保药业有限公司 | 一种口腔护理组合物及其应用 |
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