CN114409763A - 一种重组纤连蛋白肽的纯化方法 - Google Patents
一种重组纤连蛋白肽的纯化方法 Download PDFInfo
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- CN114409763A CN114409763A CN202111559558.XA CN202111559558A CN114409763A CN 114409763 A CN114409763 A CN 114409763A CN 202111559558 A CN202111559558 A CN 202111559558A CN 114409763 A CN114409763 A CN 114409763A
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Abstract
本发明涉及一种重组纤连蛋白肽的纯化方法，属于生物医药领域。所述纯化方法采用大肠杆菌作为表达系统，不添加标签蛋白或其他氨基酸，氨基酸序列与人纤连蛋白氨基酸序列一致，进行纯化；其中，所述的大肠杆菌为大肠杆菌BL21(DE3)；该方法与现有技术相比，具有生长周期短，纯化步骤简单，高纯度，高收率，低成本，适用于工业放大生产的特点。
Description
技术领域
本发明涉及生物医药领域，具体涉及一种重组纤连蛋白肽的纯化方法。
背景技术
纤连蛋白(Fibronectin，FN)是一种巨大糖蛋白，分子量约为250kDa(单体)，它广泛存在于血浆、多种细胞表面及细胞基质中，在细胞与细胞之间、细胞与基质之间的相互作用中发挥极其重要的功能。
纤连蛋白由两个几乎相同的单体通过一对二硫键连接而成。每一个纤连蛋白亚基的分子量为230-250kDa，它们由三种重复的模块(modular structures)组成，以上各种模块组成了纤连蛋白的功能性结构域。其中，第一、第二和第三重复序列(此后分别称为III-1、III-2和III-3)包含在自缔合结构域中，第四、第五和第六重复序列(此后分别称为III-4、III-5和III-6)包含在DNA结合结构域中，第八、第九和第十重复序列(此后分别称为III-8、III-9和III-10)包含在细胞结合结构域中，和第十二、第十三和第十四重复序列(此后分别称为III-12、III-13和III-14)包含在肝素结合结构域中。
现有技术中，重组纤连蛋白肽的纯化方法，常用酵母表达来实现纯化，该方法存在发酵周期长、产量低的缺点；文献中也有报导用大肠杆菌加组氨酸或其他标签蛋白表达来实现纯化，该方法存在免疫原性的缺陷；采用包涵体表达来实现纯化，其存在变性、复性的繁琐纯化步骤的的缺点。
因此，仍然需要研究重组纤连蛋白肽的纯化方法，以获得具有生长周期短，纯化步骤简单，高纯度，高收率，低成本，适用于工业放大生产的纯化方法。
发明内容
针对上述重组纤连蛋白肽的纯化方法，其存在发酵周期长、产量低、具有免疫原性，纯化步骤繁琐的技术问题，本发明提供一种纤连蛋白肽的纯化方法，该方法具有生长周期短，纯化步骤简单，高纯度，高收率，低成本，适用于工业放大生产的特点。
本发明提供一种重组纤连蛋白肽的纯化方法。重组纤连蛋白肽是具有分子量约59kDa(545个氨基酸残基)的重组蛋白，其从N-末端侧按顺序包含III-8、III-9、III-10、III-12、III-13和III-14。重组纤连蛋白肽的基因序列及氨基酸序列在本申请的序列表中显示。
为了解决上述的技术问题，本发明提供的纯化方法基于大肠杆菌系统可溶表达，且没有添加标签蛋白等其他氨基酸，氨基酸序列与人纤连蛋白氨基酸序列一致来实现重组纤连蛋白肽的纯化。本发明的目的是解决现有技术中重组纤连蛋白肽纯化中的周期长，具有免疫原性及纯化步骤繁杂的问题。
为实现上述目的，本发明采用如下的技术方案：
本发明公开了一种重组纤连蛋白肽，所述的重组纤连蛋白肽基因序列如序列表中所示。
所述的重组纤连蛋白肽氨基酸序列如序列表中所示。
本发明还提供了一种重组纤连蛋白肽的纯化方法，所述的纯化方法，包括：采有大肠杆菌可溶表达进行纯化。
在一些实施例中，所述的大肠杆菌可溶表达，包括：采用大肠杆菌作为表达系统，不添加标签蛋白或其他氨基酸，进行纯化；其中，所述的大肠杆菌为大肠杆菌BL21(DE3)。
在一些实施例中，所述的大肠杆菌可溶表达，包括：采用大肠杆菌作为表达系统，不添加标签蛋白或其他氨基酸，氨基酸序列与人纤连蛋白氨基酸序列一致，进行纯化；其中，所述的大肠杆菌为大肠杆菌BL21(DE3)。
本发明还提供了一种重组纤连蛋白肽的纯化方法，所述的纯化方法，包括如下步骤：
(1)合成纤连蛋白的Ⅲ-8、Ⅲ-9、Ⅲ-10和Ⅲ-12、Ⅲ-13、Ⅲ-14序列；
(2)将步骤(1)中的纤连蛋白转化成pET-27b(+)载体；
(3)将构建好的质粒转化大肠杆菌BL21(DE3)并筛选高表达菌株；
(4)筛选步骤(3)中的高表达菌株后进行高密度发酵、离心收集菌体、高压破菌后收集上清液；
(5)往步骤(4)中的上清液中加入中性盐进行盐析，得到初纯样品；
(6)将步骤(5)中的初纯样品用缓冲液溶解后进行纯化，所述的纯化包括：采用阴离子交换层析填料进行纯化，纯化的洗脱液为磷酸盐缓冲液和氯化钠混合溶液；
(7)收集步骤(6)中的洗脱液，得到高纯度的纤连蛋白原液。
在一些实施例中，所述步骤(5)中的中性盐选自硫酸铵、硫酸钠和氯化钠中的一种。
在一些实施例中，所述步骤(5)中的中性盐为硫酸铵、硫酸钠或氯化钠的水溶液，其浓度为0.3M至1.5M。
在一些实施例中，所述步骤(5)中的中性盐为硫酸铵，所述硫酸铵的浓度为0.3M至1.5M。
在一些实施例中，所述步骤(6)中的缓冲液为磷酸盐缓冲液；在一些实施例中，所述步骤(6)中的缓冲液浓度为10mM至50mM的磷酸盐缓冲液。
在一些实施例中，所述步骤(6)中的缓冲液为磷酸盐缓冲液，所述磷酸盐缓冲液的PH值为6.5至7.5。
在一些实施例中，所述步骤(6)中的洗脱液为磷酸盐和氯化钠的混合溶液，其中，磷酸盐浓度为10mM至50mM，氯化钠浓度为0.3M至1.0M。
在一些实施例中，所述步骤(6)中的阴离子交换层析的填料选自Q SepharoseFastFlow和DEAE Sepharose Fast Flow中的一种；更优选Q Sepharose Fast Flow。
本发明还提供了重组纤连蛋白肽在生物美容中的应用，可促进细胞往色素沉积部位迁移，并刺激细胞分泌黑色素代谢酶，从而清除色斑，同时在防晒、晒后修复、抗衰、保湿和去皱均有应用。重组纤连蛋白肽在药妆护肤品原料和成品市场有巨大潜力。所有含有所述的重组纤连蛋白肽的药妆医美护肤品原料及成品均在本发明的保护范围之内。
本发明首先合成纤连蛋白的Ⅲ-8、Ⅲ-9、Ⅲ-10和Ⅲ-12、Ⅲ-13、Ⅲ-14序列并转化pET-27b(+)载体，将构建好的质粒转化大肠杆菌BL21(DE3)，筛选高表达菌株后进行发酵、离心收集菌体、高压破菌后收集上清液，加入硫酸铵盐析，得到初纯样品，再用磷酸盐缓冲液溶解后上阴离子交换柱，收集洗脱液得到高纯度的纤连蛋白原液。
与现有技术相比，本发明具有以下几点优势：
1.构建以大肠杆菌可溶表达、且没有添加标签蛋白等其他氨基酸，氨基酸序列与人纤连蛋白氨基酸序列一致的重组纤连蛋白肽的纯化方法，与采用酵母表达纯化的方法相比，本发明具有具有生产周期短、产量高的优点、纯化步骤简单、纯度高产量高、生产成本低等优势，适用于大规模的工业化生产；
2.采用本发明的重组纤连蛋白肽的纯化方法，与用大肠杆菌加组氨酸或其他标签蛋白表达来实现纯化相比，本发明的技术方案克服了免疫原性的缺陷；
3.采用本发明的重组纤连蛋白肽的纯化方法，与采用包涵体表达来实现纯化的方法相比，本发明的技术方案不存在变性、复性的繁琐纯化步骤的缺点，纯化步骤简单；
4.综合1、2和3可知，本发明重组纤连蛋白肽的纯化方法，具有生长周期短，纯化步骤简单，高纯度，高收率，低成本，适用于工业放大生产的特点。
在本发明的描述中，需要理解的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本发明的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。
在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
附图说明
图1为实施例2的表达菌株的筛选；
图2为纯化的硫酸铵沉淀；
图3为阴离子交换柱纯化洗脱的的目的蛋白。
具体实施方式
为了使本领域的技术人员更好地理解本发明的技术方案，下面进一步披露一些非限制实施例，对本发明作进一步的详细说明。
本发明所使用的试剂均可以从市场上购得或者可以通过本发明所描述的方法制备而得。
本发明中，kDa表示、Da全称道尔顿(Dalton)，是分子量最常用的单位，由于蛋白质是大分子，所以常用KDa(千道尔顿)来表示。
本发明中，浓度中的单位M表示mol/L。
本发明中，Q Sepharose FastFlow表示快流速Q琼脂糖凝胶。
本发明中，DEAE Sepharose Fast Flow表示快流速DEAE-琼脂糖凝胶。
本发明中，重组纤连蛋白肽目的片段购自于通用生物系统(安徽)有限公司。
本发明中，载体pET-27b(+)购自于优宝生物。
实施例1构建重组纤连蛋白肽表达载体
在纤连蛋白肽的N端加入Nco I酶切位点和起始密码子，核苷酸序列为CCATGG，C端加入终止密码子和XhoI酶切位点，核苷酸序列为TAACTCGAG。
采用快切酶QuickCutNcoI和QuickCut XhoI分别对重组纤连蛋白肽目的片段和载体pET-27b(+)进行反应。反应体系及条件如下
| 试剂 | 使用量(目的片段) | 使用量(pET-27b(+)载体) |
| DNA | 10ul(≤0.2ug) | 15ul(≤2.5ug) |
| QuickCut NcoI | 1.5ul | 2.5ul |
| QuickCut XhoI | 1.5ul | 2.5ul |
| 10XQuickCutBuffer | 3ul | 5ul |
| 3dH<sub>2</sub>O | 14ul | 25ul |
| TotalVolume | 30ul | 50ul |
轻轻混匀后离心，反应条件如下：
37℃反应1.5h，双酶切反应结束后，对目的片段和pET-27b(+)载体酶切产物进行1％的琼脂糖凝胶电泳，并将酶切产物进行切胶回收。
目的片段与pET-27b(+)表达载体连接：
将上述双酶切纯化后的目的片段与pET-27b(+)载体进行连接，构建重组质粒，连接采用快速DNA连接试剂盒在室温下连接30min，反应体系如下：
| 反应组分 | 20ul反应体系 |
| 5XRapidLigationBuffer | 4ul |
| 目的片段 | 10ul |
| 载体 | 2ul |
| FastDNALigae | 1ul |
| 3dH<sub>2</sub>O | 3ul |
转化、涂板培养：
取上述6ul连接液加入到TOP10感受态细胞中，冰上放置30min，于42℃水浴热击60sec，立即冰上静置5min，向转化反应液中加入500ulSOC液体培养基，在摇床中37℃、120rpm下震荡复苏1h，取100ul复苏培养菌液涂布于含有Kan+的LB固体培养基上，于37℃培养箱中培养过夜，挑取单菌落培养并测序。
实施例2表达菌株的构建与表达鉴定
将上述测序正确的克隆，用LB液体培养基活化后提质粒(
Plasmid MiniKit I,货号：D6943)并用琼脂糖凝胶电泳检测。取上述提取的质粒5ul加入到感受态细胞BL21(DE3)(索莱宝，货号：C1400)中，冰上放置30min，于42℃水浴热击60sec，立即冰上静置5min，向转化反应液中加入800ul LB液体培养基，在摇床中37℃、120rpm下震荡复苏1h，取100ul复苏培养菌液涂布于含有Kan+的LB固体培养基上，于37℃培养箱中培养过夜，挑选单克隆接种于LB(Kan+)培养基中，37℃培养得到重组菌液。分别取经诱导的阳性重组菌液和未诱导的阳性重组菌液，10000rpm下离心5min，弃上清，沉淀用PBS重悬，加入loadingbuffer后煮沸10min，10000rpm下离心30sec，取上清进行SDS-PAGE电泳，上样量10ul，电流为100mA，电压80-120V，电泳时间1.5h，电泳完成后取下凝胶，用考马斯亮蓝染色液染色30min，然后换脱色液反复脱色之蛋白条带清晰，可以看到在60KDa附近有明显条带为目的蛋白。见图1。
实施例3重组纤连蛋白肽的高密度发酵
种子培养：在两个1L三角瓶中各装入种子培养基200ml，121℃湿热灭菌20min，冷却至室温，取上述筛选到的高表达菌种接种于种子培养基中，接种量为0.05％，30℃、120rpm下恒温震荡16-18h，测定OD600＝2.5左右，得到发酵种子培养液。种子培养基配方为酵母粉5g/L，蛋白胨10g/L，氯化钠10g/L，用纯化水配置，自然PH。
发酵罐灭菌：发酵培养基组分为酵母粉24g/L，蛋白胨12g/L，甘油4ml/L，磷酸二氢钾2.31g/L*，磷酸氢二钾12.54g/L*；补料培养基1为50％葡萄糖(115度灭菌30min)；补料培养基2为硫酸铵30g/L；其中发酵培养基的组分磷酸二氢钾2.31g/L*、磷酸氢二钾12.54g/L*和补料培养基用高压灭菌锅单独灭菌。将发酵罐清洗干净，对PH和溶氧探头进行校正装入发酵罐中，将发酵培养基溶于纯化水中搅拌均匀，转移至发酵罐中，121℃蒸汽湿热灭菌30min，冷却至37℃备用。
接种：将发酵培养基组分磷酸二氢钾、磷酸氢二钾和种子液接种至发酵罐中，用氨水调节PH在7.0左右，培养温度37℃，初始搅拌转速为200rpm。
发酵：发酵温度控制在36-38℃，转速200-600rpm，通气量20-100L/min，罐压0.02-0.05MPa，用氨水控制发酵体系PH在7.0±0.2，发酵过程中通过调节转速、通气量、罐压等参数将溶氧控制在20％以上。待溶氧反弹时开始补料，补料过程中通过溶氧反馈调整补料速度，待OD600达到10左右时，加入IPTG至终浓度0.5mM开始诱导，诱导4-5h后结束发酵，在10000rpm下离心10min，收集菌体。
实施例4重组纤连蛋白的纯化
细菌破碎：将实施例3收集到的菌体，按照1:30的量加入20mMTris缓冲液重悬菌体，采用高压均质机破碎3次，压力分别为400bar、600bar、700bar，破碎后在显微镜下镜检破碎率≥95％，将破碎液在10000rpm下离心30min，收集破碎上清液。
盐析与溶解：向破菌上清液中加入硫酸铵至终浓度1M，搅拌溶解，4℃静置2h，离心收集沉淀，沉淀如图2所示。将沉淀按照1:20的量加入20mM PB PH7.2溶解，溶解后在10000rpm下离心10min，去沉淀，收集上清液过阴离子交换柱。
柱层析：将上述收集到的样品进行Q Sepharose Fast Flow柱层析用20mM PBPH7.2缓冲液平衡层析柱，流速150cm/小时，平衡5倍柱体积样品上样，流速150cm/小时，用20mM PB PH7.2缓冲液平衡层析柱，流速150cm/小时，平衡3倍柱体积，用20mM PB PH7.2+0.5MNaCl洗脱，流速150cm/小时，收集目标蛋白锋，如图3所示。
实施例5重组纤连蛋白的纯化
细菌破碎：将实施例3收集到的菌体，按照1:30的量加入20mM PB缓冲液重悬菌体，采用高压均质机破碎3次，压力分别为400bar、600bar、700bar，破碎后在显微镜下镜检破碎率≥95％，将破碎液在10000rpm下离心30min，收集破碎上清液
盐析与溶解：向破菌上清液中加入硫酸铵至终浓度1M，搅拌溶解，4℃静置2h，离心收集沉淀，将沉淀按照1:20的量加入20mM PB PH7.2+1M脲溶解，溶解后在10000rpm下离心10min，去沉淀，收集上清液过阴离子交换柱。
柱层析：将上述收集到的样品进行Q Sepharose Fast Flow柱层析用20mM PBPH7.2+1M脲缓冲液平衡层析柱，流速150cm/小时，平衡5倍柱体积样品上样，流速150cm/小时，用20mM PB PH7.2+1M脲缓冲液平衡层析柱，流速150cm/小时，平衡3倍柱体积，用20mMPB PH7.2+0.5M NaCl洗脱，流速150cm/小时，收集目标蛋白锋。
实施例6重组纤连蛋白的纯化
细菌破碎：将实施例3收集到的菌体，按照1:30的量加入20mM PB缓冲液重悬菌体，采用高压均质机破碎3次，压力分别为400bar、600bar、700bar，破碎后在显微镜下镜检破碎率≥95％，将破碎液在10000rpm下离心30min，收集破碎上清液。
盐析与溶解：向破菌上清液中加入硫酸铵至终浓度1M，搅拌溶解，4℃静置2h，离心收集沉淀，将沉淀按照1:20的量加入20mM PB PH7.2+4M脲溶解，搅拌下再缓慢滴加PB(20mM，PH7.2)稀释4倍至脲浓度为1M，溶解后在10000rpm下离心10min，去沉淀，收集上清液过阴离子交换柱。
柱层析：将上述收集到的样品进行Q Sepharose Fast Flow柱层析，用20mM PBPH7.2+1M脲缓冲液平衡层析柱，流速150cm/小时，平衡5倍柱体积，样品上样，流速150cm/小时，用20mM PB PH7.2+1M脲缓冲液平衡层析柱，流速150cm/小时，平衡3倍柱体积，用20mMPB PH7.2+0.5M NaCl洗脱，流速150cm/小时，收集目标蛋白锋。
本发明的方法已经通过较佳实施例进行了描述，相关人员明显能在本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明内。
序列表
<110> 湖南华腾制药有限公司
<120> 一种重组纤连蛋白肽的纯化方法
<130> 2021.12.06
<141> 2021-12-20
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1635
<212> DNA
<213> 人工合成 人(person)
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ccgatttttg aagattttgt tgatagcagt gtgggctatt ataccgtgac cggcctggaa 180
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ctggtgcgtt atagcccggt taaaaatgaa gaagatgttg ccgaactgag tattagcccg 420
agcgataatg ccgtggtgct gaccaatctg ctgccgggca ccgaatatgt ggttagtgtt 480
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gatagcccga ccggcattga ttttagcgat attaccgcca atagttttac cgttcattgg 600
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ctgctgattg gtcagcagag caccgttagc gataaaccga gccagatgca ggtgaccgat 840
gtgcaggata atagcattag tgttaaatgg ctgccgagca gcagtccggt taccggttat 900
cgtgtgacca ccaccccgaa aaatggccct ggtccgacca aaaccaaaac cgccggtccg 960
gatcagaccg aaatgaccat tgaaggtctg cagccgaccg ttgaatatgt ggtgagcgtt 1020
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attcatgaac tgtttccggc cccggatggc gaagaagata ccgcagaact gcagggcctg 1260
cgccctggta gcgaatatac cgttagcgtg gttgccctgc atgatgatat ggaaagtcag 1320
cctctgattg gcacccagag taccgcaatt ccggcaccga ccgatcttaa attcactcag 1380
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Claims (10)
1.一种重组纤连蛋白肽的纯化方法，包括，采用大肠杆菌作为表达系统，不添加标签蛋白或其他氨基酸，氨基酸序列与人纤连蛋白氨基酸序列一致，进行纯化；其中，所述的大肠杆菌为大肠杆菌BL21(DE3)。
2.根据权利要求1中所述的纯化方法，其包括如下步骤：
步骤1：合成纤连蛋白的Ⅲ-8、Ⅲ-9、Ⅲ-10和Ⅲ-12、Ⅲ-13、Ⅲ-14序列；
步骤2：将步骤1中的纤连蛋白转化成pET-27b(+)载体；
步骤3：将构建好的质粒转化大肠杆菌BL21(DE3)并筛选高表达菌株；
步骤4：筛选步骤3中的高表达菌株后进行高密度发酵、离心收集菌体、高压破菌后收集上清液；
步骤5：往步骤4中的上清液中加入中性盐进行盐析，得到初纯样品；
步骤6：将步骤5中的初纯样品用缓冲液溶解后进行纯化，所述的纯化包括：采用阴离子交换层析填料进行纯化，纯化的洗脱液为磷酸盐缓冲液和氯化钠混合溶液；
步骤7：收集步骤6中的洗脱液，得到高纯度的纤连蛋白原液。
3.根据权利要求2中所述的纯化方法，步骤5中所述的中性盐选自硫酸铵、硫酸钠和氯化钠中至少的一种。
4.根据权利要求3中所述的纯化方法，步骤5中所述的中性盐水溶液的浓度为0.3mol/L至1.5mol/L。
5.根据权利要求2中所述的纯化方法，步骤5中所述的中性盐为硫酸铵，所述硫酸铵水溶液的浓度为0.3mol/L至1.5mol/L。
6.根据权利要求2中所述的纯化方法，所述步骤6中的缓冲液为磷酸盐缓冲液。
7.根据权利要求6中所述的纯化方法，所述磷酸盐缓冲液浓度为10mmol/L至50mmol/L的磷酸盐缓冲液。
8.根据权利要求2中所述的纯化方法，所述步骤6中的缓冲液为磷酸盐缓冲液，所述磷酸盐缓冲液的PH值为6.5至7.5。
9.根据权利要求2中所述的纯化方法，所述步骤6中的洗脱液为磷酸盐和氯化钠的混合溶液，其中，磷酸盐浓度为10mmol/L至50mmol/L，氯化钠浓度为0.3mol/L至1.0mol/L。
10.根据权利要求2中所述的纯化方法，所述步骤6中的阴离子交换层析的填料选自QSepharose Fast Flow和DEAE Sepharose Fast Flow中的一种。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117143224A (zh) * | 2023-10-26 | 2023-12-01 | 广州暨南大学医药生物技术研究开发中心有限公司 | 一种重组人纤连蛋白截短肽及其制备方法与应用 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0399806A1 (en) * | 1989-05-26 | 1990-11-28 | Takara Shuzo Co. Ltd. | Fibronectin derivative |
| JP2002522513A (ja) * | 1998-08-11 | 2002-07-23 | アメリカ合衆国 | 哺乳動物細胞を形質導入する方法およびそれに関連する産物 |
| CN109762061A (zh) * | 2019-03-15 | 2019-05-17 | 辽宁何氏医学院 | 一种简单高效的单克隆抗体的纯化方法 |
| CN113150120A (zh) * | 2021-05-21 | 2021-07-23 | 江南大学 | 发酵液中猪肌红蛋白的分离纯化方法 |
-
2022
- 2022-03-15 CN CN202111559558.XA patent/CN114409763A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0399806A1 (en) * | 1989-05-26 | 1990-11-28 | Takara Shuzo Co. Ltd. | Fibronectin derivative |
| JP2002522513A (ja) * | 1998-08-11 | 2002-07-23 | アメリカ合衆国 | 哺乳動物細胞を形質導入する方法およびそれに関連する産物 |
| CN109762061A (zh) * | 2019-03-15 | 2019-05-17 | 辽宁何氏医学院 | 一种简单高效的单克隆抗体的纯化方法 |
| CN113150120A (zh) * | 2021-05-21 | 2021-07-23 | 江南大学 | 发酵液中猪肌红蛋白的分离纯化方法 |
Non-Patent Citations (5)
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117143224A (zh) * | 2023-10-26 | 2023-12-01 | 广州暨南大学医药生物技术研究开发中心有限公司 | 一种重组人纤连蛋白截短肽及其制备方法与应用 |
| CN117143224B (zh) * | 2023-10-26 | 2024-01-30 | 广州暨南大学医药生物技术研究开发中心有限公司 | 一种重组人纤连蛋白截短肽及其制备方法与应用 |
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