CN113004425A - 人表皮生长因子融合功能多肽的蛋白及其制备方法与应用 - Google Patents
人表皮生长因子融合功能多肽的蛋白及其制备方法与应用 Download PDFInfo
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- CN113004425A CN113004425A CN202110328860.8A CN202110328860A CN113004425A CN 113004425 A CN113004425 A CN 113004425A CN 202110328860 A CN202110328860 A CN 202110328860A CN 113004425 A CN113004425 A CN 113004425A
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Abstract
本发明属于生物医药领域,具体涉及一种人表皮生长因子融合功能多肽的蛋白及其制备方法与应用。所述人表皮生长因子融合功能多肽的蛋白包括4种活性功能的多肽和人表皮生长因子,4种具有活性功能的多肽分别为:纤维粘连蛋白的结合胶原蛋白的结构域序列(fnCBD)、弹性蛋白重复序列(E6)、RGD肽和Tag标签;4种具有活性功能的多肽和人表皮生长因子以任意顺序排列组合。该蛋白是基于4种具有活性功能的多肽和人表皮生长因子进行融合,通过原核系统表达制备、纯化,该融合蛋白组合既能保证hEGF的促进细胞增殖的功能,又能有效增加hEGF附着到上皮细胞的能力,从而进一步增强hEGF生物活性。
Description
技术领域
本发明属于生物医药领域,具体涉及一种人表皮生长因子融合功能多肽的蛋白及其制备方法与应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
人天然的表皮生长因子(Human Epidermal Growth Factorh,hEGF)是人生长因子的一种,由多种细胞分泌,有53个氨基酸组成,相对分子质量为6045小分子多肽。多肽链N-端为天冬酰酸,C-端为精氨酸,等电点4.12。hEGF具有较高的稳定性,100℃中性水中煮30分钟仍能保持活性,而且能抵抗蛋白酶消化,-20℃可长期保持活性。三个二硫键是hEGF生物活性必须的结构。二级结构只有β-折叠,1-33氨基酸残基构成N-端结构域和34-53氨基酸残基构成C-端结构域。多肽链上几乎所有芳香侧链残基的都在分子表面成簇存在,形成疏水的袋。
研究表明,hEGF控制正常细胞生长和伤口愈合的分子机理是通过特异性的与跨细胞膜表面的EGF受体(EGFR)结合,刺激EGFR复合物中的酪氨酸激酶的活性,通过EGFR复合物的自身磷酸化作用,在细胞内形成快速的信息传递网络,激活蛋白酶和磷酸酯酶等的一系列生化反应,促进体内Ca2+、K+和糖等低分子物大量进入细胞内(主动运输),糖酵解量增大,RNA与蛋白质合成增多,作用一段时间后,EGFR复合物开始促进DNA合成,并由此趋向刺激内皮细胞、单核细胞等多种细胞分裂、增殖和分化,使之向创伤部位迁移,加速启动创伤组织再生、修复和胞外间质形成。另一方面,hEGF能增加其他内源性生长因子,促进羟脯氨酸合成,调节胶原酶和胶原的合成、分泌和沉淀,调节胶原降解,使胶原纤维以线性方式排列,增强创面抗张程度,减少疤痕形成,提高愈合质量。另有研究表明,内、中、外3个胚层的细胞均有EGFR存在。人的角质细胞、层细胞和纤维细胞等正常细胞均有特异性的EGFR。人的表皮成纤维细胞有4-10×105个EGFR结合部位,从理论上为人体皮肤细胞吸收hEGF提供依据。但这种作用具有靶向性(靶细胞为上皮细胞、内皮细胞和成纤维细胞等)和实时性。
由于hEGF没有糖基化位点,非常适合原核表达系统进行蛋白表达,但hEGF属于小分子量多肽直接表达会影响表达、纯化和检测。现有技术中公开了一种基于抗菌肤的双功能融合蛋白,通过连接蛋白将天蚕素B 的C端与人表皮生长因子的N端连接而构成,该方法使得小分子hEGF转化为大分子多肽进行融合表达,方便了表达、纯化和检测的过程,且同时实现了抗菌和促表皮细胞生长的双功能,但hEGF本身难以主动附着到上皮细胞上,从而影响了hEGF更好地发挥促表皮细胞生长的生物活性。
发明内容
为了解决现有技术的不足,本发明提供一种人表皮生长因子融合功能多肽的蛋白及其制备方法与应用,该蛋白是基于4种具有活性功能的多肽和人表皮生长因子进行融合,通过原核系统表达进行制备、纯化,该融合蛋白组合既能保证hEGF的促进细胞增殖的功能,又能有效增加hEGF附着到上皮细胞的能力,从而进一步增强hEGF生物活性。
为了实现上述目的,本发明第一方面提供一种人表皮生长因子融合功能多肽的蛋白,具体为:包括4种具有活性功能的多肽和人表皮生长因子,4种具有活性功能的多肽分别为:纤维粘连蛋白的结合胶原蛋白的结构域序列(fnCBD)、弹性蛋白重复序列(E6)、RGD肽和Tag标签;4种活性功能的多肽和人表皮生长因子以任意顺序排列组合;
本发明第二方面提供一种编码上述人表皮生长因子融合功能多肽的蛋白的基因。
本发明第三方面提供一种上述人表皮生长因子融合功能多肽的蛋白的制备方法,包括以下步骤:
(1)将编码4种活性功能的多肽的碱基序列和编码人表皮生长因子的基因序列进行核苷酸(或碱基)组合排列,并在5’-和3’-末端分别引入限制核酸内切酶碱基序列;
(2)将人表皮生长因子融合功能多肽核苷酸序列(基因)插入到原核表达载体中,构建成重组表达载体;
(3)将重组表达载体转化到大肠杆菌菌株,培养并收集细菌细胞;
(4)将细菌细胞破碎、纯化,得到目的蛋白组分。
本发明第三方面提供一种上述人表皮生长因子融合功能多肽的蛋白在制备化妆品或其他促进上皮细胞分裂制品中的应用。
本发明的一个或多个实施方式至少具有以下有益效果:
(1)本发明构建的人表皮生长因子融合功能多肽的蛋白,将4种具有活性功能的多肽和人表皮生长因子进行融合,使得hEGF能够更好的发挥生物活性。其中,hEGF属于小分子量多肽,直接表达会影响表达、纯化和检测,在hEGF基础上进行融合4种具有活性功能的多肽进行表达,融合的多肽一方面可以增大分子量便于表达纯化和检测,解决了纯化困难的问题,另一方面可根据通过功能多肽的引入使hEGF获得与人体皮肤间的高亲和作用。
(2)本发明所构建的人表皮生长因子融合功能多肽的蛋白,不局限于某一种特定的组合排序,人表皮生长因子可与功能多肽以任意顺序排列组合,使得融合蛋白的构建过程更加灵活。
(3)本发明所构建的人表皮生长因子融合功能多肽的蛋白应用广泛,能够用于化妆品添加剂或其他促进上皮细胞分裂制品的添加剂。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为本发明实施例1中融合蛋白的基因构建示意图;
图2为本发明实施例1中Fere-EGF的SDS-PAGE电泳检测图;
图3为本发明实施例1中Fere-EGF纯化SDS-PAGE电泳检测图;
图4为本发明实施例1中阴性对照免疫组化检测结果图;
图5为本发明实施例1中Fere-EGF特异性结合细胞实验组免疫组化检测结果图;
图6为本发明实施例1中细胞增殖实验结果示意图。其中横坐标为实验分组组别,纵坐标为细胞增殖倍数。
具体实施方式
应该指出,以下详细说明都是示例性的,旨在对本发明提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
正如背景技术所介绍的,现有技术中,hEGF本身难以主动附着到上皮细胞上,从而影响了hEGF更好地发挥促表皮细胞生长的生物活性。
为了解决如上的技术问题,本发明提出了本发明第一方面提供一种人表皮生长因子融合功能多肽的蛋白,具体为:包括4种具有活性功能的多肽和人表皮生长因子,4种具有活性功能的多肽分别为:纤维粘连蛋白的结合胶原蛋白的结构域序列(fnCBD)、弹性蛋白重复序列(E6)、RGD肽和Tag标签;
本发明设计上述人表皮生长因子融合功能多肽的蛋白时,充分考虑到如何能使hEGF更好地发挥促表皮细胞生长的生物活性,基于该目的,本发明使hEGF与其他功能多肽融合,来获得hEGF与人体皮肤间高亲和作用,从而更有效地促进表皮细胞的增殖。
其中,fnCBD、E6用于结合上皮细胞,RGD起到粘附细胞作用,Tag是为了使目的蛋白快速高效纯化。该融合蛋白组合既能保证hEGF的促进细胞增殖功能,又能有效增加EGF附着到上皮细胞的能力,从而增强hEGF生物活性。
本发明所构建的人表皮生长因子融合功能多肽的蛋白,4种活性功能的多肽和人表皮生长因子以任意顺序排列组合,不局限于某一种特定的组合排序,因此使得融合蛋白的构建过程更加灵活。
在本发明的一个或多个实施方式中,所述fnCBD的氨基酸序列如SEQ ID NO. 1所示;
在本发明的一个或多个实施方式中,所述E6为一段六个氨基酸组成的弹性蛋白重复序列,氨基酸序列如SEQ ID NO.2所示;
在本发明的一个或多个实施方式中,所述RGD肽为三个氨基酸组成的序列,氨基酸序列如SEQ ID NO. 3所示;
在本发明的一个或多个实施方式中,所述Tag标签由六个连续的组氨酸组成,氨基酸序列如SEQ ID NO. 5所示;
在本发明的一个或多个实施方式中,所述人表皮生长因子融合功能多肽的蛋白的两端设置有粘性末端,用于链接目的基因和表达载体。
在本发明的一个或多个实施方式中,所述人表皮生长因子融合功能多肽基因工程蛋白由168氨基酸残基构成,理论相对分子量约17kDa,理论pI为5.83。
本发明第二方面提供一种编码上述人表皮生长因子融合功能多肽的蛋白的基因;
优选的,该基因序列如SEQ ID NO. 6所示。
本发明第三方面提供一种上述人表皮生长因子融合功能多肽的蛋白的制备方法,包括以下步骤:
(1)将编码4种活性功能的多肽的碱基序列和编码人表皮生长因子的基因序列进行核苷酸(或碱基)组合排列,并在5’-和3’-末端分别引入限制核酸内切酶碱基序列;
(2)将人表皮生长因子融合功能多肽核苷酸序列(基因)插入到原核表达载体中,构建成重组表达载体;
(3)将重组表达载体转化到大肠杆菌菌株,培养并收集细菌细胞;
(4)将细菌细胞破碎、纯化,得到目的蛋白组分。
优选的,所述内切酶碱基序列为EcoR I和BamH I碱基序列;
优选的,步骤(2)中,原核表达载体pBV220;pBV220作为蛋白表达载体,属于温度诱导型原核表达载体,避免了中间添加化学诱导剂繁琐环节,且还可以降低成本。另外,表达量也比较高。
优选的,步骤(3)中,使用含有氨苄的LB培养基将大肠杆菌菌株37℃空气震荡培养至对数期,42℃诱导培养约12-20小时,25℃下5000rmp;
优选的,步骤(4)中,将细菌细胞悬浮于缓冲液中,冰浴破碎细胞,4-8℃下12000rmp离心得到沉淀(包涵体)和上清液,上清可直接、包涵体需经8M尿素缓冲液溶解后上镍柱,进行亲和层析纯化得到分子量约为17kDa的目的蛋白组分。
本发明第三方面提供一种上述人表皮生长因子融合功能多肽的蛋白在制备化妆品或其他促进上皮细胞分裂制品中的应用。
本发明所涉及的核酸和氨基酸序列如下表所示:
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例与对比例详细说明本发明的技术方案。
实施例1
(1)根据文献报道的人EGF序列及检索的功能结构域序列,确定氨基酸序列组合,并确定相关其基因序列组合,根据原核表达系统的密码子偏好性对个别碱基调整后,得出基因序列(SEQ ID NO. 5);
人表皮生长因子融合功能多肽的蛋白的
(3)依据设计的基因序列,提交上海生物工程有限公司进行合成,并连接到克隆载体pUC57上,经EcoR I/BamH I双酶切后将基因连接到pBV220表达载体的EcoR I/BamH I克隆位点上形成表达载体pBV-Fere-EGF,并转化到宿主菌HB101中,挑取单克隆构建成工程菌,经过北京华大基因2次测序证实载体构建正确。
(4)工程菌经LB培养基,37°C培养至对数期,42°C诱导表达过夜,离心收集菌体,超声波冰浴破碎细胞,高速离心获得包涵体,经1M NaCl清洗1次后,用8M尿素溶解包涵体蛋白溶液,离心取上清,上清经鎳柱特异性亲和层析纯化,得到目的蛋白Fere-EGF,经SDS-PAGE电泳分析,分子量约为17kDa,与理论分子量相当。纯化的蛋白溶液经过含6M、4M和2M尿素的20mM磷酸盐缓冲液(pH7.4)3次递减式透析,得到复性的目的蛋白。紫外法测定蛋白浓度为3mg/ml,每100mlLB发酵液制备了约10mg目的蛋白,其氨基酸序列如SEQ ID NO. 6所示。
具体为:
步骤1:将工程菌原种接种10μL于3-4mlLB试管中,37℃震荡培养过夜,得到复苏菌,按接种量1:100比例转接到50LB(Amp+)培养基中,37℃放大培养过夜。
步骤2:将放大培养的菌种分别按1ml接种到120mlLB(Amp+)的500ml三角瓶(每瓶120ml左右的LB),共计6瓶,200rmp转速30-32℃培养到对数期(约2-3小时左右,A600值约0.5),将对数期工程菌放置在42℃水浴摇床,150rmp振荡培养,诱导过夜,约18小时左右。
步骤3:收集发酵液,4℃ 5000rmp 10min离心,收集工程菌沉淀,并合并在一个离心管中。离心前留取1ml发酵菌液,4℃放置备用。
步骤4:用约50ml 20mM磷酸盐缓冲液(pH7.4)悬菌,冰浴超声破碎(15s超声,30s停止,超声40min),4℃ 12000rmp 20min离心收集包涵体沉淀。
步骤5:用40ml 1MNaCl溶液洗包涵体1次,20mM磷酸盐缓冲液(pH7.4)洗包涵体1次,每次洗涤包含体时要充分吹打,形成均一悬液,离心(4℃,12000rmp,20min)收集包涵体沉淀。加入适量的20mM磷酸盐缓冲液(pH7.4)悬起包涵体,放置4℃保存备用。
步骤6:包涵体悬液添加1%的巯基乙醇后,逐渐添加尿素,边加边搅,直到澄清,25℃12000rmp 20min离心,取澄清的上清溶液,用于进一步的蛋白纯化。保留1ml溶解的包涵体4℃保存备用。
步骤7:镍柱装柱8ml-10ml,蒸馏水洗200ml,20mM磷酸盐缓冲液(pH7.4)洗100ml,含20mM咪唑6M尿磷酸盐缓冲液(pH7.4)洗50ml。
步骤8:将尿素溶解的60ml包涵体溶液上镍柱,收集上样后流出的穿过峰组分约60ml。
用20mM咪唑6M尿的磷酸盐缓冲液洗40ml体积,并收集该组分为20mM咪唑浓度特异性洗脱组分约40ml。分别用40ml 100mM、250mM、500mM咪唑磷酸盐缓冲液洗镍柱,并收集相关组分,各组分4℃保存备用。最后用100ml 6M尿的磷酸盐缓冲液彻底清洗镍柱。
步骤9:将所有组分进行SDS-PAGE电泳检测。12%分离胶,4%浓缩胶,14V预电泳5min,160V电泳,约45-60min,结束,染色过夜,脱色看结果(图2-3)。观察目的蛋白所在咪唑洗脱组分,并得到纯化。
步骤10:对目的蛋白组分分别用4M,2M尿磷酸盐缓冲液透析,进行蛋白透析和复性每4小时换次透析液。
步骤11:对于透析后的蛋白进行浓度测定,紫外分光光度法测定的纯化蛋白浓度约为1.5mg/ml,共100ml,相当于100mlLB发酵液得到25mg的蛋白质制品。
步骤12:按照《国家药典》(2015版)的方法对表达制备的人表皮生长因子融合功能多肽进行了特异性结合活性和生物功能测定,结果见图4-6和表1。
结果分析
1、目的蛋白表达和纯化鉴定结果
图3 为Fere-EGF纯化SDS-PAGE电泳图,其中:
M:标准分子量蛋白;1:未诱导全工程菌;2:诱导全工程菌;3:破碎细胞上清组分;4:破碎细胞沉淀组分;5:穿过组分;6: 20mM咪唑洗脱组分;7:100mM咪唑洗脱组分;8:250mM咪唑洗脱组分。从该图可以看出,目的蛋白在工程菌种以包涵体形式表达,经镍柱亲和层析纯化,主要在100mM咪唑洗脱组分中。
、免疫组化特异性结合检测
从图4可以看出,仅加入PBS缓冲液,未添加Fere-EGF融合多肽蛋白的Balb/c 3T3细胞株,经一抗-兔抗EGF抗体(1:100)和二抗-FITC标记驴抗兔抗体(1:100)处理后,在普通光源照射下的显微照片(a)和492nm激发光下的显微照片(b)(放大倍数10×10),没有出现明显的荧光斑点,说明细胞不能特异性结合上一抗和二抗(b)。
图5为Fere-EGF特异性结合细胞实验组免疫组化检测结果,与20μg/mL Fere-EGF孵育后的Balb/c 3T3细胞,经一抗-兔抗EGF抗体(1:100)和二抗-FITC标记驴抗兔抗体(1:100)分别反应后,在普通光源照射下的显微照片(a)和492nm激发光下的显微照片(b)(放大倍数10×10),从图中可以看出,Fere-EGF能特异性结合到细胞膜的EGF受体上,并能特异性结合上一抗和二抗,显示出高亮度的荧光斑点(b),证实基因工程表达纯化的Fere-EGF具有天然EGF的天然活性构象,可以特异性与细胞膜上受体结合。
、Fere-EGF促细胞增值作用检测结果
Fere-EGF促细胞增值细胞计数结果示于表1:
表1
其中,添加不同浓度的Fere-EGF至Balb/c 3T3细胞培养液中,37℃,5%CO2培养36h后,使用血球计数板进行细胞直接计数,结果证实随着Fere-EGF浓度增加,细胞数量明显增加,说明基因工程表达纯化后的Fere-EGF具有促进细胞增殖的作用。
图6与表1对应,1、2、3、4分别表示为四个实验分组,分别表示0、100、200、500ng/ml浓度的Fere-EGF对细胞增值的影响,如表1所示,与1号对照组相比,添加了100、200、500ng/ml浓度的Fere-EGF能使细胞增殖增加0.41、0.67和1.21倍。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 山东林森生物制品股份有限公司
<120> 人表皮生长因子融合功能多肽的蛋白及其制备方法与应用
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Claims (10)
1.一种人表皮生长因子融合功能多肽的蛋白,其特征在于:具体为:包括4种活性功能的多肽和人表皮生长因子,4种具有活性功能的多肽分别为:纤维粘连蛋白的结合胶原蛋白的结构域序列fnCBD、弹性蛋白重复序列E6、RGD肽和Tag标签;4种具有活性功能的多肽和人表皮生长因子以任意顺序排列组合。
2.如权利要求1所述的人表皮生长因子融合功能多肽的蛋白,其特征在于:所述fnCBD的氨基酸序列如SEQ ID NO. 1所示。
3.如权利要求1所述的人表皮生长因子融合功能多肽的蛋白,其特征在于:所述E6为一段六个氨基酸组成的弹性蛋白重复序列,氨基酸序列如SEQ ID NO.2所示。
4.如权利要求1所述的人表皮生长因子融合功能多肽的蛋白,其特征在于:所述RGD肽为三个氨基酸组成的序列,氨基酸序列如SEQ ID NO. 3所示。
5.如权利要求1所述的人表皮生长因子融合功能多肽的蛋白,其特征在于:所述Tag由六个连续的组氨酸组成,氨基酸序列如SEQ ID NO. 5所示。
6.如权利要求1所述的人表皮生长因子融合功能多肽的蛋白,其特征在于:所述人表皮生长因子融合功能多肽的蛋白的两端设置有粘性末端。
7.如权利要求1所述的人表皮生长因子融合功能多肽的蛋白,其特征在于:所述人表皮生长因子融合功能多肽基因工程蛋白由168氨基酸残基构成,理论相对分子量为17kDa,理论pI为5.83。
8.一种编码权利要求1-7任一项所述的人表皮生长因子融合功能多肽的蛋白的基因;
优选的,该基因序列如SEQ ID NO. 6所示。
9.权利要求1-7任一项所述的人表皮生长因子融合功能多肽的蛋白的制备方法,其特征在于:包括以下步骤:
(1)将编码4种活性功能的多肽的碱基序列和编码人表皮生长因子的基因序列进行核苷酸组合排列,并在5’-和3’-末端分别引入限制核酸内切酶碱基序列;
(2)将人表皮生长因子融合功能多肽核苷酸序列插入到原核表达载体中,构建成重组表达载体;
(3)将重组表达载体转化到大肠杆菌菌株,培养并收集细菌细胞;
(4)将细菌细胞破碎、纯化,得到目的蛋白组分;
优选的,所述内切酶碱基序列为EcoR I和BamH I碱基序列;
优选的,步骤(2)中,原核表达载体pBV220;
优选的,步骤(3)中,使用含有氨苄的LB培养基将大肠杆菌菌株37℃空气震荡培养至对数期,42℃诱导培养12-20小时,25℃下5000rmp;
优选的,步骤(4)中,将细菌细胞悬浮于缓冲液中,冰浴破碎细胞,4-8℃下12000rmp离心得到沉淀和上清液,上清可直接、包涵体需经8M尿素缓冲液溶解后上镍柱,进行亲和层析纯化得到分子量为17kDa的目的蛋白组分。
10.一种权利要求1-7任一项所述的人表皮生长因子融合功能多肽的蛋白在制备化妆品或其他促进上皮细胞分裂制品中的应用。
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CN115028736A (zh) * | 2022-05-10 | 2022-09-09 | 南京大学 | 一种靶向分子探针及应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101698682A (zh) * | 2009-10-26 | 2010-04-28 | 陕西省微生物研究所 | 一种基于抗菌肽的双功能融合蛋白及其制备方法和用途 |
US20130274124A1 (en) * | 2012-03-09 | 2013-10-17 | Massachusetts Institute Of Technology | Adhesion signatures |
CN106432509A (zh) * | 2016-09-13 | 2017-02-22 | 河南师范大学 | 一种治疗代谢疾病的重组人成纤维细胞生长因子21融合蛋白及其制备方法和应用 |
CN111620953A (zh) * | 2020-06-24 | 2020-09-04 | 亘元(天津)生物医药科技有限公司 | 类胶原融合蛋白组合物及制备方法 |
-
2021
- 2021-03-27 CN CN202110328860.8A patent/CN113004425A/zh not_active Withdrawn
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101698682A (zh) * | 2009-10-26 | 2010-04-28 | 陕西省微生物研究所 | 一种基于抗菌肽的双功能融合蛋白及其制备方法和用途 |
US20130274124A1 (en) * | 2012-03-09 | 2013-10-17 | Massachusetts Institute Of Technology | Adhesion signatures |
CN106432509A (zh) * | 2016-09-13 | 2017-02-22 | 河南师范大学 | 一种治疗代谢疾病的重组人成纤维细胞生长因子21融合蛋白及其制备方法和应用 |
CN111620953A (zh) * | 2020-06-24 | 2020-09-04 | 亘元(天津)生物医药科技有限公司 | 类胶原融合蛋白组合物及制备方法 |
Non-Patent Citations (3)
Title |
---|
ELLOUMI HANNACHI IMEN等: "Construction of multifunctional proteins for tissue engineering: Epidermal growth factor with collagen binding and cell adhesive activities", 《JOURNAL OF BIOTECHNOLOGY》 * |
TETSUYA ISHIKAWA等: "Production of a Biologically Active Epidermal Growth Factor Fusion Protein with High Collagen Affinity", 《J.BIOCHEM》 * |
毛芸: "类弹性蛋白-表皮生长因子(ELPs-EGF)融合蛋白的制备及其对皮肤损伤修复的影响", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115028736A (zh) * | 2022-05-10 | 2022-09-09 | 南京大学 | 一种靶向分子探针及应用 |
CN115028736B (zh) * | 2022-05-10 | 2023-07-25 | 南京大学 | 一种靶向分子探针及应用 |
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