CN113845574A - 一种穿透肽tdp、融合穿透肽蛋白及其制备方法和应用 - Google Patents
一种穿透肽tdp、融合穿透肽蛋白及其制备方法和应用 Download PDFInfo
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Abstract
本申请涉及小分子肽的应用领域,尤其涉及一种穿透肽TDP、融合穿透肽蛋白及其制备方法和应用;所述穿透肽TDP的氨基酸序列为KETWWETWWTEWSQPKKRKKV;所述融合穿透肽蛋白包括穿透肽TDP、连接肽和生长因子;所述方法包括:得到细胞生长因子成熟肽的氨基酸序列;分别在氨基酸序列的氨基端和羧基端设计穿透肽和连接肽;根据穿透肽、连接肽和氨基酸序列,得到核苷酸序列;将核苷酸序列加上内切酶位点,得到目标DNA片段;将目标DNA片段构建转化子;根据核苷酸序列,判断转化子的核苷酸序列是否合格;将表达菌体进行培养和纯化,得到融合穿透肽蛋白;通过设计穿透肽的氨基酸序列,提高细胞生长因子的表皮吸收和透皮效果。
Description
技术领域
本申请涉及小分子肽的应用领域,尤其涉及一种穿透肽TDP、融合穿透肽蛋白及其制备方法和应用。
背景技术
细胞生长因子能够通过调节组织形态发生、血管生成和神经突增生来增强细胞增殖、细胞生长和细胞分化,生长因子通常作为细胞间信号分子来调节多种细胞过程,其活动通过结合到跨膜受体来调节,同时一些生长因子及其受体的调节也涉及到肿瘤的形成。
从分子结构来看,细胞生长因子都是小分子的多肽,多数由100个左右氨基酸组成,细胞生长因子都是通过与靶细胞表面的细胞生长因子受体特异结合后才能发挥其生物学效应,而随着分子生物技术的发展,为高产量、低成本的获得高纯度的细胞生长因子成为可能,但是由于细胞生长因子为大分子物质,并且由于机体皮肤及生物膜的屏障作用,因此难以被应用,而目前透皮给药主要采用化学促渗剂、物理促渗装置、制剂学或结构改造等方法扩大透皮给药的应用范围,但是这些方法都有各自的局限性;同时将生物大分子导入到活细胞中,主要采用scrapa loading法、显微注射法、电穿孔法、脂质体法、细菌毒素及红细胞或受体介导的胞吞作用等,但是这些方法都有各自的弊端。
因此如何提高细胞生长因子表皮吸收和透皮效果,是目前亟需解决的技术问题。
发明内容
本申请提供了一种穿透肽TDP、融合穿透肽蛋白及其制备方法和应用,以解决现有技术中细胞生长因子的表皮吸收和透皮效果难以提高的技术问题。
第一方面,本申请提供了一种穿透肽TDP,所述穿透肽的氨基酸序列为KETWWETWWTEWSQPKKRKKV,所述穿透肽的氨基酸序列具体如SEQ ID NO.1所示。
第二方面,本申请提供了一种融合穿透肽蛋白,所述穿透肽蛋白包括第一方面所述的穿透肽TDP、连接肽和生长因子,所述穿透肽蛋白的组合通式为:穿透肽+连接肽+细胞生长因子,
或细胞生长因子+链接肽+穿透肽,
或穿透肽+连接肽+细胞生长因子+链接肽+穿透肽。
可选的,所述细胞生长因子包括:转化生长因子-β、表皮生长因子、血管内皮生长因子、成纤维细胞生长因子、神经生长因子、血小板衍生的生长因子、肝细胞生长因子、肥大细胞生长因子、干扰素和白介素中至少一种。
可选的,所述连接肽包括:甘氨酸、丝氨酸或甘氨酸和丝氨酸的组合物。
可选的,所述融合穿透肽蛋白的表达载体包括细菌载体、酵母菌载体或哺乳动物细胞载体。
第三方面,本申请提供了一种融合穿透肽蛋白的制备方法,所述方法包括:
得到细胞生长因子成熟肽的氨基酸序列;
在所述氨基酸序列的氨基端设计第一穿透肽和第一连接肽;
在所述氨基酸序列的羧基端设计第二穿透肽和第二连接肽;
根据所述第一穿透肽、所述第一连接肽、所述第二穿透肽、所述第二连接肽和所述氨基酸序列,得到核苷酸序列;
将所述核苷酸序列的5’端和3’端分别加上内切酶位点,得到目标DNA片段;
将所述目标DNA片段构建到酶切载体上,后转化受体菌,得到转化子;
根据所述核苷酸序列,判断所述转化子的核苷酸序列是否合格;
若是,则将所述转化子进行诱导验证,后进行放大培养得到表达菌体;
将所述表达菌体进行培养和纯化,得到融合穿透肽蛋白。
可选的,所述第一连接肽的氨基酸序列为MKETWWETWWTEWSQPKKRKKVGGGGS,所述第一连接肽的氨基酸序列具体如SEQ ID NO.2所示。
可选的,所述第二连接肽的氨基酸序列为GGGGSKETWWETWWTEWSQPKKRKKV,所述第二连接肽的氨基酸序列具体如SEQ ID NO.3所示。
第四方面,本申请提供了一种外用制剂的组合物,所述组合物为包裹有第一方面所述的穿透肽和细胞生长因子的组合物的脂质体,或包裹有第二方面所述的融合穿透肽蛋白的脂质体,其中,所述脂质体的原料为天然或合成的磷脂。
第五方面,本申请提供了一种融合穿透肽蛋白的应用,将所述融合穿透肽蛋白用于临床治疗和医疗美容中。
本申请实施例提供的上述技术方案与现有技术相比具有如下优点:
本申请实施例提供的一种穿透肽TDP,通过对穿透肽TDP的氨基酸序列进行设计,能较好的穿过皮肤的角质层细胞和皮肤细胞,从而能作为细胞生长因子的载体,将细胞生长因子穿过皮肤角质层和皮肤细胞,提高细胞生长因子的表皮吸收和透皮效果。
附图说明
此处的附图被并入说明书中并构成本说明书的一部分,示出了符合本发明的实施例,并与说明书一起用于解释本发明的原理。
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,对于本领域普通技术人员而言,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1为本申请实施例提供的方法的流程示意图;
图2为本申请实施例提供的表达载体PBV220TDPaFGF和PBV220TDPbFGF的酶切结果鉴定图;
图3为本申请实施例提供的含有FGF的融合穿透肽蛋白的SDS-PAGE电泳检测前后的对比图;
图4为本申请实施例提供的融合蛋白包涵体鉴定结果图;
图5为本申请实施例提供的融合蛋白的纯品鉴定结果图。
具体实施方式
为使本申请实施例的目的、技术方案和优点更加清楚,下面将结合本申请实施例中的附图,对本申请实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本申请的一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都属于本申请保护的范围。
本申请的创造性思路为:细胞生长因子中成纤维细胞生长因子是一种能促进成细胞生长的多肽类物质,在促进成纤维细胞有丝分裂、中胚层细胞和神经细胞的生长,还可刺激血管形成,创伤愈合及肢体再生中发挥作用,具有深层修复作用,再现代临床医学及外科手术和美容手术中发挥着不可估量的巨大作用。
细胞生长因子对来源于中胚层和神经外胚层的多种细胞具有促进分裂的作用,可用于外伤,溃疡等的治疗,目前已应用于皮肤组织的各种损伤,去除暗疮、痊疮、去痣后的小缺损,换肤后的脱皮、发红,果酸等导致的皮肤灼伤,磨削后的表皮损伤修复等具有显著效果,FGF能够加快创伤愈合,修复损伤组织,减少疤痕收缩和皮肤的畸形增生,促进细胞间质的形成,促进胶原蛋白的合成和分泌,促进弹性纤维合成和分泌,促进细胞间基质的增加,使皮肤细嫩健美、饱满有弹性,从而消除皱纹,从而缩短创伤愈合时间以及减少疤痕形成的作用。
透皮机制研究发现,影响细胞穿透肽/被载物质复合物进入细胞的因素包括被载物质性质、细胞穿透肽、细胞穿透肽浓度等,目前常用非胞吞或能量无关的途径和内吞途径来解释为何细胞穿透肽可促使各种大分子进入细胞,虽然不同类型的细胞穿透肽与被载分子的内化机制不同,但内吞作用为多数细胞穿透肽的细胞易位机制却有一致共识,皮肤的角质层细胞为死细胞,形成具有代谢活性的环境,但没有内吞作用。此外,角质层和细胞膜不仅脂质组成不同,而且水含量和脂质/蛋白质比也不同。因此, 细胞穿透肽在皮肤中的渗透可能不同于他们穿过细胞膜的机制。研究表明, 细胞穿透肽的皮肤促渗机制有以下几种:①角质层死细胞的代谢活性直接参与细胞穿透肽的传输;②脂类和细胞穿透肽之间的相互作用会破坏皮肤细胞间的紧密连接,也可能在透过角质层过程中发挥重要作用;③胞饮作用;④通过毛孔等附属器途径促渗;此外,当渗透到皮肤的不同层时,形成的浓度梯度也可能是细胞穿透肽的促渗驱动力。
在本申请一个实施例中,提供一种穿透肽TDP,所述穿透肽的氨基酸序列为KETWWETWWTEWSQPKKRKKV,所述穿透肽的氨基酸序列具体如SEQ ID NO.1所示。
在本申请一个实施例中,提供一种融合穿透肽蛋白,所述穿透肽蛋白包括所述穿透肽TDP、连接肽和生长因子,所述穿透肽蛋白的组合通式为:穿透肽+连接肽+细胞生长因子,
或细胞生长因子+链接肽+穿透肽,
或穿透肽+连接肽+细胞生长因子+链接肽+穿透肽。
本申请中,通过设计的穿透肽和连接肽,穿透肽通过连接肽连接到细胞生长因子的两端,从而通过穿透肽增加细胞生长因子的穿透效果,提高细胞生长因子的表皮吸收和透皮效果。
作为一个可选的实施方式,所述细胞生长因子可以是转化生长因子-β(TGF-β)、表皮生长因子(EGF)、血管内皮生长因子(VEGF)、成纤维细胞生长因子(FGF)、神经生长因子(NGF)、血小板衍生的生长因子(PDGF)、肝细胞生长因子(HGF)、肥大细胞生长因子(SCF)、干扰素和白介素中至少一种。
本申请中,通过限定细胞生长因子的范围,能涵盖大部分通用的细胞生长因子,从而使本申请的穿透肽具有普遍的适用性。
作为一个可选的实施方式,所述连接肽包括:甘氨酸、丝氨酸或甘氨酸和丝氨酸的组合物。
本申请中,通过限定连接肽中含有甘氨酸和丝氨酸,使设计的穿透肽能有效的通过连接肽连接细胞生长因子,从而提高细胞生长因子的表皮吸收和透皮效果。
作为一个可选的实施方式,所述融合穿透肽蛋白的表达载体包括细菌载体、酵母菌载体或哺乳动物细胞载体。
本申请中,通过限定融合穿透肽蛋白的表达载体,使融合穿透肽蛋白能通过载体进入到受体菌中,从而方便转化的进行,提高细胞生长因子的转化效率。
在本申请一个实施例中,如图1所示,提供一种融合穿透肽蛋白的制备方法,所述方法包括:
S1.得到细胞生长因子成熟肽的氨基酸序列;
S2.在所述氨基酸序列的氨基端设计第一穿透肽和第一连接肽,得到TDPaFGF、TDPbFGF、TDPEGF和TDPKGF;
S3.在所述氨基酸序列的羧基端设计第二穿透肽和第二连接肽,得到aFGFTDP、bFGFTDP、EGFTDP和KGFTDP;
S4.根据所述第一穿透肽、所述第一连接肽、所述第二穿透肽、所述第二连接肽和所述氨基酸序列,得到核苷酸序列;
S5.将所述核苷酸序列的5’端和3’端分别加上内切酶位点EcoRI和SalI,得到目标DNA片段;
S6.将所述目标DNA片段构建到酶切载体pBV220上,后转化受体菌DH5α,得到转化子;
S7.根据所述核苷酸序列,判断所述转化子的核苷酸序列是否合格;
若是,则将所述转化子进行诱导验证,后进行放大培养得到表达菌体;
若否,则重新设计第一穿透肽、第一连接肽、第二穿透肽和第二连接肽;
S8.将所述表达菌体进行培养和纯化,得到融合穿透肽蛋白。
本申请中,分别通过对细胞生长因子的氨基端和羧基端进行设计,从而形成包括第一穿透肽+第一连接肽+细胞生长因子+第二连接肽+第二穿透肽的融合穿透肽蛋白的设计结构,再通过对设计的融合穿透肽蛋白进行核苷酸序列的构建和转化子的构建,从而方便融合穿透肽的表达。
作为一个可选的实施方式,所述第一连接肽的氨基酸序列为MKETWWETWWTEWSQPKKRKKVGGGGS,所述第一连接肽的氨基酸序列具体如SEQ ID NO.2所示。
本申请中,通过设计的第一连接肽的氨基酸序列,可具体的将细胞生长因子的氨基端同第一穿透肽连接在一起,从而方便细胞生长因子进入细胞中,提高细胞生长因子的的表皮吸收和透皮效果。
作为一个可选的实施方式,所述第二连接肽的氨基酸序列为GGGGSKETWWETWWTEWSQPKKRKKV,所述第二连接肽的氨基酸序列具体如SEQ ID NO.3所示。
本申请中,通过设计的第二连接肽的氨基酸序列,可具体的将细胞生长因子的氨基端同第二穿透肽连接在一起,从而方便细胞生长因子进入细胞中,提高细胞生长因子的的表皮吸收和透皮效果。
在本申请一个实施例中,提供一种外用制剂的组合物,所述组合物为包裹有第一方面所述的穿透肽和细胞生长因子的组合物的脂质体,或包裹有第二方面所述的融合穿透肽蛋白的脂质体,其中,所述脂质体的原料为天然或合成的磷脂。
本申请中,通过采用脂质体包裹含有穿透肽的组合物或融合穿透肽蛋白,利用脂质体与细胞膜的相似性,进一步提高含有穿透肽和细胞生长因子的物质的透皮转运速率和透过率。
在本申请一个实施例中,提供一种融合穿透肽蛋白的应用,将所述融合穿透肽蛋白用于临床治疗和医疗美容。
实施例1
一种穿透肽TDP,穿透肽的氨基酸序列为KETWWETWWTEWSQPKKRKKV,穿透肽的氨基酸序列具体如SEQ ID NO.1所示。
一种融合穿透肽蛋白,穿透肽蛋白包括所述的穿透肽TDP、连接肽和生长因子,穿透肽蛋白的组合通式为:穿透肽+连接肽+细胞生长因子+链接肽+穿透肽。
细胞生长因子为成纤维细胞生长因子。
如图1所示,一种融合穿透肽蛋白的制备方法,方法包括:
S1.得到细胞生长因子成熟肽的氨基酸序列;
S2.在氨基酸序列的氨基端设计第一穿透肽和第一连接肽,得到TDPaFGF;
S3.在氨基酸序列的羧基端设计第二穿透肽和第二连接肽,得到aFGFTDP;
S4.根据第一穿透肽、第一连接肽、第二穿透肽、第二连接肽和氨基酸序列,得到核苷酸序列;
S5.将核苷酸序列的5’端和3’端分别加上内切酶位点EcoRI和SalI,得到目标DNA片段;
S6.将目标DNA片段构建到酶切载体pBV220上,后转化受体菌DH5α,得到转化子;
S7.根据核苷酸序列,判断转化子的核苷酸序列是否合格;
若是,则将转化子进行诱导验证,后进行放大培养得到表达菌体;
S8.将表达菌体进行培养和纯化,得到融合穿透肽蛋白;
其中,得到融合穿透肽蛋白后,通过SDS电泳验证,可确定生物功能活性。
第一连接肽的氨基酸序列为MKETWWETWWTEWSQPKKRKKVGGGGS,第一连接肽的氨基酸序列具体如SEQ ID NO.2所示。
第二连接肽的氨基酸序列为GGGGSKETWWETWWTEWSQPKKRKKV,第二连接肽的氨基酸序列具体如SEQ ID NO.3所示。
一种外用制剂的组合物,组合物为包裹穿透肽和细胞生长因子的组合物的脂质体,或包裹有融合穿透肽蛋白的脂质体,其中,脂质体的原料为天然或合成的磷脂。
实施例2
将实施例2和实施例1相对比,实施例2和实施例1的区别在于:
选用人酸性成纤维细胞生长因子和碱性成纤维细胞生长因子的全长核苷酸序列,分别得到TDPaFGF和TDPbFGF,再通过EcoRI和SalI酶切连接到表达载体PBV220中,构建含有pBV-TDPaFGF和pBV-TDPbFGF的表达载体,再转入大肠杆菌DH5α,筛选并挑取阳性克隆,经限制性内切酶的酶谱分析和DNA酶谱分析两种方法进行鉴定,通过表达验证,如图2所示,判定出正确构建稳表达稳定的含有pBV-TDPaFGF/DH5α和pBV-TDPbFGF/DH5α的工程菌种。
实施例3
将实施例3和实施例2相对比,实施例3和实施例2的区别在于:
将获得的含有pBV-TDPaFGF/DH5α和pBV-TDPbFGF/DH5α的工程菌种按照质量体积比1g:100mL接种到LB培养基中,在温度为35℃条件下按照转速200rpm进行培养至培养液中的OD值在600nm的紫外波长下为0.6~0.8为止,再后续升温到40℃后继续培养4h,停止培养,后离心收集菌体,完成诱导验证,分别对接种前的工程菌体和接种诱导培养后的工程菌体的样品进行SDS-PAGE电泳检测,如图3和图4所示,证明融合穿透肽蛋白为包涵体形式表达,其中,LB培养基的成分为10g/L的胰蛋白胨(Tryptone),5g/L的酵母提取物(Yeastextract) 5g/L的氯化钠(NaCl),并且用NaOH调节LB培养基的pH至7.0为止。
实施例4
将实施例4和实施例3相对比,实施例4和实施例3的区别在于:
将接种诱导培养后工程菌体超声破碎后,再通过含EDTA和尿素的去污剂变性,采用梯度透析后,肝素亲和纯化,后经过阳离子交换树脂,如图5所示,能得到纯化的融合穿透肽蛋白,其中,梯度透析为依次更换外液尿素浓度6mol、4mol、3mol、2mol、1mol,并加入20mmol的磷酸缓冲液,同时维持外液尿素的pH为7.4;
在肝素亲和纯化中,采用浓度为0.1mol、0.5mol、1.0mol、1.5mol的NaCl溶液,依次加入20mmol的Tris-HCl和1mmol的EDTA-2Na,并调节混合溶液的pH至7.4,形成缓冲液,后进过洗脱,收集1.5mol的NaCl蛋白峰。
相关实验:
将实施例得到的融合蛋白分别进行体外透皮实验和体内透皮实验,实验原理参照Mosmann 和 Alley 等的方法进行,该方法是基于黄色的二苯基四氮唑溴盐(MTT)可以被代谢活跃的细胞裂解为紫色的甲月替结晶,随着活细胞的增加,对应形成的紫色结晶也会增加。
体外透皮实验:抽取若干只Sprague Dawley(SD)大鼠,随机分成两组,脱毛后,从同一动物身上取两块皮肤,分别做不含穿透肽的细胞生长因子bfGF 组和含穿透肽的细胞生长因子TDP-bFGF混合物组,将皮肤安装在透皮槽上,角质面加1mL的bFGF药物,真皮面加4mL的Hepes Buffer接受液,每组给药30μg,给药后立即开始计时,分别于2h、4h、16h 吸取收集液,并取100μL 收集液加入ELISA 96孔板中检测。
将人皮肤离体细胞培养后,得到人工皮肤,将人工皮肤分别做不含穿透肽的细胞生长因子bfGF 组和含穿透肽的细胞生长因子TDP-bFGF组,将人工皮肤分别在蛋白量为400μg和溶剂为生理盐水的500μL的透皮槽中,并置于37℃水浴中固定2h,收集样品溶液,用bFGF ELISA Kit 进行检测。
体内透皮实验:取昆明小鼠60只,雄雌各半,随机分组。分别用生理盐水配置50μg/mL的bfGF, 在50μg/mL的bfGF中分别加入50μg/mL的TDP肽和TDP-bFGF融合穿透肽蛋白样品,混匀后,待用。
剪去小鼠腹部鼠毛,面积为6cm×6cm,将配制的药物分别涂抹于去毛表面,开始计时,8h后麻醉处死,剥离腹部去毛皮肤,去除皮下组织和脂肪,生理盐水冲洗干净,获得完整的小鼠腹部皮肤,剪取有效扩散面积以内的皮肤称重,剪碎,匀浆后离心(5000rpm),用生理盐水反复冲洗沉淀后离心,将上清液合并。以ELISA方法分别测定各组动物皮肤组织上清液中bFGF含量。
上述实验的结果如表1所示。
表1
组别 | bFGF含量 |
空白组 | |
bFGF组 | (8.07±3.66)% |
TDP-bFGF混合物组 | (16.05±2.43)% |
TDP-bFGF融合穿透肽组 | (47.68±10.45)% |
表1具体分析,
由表1可知,含有穿透肽TDP的混合物组和融合穿透组的透皮效果都较好,说明了本申请的穿透肽TDP能良好的促进细胞生长因子的透皮效果。
本申请实施例中的一个或多个技术方案,至少还具有如下技术效果或优点:
(1)本申请实施例提供的穿透肽,再通过利用该穿透肽进行融合穿透肽蛋白的设计,将细胞生长因子、连接肽和穿透肽设计成一体,从而利用穿透肽增强细胞生长因子的穿过角质细胞和皮肤细胞的能力,提高细胞生长因子的表皮吸收和透皮效果。
(2)本申请实施例提供的融合穿透肽蛋白,通过在细胞生长因子的氨基端和羧基端分别涉及连接肽和穿透肽,从而能定向的增强细胞生长因子的表皮吸收和透皮效果。
(3)本申请实施例提供的方法,通过先对细胞生长因子进行目标融合穿透肽蛋白的设计,再反推出核苷酸序列,最后经过对表达载体和受体菌的构建,从而得到转化子,最后进行扩大培养,即能得到所需要的融合穿透肽蛋白,从而能实现产业化生产。
(4)本申请还提供一种bFGF凝胶剂的制备方法,具体包括:
a)取1g卡波姆940,加蒸馏水适量,搅拌均匀后放置充分溶胀,得卡波姆凝胶溶液,然后取10单位的bFGF蛋白,加入2mL的PBS缓冲液中,然后采用固相合成法合成穿透肽TDP,取1g穿透肽,溶解于2mL的PBS缓冲液中,得TDP穿透肽溶液。
b)将5g的丙二醇、10g的乙醇加入至上述卡波姆凝胶溶液,边加边搅拌,以三乙醇胺调pH至6.0~7.0,再以蒸馏水加至混合液的总重为90g时,将bFGFPBS溶液和TDP穿透肽溶液加入至卡波姆凝胶溶液中,充分搅拌混匀后,以蒸馏水加至总重为100g,即得bFGF凝胶剂。
附图解释:
图2为本申请实施例提供的表达载体PBV220-TDPaFGF和PBV220-TDPbFGF的酶切结果鉴定图,由图2可知,PBV220-TDPaFGF和PBV220-TDPbFGF经过内切酶EcoRI和SaLI消化后,再进过琼脂糖电泳,图中从左向右,第一道为PBV220-TDPbFGF酶切结果,第三道为PBV220-TDPaFGF的酶切结果,第二道为DNA标准分子量marker,从上至下分别为5000bp,4000bp,3000bp,2000bp,1500bp,1000bp和600bp,因此说明了构建的表达载体成功。
图3为本申请实施例提供的含有FGF的融合穿透肽蛋白的SDS-PAGE电泳检测前后的对比图,由图3可知,所得凝胶经过考马斯亮蓝染色后的结果图,从左到右,第一道为TDPaFGF诱导前全菌,第二道为TDPaFGF诱导后全菌,第三道为TDPbFGF诱导前全菌,第四道为TDPbFGF诱导后全菌,第五道为蛋白分子量标准,从上至下分别为95kD,66kD,43kD,22kD,14kD,由图3可知含有TDPaFGF和TDPbFGF的工程菌表达成功。
图4为本申请实施例提供的融合蛋白包涵体鉴定结果图,由图4可知,从左向右,第一道为TDPaFGF全菌表达组,第二道为TDPaFGF包涵体组,第三道为上清组,第四道为空白组,第五道为TDPbFGF全菌破碎上清组,第六道为TDPbFGF全菌破碎沉淀组,第七道为TDPbFGF包涵体,第八道为蛋白分子量标准组,从上至下分别为95kD,66kD,43kD,22kD,14kD,由图4可知含有TDPaFGF和TDPbFGF的工程菌的表达蛋白形成包涵体。
图5为本申请实施例提供的融合蛋白的纯品鉴定结果图,由图5可知,从左向右,第一道为融合蛋白TDPaFGF的非还原buffer中的蛋白鉴定结果,第二道为融合蛋白TDPbFGF的非还原buffer中的蛋白鉴定结果,第三道为融合蛋白TDPaFGF的还原buffer中的蛋白鉴定结果,第四道为融合蛋白TDPbFGF的还原buffer中的蛋白鉴定结果,第五道为蛋白分子量标准,从上至下分别为95kD,66kD,43kD,22kD,14kD,由图5可知含有TDPaFGF和TDPbFGF的工程菌表达蛋白已经被成功纯化。
需要说明的是,在本文中,诸如“第一”和“第二”等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下,由语句“包括一个……”限定的要素,并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
以上所述仅是本发明的具体实施方式,使本领域技术人员能够理解或实现本发明。对这些实施例的多种修改对本领域的技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所申请的原理和新颖特点相一致的最宽的范围。
序列表
<110> 百益美恒(北京)科技有限公司
<120> 一种穿透肽TDP、融合穿透肽蛋白及其制备方法和应用
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Lys Arg Lys Lys Val
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<210> 2
<211> 27
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro
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Lys Lys Arg Lys Lys Val Gly Gly Gly Gly Ser
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<210> 3
<211> 26
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<213> 人工序列(Artificial Sequence)
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Gly Gly Gly Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu
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Claims (10)
1.一种穿透肽TDP,其特征在于,所述穿透肽的氨基酸序列为KETWWETWWTEWSQPKKRKKV,所述穿透肽的氨基酸序列具体如SEQ ID NO.1所示。
2.一种融合穿透肽蛋白,其特征在于,所述穿透肽蛋白包括如权利要求1所述的穿透肽TDP、连接肽和生长因子,所述穿透肽蛋白的组合通式为:穿透肽+连接肽+细胞生长因子,
或细胞生长因子+链接肽+穿透肽,
或穿透肽+连接肽+细胞生长因子+链接肽+穿透肽。
3.根据权利要求2所述的融合穿透肽蛋白,其特征在于,所述细胞生长因子包括:转化生长因子-β、表皮生长因子、血管内皮生长因子、成纤维细胞生长因子、神经生长因子、血小板衍生的生长因子、肝细胞生长因子、肥大细胞生长因子、干扰素和白介素中至少一种。
4.根据权利要求2所述的融合穿透肽蛋白,其特征在于,所述连接肽包括:甘氨酸、丝氨酸或甘氨酸和丝氨酸的组合物。
5.根据权利要求2所述的融合穿透肽蛋白,其特征在于,所述融合穿透肽蛋白的表达载体包括细菌载体、酵母菌载体或哺乳动物细胞载体。
6.一种制备如权利要求2-4任一项所述的融合穿透肽蛋白的方法,其特征在于,所述方法包括:
得到细胞生长因子成熟肽的氨基酸序列;
在所述氨基酸序列的氨基端设计第一穿透肽和第一连接肽;
在所述氨基酸序列的羧基端设计第二穿透肽和第二连接肽;
根据所述第一穿透肽、所述第一连接肽、所述第二穿透肽、所述第二连接肽和所述氨基酸序列,得到核苷酸序列;
将所述核苷酸序列的5’端和3’端分别加上内切酶位点,得到目标DNA片段;
将所述目标DNA片段构建到酶切载体上,后转化受体菌,得到转化子;
根据所述核苷酸序列,判断所述转化子的核苷酸序列是否合格;
若是,则将所述转化子进行诱导验证,后进行放大培养得到表达菌体;
将所述表达菌体进行培养和纯化,得到融合穿透肽蛋白。
7.根据权利要求6所述的方法,其特征在于,所述第一连接肽的氨基酸序列为MKETWWETWWTEWSQPKKRKKVGGGGS,所述第一连接肽的氨基酸序列具体如SEQ ID NO.2所示。
8.根据权利要求6所述的方法,其特征在于,所述第二连接肽的氨基酸序列为GGGGSKETWWETWWTEWSQPKKRKKV,所述第二连接肽的氨基酸序列具体如SEQ ID NO.3所示。
9.一种外用制剂的组合物,其特征在于,所述组合物为包裹有权利要求1所述的穿透肽和细胞生长因子的组合物的脂质体,或包裹有如权利要求2-4任一项所述的融合穿透肽蛋白的脂质体,其中,所述脂质体的原料为天然或合成的磷脂。
10.一种如权利要求2-4任一项所述的融合穿透肽蛋白的应用,其特征在于,将所述融合穿透肽蛋白用于临床治疗和医疗美容中。
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