CN108752426A - 带中性电荷的细胞穿透肽及作为细胞内运送载体的用途 - Google Patents
带中性电荷的细胞穿透肽及作为细胞内运送载体的用途 Download PDFInfo
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Abstract
一类带中性电荷细胞穿透肽，在生理条件下净电荷为0，并提供具体的氨基酸序列。并提供中性电荷细胞穿透肽作为细胞内运送载体的用途以及带中性电荷细胞穿透肽作为治疗药物在细胞内的运送载体的医药用途及作为造影剂在细胞内的运送载体的医学成像技术的用途。此外，中性电荷细胞穿透肽的同源物也具有细胞穿透的性能，可以作为细胞内运送载体及作为治疗药物在细胞内的运送载体的医药用途。
Description
技术领域
本发明涉及生物医药领域，特别涉及一类带中性电荷的细胞穿透肽及其作为哺乳动物细胞内运送载体在生物学和医药领域的应用。
技术背景
细胞膜是细胞与细胞外环境间的半透性屏障，具有选择性通透作用从而使细胞保持恒定的内环境。虽然这种磷脂双分子层对于细胞的存活和功能必不可少，它却给细胞内外货物分子的交换提出了挑战。由于蛋白、多肽、核苷酸等药物大分子物质和显影剂必须要达到细胞内部才能发挥相应的作用，因此实现这些物质的跨膜转运变得非常必要。
目前，能够向细胞内转运大分子的技术主要有电穿孔(electroporation)、显微注射(microinjection)、脂质体转染(transfection)和病毒载体(viralvector)等，但这些技术由于效率低下、安全性差等原因，难以应用于临床诊断和治疗药物的研发。近年来，有关细胞穿透肽(cell-penetration peptides，CPPs)的研究为解决上述问题提供了一个良好的机遇。关于CPPs的首次报道出现在1988年，Frankel等发现来源于人免疫缺陷病毒HIV-1转录激活因子的蛋白Tat具有穿透细胞膜和核膜，并锚定于特异基因调控区的能力(Cell,1988,55:1189-1193)。随后研究还发现，Tat蛋白中的一段长度为11个氨基酸残基的碱性氨基酸序列YGRKKRRQRRR发挥了蛋白转运的作用(J Biol Chem,1997,272:16010-16017)。经过二三十年的探索，人们逐渐认识到，CPPs是一类长约9～16个氨基酸的小分子多肽，富含碱性氨基酸。作为一种有效的运输载体，CPPs在体内外可以将多肽、蛋白、核酸、肽核酸、脂质体、显像剂、纳米大小颗粒等多种物质有效地运送入细胞，适用于几乎所有的真核细胞，并且具有蛋白转导发生迅速、操作简单，毒性、副作用较小等优点。
由于CPPs的发现为细胞膜的结构和功能提供了极佳的研究工具，因而对于其穿膜机制的研究也得到了大量的关注。以研究最多的Tat为例，目前普遍认为，CPPs穿透细胞膜的过程基本上可分为两个阶段：首先通过电荷作用与细胞表面结合；然后穿透细胞膜进入细胞，释放进入胞浆乃至胞核。在第一阶段中，Tat能通过精氨酸残基的胍基与细胞表面上带有阴离子的胺聚糖、膜磷脂头部等发生强烈的相互作用，从而在细胞膜表面聚集。因此，氨基酸序列中含有较多的正电荷是目前穿透肽的主要序列特征之一。肝素(Heparin)是硫酸肝素蛋白多糖(heparan sulfate proteoglycan,HSPG)上硫酸肝素(heparin sulfate,HS)的结构类似物，当与HS同时存在时，起到竞争性与配体结合的作用，而肝素酶Ⅲ(HeparinaseIII)可以特异性降解细胞膜表面的HS链，这两种抑制剂通过不同的机制都可以阻断细胞膜上HSPG的HS链与配体的相互作用，进而阻断正电荷CPPs的内化过程。在第二阶段中，CPPs内化的机制主要涉及三条途径(Cell Mol Life Sci.2005,62:1839-1849)：一是CPPs通过细胞脂质双分子层直接进入细胞内；二是CPPs在细胞表面形成特定的跨膜结构实现转运，比如微团模式，打孔模式等；三是内吞介导的内化模式，其中又可能涉及网格蛋白、胞膜窖和巨胞饮介导等具体内吞方式。上述三种途径中，内吞介导的内化模式目前被认为是大多数CPPs进入细胞的主要途径，也是研究最深入的一条途径。但是对于某一条CPPs(例如研究最为透彻的Tat)的穿膜途径而言，由于实验方法和条件的不一致，不同的研究机构和组织往往得到了不同的结论。另外，有研究发现，CPPs乃至货物分子的长度、电荷等都会对其具体的穿膜机制产生影响。由于上述因素的存在，加之研究手段的落后和实验条件的差异，使得对于CPPs的穿膜机制这一问题尚未取得统一的认识。另一方面，关于CPPs内吞进入细胞后的运输问题逐渐得到越多越多的关注和研究。有数据显示，目前已知的以正电荷为主的CPPs中，有相当数量的成员内化后会被包裹在内体中，直接导致其携带的药物分子无法得到有效的释放。该现象随着货物分子量的增加而更为明显。有学者提出CPPs转运药物的限速步骤就在于其逃离胞吞囊泡的过程(NatMed,2004,10(3):310-315)。他们发现，大多数的Tat(48-57)-Cre通过脂筏发生胞吞后，可滞留在胞吞中长达24小时，同时这一逃离过程的效率是非常低下的。
尽管目前对于CPPs如何作用于细胞脂膜，穿透脂膜的机理目前尚不完全清楚，细胞内药物转运释放效率尚不尽如人意，但其在生物和医药领域的诱人应用前景却是毋庸置疑的。由于CPPs能够显著提高货物分子的通透性，因而能够携带显影剂和放射性同位素标记的抗体分子穿透如血脑屏障等特殊生理结构，达到细胞的内部，从而为中枢系统疾病部位的可视化提供了途径。S.Santra等用微导管将和Tat共轭结合的量子点通过颈动脉导入大鼠体内(Chem.Commun.(Camb.),2005,144-3146)后，发现Tat能够成功和迅速地将量子点运送到脑部实质，通过手持式的紫外灯照射就能够达到脑部组织的荧光可视化，而无需破坏血脑屏障的完整结构。此外，在肿瘤治疗方面，CPPs也成为了重要的工具，用于运输化疗药物和促凋亡蛋白至肿瘤细胞内部。为了达到靶向性治疗的目的，研究者设计将Tat与抗Her-2/neu的模拟肽AHNP相连接(Cancer Res.2006,66:3764-3772.)。该模拟肽能够特异性与表皮生长因子受体ErB2相结合，而在30％的乳腺癌患者中受体ErB2都出现过表达。经过改造后，癌细胞特异性药物转运体系能够大大提高肿瘤细胞内部药物的浓度，从而达到改善肿瘤治疗效果的目的。
综上所述，目前已知的CPPs分子序列中大都含有较多的碱性氨基酸(如精氨酸和赖氨酸)，其内化强烈依赖于生理条件下带正电荷的碱性氨基酸与细胞膜外带负电硫酸肝素的相互作用，同时多数内化后会被限制在内吞囊泡中，胞内释放过程效率低下。考虑到对于经典的CPPs而言，序列中的正电荷为其发生内化所必须具备的条件，带有中性电荷CPPs可能代表着一类尚未被人们所认识的新家族，并可能成为大分子研究工具和治疗药物的转运载体在细胞生物学和医药领域得到应用。
发明内容
本发明目的在于提供一类新的带有中性电荷的细胞穿透肽序列及其同源物、类似物和衍生物，以及该类穿透肽作为大分子研究工具和治疗药物的转运载体在细胞生物学和医药领域的应用。
本发明的上述目的通过如下技术手段实现：
提供一种带中性电荷细胞穿透肽，在生理条件下净电荷为0。
上述带中性电荷的细胞穿透肽，氨基酸序列为：PNEKRDH、NEKRDHM、HGENNRY、RDHTTPN、QMSKQHE、DPKASQQ、QERASQG、THERMHY、TATKNND、SNHQYSS、WPPPTQQ、DTPRGAH、TWKEHPM、NYNSHNV、WHAPSHH、PAHGTQQ、QSNQHTV、RTSDSHF、HDGRLPW、TTSHNMH、SSTYPHY、GNPSLNQ、HHFSAQH、NHYLSTQ、ANHGVRE、WPNSTST、QDSRFHV、HGVWSQQ、NWYQSLT、PTLHSSQ、EAHKTLS、SSTKDSV、QPTTSYF、TAASHWN、TTTTSNA、AREPCLN、HSGVPHG、HNVSGGS、HPETLVK、NTPYLTS、DATYGKL、GQGVWPY、ERAGGLL、NSHNVYI、HAPPSSL、DAVRLHW、PILPHGQ、HGAGTAS、APLGTPH、VHNQLQL、PYSSAAS、GPGTSFG、STQSALS、NPGLSSF、STSTAMW、AINGHAS、VPTPSAS、NLGSSYL、WSVSLYH、TSMPLST、PVPWPTL、TLTPTSC、PSLANPL、LPSFIHQ、TTYGAGV、LDGVKPV、LTGPAHL、TVLPYWA、WFISSSA、VPWPTLV、LGAGAAT、TAAGITL、NFIAVSA或者GVVPILV。
本发明还提供上述带中性电荷细胞穿透肽作为细胞内运送载体的用途。
优选的，上述细胞为哺乳动物细胞。
本发明提供上述的带中性电荷细胞穿透肽作为治疗药物在细胞内的运送载体的医药用途。
优选的，上述的带中性电荷细胞穿透肽作为用于在细胞内运送治疗药物载体的医药用途。
优选的，上述述的带中性电荷细胞穿透肽作为用于在细胞内运送治疗药物载体的医药用途，包括作为运送治疗中枢退行性疾病、脑缺血、药物成瘾、心肌梗死、恶性肿瘤、感染、炎症性相关疾病的药物进入动物细胞方面的医药用途。
优选的，上述的带中性电荷细胞穿透肽作为造影剂在细胞内的运送载体的医学成像技术的用途。
本发明提供一种带中性电荷细胞穿透肽的同源物，
所述同源物为包含中电荷细胞穿透肽的任意多肽或蛋白；或者
所述同源物为包含带中电荷细胞穿透肽的同源物的任意多肽或蛋白。
优选的，上述同源物为氨基酸序列同源性大于60％的任意七肽。
优选的，上述同源物作为细胞内运送载体及作为在细胞内运送治疗药物的载体及作为造影剂在细胞内的运送载体的医学成像技术的用途。
本发明提供了一类带中性电荷细胞穿透肽，在生理条件下净电荷为0。并提供中性电荷细胞穿透肽作为细胞内运送载体的用途以及带中性电荷细胞穿透肽作为治疗药物在细胞内的运送载体的医药用途及作为造影剂在细胞内的运送载体的医学成像技术的用途。此外，中性电荷细胞穿透肽的同源物也具有细胞穿透的性能，可以作为细胞内运送载体及作为治疗药物在细胞内的运送载体的医药用途。
附图说明
结合附图对本发明作进一步说明，但附图中的内容不构成对本发明的限制。
图1为FITC-PNEKRDH和FITC-THERMHY内化不同哺乳动物细胞的实验结果。5μmol/L的FITC-PNEKRDH和FITC-THERMHY与人前列腺癌细胞株PC-3、人神经母细胞瘤细胞系SH-SY-5Y、人肾上皮细胞系HEK-293A、小鼠胚胎成纤维细胞系3T3、人脐静脉内皮细胞系ECV304在内的多种哺乳动物细胞系HUVEC和新生大鼠心肌细胞Neonatal rat cardiomyocytes孵育1小时后的荧光图像；相同浓度的FITC-Tat作为对照。
图2为PNEKRDH和THERMHY携带SOD1分子内化提高了亚砷酸钠氧化刺激后A549细胞的存活率。相同浓度的SOD1分子作为对照。
具体实施方案
以下实施例将有助于本领域的普通技术人员进一步理解本发明，但不以任何形式限制本发明。
实施例1。
一种带中性电荷细胞穿透肽，在生理条件下净电荷为0，氨基酸序列为：PNEKRDH、NEKRDHM、HGENNRY、RDHTTPN、QMSKQHE、DPKASQQ、QERASQG、THERMHY、TATKNND、SNHQYSS、WPPPTQQ、DTPRGAH、TWKEHPM、NYNSHNV、WHAPSHH、PAHGTQQ、QSNQHTV、RTSDSHF、HDGRLPW、TTSHNMH、SSTYPHY、GNPSLNQ、HHFSAQH、NHYLSTQ、ANHGVRE、WPNSTST、QDSRFHV、HGVWSQQ、NWYQSLT、PTLHSSQ、EAHKTLS、SSTKDSV、QPTTSYF、TAASHWN、TTTTSNA、AREPCLN、HSGVPHG、HNVSGGS、HPETLVK、NTPYLTS、DATYGKL、GQGVWPY、ERAGGLL、NSHNVYI、HAPPSSL、DAVRLHW、PILPHGQ、HGAGTAS、APLGTPH、VHNQLQL、PYSSAAS、GPGTSFG、STQSALS、NPGLSSF、STSTAMW、AINGHAS、VPTPSAS、NLGSSYL、WSVSLYH、TSMPLST、PVPWPTL、TLTPTSC、PSLANPL、LPSFIHQ、TTYGAGV、LDGVKPV、LTGPAHL、TVLPYWA、WFISSSA、VPWPTLV、LGAGAAT、TAAGITL、NFIAVSA或者GVVPILV。
实验发现，该带中性电荷细胞穿透肽可以作为哺乳动物细胞内运送载体的用途。由于其具有良好的细胞内化性能，可以作为用于在细胞内运送治疗药物载体的医药用途，包括作为运送治疗中枢退行性疾病、脑缺血、药物成瘾、心肌梗死、恶性肿瘤、感染、炎症性相关疾病的药物进入动物细胞方面的医药用途。此外，该带中性电荷细胞穿透肽还可作为造影剂在细胞内的运送载体的医学成像技术的用途。
可能用于中枢退行性疾病如老年性痴呆和帕金森氏症、脑缺血如中风、心肌梗死、恶性肿瘤、抗菌和炎性疾病等的治疗以及医学成像技术的开发上。
①中枢神经系统疾病：实验发现，此类带中性电荷细胞穿透肽可能具有透过生理屏障(包括血脑屏障)的能力，可以提高药物从血液运送到中枢神经系统的效率，更可进入神经细胞内发挥作用。此类中性电荷细胞穿透肽可用于老年性痴呆和帕金森氏症等中枢退行性疾病、脑缺血(中风)、药物成瘾等的治疗。
②心血管系统疾病：由于心肌缺血和脑缺血机制具有相似性，此类中性电荷细胞穿透肽亦可携带抗凋亡或激酶等活性分子进入心肌细胞和血管内皮细胞中，发挥保护作用，用于抑制由缺血或再灌注引起的心肌损伤，组织微细血管破坏和微血栓的形成。此类中性电荷细胞穿透肽可用于心肌梗死等心血管系统疾病的治疗。
③肿瘤治疗：实验发现，此类带中性电荷细胞穿透肽可能与表皮生长因子受体或肿瘤归巢结构域等特异性识别分子相连接，实现癌症的靶向治疗，并有效提高细胞内治疗药物浓度；可通过引导放射免疫试剂，提高抗体其在肿瘤的吸收和保留能力；可以与化疗药物设计形成复合物，以主动方式进入肿瘤细胞内，减小化疗药物耐药性。此外，它们中的一些还可能具备肿瘤细胞毒性，穿透细胞膜的同时可直接抑制肿瘤细胞的增殖。此类中性电荷细胞穿透肽可用于恶性肿瘤疾病的治疗。
④炎症相关疾病：实验发现，此类带中性电荷细胞穿透肽可能提高抗炎药物对皮肤的穿透效率，帮助抗炎药物有效抵达真皮T淋巴细胞，提高抗炎药物局部给药的疗效；可能对屏障功能发生障碍的炎症上皮细胞具有更高的内化能力。此类中性电荷穿透肽可用于抗炎药物的载体开发。
⑤医学成像技术：实验发现，此类带中性电荷细胞穿透肽可能提高造影剂对器官和组织的渗透性，提高造影剂对血脑屏障的通透性，实现中枢神经系统的可视化；提高量子点和抗体等大分子在细胞内的浓度，用于肿瘤光学成像和放射免疫治疗技术的开发。此类中性电荷穿透肽可用于医学成像技术的发展。
可见，本发明的带中性电荷细胞穿透肽可以突破现有技术中的正性电荷细胞穿透肽的局限，作为一种新的使用用途。
实施例2。
本实施例通过噬菌体展示肽库对人宫颈腺癌Hela细胞采用全细胞进行三轮筛选，得到152条细胞穿透肽基因序列，其中带中性电荷的穿透肽74条，获取带中性电荷的穿透肽氨基酸序列，并验证其内化性能。详细过程如下：
1、细胞穿透肽的噬菌体展示技术筛选
Hela(人宫颈腺癌)细胞培养在含5％BSA(胎牛血清)－DMEM(Dulbecco改良的Eagle培养基)完全培养基中，在37℃下，5％CO2培养箱里培养。
筛选前一天接种于10cm皿中，待细胞生长良好至单层贴壁铺满板底即可用于筛选。弃去10cm皿中的培养液，用DMEM培养基冲洗贴壁的Hela细胞3次，加入含0.1％BSA的DMEM培养基于37℃孵育1小时。加入Ph.D.-C7C噬菌体展示肽库原液10μl(滴度1.76×1011pfu)，放入37℃摇床，70rpm温和振摇孵育15分钟，继续在细胞培养箱中孵育1.5小时。立即将培养板于冰上放置5分钟，终止噬菌体的内化。弃去未结合的噬菌体上清，用含0.1％BSA的5ml的DMEM培养基室温洗涤细胞10次。用1.5ml PBS(9.0g/LNaCl，0.15g/LNa2HPO4·12H2O，0.2g/L KH2PO4,pH7.2)和1.5ml0.25％胰酶混合液消化Hela细胞，置于培养箱中保温，至镜下观察细胞圆缩脱离皿壁。4℃，1,000rpm离心2分钟，去上清，再加入3ml PBS轻柔重悬细胞，离心，反复洗涤细胞5次。细胞沉淀置于液氮和37℃恒温水浴锅中反复冻融5次，并充分震荡。冻融物中加入2ml含1％Triton X-100(曲拉通X-100)的PBS，室温作用2小时，以裂解细胞。4℃，5,000rpm离心10分钟。离心后的上清即为淘选分离液。取100μl淘选分离液测定噬菌体滴度，其余裂解液加入锥形瓶中对数期大肠杆菌中，再加入50ml LB(含1/1000四环素)，37℃剧烈振摇培养4.5小时，将培养物转入一新离心管中，4℃，10,000rpm离心10分钟。上清液转入另一离心管中，再次离心。将80％的上清液上部转入一新离心管中，加入1/6体积的PEG/NaCl，混匀，4℃沉淀过夜。沉淀于4℃，10,000离心15分钟，弃上清液，再短暂离心，吸去残留上清液。沉淀物重悬于1ml TBS(Tris缓冲盐溶液)中，悬液转入离心管中，4℃，10,000rpm离心5分钟。上清转入另一新离心管，用1/6体积的PEG/NaCl进行二次沉淀。冰上孵育40分钟，4℃，10,000rpm离心10分钟，弃上清，再短暂离心，吸去残余上清。沉淀物重悬于200μl含0.02％NaN3(叠氮钠)/TBS缓冲液中。4℃，1,0000rpm离心5分钟，上清转入新离心管，此即为噬菌体扩增液。
利用第1轮噬菌体扩增液重复以上步骤进行第2轮筛选，再利用第2轮筛选得到的噬菌体扩增液重复以上步骤进行第3轮筛选，再利用第3轮筛选得到的噬菌体扩增液重复上述步骤进行4轮筛选。
2、细胞穿透肽原核表达文库的构建
将经过纯化的第三轮筛选噬菌体离心，取100μl上清转入离心管中，加入40μlPEG/NaCl，混匀后冰上放置1小时。于4℃，10,000rpm，离心10分钟，弃上清。10秒钟短暂离心，小心吸去残余上清。将沉淀彻底重悬于1ml碘化物缓冲液中，加入2.5ml无水乙醇，室温温育10分钟。于4℃，10,000rpm，离心10分钟，弃上清。用70％乙醇洗涤沉淀，短暂干燥，沉淀重悬于30μl TBS中。
用含有酶切位点的上下游引物，以提取的噬菌体总DNA为模板，扩增目的片段，插入载体pET-14bMCStop/EGFP中。上游引物为5'-CCCGGCCCATATGGTGGTGCCTTTCTATTCCACTCTGCTTGT-3'，其中CATATG为Nde1酶切位点，GTGGTGCCTTTCTATTCCACTCTG CTTGT为噬菌体载体上的共有序列；下游引物为5'-TTAGGTACCAGTT TCGGCAGAACCTCCACCGCA-3'，其中GGTACC为Kpn1酶切位点，AGTTTCGGCAGAACCTCCACCGCA为噬菌体载体上的共有序列。用Taq酶进行PCR反应的条件为：94℃30s、58℃30s、72℃30s为一个循环，共进行18个循环。将PCR产物和载体pET-14bMCStop/EGFP分别用Nde I和KpnI进行双酶切，凝胶电泳并回收酶切产物。回收产物用T4DNA连接酶于16℃连接16小时，-20℃保存连接产物。
其中，pET-14bMCStop/EGFP载体用于构建细胞穿透肽原核表达文库，是在pET-14bMCStop载体中亚克隆入EGFP序列改造完成。EGFP的DNA片段是由BD Biosciences公司的载体pEGFP-C2作为模板，通过PCR方法得到。而pET-14bMCStop载体是在pET14b载体多克隆位点Nde I和Xho I之间加入四个酶切位点Apa I(GGGCCC)、Kpn I(GGTACC)、Sma I(CCCGGG)和Not I(CGGCCG)所改造完成。
从新鲜的琼脂板上挑取一个大肠杆菌BL21(DE3)单菌落，接种于50ml的LB培养基中，37℃剧烈震荡培养过夜。过夜培养物以1:50接种于500ml预热的新鲜LB液体培养基中(置于2L的锥形瓶中)，于37℃剧烈震荡(275rpm)培养，每隔20分钟测量一次OD600值。当OD600值达到0.4-0.5左右时，迅速将培养基置于冰水浴中30分钟，同时不断缓慢摇匀以保证内容物充分冷却。并将离心管置于冰上预冷。将细菌转移至冰冷的离心管中，4℃，1,000rpm，离心15分钟，收集细胞。弃去上清，沉淀菌体用500ml含10％甘油的冰冷去离子水重悬。4℃，1,000rpm，离心20分钟收集细胞。弃去上清，沉淀菌体用250ml含10％甘油的冰冷去离子水重悬。4℃，1,000rpm，离心20分钟收集细胞。弃去上清，沉淀菌体用0.5ml冰冷的10％甘油重悬。100倍稀释上述悬液后，测量OD600值，用冰冷的10％甘油将其稀释至浓度为2×1010-3×1010个细胞/ml，按40μl/管分装至PCR管中。
每40μl细胞悬液中加入1μl对照质粒或者连接产物，轻柔吹打混匀后，转入预冷后的电转杯中，轻击液体以确保细菌与DNA悬液位于电转杯的底部。混合后冰浴不超过5分钟。用卷纸擦干电转槽及电转杯外侧的冷凝水，将电转杯置于电转仪中。调节电转仪参数：电脉冲为25μF，电压为2.5/2.0kV，电阻为200Ω。启动电脉冲，仪器应显示4-5ms具有12.5Kv/cm的电场强度。电击结束后，室温下立即向电转杯中加入800ml温热的LB培养基，用移液器混匀，尽量吸出杯中的菌液，装于干净无菌的EP管中，即得到穿透肽原核表达文库。
3、细胞穿透肽的验证
取步骤2中经电转化得到的文库分装保存液一支，加入5ml LB液体培养基，37℃轻柔复苏1小时。取200μl培养物涂布于Amp+(100μg/ml)的LB琼脂平板上，37℃培养10小时。次晨挑取若干个单克隆分别扩大培养并表达纯化蛋白。
验证实验的前一天，将Hela细胞接种于96孔板中，待细胞生长良好至单层贴壁铺满板底即可。将纯化好的蛋白分别加入96孔板的细胞内，37℃孵育2小时，预热的PBS洗涤细胞2次后，使用Zeiss显微镜观察。观察指标为荧光显微镜下肉眼可见细胞内任何部位包括细胞浆以及细胞器等有绿色荧光聚集，即可认定为阳性结果。
该实施例验证出152条具有细胞内化功能的细胞穿透肽，经序列净电荷分析，共有74条序列在生理条件下净电荷为0。
74条中性细胞穿透肽的氨基酸序列分别为：PNEKRDH、NEKRDHM、HGENNRY、RDHTTPN、QMSKQHE、DPKASQQ、QERASQG、THERMHY、TATKNND、SNHQYSS、WPPPTQQ、DTPRGAH、TWKEHPM、NYNSHNV、WHAPSHH、PAHGTQQ、QSNQHTV、RTSDSHF、HDGRLPW、TTSHNMH、SSTYPHY、GNPSLNQ、HHFSAQH、NHYLSTQ、ANHGVRE、WPNSTST、QDSRFHV、HGVWSQQ、NWYQSLT、PTLHSSQ、EAHKTLS、SSTKDSV、QPTTSYF、TAASHWN、TTTTSNA、AREPCLN、HSGVPHG、HNVSGGS、HPETLVK、NTPYLTS、DATYGKL、GQGVWPY、ERAGGLL、NSHNVYI、HAPPSSL、DAVRLHW、PILPHGQ、HGAGTAS、APLGTPH、VHNQLQL、PYSSAAS、GPGTSFG、STQSALS、NPGLSSF、STSTAMW、AINGHAS、VPTPSAS、NLGSSYL、WSVSLYH、TSMPLST、PVPWPTL、TLTPTSC、PSLANPL、LPSFIHQ、TTYGAGV、LDGVKPV、LTGPAHL、TVLPYWA、WFISSSA、VPWPTLV、LGAGAAT、TAAGITL、NFIAVSA和GVVPILV，中性细胞穿透肽的多肽名称、氨基酸序列和核苷酸编码序列如表一所示，根据氨基酸序列转成计算机可读形式的基因序列表对应见说明书的序列表部分。
表一中性电荷穿透肽序列：
实验结果显示，此74条中性细胞穿透肽具有细胞内化特性，可以作为哺乳动物细胞内运送载体的用途。基于其内化特性，可作为治疗药物在细胞内的运送载体在医药方面获得良好的用途。
图1示意出了FITC-PNEKRDH和FITC-THERMHY内化不同哺乳动物细胞的实验结果。5μmol/L的FITC-PNEKRDH和FITC-THERMHY与人前列腺癌细胞株PC-3、人神经母细胞瘤细胞系SH-SY-5Y、人肾上皮细胞系HEK-293A、小鼠胚胎成纤维细胞系3T3、人脐静脉内皮细胞系ECV304在内的多种哺乳动物细胞系HUVEC和新生大鼠心肌细胞Neonatal rateCardiomyocytes孵育1小时后的荧光图像；相同浓度的FITC-Tat作为对照。从结果可以看出，细胞穿透肽PNEKRDH和THERMHY具有良好的内化性能。实验结果发现，本发明提出的其它带中性电荷的细胞穿透肽及其同源物也具有良好的内化性能，在此不一一展示实验结果。
实施例3。
本实施例提供如实施例1或2的带中性电荷细胞穿透肽的同源物，同源物为氨基酸序列同源性大于60％的任意七肽；或者同源物为包含带中性电荷细胞穿透肽的任意多肽或蛋白；或者同源物为包含带中性电荷细胞穿透肽的同源物的任意多肽或蛋白；或者同源物为以带中性电荷细胞穿透肽为功能骨架的多肽。
实验发现，该带中性电荷细胞穿透肽的同源物可以作为哺乳动物细胞内运送载体的用途。由于其具有良好的细胞内化性能，可以作为用于在细胞内运送治疗药物载体的医药用途，包括作为运送治疗中枢退行性疾病、脑缺血、药物成瘾、心肌梗死、恶性肿瘤、感染、炎症性相关疾病的药物进入动物细胞方面的医药用途。此外，该带中性电荷细胞穿透肽的同源物还可作为造影剂在细胞内的运送载体的医学成像技术的用途。
该带中性电荷细胞穿透肽的同源物可用于中枢退行性疾病如老年性痴呆和帕金森氏症、脑缺血如中风、心肌梗死、恶性肿瘤、抗菌和炎性疾病等的治疗以及医学成像技术的开发上。
实施例4。
于96孔培养板中铺入5×103个/孔HEK293细胞，12小时后分别用0、100、200、400和800μM的亚砷酸钠处理3小时。实验组细胞提前1小时加入抗氧化保护剂SOD1-PNEKRDH、SOD1-THERMHY或SOD1预处理。处理完毕后的细胞经CCK-8试剂盒染色后通过微孔板阅读器获得细胞存活率数值。每组实验分别设立5个平行复孔，该实验重复三次。
结果如图2所示，实验结果显示，经抗氧化保护剂处理后的三组细胞存活率高于未保护组细胞。其中，SOD1分子由于无法穿透细胞效率低下，因而抗氧化保护作用有限，200μM的亚砷酸钠处理组的细胞存活率即出现明显下降。而SOD1-PNEKRDH或SOD1-THERMHY处理组细胞对抗氧化损伤能力明显提高，高浓度亚砷酸钠处理组仍显示较高的细胞存活率。提示，带中性电荷细胞穿透肽PNEKRDH和THERMHY能够提高SOD1在细胞内的浓度，并以活性形式存在，能够显著提高抗氧化分子对抗氧化损伤的保护效能。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
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<110> 南方医科大学
<120> 带中性电荷的细胞穿透肽及作为细胞内运送载体的用途
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<213> 人工序列(artificial)
<400> 42
Gly Gln Gly Val Trp Pro Tyr
1 5
<210> 43
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 43
Glu Arg Ala Gly Gly Leu Leu
1 5
<210> 44
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 44
Asn Ser His Asn Val Tyr Ile
1 5
<210> 45
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 45
His Ala Pro Pro Ser Ser Leu
1 5
<210> 46
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 46
Asp Ala Val Arg Leu His Trp
1 5
<210> 47
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 47
Pro Ile Leu Pro His Gly Gln
1 5
<210> 48
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 48
His Gly Ala Gly Thr Ala Ser
1 5
<210> 49
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 49
Ala Pro Leu Gly Thr Pro His
1 5
<210> 50
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 50
Val His Asn Gln Leu Gln Leu
1 5
<210> 51
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 51
Pro Tyr Ser Ser Ala Ala Ser
1 5
<210> 52
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 52
Gly Pro Gly Thr Ser Phe Gly
1 5
<210> 53
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 53
Ser Thr Gln Ser Ala Leu Ser
1 5
<210> 54
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 54
Asn Pro Gly Leu Ser Ser Phe
1 5
<210> 55
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 55
Ser Thr Ser Thr Ala Met Trp
1 5
<210> 56
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 56
Ala Ile Asn Gly His Ala Ser
1 5
<210> 57
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 57
Val Pro Thr Pro Ser Ala Ser
1 5
<210> 58
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 58
Asn Leu Gly Ser Ser Tyr Leu
1 5
<210> 59
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 59
Trp Ser Val Ser Leu Tyr His
1 5
<210> 60
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 60
Thr Ser Met Pro Leu Ser Thr
1 5
<210> 61
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 61
Pro Val Pro Trp Pro Thr Leu
1 5
<210> 62
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 62
Thr Leu Thr Pro Thr Ser Cys
1 5
<210> 63
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 63
Pro Ser Leu Ala Asn Pro Leu
1 5
<210> 64
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 64
Leu Pro Ser Phe Ile His Gln
1 5
<210> 65
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 65
Thr Thr Tyr Gly Ala Gly Val
1 5
<210> 66
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 66
Leu Asp Gly Val Lys Pro Val
1 5
<210> 67
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 67
Leu Thr Gly Pro Ala His Leu
1 5
<210> 68
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 68
Thr Val Leu Pro Tyr Trp Ala
1 5
<210> 69
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 69
Trp Phe Ile Ser Ser Ser Ala
1 5
<210> 70
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 70
Val Pro Trp Pro Thr Leu Val
1 5
<210> 71
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 71
Leu Gly Ala Gly Ala Ala Thr
1 5
<210> 72
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 72
Thr Ala Ala Gly Ile Thr Leu
1 5
<210> 73
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 73
Asn Phe Ile Ala Val Ser Ala
1 5
<210> 74
<211> 7
<212> PRT
<213> 人工序列(artificial)
<400> 74
Gly Val Val Pro Ile Leu Val
1 5
Claims (10)
1.带中性电荷细胞穿透肽，其特征在于：在生理条件下净电荷为0。
2.根据权利要求1所述的带中性电荷的细胞穿透肽，其特征在于：氨基酸序列为，
PNEKRDH、NEKRDHM、HGENNRY、RDHTTPN、QMSKQHE、DPKASQQ、QERASQG、THERMHY、TATKNND、SNHQYSS、WPPPTQQ、DTPRGAH、TWKEHPM、NYNSHNV、WHAPSHH、PAHGTQQ、QSNQHTV、RTSDSHF、HDGRLPW、TTSHNMH、SSTYPHY、GNPSLNQ、HHFSAQH、NHYLSTQ、ANHGVRE、WPNSTST、QDSRFHV、HGVWSQQ、NWYQSLT、PTLHSSQ、EAHKTLS、SSTKDSV、QPTTSYF、TAASHWN、TTTTSNA、AREPCLN、HSGVPHG、HNVSGGS、HPETLVK、NTPYLTS、DATYGKL、GQGVWPY、ERAGGLL、NSHNVYI、HAPPSSL、DAVRLHW、PILPHGQ、HGAGTAS、APLGTPH、VHNQLQL、PYSSAAS、GPGTSFG、STQSALS、NPGLSSF、STSTAMW、AINGHAS、VPTPSAS、NLGSSYL、WSVSLYH、TSMPLST、PVPWPTL、TLTPTSC、PSLANPL、LPSFIHQ、TTYGAGV、LDGVKPV、LTGPAHL、TVLPYWA、WFISSSA、VPWPTLV、LGAGAAT、TAAGITL、NFIAVSA或者GVVPILV。
3.如权利要求1至2任意一项所述的带中性电荷细胞穿透肽作为细胞内运送载体的用途。
4.根据权利要求1至2任意一项所述的带中性电荷细胞穿透肽作为治疗药物在细胞内的运送载体的医药用途。
5.根据权利要求4所述的带中性电荷细胞穿透肽作为用于在细胞内运送治疗药物载体的医药用途，包括作为运送治疗中枢退行性疾病、脑缺血、药物成瘾、心肌梗死、恶性肿瘤、感染、炎症性相关疾病的药物进入动物细胞方面的医药用途。
6.根据权利要求1至2任意一项所述的带中性电荷细胞穿透肽作为造影剂在细胞内的运送载体的医学成像技术的用途。
7.如权利要求1至2任意一项所述的带中性电荷细胞穿透肽的同源物。
8.根据权利要求7所述的带中性电荷细胞穿透肽的同源物，其特征在于：
所述同源物为包含中电荷细胞穿透肽的任意多肽或蛋白；或者
所述同源物为包含带中电荷细胞穿透肽的同源物的任意多肽或蛋白。
9.根据权利要求8所述的所述的带中性电荷细胞穿透肽的同源物，其特征在于：所述同源物为氨基酸序列同源性大于60％的任意七肽。
10.根据权利要求9所述的所述的带中电荷细胞穿透肽的同源物，其特征在于：所述同源物作为细胞内运送载体及作为在细胞内运送治疗药物的载体及作为造影剂在细胞内的运送载体的医学成像技术的用途。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112851759A (zh) * | 2021-02-23 | 2021-05-28 | 李婧炜 | 噬菌体展示技术筛选可穿越血-脑脊液屏障的多肽 |
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|---|---|---|---|---|
| CN1733806A (zh) * | 2005-09-01 | 2006-02-15 | 南方医科大学 | 一种多肽随机文库及其构建方法和从该文库中筛选细胞穿透多肽的方法 |
| US20140065175A1 (en) * | 2011-02-18 | 2014-03-06 | INSTITUTO NACIONAL DE INVESTIGACION Y TECNOLOGIA AGRARIA Y ALIMENTARIA (INIA) a corporation | Carrier peptides for cell delivery |
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2018
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| CN1733806A (zh) * | 2005-09-01 | 2006-02-15 | 南方医科大学 | 一种多肽随机文库及其构建方法和从该文库中筛选细胞穿透多肽的方法 |
| US20140065175A1 (en) * | 2011-02-18 | 2014-03-06 | INSTITUTO NACIONAL DE INVESTIGACION Y TECNOLOGIA AGRARIA Y ALIMENTARIA (INIA) a corporation | Carrier peptides for cell delivery |
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| Title |
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| 吴向玲: "Hela细胞穿透肽的筛选和初步鉴定", 《中国优秀硕士学位论文全文数据《中国优秀硕士学位论文全文数据库 医药卫生科技辑》库 医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112851759A (zh) * | 2021-02-23 | 2021-05-28 | 李婧炜 | 噬菌体展示技术筛选可穿越血-脑脊液屏障的多肽 |
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