CN113166216A - 源自人lrrc24蛋白的跨膜结构域 - Google Patents
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Abstract
本发明涉及源自人LRRC24蛋白的跨膜结构域。更具体地，本发明涉及源自人LRRC24蛋白的跨膜结构域(LRRC24P跨膜结构域)或细胞穿透肽，以及包含它们的细胞内递送系统。本发明的源自人LRRC24蛋白的跨膜结构域可以用于将货物材料如化合物、生物分子和各种聚合物材料递送到细胞中。由于与常规的细胞穿透肽相比，本发明的LRRC24P跨膜结构域表现出更高的细胞穿透效率，并且源自人蛋白，因此避免了由源自外源蛋白的肽引起的副作用和免疫应答，它可以有效地用作应用于人体的化合物、生物分子和各种聚合物材料的有效细胞内递送方法。
Description
发明领域
本发明涉及源自人LRRC24蛋白的跨膜结构域和包含所述跨膜结构域的细胞内递送系统。
发明背景
用于治疗疾病或减轻症状的药物主要分为低分子量化合物药物和大分子生物药物，生物药物包括治疗性蛋白、抗体、用于预防或治疗的疫苗、基因疗法和细胞疗法。特别地，重组蛋白药物是使用难以在体内产生的治疗性蛋白的基因重组技术大量生产的。在二十世纪八十年代早期，在美国FDA首次批准了用于糖尿病治疗的重组人胰岛素之后，已经放开了各种重组蛋白药物，如生长激素、促红细胞生成素、干扰素、集落刺激因子(CSF)和血液因子。然而，与通过细胞膜并进入细胞以相对容易地显示其活性的低分子化合物药物相比，大多数大分子物质(包括重组蛋白和核酸)非常难以递送到细胞中，从而使其难以显示作为药物的功效。因此，使用电穿孔、显微注射、阳离子脂质囊泡、病毒载体、病毒样颗粒等的方法已经被用于或研究用于大分子物质的细胞内递送。然而，上述方法难以直接用于人体，或者难以在递送到从人体分离的细胞之后再次注射入人体，因此，在增加时间和成本方面存在困难。此外，它可能导致在体内诱导细胞凋亡或免疫应答的副作用，这在其应用中具有很大的局限性。
正在积极进行可以克服上述问题的新的药物递送系统和大分子生物药物的开发研究，其中的代表性方法是称为蛋白质转导结构域(PTD)或细胞穿透肽(CPP)的载体。首先发现了细胞穿透肽具有通过HIV病毒的TAT蛋白的细胞膜的性质和TAT蛋白中存在的多肽(GRKKRRQRRRPPG、RKKRRQRRR、YGRKKRRQRRR)，之后，开发了新的细胞穿透肽，如源自果蝇的Penetratin(RQIKIWFQNRRMKWKK)、源自HIV糖蛋白41的MPG(GALFLGFLGAAGSTMGAWSQPKKKRKV)、源自牛朊病毒蛋白的BPrPr(MVKSKIGSWILVLFVAGPSDVGLCKKRP)、源自人Hph-1蛋白的Hph-1(YARVRRRGPRR)和源自人NLBP蛋白的NP2(KIKKVKKKGRK)。从那时起，已经积极地进行了使用上述细胞穿透肽的研究，并且已经发现诸如DNA、siRNA、PNA(肽核酸)、蛋白质和脂质体纳米颗粒的货物物质可以被有效地递送到细胞中。
先前的研究已经显示，细胞穿透肽通过直接穿透是能量非依赖性的，并且即使在低温下也可以将大分子物质递送到细胞中，并且已经发现，通过胞吞作用渗透也是可能的。
目前，正在开发细胞穿透肽，用于使用TAT-JBD20进行脑缺血和阿尔茨海默病治疗，正在进行使用TAT-BH4进行肌萎缩性侧索硬化症治疗的开发以及使用MPG-8/siRNA和TAT-DRBD/siRNA进行诸如癌症治疗的临床前实验。此外，使用TAT-δPKC抑制剂治疗心肌梗塞、使用TAT-JBD20治疗听力损失和炎症以及使用p28治疗脑肿瘤的开发正在临床试验中。
如上所述，对细胞穿透肽的研究和使用细胞穿透肽的各种候选药物的开发正在积极地进行，但存在待克服的问题，为此目的，有必要开发新的细胞穿透肽，其能够提高细胞穿透效率并且使由细胞穿透肽和与其结合的货物材料导致的体内副作用和非预期的免疫应答最小化。
因此，为了克服现有的细胞穿透肽主要是从源自非人(如病毒和果蝇)的蛋白质发明的，或者具有低穿透效率的缺点，在人来源的肽中，发明了具有高穿透效率的细胞穿透肽，即源自人LRRC24(富含亮氨酸重复域24)蛋白的细胞穿透肽，鉴定人来源的蛋白质中存在的肽与现有的细胞穿透肽的相似性，并通过此完成了本发明。
作为现有技术文献，参考了以下非专利文献。
1.Mutational analysis of a human immunodeficiency virus type 1Tatprotein transduction domain which is required for delivery of an exogenousprotein into mammalian cells.J.Gen.Virol.,83(2002),pp.1173-1181
2.The third helix of the Antennapedia homeodomain translocatesthrough biological membranes.J.Biol.Chem.,269(1994),pp.10444-10450
3.A new peptide vector for efficient delivery of oligonucleotidesinto mammalian cells.Nucleic Acids Res.14(1997)pp.2730-2736.
4.N-terminal peptides from unprocessed prion proteins enter cells bymacropinocytosis.Biochem.Biophys.Res.Commun.,348(2006),pp.379-385
5.Intranasal delivery of the cytoplasmic domain of CTLA-4using anovel protein transduction domain prevents allergic inflammation.Nat Med,12(2006),pp.574-579
6.dNP2 is a blood-brain barrier-permeable peptide enabling ctCTLA-4protein delivery to ameliorate experimental autoimmune encephalomyelitis.NatCommun.8244(2015),pp.1-13
发明详述
[概述]
本发明的一个目的是提供能够使副作用最小化并且表现出高的细胞穿透效率的新型跨膜结构域。
本发明的另一个目的是提供包含跨膜结构域的细胞内递送系统，细胞内运送目标的货物与肽的末端结合。
本发明的另一个目的是提供将货物运送到细胞中的方法，其包括接触跨膜结构域的步骤，其中细胞内运送目标的货物与肽的末端结合。
[技术方案]
为了实现上述目的，本发明提供了跨膜结构域，其包含来源于人LRRC24(富含亮氨酸重复域24)蛋白的SEQ ID NO:1-22中任一个的多肽。
在本发明中，术语“细胞穿透肽(CPP)”是指具有在体外和/或体内将待运送目标的货物运送到细胞中的能力的肽，并且可与“跨膜结构域”互换使用。
此外，在本发明中，术语“肽”是指通过肽键将4至1000个氨基酸残基彼此键合而形成的链形式的大分子，并且可与“多肽”互换使用。
在本发明中，与源自现有病毒或其它物种的细胞穿透肽不同，为了使货物材料通过细胞穿透肽递送到人体时发生的副作用最小化，由于它是存在于人来源蛋白中的肽，与现有的细胞穿透肽相比，它提供了新的人来源的跨膜结构域或细胞穿透肽，其具有非常高的递送到细胞中的效率，并且当处理至人体时可以使副作用最小化。
本发明提供了具有改善的细胞膜渗透性的重组货物，其包含与跨膜结构域的N-末端或C-末端融合的货物和跨膜结构域。
货物可以是蛋白质、核酸、脂质或化合物的重组货物。此外，货物可以是佐剂或抗原。
根据一个实施方案，货物可以是选自以下的重组货物：激素、免疫球蛋白、抗体、结构蛋白、信号肽、储存肽、膜肽、跨膜肽、内部肽、外部肽、分泌肽、病毒肽、天然肽、糖基化蛋白、片段化蛋白、二硫化物肽、重组蛋白、化学修饰的蛋白和朊病毒。
此外，本发明中的术语“重组货物”是指由跨膜结构域和一个或多个货物通过遗传融合或化学键合重组而形成的复合物。
此外，本发明中的术语“接触”意指货物与真核或原核细胞接触，并且通过这种接触，货物被转移到真核或原核细胞中。
此外，在本发明中，将蛋白质、肽等引入细胞可与转运、穿透、转运、递送或通过的表述互换使用。
此外，根据一个实施方案，货物可以是选自以下的重组货物：核酸、编码核酸序列、mRNA、反义RNA分子、碳水化合物、脂质和糖脂。
此外，根据一个实施方案，货物可以是造影材料、药物或化学物质的重组货物。
在本发明中，术语“造影材料”意指用于医学成像中活体结构或流体的造影的所有材料。造影材料可以包括不透射线的造影材料、顺磁造影材料、超顺磁造影材料、CT造影材料或其它造影材料，但不限于此。实例可以包括不透射线的金属及其盐(例如银、金、铂等)和其它不透射线的化学品(例如钙盐、钡盐如硫酸钡、钽和氧化钽)。顺磁造影材料(用于MR成像)可以包括二亚乙基三胺五乙酸钆(gadolinium dientylene triaminepentaaceticacid)及其衍生物和其它钆、锰、铁、镝、铜、铕、铒、铬、镍和钴络合物羟基苄基乙二胺二乙酸(HBED)。超顺磁造影材料(用于MR成像)可以包括磁铁矿、超顺磁氧化铁、超细氧化铁、超顺磁氧化铁和单晶氧化铁。其它合适的造影材料可以包括碘化和非碘化，离子和非离子CR组合物，以及诸如自旋标记物或其它诊断活性物质的造影材料。当在细胞中表达时，它可以含有编码容易检测到的蛋白质的标志物基因。可以使用各种标记物，如放射性核素、荧光物质、酶辅因子、酶抑制剂等。
例如，当根据本发明的货物是蛋白质或肽时，通过将表达转移肽的DNA与表达转移靶标的DNA结合，然后进行表达，转移肽和转移靶标可以以转移靶标和肽的融合蛋白的形式组合。通过融合蛋白结合的具体实例如下：当使引物产生融合蛋白时，在将编码转运肽的核苷酸连接在表达转移目标的核苷酸之前之后，用限制酶将获得的核苷酸插入载体(例如PET载体)中，并且转化到BL-21(DE3)细胞中用于表达。此时，当用表达诱导物如IPTG(异丙基β-D-1-硫代吡喃半乳糖苷)处理以纯化融合蛋白，然后用PBS处理时，转运肽可与染色物质或荧光物质，特别是异硫氰酸荧光素(FITC)、荧光素酶(Luciferase，Luc)、绿色荧光蛋白(GFP)、增强型绿色荧光蛋白(EGFP)、TagGFP、Superfolder GFP、PA GFP、AcGFP、PS-GFP2，黄色荧光蛋白(YFP)，增强型黄色荧光蛋白(EYFP)、SYFP、TagYFP、PhiYFP、蓝铜矿(Azurite)、mKalamal、青色荧光蛋白(CFP)、增强型青色荧光蛋白(ECFP)、TagCFP、PS-CFP2、红色荧光蛋白(RFP)、TagRFP、TagRFP657、mRFP1、PaTagRFP、Turbo RFP、RFP693、tdRFP，蓝色荧光蛋白(BFP)、mTag BFP、黄绿色荧光蛋白、mNeongreen、明亮的单体荧光蛋白、Scarlet-i、mplum，单体樱桃红荧光蛋白(monomeric cherry fluorescent protein)、mcherry、PAmcherry、单体草莓色荧光蛋白(monomeric strawberry fluorescent proteins)、mStrawberry、单体橙色荧光蛋白、mOrange、PSmOrange、mRasberry、mKate、mKate2、单体青绿色荧光蛋白(monomeric teal fluorescent protein)、mTFP、mNeptune、mRubby、mRubby2、mBanana、香菇珊瑚红色荧光蛋白(Discosoma sp、red fluorescent protein，DSRed)、mCitrine、Emerald、T-Sapphire、mApple、mgrape、Venus、Topaz、J-Red、tdTomato、mTurquoise、mTurquosie2、mKO、mKO2、mUKG.IFP1.4、mEos2、mEos4、mHoneydew、Dronpa、katushka、Ypet、CyPet、Clover、kaede、KikGR、mTangerine、Zsgreen、ZsYellow、Cerulean、β-半乳糖苷酶、LacZ、β-内酰胺酶、BLA、β-葡糖醛酸酶、GUS、碱性磷酸酶、氯霉素乙酰转移酶或过氧化物酶组合。
根据本发明的另一方面，本发明提供了包含编码跨膜结构域的多核苷酸的基因构建体。
此外，本发明提供了用于表达具有改善的细胞穿透效率的重组货物蛋白的表达载体，其包括基因构建体。
本发明还提供了用于预防或治疗诸如宫颈癌、前列腺癌、卵巢癌、子宫内膜癌、子宫癌、膀胱癌、食道癌、头颈癌、威尔姆氏瘤(Wilms tumor)、软组织肉瘤、胃癌、胰腺癌、乳腺癌、支气管癌等的疾病的方法，其包括向个体施用药物组合物的步骤，所述药物组合物包含其中生理学活性肽与跨膜结构域结合的缀合物和药学上可接受的载体。
根据本发明的药物组合物可以根据常规方法配制成口服剂型，如散剂、颗粒剂、片剂、胶囊剂、混悬剂、乳剂、糖浆剂、气雾剂等，或外用制备物，栓剂和无菌可注射溶液的形式。
可以包含在组合物中的载体、赋形剂和稀释剂包括乳糖、Textrose、蔗糖、山梨糖醇、甘露糖醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、阿拉伯树胶、藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、甲基氰化物氧基苯甲酸酯(methylhydride Oxybenzoate)、羟基苯甲酸丙酯、滑石、硬脂酸镁和矿物油。
在制剂的情况下，使用通常使用的稀释剂或赋形剂，如填充剂、增量剂、粘合剂、润湿剂、崩解剂和表面活性剂进行制备。用于口服施用的固体制备物包括片剂、丸剂、散剂、颗粒剂、胶囊等，并且这些固体制备物通过将至少一种赋形剂如淀粉、碳酸钙、蔗糖或乳糖、明胶等与提取物混合来制备。除了简单的赋形剂之外，还使用润滑剂，如硬脂酸镁和滑石。用于口服使用的液体制备物包括混悬剂、液体溶液、乳剂、糖浆等，并且除了作为通常使用的简单稀释剂的水和液体石蜡之外的各种赋形剂可以包括湿润剂、甜味剂、香料和防腐剂。用于肠胃外施用的制剂包括无菌水溶液、非水溶液、混悬剂、乳剂、冻干制剂和栓剂。
作为非水溶剂混悬剂，可以使用丙二醇，聚乙二醇，植物油如橄榄油和可注射酯如油酸乙酯。作为栓剂的基质，可以使用witepsol、聚乙二醇(macrogol)、吐温61、可可脂、laurinji、甘油明胶等。
本发明组合物的优选剂量根据患者的状况和体重、疾病的严重程度、药物的形式、施用途径和持续时间而变化，但也可以由本领域技术人员适当选择。然而，为了达到理想的效果，本发明的组合物优选以每天0.0001-500mg/kg，优选0.001-250mg/kg施用。施用可以以一天一次施用，或者可以分成几次。上述剂量不以任何方式限制本发明的范围。
根据本发明的另一方面，本发明提供了将货物递送到细胞中的方法，其包括制备重组货物；以及使制备的重组货物与分离的细胞接触的步骤，在所述重组货物中，货物与跨膜结构域的N-末端或C-末端融合。
根据本发明的另一方面，本发明提供了将货物递送到动物中的方法，所述方法包括制备重组货物；以及将制备的重组货物施用于除人以外的动物的步骤，在所述重组货物中，货物与跨膜结构域的N-末端或C-末端融合。
对于跨膜结构域与细胞膜之间的接触没有特别需要的条件，如有限的时间、温度和浓度，并且可以在本领域中应用于细胞穿透的一般条件下进行。
[发明的效果]
与现有的肽相比，本发明的细胞穿透肽表现出显著改善的细胞穿透效率或细胞渗透性，从而将运送的货物或生物活性分子引入细胞，有效地保持其活性，并且可以大大降低成本。
此外，可以增加通过细胞穿透肽或跨膜结构域递送的货物材料的效果。
此外，由于细胞穿透肽源自人蛋白，因此可以使由源自非人蛋白的肽引起的副作用最小化。
然而，本发明的效果不限于上述效果，并且本领域技术人员从以下描述将清楚地理解未提及的其它效果。
附图简述
图1显示了用于进行表达LRRC24P-EGFP重组融合蛋白的基因合成的基因合成的示意图。
图2显示了通过琼脂糖凝胶电泳对基因组合物的观察结果，所述基因组合物表达来源于人LRRC24蛋白的细胞穿透肽LRRC24P和增强型绿色荧光蛋白(EGFP)融合的蛋白。
图3显示了将LRRC24P-EGFP基因化合物插入蛋白表达载体pET28a+中，并且通过用限制酶处理结合的基因化合物确认所得物。
图4显示了将LRRC24P-EGFP-pET28a+载体转化到Rosetta(DE3)感受态细胞中，然后诱导蛋白质产生和进行超声消化，然后进行聚丙烯酰胺凝胶电泳。
图5显示了将使用Rosetta(DE3)感受态细胞产生的LRRC24P-EGFP重组融合蛋白纯化并通过聚丙烯酰胺凝胶电泳观察。
图6显示了在用重组蛋白(其中常规的细胞穿透肽与EGFP融合)和LRRC24P-EGFP重组蛋白处理人T免疫细胞系(Jurkat)之后，通过流式细胞术进行的细胞内穿透效率的比较。
图7显示了在按浓度用与细胞穿透肽融合的重组蛋白处理之后，通过流式细胞术进行的细胞内穿透效率的比较。
图8显示了在按时间用与细胞穿透肽融合的重组蛋白处理之后，通过流式细胞术进行的细胞内穿透效率的比较。
图9显示了在用LRRC24P-EGFP重组蛋白处理宫颈癌细胞系(HeLa)之后，使用共聚焦显微镜进行的渗透到细胞中的LRRC24P-EGFP重组蛋白的分布模式。
图10显示了确认本发明的LRRC24P突变体-EGFP融合蛋白在处理人T免疫细胞系(Jurkat)之后，然后使用流式细胞仪(FACS)进行的细胞内转导效率的结果。
详述
除非本文另有定义，否则所用的所有技术和科学术语具有与本领域普通技术人员通常理解的相同的含义。包括本文所包括的术语在内的多种科学事件是本领域熟知的和可获得的。
本发明提供了跨膜结构域或细胞穿透肽，其包含源自人LRRC24(富含亮氨酸重复域24)蛋白的SEQ ID NO:1-22中任一个的多肽。
常规的细胞穿透肽主要是由源自非人，如病毒和果蝇的蛋白质发明的，或者具有低穿透效率的缺点。为了克服这一点，本申请的发明人在人LRRC24蛋白中发现了由氨基酸序列427-436组成的LRRC24P肽，预期其在人来源的肽中具有细胞渗透性。
SEQ ID NO:1表示的细胞穿透肽是由人LRRC24蛋白中的氨基酸序列427-436组成的LRRC24P的细胞穿透肽，用于本发明的氨基酸和核苷酸序列信息如下。
1.LRRC24P肽氨基酸序列(SEQ ID NO:1)：RRRRRRKKAR
2.LRRC24P表达基因核酸序列(SEQ ID NO:23)：cgccggcgccgcaggcgaaaaaaggcgcgg
3.LRRC24P基因合成核酸序列(SEQ ID NO:24)：cgccgccgtagaagacgtaaaaaggcaaga。
在基因合成的情况下，对于密码子优化，如上所述使用转换。为了验证细胞渗透性，表达连接在表达LRRC24P肽(253个氨基酸)的核酸序列后面的EGFP(增强型绿色荧光蛋白)的LRRC24P-EGFP融合蛋白(253个氨基酸)的核酸序列信息与核酸序列25相同，并且在SEQ ID NO：26所述的实施例中使用的LRRC24P细胞穿透肽或跨膜结构域与EGFP蛋白之间的接头部分的核酸序列是ggaggtgggggctcg。LRRC24P细胞穿透肽具有10个氨基酸，是亲水性的并且具有正电荷。
本发明的由SEQ ID NO:2-22表示的细胞穿透肽或跨膜结构域是LRRC24P细胞穿透肽的突变细胞穿透肽，并且是其中丙氨酸(LRRC24P的第9个氨基酸)被另一个氨基酸取代的LRRC24P突变体，以及其中N-末端和C-末端氨基酸分别逐个缺失的LRRC24P的突变的跨膜结构域或细胞穿透肽。本发明的人LRRC24蛋白来源的跨膜结构域或细胞穿透肽显示在下表1中。
表1：人LRRC24蛋白来源的跨膜结构域或细胞穿透肽
[表1]
参考以下详细描述的实施方案，本发明的优点和特征以及实现它们的方法将变得显而易见。在下文中，将通过实施例更详细地描述本发明。然而，以下实施例仅是对本发明的说明，而并非旨在将本发明的内容限制于以下实施例。
<实施例1>表达人LRRC24蛋白来源的细胞穿透肽(LRRC24P)和EGFP荧光蛋白融合的蛋白的质粒载体的构建
为了测试LRRC24的细胞穿透效率，如下进行人工基因合成。如图1所示，合成了5’-NheI限制酶识别序列-LRRC24P核酸序列-接头核酸序列-EGFP蛋白核酸序列-终止密码子-XhoI限制酶识别序列-3’。
特别地，当合成人工基因时，通过密码子优化将LRRC24P核酸序列转换为表达相同肽的核酸序列，并且为了验证细胞渗透性，将表达EGFP荧光蛋白的基因连接到LRRC24P细胞穿透肽的核酸序列上，然后合成(图2)。此外，在细胞穿透肽与EGFP蛋白之间添加接头序列以增加灵活性。在用NheI限制酶和XhoI限制酶处理人工合成基因之后，通过T4连接酶将其引入到用NheI限制酶和XhoI限制酶类似处理的pET28a+质粒载体中。本文所用的pET-28a质粒载体在LRRC24P-EGFP融合蛋白前面的N-末端处表达6个组氨酸(6×His)，使得能够通过Ni-NTA树脂进行蛋白纯化。将引入了LRRC24P-EGFP融合蛋白人工基因的pET28a+质粒载体转化到Top 10感受态细胞中，然后在含有卡那霉素的LB琼脂平板上培养，并且将生长的菌落在LB培养基中培养约16小时。之后，通过DNA提取实验获得质粒，用NheI限制酶和XhoI限制酶处理，然后通过琼脂糖凝胶电泳确认了LRRC24P-EGFP融合蛋白的人工基因成功地引入到pET-28a质粒载体中(图3)。
<实施例2>人LRRC24蛋白来源的细胞穿透肽(LRRC24P)和EGFP荧光蛋白融合的蛋白的表达条件和高纯度纯化的建立
为了建立源自人LRRC24蛋白的细胞穿透肽(LRRC24P)和EGFP荧光蛋白融合的蛋白的表达条件，将实施例1中制备的质粒载体转化到Rosetta(DE3)感受态细胞中，然后在含有卡那霉素的LB培养基中孵育约16小时。在向其中加入100倍的LB培养基并孵育直至OD值达到0.4-0.6之后，加入IPTG(异丙基β)至终浓度为1mM，在37℃孵育5小时或在20℃孵育16小时。之后，将通过离心获得的细胞进行超声处理，然后通过离心分成上清液和细胞沉淀，并且通过聚丙烯酰胺凝胶电泳和考马斯蓝染色确认蛋白质的表达量和水溶性(图4)。
通过这一点，确认了LRRC24P-EGFP融合蛋白在加入IPTG之后在20℃孵育16小时的条件下增加了蛋白的水溶性和表达效率。因此，为了纯化高纯度LRRC24P-EGFP融合蛋白，将转化的Rosetta(DE3)感受态细胞在相同条件下培养，将培养基离心以获得细胞沉淀，并悬浮于裂解缓冲液(50mM NaH2PO4,300mM NaCl,10mM咪唑)中。
通过超声分解并再次离心收集上清液，通过45μm过滤器过滤，然后使上清液通过Ni-NTA琼脂糖柱以结合重组蛋白。为了去除非特异性结合到Ni-NTA琼脂糖柱上的蛋白，用洗涤缓冲液(50mM NaH2PO4,300mM NaCl,20mM咪唑)洗涤Ni-NTA琼脂糖柱，然后，为了回收结合到Ni-NTA琼脂糖柱上的重组蛋白，使用洗脱缓冲液(50mM NaH2PO4,300mM NaCl,500mM咪唑)洗脱重组蛋白。在使用PD-10脱盐柱用PBS替代含有LRRC24P-EGFP融合蛋白的溶液之后，通过聚丙烯酰胺凝胶电泳和考马斯蓝染色确认(图5)。
<实施例3>LRRC24P-EGFP融合蛋白的细胞内递送效率的确认
为了研究LRRC24P-EGFP融合蛋白的细胞内递送效率，以与表达LRRC24P-EGFP融合蛋白的质粒载体相同的方式构建了表达蛋白的质粒载体，其中EGFP荧光蛋白分别融合至细胞穿透肽，即先前已知的细胞穿透肽(CPP)TAT(氨基酸序列：YGRKKRRQRRR,Diabetes2001Aug；50(8):1706-1713,Proteins Linked to a Protein Transduction DomainEfficiently Transduce Pancreatic Islets)、Hph-1(氨基酸序列：YARVRRRGPRR,NatureMedicine 12(5):574-9,2006年6月,Intranasal delivery of the cytoplasmic domainof CTLA-4using a novel protein transduction domain prevents allergicinflammation)和NP2(氨基酸序列：KIKKVKKKGRK,Nature Communications 6卷,文章号:8244(2015)，dNP2是能使ctCTLA-4蛋白递送以改善实验性自身免疫性脑脊髓炎的血脑屏障可渗透的肽)。现有CPP-EGFP融合蛋白的表达和纯化也以与LRRC24P-EGFP融合蛋白相同的方式进行。对于细胞实验，在将Jurkat细胞1.5×106个/孔分配到24孔板中之后，通过漂浮在含有10％胎牛血清和1％青霉素/链霉素的2ml RPMI 1640培养基中，处理终浓度为5μM的CPP-EGFP融合蛋白，并在保持在37℃的5％CO2培养箱中孵育2小时。此后，将含有细胞的培养基转移到5ml管中，离心去除上清液，并用PBS溶液重复洗涤三次。此后，将Jurkat细胞悬浮在650μl PBS中，并通过流式细胞仪测量引入细胞的CPP-EGFP融合蛋白的荧光以确认细胞内引入效率(图6)。通过这一点，确认了与现有细胞穿透肽相比，LRRC24P细胞穿透肽表现出约3-5倍的效率(图6)。
表2：实施例3中使用的重组蛋白
[表2]
为了通过浓度确认LRRC24P-EGFP融合蛋白的引入效率，使用EGFP蛋白和TAT与EGFP的融合蛋白(其是目前最广泛使用的细胞穿透肽)进行了比较实验。将EGFP、TAT-EGFP和LRRC24P-EGFP融合蛋白以0.5、1、2、5和10μM的终浓度处理Jurkat细胞，并通过与先前实验相同的过程确认引入效率(图7)。通过这一点，确认了LRRC24P-EGFP融合蛋白以浓度依赖性的方式被引入细胞中，并且与TAT-EGFP融合蛋白相比，确认了在所有浓度下的递送效率显著更高。
然后，为了检查LRRC24P-EGFP融合蛋白随时间的引入效率，使用EGFP和TAT-EGFP融合蛋白进行比较实验。将EGFP、TAT-EGFP和LRRC24P-EGFP融合蛋白以5μM的终浓度分别处理Jurkat细胞，分别培养0.5小时、1小时、2小时、4小时和6小时，并通过与先前实验相同的程序确认引入效率(图8)。通过这一点，确认了LRRC24P-EGFP融合蛋白以时间依赖性的方式被引入到细胞中，并且与TAT-EGFP融合蛋白相比，确认了递送效率始终显著更高。
<实施例4>LRRC24P-EGFP融合蛋白的细胞内递送的确认
为了用共聚焦显微镜确认引入细胞的LRRC24P-EGFP融合蛋白，首先将HeLa细胞以2×104个/孔分配到24孔细胞培养载玻片中，然后将其悬浮在500μl含有10％胎牛血清和1％青霉素/链霉素的DMEM培养基中，并在保持在37℃的5％CO2培养箱中孵育18小时。此后，分别用终浓度为1μM的EGFP和LRRC24P-EGFP融合蛋白处理细胞，并培养1小时。此后，去除培养液，用PBS溶液洗涤3次，然后在室温下用4％PFA溶液处理30分钟以固定。在去除PFA溶液之后，再次用PBS溶液洗涤3次，用封片介质处理，覆盖盖玻片，并用共聚焦显微镜观察(图9)。通过这一点，确认了LRRC24P-EGFP融合蛋白被有效地引入HeLa细胞中。
<实施例5>LRRC24P突变体-EGFP融合蛋白的细胞内递送效率的确认
为了确认LRRC24P细胞穿透肽突变体的细胞内递送效率，在设计了丙氨酸(LRRC24P的第9个氨基酸)被另一个氨基酸取代的LRRC24P突变体(突变)以及N-末端和C-末端处的氨基酸逐个缺失的LRRC24P突变体之后，构建了表达LRRC24突变体-EGFP融合蛋白的质粒载体，然后表达并纯化该蛋白。在本发明的LRRC24P细胞穿透肽突变中，用序列名称为LRRC24P M1-M8(SEQ ID NO:2-7或SEQ ID NO:21和28)的肽进行细胞实验。对于细胞实验，将Jurkat细胞以1.5×106个/孔分配到24孔板中，悬浮在2ml含有10％胎牛血清和1％青霉素/链霉素的RPMI 1640培养基中，并用终浓度为5μM的LRRC24P-EGFP和LRRC24P突变体-EGFP融合蛋白处理，然后在保持在37℃的5％CO2培养箱中孵育2小时。此后，将含有细胞的培养基转移到5ml试管中，离心去除上清液，并用PBS溶液重复洗涤3次。此后，将Jurkat细胞悬浮在650μl PBS中，并通过流式细胞仪测量引入细胞的LRRC24P突变体-EGFP融合蛋白的荧光以确认细胞内引入效率(图10)。通过这一点，确认了LRRC24P突变体显示了通过突变导致的引入效率的变化，但保持了细胞穿透能力(图10)。
序列表
<110> KRICT
<120> 源自人LRRC24蛋白的跨膜结构域
<130> M18-5794-A
<150> KR 10-2018-0165438
<151> 2018-12-19
<160> 26
<170> KoPatentIn 3.0
<210> 1
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P
<400> 1
Arg Arg Arg Arg Arg Arg Lys Lys Ala Arg
1 5 10
<210> 2
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-M1
<400> 2
Arg Arg Arg Arg Arg Arg Lys Lys Ser Arg
1 5 10
<210> 3
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-M2
<400> 3
Arg Arg Arg Arg Arg Arg Lys Lys Val Arg
1 5 10
<210> 4
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-M3
<400> 4
Arg Arg Arg Arg Arg Arg Lys Lys Tyr Arg
1 5 10
<210> 5
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-M4
<400> 5
Arg Arg Arg Arg Arg Arg Lys Lys Glu Arg
1 5 10
<210> 6
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-M5
<400> 6
Arg Arg Arg Arg Arg Arg Lys Lys Gln Arg
1 5 10
<210> 7
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-M6
<400> 7
Arg Arg Arg Arg Arg Arg Lys Lys Arg Arg
1 5 10
<210> 8
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-MN
<400> 8
Arg Arg Arg Arg Arg Arg Lys Lys Asn Arg
1 5 10
<210> 9
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-MD
<400> 9
Arg Arg Arg Arg Arg Arg Lys Lys Asp Arg
1 5 10
<210> 10
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-MC
<400> 10
Arg Arg Arg Arg Arg Arg Lys Lys Cys Arg
1 5 10
<210> 11
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-MG
<400> 11
Arg Arg Arg Arg Arg Arg Lys Lys Gly Arg
1 5 10
<210> 12
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-MH
<400> 12
Arg Arg Arg Arg Arg Arg Lys Lys His Arg
1 5 10
<210> 13
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-MI
<400> 13
Arg Arg Arg Arg Arg Arg Lys Lys Ile Arg
1 5 10
<210> 14
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-ML
<400> 14
Arg Arg Arg Arg Arg Arg Lys Lys Leu Arg
1 5 10
<210> 15
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-MK
<400> 15
Arg Arg Arg Arg Arg Arg Lys Lys Lys Arg
1 5 10
<210> 16
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-MM
<400> 16
Arg Arg Arg Arg Arg Arg Lys Lys Met Arg
1 5 10
<210> 17
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-MF
<400> 17
Arg Arg Arg Arg Arg Arg Lys Lys Phe Arg
1 5 10
<210> 18
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-MP
<400> 18
Arg Arg Arg Arg Arg Arg Lys Lys Pro Arg
1 5 10
<210> 19
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-MT
<400> 19
Arg Arg Arg Arg Arg Arg Lys Lys Thr Arg
1 5 10
<210> 20
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-MW
<400> 20
Arg Arg Arg Arg Arg Arg Lys Lys Trp Arg
1 5 10
<210> 21
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-M7
<400> 21
Arg Arg Arg Arg Arg Lys Lys Ala Arg
1 5
<210> 22
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P-M8
<400> 22
Arg Arg Arg Arg Arg Arg Lys Lys Ala
1 5
<210> 23
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P表达核酸
<400> 23
cgccggcgcc gcaggcgaaa aaaggcgcgg 30
<210> 24
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P合成核酸
<400> 24
cgccgccgta gaagacgtaa aaaggcaaga 30
<210> 25
<211> 720
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> LRRC24P EGFP核酸
<400> 25
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
720
<210> 26
<211> 15
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221>
<222>
<223> 接头
<400> 26
ggaggtgggg gctcg 15
Claims (10)
1.跨膜结构域，其包含源自人LRRC24(富含亮氨酸重复域24)蛋白的SEQ ID NO:1-22的多肽中的任一个。
2.具有改善的细胞膜渗透性的重组货物，其包含与权利要求1所述的跨膜结构域的N-末端或C-末端融合的货物和跨膜结构域。
3.如权利要求2所述的重组货物，其中所述货物是蛋白质、核酸、脂质或化合物。
4.如权利要求2所述的重组货物，其中所述货物是选自以下的任一种：激素、免疫球蛋白、抗体、结构蛋白、信号肽、储存肽、膜肽、跨膜肽、内部肽、外部肽、分泌肽、病毒肽、天然肽、糖基化蛋白、片段化蛋白、二硫化物肽、重组蛋白、化学修饰的蛋白和朊病毒。
5.如权利要求2所述的重组货物，其中所述货物是选自以下的任一种：核酸、编码核酸序列、mRNA、反义RNA分子、碳水化合物、脂质和糖脂。
6.如权利要求2所述的重组货物，其中所述货物是造影材料、药物或化学品。
7.基因构建体，其包含编码权利要求1所述的跨膜结构域的多核苷酸。
8.用于表达具有改善的细胞膜渗透性的重组货物蛋白的表达载体，其包含权利要求7所述的基因构建体。
9.将货物递送到细胞中的方法，其包括制备重组货物的步骤；以及使制备的重组货物与分离的细胞接触的步骤，在所述重组货物中，货物与权利要求1所述的跨膜结构域的N-末端或C-末端融合。
10.将货物递送到动物中的方法，其包括制备重组货物的步骤；以及将制备的重组货物施用至除人以外的动物的步骤，在所述重组货物中，货物与权利要求1所述的跨膜结构域的N-末端或C-末端融合。
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2019
- 2019-11-13 KR KR1020190144779A patent/KR102351041B1/ko active IP Right Grant
- 2019-12-16 JP JP2021531077A patent/JP7193633B2/ja active Active
- 2019-12-16 CA CA3122428A patent/CA3122428A1/en active Pending
- 2019-12-16 AU AU2019403533A patent/AU2019403533A1/en not_active Abandoned
- 2019-12-16 SG SG11202105169WA patent/SG11202105169WA/en unknown
- 2019-12-16 CN CN201980079173.8A patent/CN113166216A/zh active Pending
- 2019-12-16 US US17/296,622 patent/US20220306718A1/en active Pending
- 2019-12-16 EP EP19898351.2A patent/EP3875113A4/en active Pending
- 2019-12-16 BR BR112021012019A patent/BR112021012019A2/pt not_active IP Right Cessation
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2021
- 2021-05-20 PH PH12021551150A patent/PH12021551150A1/en unknown
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| WO2013129698A1 (ja) * | 2012-02-27 | 2013-09-06 | 独立行政法人理化学研究所 | 植物細胞に核酸を導入する方法 |
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| KR20140042747A (ko) * | 2012-09-28 | 2014-04-07 | 주식회사 카엘젬백스 | 줄기세포 검출을 위한 텔로머라제 유래 펩티드 및 조영물질의 컨쥬게이트 및 이를 포함하는 조영제 |
| WO2014086835A1 (en) * | 2012-12-05 | 2014-06-12 | Ruprecht-Karls-Universität Heidelberg | Conjugates of proteins and multivalent cell-penetrating peptides and their uses |
| US20150329640A1 (en) * | 2012-12-20 | 2015-11-19 | Bluebird Bio, Inc. | Chimeric antigen receptors and immune cells targeting b cell malignancies |
| CN107428845A (zh) * | 2015-03-16 | 2017-12-01 | 阿尔勒治疗公司 | 包括细胞穿透肽、货物和tlr肽激动剂的新型复合物 |
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Also Published As
| Publication number | Publication date |
|---|---|
| EP3875113A1 (en) | 2021-09-08 |
| KR102351041B1 (ko) | 2022-01-13 |
| PH12021551150A1 (en) | 2021-11-03 |
| KR20200076582A (ko) | 2020-06-29 |
| EP3875113A4 (en) | 2022-08-31 |
| CA3122428A1 (en) | 2020-06-25 |
| AU2019403533A1 (en) | 2021-06-24 |
| SG11202105169WA (en) | 2021-06-29 |
| JP7193633B2 (ja) | 2022-12-20 |
| JP2022510309A (ja) | 2022-01-26 |
| US20220306718A1 (en) | 2022-09-29 |
| BR112021012019A2 (pt) | 2021-11-03 |
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