CN113121702A - 用于胞内递送分子的多聚化递送系统 - Google Patents
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Abstract
本发明涉及分子生物学领域,更具体的说,涉及一种多聚化递送系统,其可用于胞内递送货物分子。本发明的多聚化递送系统能够实现货物分子高效内吞以及从内吞小泡的高效释放,显著提升货物分子的细胞质递送效率。一旦在细胞质中可获得货物分子时,它们就可发挥与其相关的任何作用。因此,本发明的多聚化递送系统提供了用于影响细胞的生物学机制和途径的有效手段,其可用于研究、治疗、诊断等诸多领域。
Description
技术领域
本发明涉及分子生物学领域,更具体的说,涉及用于胞内递送货物分子的多聚化递送系统。
背景技术
细胞膜作为选择性透过性屏障对细胞存活和功能至关重要,虽然小分子物质可以通过细胞的天然过程或脂质双层的直接扩散穿过细胞膜,但是在大多数情况下,外源性的活性生物大分子等细胞内载物从细胞质膜的有效通过,仍然是细胞运送过程的主要障碍。因此,一种能够有效提高细胞内载物向活细胞运输效率的分子转运工具对于其在生物医药等领域的应用极为重要。
目前,利用细胞穿膜肽(Cell-penetrating peptides,CPPs)介导生物大分子入胞是生物医药领域的研究热点之一。CPPs一般为5-30个氨基酸的多肽,可以通过化学交联、融合表达或非共价结合的方式介导包括蛋白质在内的生物大分子穿过细胞膜进入胞内。由于在介导生物大分子入胞时具有用量低、转运所需时间短、剂量可控、操作简单、免疫反应及毒副作用低等优点,细胞穿膜肽已经被应用于生物医药的各个领域。
尽管CPPs在转导生物大分子上具有如上诸多优势,但仍存在一些局限性,主要包括其本身较低的递送效率以及在血清存在条件下进一步降低的递送效率。
已有研究表明,CPPs携带的生物大分子仅有1%能在内吞小泡逃逸成功,大部分最终导向溶酶体而被降解。因此,细胞内载物的小泡逃逸效率也是CPPs胞内递送过程的关键限制因素,其效率决定了总体递送效率。已有大量研究致力于解决CPPs递送效率较低的问题,在提升内吞小泡逃逸效率方面,主要策略为在成熟酸化的过程中破坏小泡囊膜的完整性,从而使其内容物(包括被递送的细胞内载物)释放到胞浆中。目前最有效的方式是利用来源于病毒、细菌、动植物或者人类的pH敏感肽(pH sensitive fusogenic peptides)。pH敏感肽含有一定比例的疏水氨基酸,且在低pH值的情况下会发生构象的剧烈改变。在CPPs介导细胞内吞后,内吞小泡在成熟酸化的过程中,一旦pH值下降到临界点,与CPPs偶联的pH敏感肽发生构象改变并与小泡囊膜的脂双层结合从而剧烈扰动磷脂双层膜的完整性,在其上形成小孔或导致囊膜裂解,最后将被转运的生物大分子释放进入胞浆。然而随后的研究发现,虽然融合pH敏感肽后的CPPs大分子胞内递送系统的内吞小泡逃逸效率确实得到提高,但相当一部分的被转运大分子仍留存在内吞小泡内(以荧光蛋白作为被转运大分子可见明显的点状分布)。
另一个限制穿膜肽应用的因素是血清不耐受性,即在高浓度血清条件下,CPPs会因其带有的正电荷与血红蛋白上的负电荷之间的静电作用,导致其不能与细胞膜充分接触,从而导致其递送效率急剧下降,这也严重限制了CPPs在体内的广泛应用。
综上所述,虽然针对CPPs介导大分子入胞效率的提升已开展了大量的工作,但效果仍不尽如人意。入胞效率仍然是将CPPs广泛应用于靶向胞内靶标的生物大分子药物开发领域的主要限制因素。同时,血清耐受性也是CPPs用于药物开发必需解决的问题,而目前尚无针对这一问题的研究报道。
发明内容
本申请的发明人经过大量实验和反复摸索,出人意料地发现通过引入多聚化结构域能够显著提高货物分子的内吞效率,并且显著提升血清耐受性。此外,将pH敏感肽与特定蛋白酶识别序列联用又能够显著提高货物分子从内吞小泡的释放,进一步提升货物分子的细胞质递送效率,使货物分子充分发挥其相应的生物学功能。基于这些发现,本发明人开发了能够实现高效细胞质递送的多聚化递送系统。
融合多肽
因此,在第一方面,本发明提供了一种融合多肽,其包含多聚化结构域序列、细胞穿膜肽、pH敏感融合肽(pH-sensitive fusogenic peptide)以及蛋白酶识别序列。
在本发明中,术语“多聚化结构域”是指,能够使本文所述的融合多肽的若干拷贝多聚化(即形成生物分子复合物)的任何多肽或蛋白质。在某些实施方案中,所述多聚化结构域是同源多聚化结构域,其将相同的融合多肽聚集并形成多聚体。
在某些实施方案中,所述多聚化结构域可以是二聚化结构域、三聚化结构域、四聚化结构域,或基本上任何更高级别的多聚化结构域,只要该结构域能够促进至少两个结构域(和它们形成部分的多肽)之间的相互作用。
在某些实施方案中,所述多聚化结构域选自:亮氨酸拉链、NOE(SEQ ID NO:3)、GCN4-P1(SEQ ID NO:4)、Delta(SEQ ID NO:5)及其任何组合。
在某些实施方案中,所述多聚化结构域选自亮氨酸拉链。
在某些实施方案中,所述多聚化结构域序列包含SEQ ID NO:1或2所示的序列。
在本发明中,术语“pH敏感融合肽(pH-sensitive fusogenic peptide)”与“pH敏感肽”可互换使用,其是指一类能够在酸性条件(例如,pH<6.5)下发生构象改变从而促进与内吞小泡囊膜融合的多肽。当pH敏感肽被细胞内吞后,内吞小泡在成熟酸化的过程中,一旦pH值下降到临界点,此类多肽发生构象改变并与小泡囊膜的脂双层结合从而剧烈扰动磷脂双层膜的完整性,在其上形成小孔或导致囊膜裂解,从而将被转运的生物大分子释放进入胞浆。这类多肽是本领域熟知的,并描述于例如,Varkouhi,Amir K.,et al.Journal ofControlled Release 151.3(2011):220-228;Erazo-Oliveras A,Muthukrishnan N,BakerR,et al.Pharmaceuticals,2012,5(11):1177-1209,其全部通过引用并入本文。
能够用于本发明的融合蛋白的pH敏感肽可以选自下列的蛋白或多肽,或者源自选自下列蛋白或多肽:
病毒蛋白来源:HA2(流感病毒)及其突变体KALA,GALA;五邻体蛋白(penton base)(腺病毒或鼻病毒),gp41(HIV),L2(乳头瘤病毒),包膜蛋白(西尼罗河病毒(West Nilevirus));
细菌蛋白来源:单增李斯特氏菌溶血素O(Listeriolysin O(LLO)),肺炎球菌溶血素(Pneumococcal pneumolysin(PLO)),链球菌溶血素O(Streptococcal streptolysin O(SLO)),白喉毒素(Diphtheria toxin),铜绿假单胞菌外毒素A(Pseudomonas aeruginosaexotoxin A),志贺毒素(Shiga toxin),霍乱毒素(Cholera toxin);
植物蛋白来源:蓖麻毒素(Ricin),皂草素(Saporin),Gelonin毒素;
人类/动物蛋白来源:人降钙素,成纤维细胞生长因子受体,蜂毒素(Melittin);
人工合成多肽:(R-Ahx-R)(4)AhxB,穿透素(Penetratin(pAntp)),EB1,牛朊蛋白(Bovine prion protein(bPrPp)),甜箭肽(Sweet Arrow Peptide(SAP)),聚L-组氨酸(Poly(L-histidine)),脯氨酸富含肽(Proline-rich)。
在某些实施方案中,所述pH敏感融合肽选自流感病毒HA2(SEQ ID NO:36)或其突变体、蜂毒素(Melittin)(SEQ ID NO:39),及其任意组合。在某些实施方案中,所述流感病毒HA2的突变体选自INF7(SEQ ID NO:12)、KALA(SEQ ID NO:37)或GALA(SEQ ID NO:38)。
在某些实施方案中,所述pH敏感融合肽包含INF7。在某些实施方案中,所述pH敏感融合肽包含下述序列或由其组成:SEQ ID NO:12。
在本发明中,术语“细胞穿膜肽(cell penetrating peptide,CPP)”又称为“细胞穿透肽”、“蛋白质转导域(protein translocation domain,PTD)、“Trojan horsepeptides”或“转导肽(transduction peptide)”等,其是指,能够促进各种分子(例如,各种大分子包括蛋白或核酸)的细胞摄取的多肽。这类多肽是本领域熟知的,并描述于例如,Stewart KM,et al.Org Biomol Chem.2008Jul 7;6(13):2242-55以及中国专利申请CN101490081A(其全部通过引用并入本文);或者可以通过本领域已知的方法获得,例如详细描述于美国专利申请US 2008/0234183中的方法,其全部通过引用并入本文。
能够用于本发明的融合蛋白的CPP包括但不限于:阳离子型:Penetratin、HIV-TAT-47-57、HIV-1Rev 34–50、FHV coat-35-49、低聚精氨酸(Oligoarginines)(R9–R12)、CCMV Gag-7-25、S413-PV、VP22、BP16、DPV3、DPV6、FAH外壳蛋白、鱼精蛋白1(Protamine 1)、人cJun、Engrailed-2、Islet-1、HoxA-13、TP10等;两亲性型:转运肽(transportan)、Transportan 10、Pep-1、MPGα、MPGβ、CADY、Pepfect6、Pepfect14、Pepfect15、NickFect、Hel、sC18、pVEC、ARF(1-22)、YTA2、PAR1(Palmitoyl-SFLLRN)、F2Pal10(Palmitoyl-SFLLRN)、BPrPp(1-30)、hLF肽(19–40)、Buforin 2、Crotamine、Azurin p18、hCT肽(18-32)、S413-PVrev等;疏水型:Kaposi's肉瘤成纤维细胞生长因子、源自Caiiman crocodylus的Ig轻链的信号肽、整合素β3片段、Grb2-SH2结构域、HIV-1gp41(1-23)、HBV移位基序(translocation motif)、精卵融合蛋白(89–111)、人降钙素(9–32)、Pep-7、C105Y、K-FGF等。
此外,用作本发明的融合蛋白中的CPP还可以选自与如上所述的任何多肽序列具有大约60、70、80、90、95、99%或100%的序列同一性的多肽序列,只要该多肽序列仍然保留其生物学活性,即,促进分子的细胞摄取。
在某些实施方案中,所述细胞穿膜肽选自穿透素(Penetratin)(SEQ ID NO:40)、Tat衍生肽、Rev(34-50)(SEQ ID NO:42)、VP22(SEQ ID NO:43)、转运肽(transportan)(SEQID NO:44)、Pep-1(SEQ ID NO:45)、Pep-7(SEQ ID NO:46),及其任意组合。在某些实施方案中,所述Tat衍生肽选自Tat(48-60)(SEQ ID NO:14)或Tat(47-57)(SEQ ID NO:41)。
在某些实施方案中,所述细胞穿膜肽包含Tat衍生肽,例如Tat(48-60)。在某些实施方案中,所述细胞穿膜肽包含下述序列或由其组成:SEQ ID NO:14。
在某些实施方案中,所述蛋白酶选自弗林蛋白酶和/或溶酶体半胱氨酸蛋白酶。在某些实施方案中,所述蛋白酶识别序列选自弗林蛋白酶识别序列、溶酶体半胱氨酸蛋白酶识别序列及其组合。
在某些实施方案中,所述弗林蛋白酶识别序列包含下述序列或由其组成:R-X1-X2-R↓(SEQ ID NO:47),其中,X1为任意氨基酸,X2为K或R,↓表示切割位点。
在某些实施方案中,所述弗林蛋白酶识别序列包含下述序列或由其组成:R-R-X1-X2-R↓(SEQ ID NO:48)。
在某些实施方案中,X1选自丙氨酸(A)、精氨酸(R)、天冬氨酸(D)、半胱氨酸(C)、谷氨酰胺(Q)、谷氨酸(E)、组氨酸(H)、异亮氨酸(I)、甘氨酸(G)、天冬氨酸(N)、亮氨酸(L)、赖氨酸(K)、甲硫氨酸(M)、苯丙氨酸(F)、辅氨酸(P)、丝氨酸(S)、苏氨酸(T)、色氨酸(W)、酪氨酸(Y)和缬氨酸(V)。
在某些实施方案中,所述弗林蛋白酶识别序列包含下述序列或由其组成:RRHKR↓(SEQ ID NO:49)。
在某些实施方案中,所述弗林蛋白酶识别序列包含下述序列或由其组成:QSVASSRRHKR↓FAGV(SEQ ID NO:8)。
在某些实施方案中,所述溶酶体半胱氨酸蛋白酶选自组织蛋白酶B、组织蛋白酶C、组织蛋白酶X、组织蛋白酶S、组织蛋白酶L、组织蛋白酶D或组织蛋白酶H。
在某些实施方案中,所述溶酶体半胱氨酸蛋白酶是组织蛋白酶L。
在某些实施方案中,所述组织蛋白酶L识别序列包含下述序列或由其组成:NNTHDLVGDVRLAGV(SEQ ID NO:10)。
在某些实施方案中,所述蛋白酶识别序列包含弗林蛋白酶识别序列和组织蛋白酶L识别序列。在某些实施方案中,所述蛋白酶识别序列是单链多肽,其从N端至C端包含弗林蛋白酶识别序列和组织蛋白酶L识别序列,或者其从N端至C端包含组织蛋白酶L识别序列和弗林蛋白酶识别序列。
在某些实施方案中,所述蛋白酶识别序列包含RRHKR(SEQ ID NO:49)和NNTHDLVGDVRLAGV(SEQ ID NO:10)。在某些实施方案中,所述蛋白酶识别序列包含SEQ IDNO:8和SEQ ID NO:10。
在一些实施方案中,所述融合多肽从N端至C端包含:所述细胞穿膜肽、pH敏感融合肽、蛋白酶识别序列,并且所述多聚化结构域序列位于所述融合多肽的N端或C端,或者位于上述任何两个相邻结构域之间。在某些示例性实施方案中,所述多聚化结构域序列位于所述融合多肽的N端,或者位于所述融合多肽的C端。
在另一些实施方案中,所述融合多肽从N端至C端包含:所述pH敏感融合肽、细胞穿膜肽、蛋白酶识别序列,并且所述多聚化结构域序列位于所述融合多肽的N端或C端,或者位于上述任何两个相邻结构域之间。在某些示例性实施方案中,所述多聚化结构域序列位于所述融合多肽的N端,或者位于所述融合多肽的C端。
在某些实施方案中,所述蛋白酶识别序列从N端至C端包含所述弗林蛋白酶识别序列和组织蛋白酶L识别序列。在某些实施方案中,所述蛋白酶识别序列从N端至C端包含所述组织蛋白酶L识别序列和弗林蛋白酶识别序列。
在某些实施方案中,所述融合多肽中所包含的任意相邻结构域之间任选地通过肽接头连接。在某些实施方案中,所述肽接头是(GmS)n,其中m选自1-4的整数(例如,1,2,3或4),n选自1-3的整数(例如,1,2或3)。在某些实施方案中,所述肽接头是SEQ ID NO:50。在某些实施方案中,任意相邻结构域之间的肽接头可以相同或不同。
在某些实例性实施方案中,所述融合多肽包含SEQ ID NOs:16-18任一项所示的序列。
在第二方面,本发明提供了第一方面所述的融合多肽的多聚体。
在某些实施方案中,所述多聚体是二聚体、三聚体或四聚体。在某些实施方案中,所述多聚体是二聚体。
在某些实施方案中,所述多聚体是同源多聚体。
融合蛋白
在第三方面,本发明提供了一种融合蛋白,其包含第一方面所述的融合多肽,以及另外的多肽。
在某些实施方案中,所述另外的多肽包含可检测的标记,例如酶、荧光蛋白或生物素等。
在某些实施方案中,所述另外的多肽包含表位标签(epitope tag)、报告基因编码蛋白序列和/或核定位信号(NLS)序列。
能够用于本发明的表位标签是本领域技术人员熟知的,其实例包括但不限于His、V5、FLAG、HA、Myc、VSV-G、Trx等,并且本领域技术人员已知如何根据期望目的(例如,纯化、检测或示踪)选择合适的表位标签。在某些实例性实施方案中,所述另外的多肽包含His标签。
能够用于本发明的报告基因序列是本领域技术人员熟知的,其实例包括但不限于GST、HRP、CAT、GFP、HcRed、DsRed、CFP、YFP、BFP等。
能够用于本发明的核定位信号(NLS)序列是本领域技术人员熟知的,其实例包括但不限于SV40病毒大T抗原的NLS。在某些实例性实施方案中,所述NLS序列如SEQ ID NO:22所示。
在某些实例性实施方案中,所述另外的多肽是锌指蛋白(例如ZFP9)、或蛋白磷酸酶(例如Ppm1b)。在某些实例性实施方案中,所述锌指蛋白包含NLS序列。
在某些实施方案中,所述另外的多肽是核酸结合结构域序列。
在某些实施方案中,所述核酸结合结构域序列是锌指蛋白(例如ZFP9)。
在某些实施方案中,所述核酸结合结构域序列包含SEQ ID NO:31所示的序列。
在某些示例性实施方案中,当所述另外的多肽为锌指蛋白(例如ZFP9)时,所述多聚化结构域序列包含SEQ ID NO:2所示的序列。
在某些示例性实施方案中,所述融合蛋白包含SEQ ID NOs:19-21任一项所示的序列。
在某些实施方案中,所述融合蛋白从N端至C端包含:所述细胞穿膜肽、pH敏感融合肽、蛋白酶识别序列和另外的多肽,并且,所述多聚化结构域序列位于所述融合蛋白的N端或C端,或者位于上述任何两个相邻结构域之间。在某些示例性实施方案中,所述多聚化结构域序列位于所述融合蛋白的N端,或者位于所述蛋白酶识别序列的C端。优选地,所述蛋白酶识别序列从N端至C端包含所述弗林蛋白酶识别序列和组织蛋白酶L识别序列,或者从N端至C端包含所述组织蛋白酶L识别序列和弗林蛋白酶识别序列。
在某些实施方案中,所述融合蛋白从N端至C端包含:所述pH敏感融合肽、细胞穿膜肽、蛋白酶识别序列和另外的多肽;并且,所述多聚化结构域序列位于所述融合蛋白的N端或C端,或者位于上述任何两个相邻结构域之间。在某些示例性实施方案中,所述多聚化结构域序列位于所述融合蛋白的N端,或者位于所述蛋白酶识别序列的C端。优选地,所述蛋白酶识别序列从N端至C端包含所述弗林蛋白酶识别序列和组织蛋白酶L识别序列,或者从N端至C端包含所述组织蛋白酶L识别序列和弗林蛋白酶识别序列。
在某些实施方案中,所述另外的多肽融合至所述融合多肽的C端。
在某些实施方案中,所述另外的多肽的分子量小于10000Da,例如小于5000Da,小于3000Da,或小于1000Da。
在第四方面,本发明提供了第三方面所述的融合蛋白的多聚体。
在某些实施方案中,所述多聚体是二聚体、三聚体或四聚体。在某些实施方案中,所述多聚体是二聚体。
在某些实施方案中,所述多聚体是同源多聚体。
多聚体的制备
本发明的融合多肽或融合蛋白可以本领域已知的各种方法来制备,例如,通过基因工程方法(重组技术)产生,也可以通过化学合成方法(例如Fmoc固相方法)产生。本发明的融合蛋白不受其产生方式的限定。
因此,在另一方面,本发明提供了一种分离的核酸分子,其包含编码本发明的融合多肽或融合蛋白的核苷酸序列。
在另一方面,本发明提供了一种载体(例如克隆载体或表达载体),其包含如上所述的分离的核酸分子。在某些实施方案中,所述载体是例如质粒,粘粒,噬菌体等。
在另一方面,本发明提供了一种宿主细胞,其包含如上所述的分离的核酸分子或载体。此类宿主细胞包括但不限于,原核细胞例如大肠杆菌细胞,以及真核细胞例如酵母细胞,昆虫细胞,植物细胞和动物细胞(如哺乳动物细胞,例如小鼠细胞、人细胞等)。
在另一方面,提供了制备本发明的融合多肽或融合蛋白的方法,其包括,在允许所述蛋白表达的条件下,培养如上所述的宿主细胞,和从培养的宿主细胞培养物中回收所述融合多肽或融合蛋白,其中所述融合多肽或融合蛋白以多聚体形式存在。
在另一方面,提供了制备本发明的多聚体的方法,其包括,在允许所述蛋白表达的条件下,培养如上所述的宿主细胞,和从培养的宿主细胞培养物中回收所述多聚体。
复合物
在第五方面,本发明提供了一种复合物,其包含第二方面所述的多聚体以及货物分子;或者,其包含第四方面所述的多聚体以及货物分子。所述货物分子可以是任意的分子。
在某些实施方案中,所述货物分子选自蛋白、核酸、糖类、脂质、化学化合物以及其任意的混合物。
在某些实施方案中,所述货物分子的分子量小于10000Da,例如小于5000Da,小于3000Da,或小于1000Da。
在某些实施方案中,所述货物分子包含可检测的标记,例如酶、放射性核素、荧光染料、化学发光物质或生物素等。
在某些实施方案中,所述货物分子包含药学活性剂。
肽或蛋白质
在一些实施方案中,所述货物分子是肽或蛋白。在某些实施方案中,所述货物分子与组成所述多聚体的融合多肽或融合蛋白是融合的。在某些实施方案中,所述复合物包含第二方面所述的多聚体。
在某些实施方案中,所述复合物包含单链多肽,其中:
(i)所述单链多肽从N端至C端包含:所述细胞穿膜肽、pH敏感融合肽、蛋白酶识别序列和货物分子;并且,所述多聚化结构域序列位于所述融合蛋白的N端或C端,或者位于上述任何两个相邻结构域之间;优选地,所述蛋白酶识别序列从N端至C端包含所述弗林蛋白酶识别序列和组织蛋白酶L识别序列,或者从N端至C端包含所述组织蛋白酶L识别序列和弗林蛋白酶识别序列;或,
(ii)所述融合蛋白包含从N端至C端:所述pH敏感融合肽、细胞穿膜肽、蛋白酶识别序列和货物分子;并且,所述多聚化结构域序列位于所述融合蛋白的N端或C端,或者位于上述任何两个相邻结构域之间;优选地,所述蛋白酶识别序列从N端至C端包含所述弗林蛋白酶识别序列和组织蛋白酶L识别序列,或者从N端至C端包含所述组织蛋白酶L识别序列和弗林蛋白酶识别序列;或,
(iii)所述货物分子融合至所述融合多肽的C端。
核酸
在另一些实施方案中,所述货物分子是核酸。在某些实施方案中,所述核酸选自DNA分子、RNA分子、siRNA、反义寡核苷酸、核酶、适体(aptamer)及其任意组合。
在某些实施方案中,所述复合物包含第四方面所述的多聚体,其中,组成所述多聚体的融合蛋白包含核酸结合结构域序列。在某些实施方案中,所述核酸结合结构域序列是锌指蛋白(例如ZFP9)。在某些实施方案中,所述核酸结合结构域序列包含SEQ ID NO:31所示的序列。在某些示例性实施方案中,当所述另外的多肽为锌指蛋白(例如ZFP9)时,所述多聚化结构域序列包含SEQ ID NO:2所示的序列。
连接方式
在某些实施方案中,所述货物分子与所述多聚体(或组成所述多聚体的融合多肽或融合蛋白)融合、化学偶联或非共价连接。
在一些实施方案中,第二方面或第四方面所述的多聚体(或组成所述多聚体的融合多肽或融合蛋白)与货物分子融合。在某些实施方案中,所述货物分子是肽或蛋白。
在一些实施方案中,第二方面或第四方面所述的多聚体(或组成所述多聚体的融合多肽或融合蛋白)与货物分子化学偶联(chemical coupling)。所述“化学偶联”指在多聚体(或组成所述多聚体的融合多肽或融合蛋白)所包含的反应基团与货物分子所包含的反应基团之间的化学反应中获得的键合,所述化学反应后两个部分通过共价键连接。在上述化学反应(偶联反应)之前,可以用接头分子在独立的反应中对多聚体、货物分子或二者进行修饰以使它们分别包含该化学偶联所需的反应基团。用于修饰多聚体或货物分子的接头分子的选择取决于所使用的偶联策略。
在某些实施方案中,所述共价键为二硫键、磷酸二酯键、硫代磷酸酯键、酰胺键、胺键、硫醚键、醚键、酯键或碳-碳键。
在某些实施方案中,所述货物分子偶联至所述多聚体的C端。
在某些实施方案中,所述货物分子是核酸。
在另一些实施方案中,第二方面或第四方面所述的多聚体与货物分子非共价连接。
在某些实施方案中,所述多聚体与货物分子通过静电作用缀合。
在某些实施方案中,所述货物分子是核酸。
复合物的制备
在一些实施方案中,当所述复合物包含化学偶联的多聚体与货物分子时,本发明的复合物可以通过下述实例性方法获得:在允许所述多聚体和货物分子所分别包含的反应基团发生化学反应的条件下,将所述多聚体与货物分子混合,以使得所述两个部分通过共价键连接。在某些实施方案中,所述方法还包括:使用接头分子对多聚体、货物分子或二者进行修饰以使它们分别包含上述化学反应所需的反应基团。在某些实施方案中,所述货物分子是核酸。
在另一些实施方案中,当所述复合物包含通过静电作用缀合的多聚体与货物分子时,本发明的复合物可以通过下述实例性方法获得:(1)使本发明的多聚体与货物分子混合,以形成混合物;和(2)培育所述混合物以使得所述多聚体与货物分子形成复合物。在某些实施方案中,所述货物分子是核酸。在此类实施方案中,组成所述多聚体的融合蛋白优选地还包含核酸结合结构域序列。
用途及方法
本发明的多聚体或复合物能够显著提高货物分子的内吞效率,使货物分子从内吞小泡中高效释放,一旦在细胞质中可获得货物分子时,它们就可发挥与其相关的任何作用。因此,本发明的多聚体或复合物可用作胞内递送试剂,从而进一步用于研究以及治疗和诊断应用。
因此,在另一方面,本发明提供了一种药物组合物,其含有本发明的融合多肽、融合蛋白、多聚体、复合物、分离的核酸分子、载体或宿主细胞,以及药学上可接受的载体和/或赋形剂。
在某些实施方案中,所述药物组合物包含第五方面所述的复合物。
在某些实施方案中,所述复合物所包含的货物分子是药学活性剂或可检测的标记。所述标记可用于诊断、用于研究药物处置(例如,吸收、分布、代谢、排泄)、用于研究治疗或药物的功效或副作用等。
在另一方面,本发明还涉及本发明的融合多肽、融合蛋白、多聚体、复合物或包含编码所述融合多肽或融合蛋白的核苷酸序列的分离的核酸分子、载体或宿主细胞,在制备用于治疗疾病的药物中的用途;其中,所述复合物中所包含的货物分子能够治疗所述疾病。
在某些实施方案中,所述疾病是与细胞程序性坏死相关的疾病,其中,所述货物分子包含蛋白磷酸酶1B(Ppm1b)。在某些实施方案中,所述与细胞程序性坏死相关的疾病包括肝损伤(例如药源性肝损伤,例如乙酰氨基酚APAP导致的急性肝损伤)、炎性疾病(例如周身性炎症反应SIRS)、缺血再灌注损伤和/或神经退行性疾病。在某些实施方案中,所述疾病是与TNF-α导致的细胞程序性坏死相关的疾病,例如TNF-α导致的周身性炎症反应SIRS。
本发明的融合多肽、融合蛋白、多聚体、复合物或药物组合物可以为医学领域已知的任何形式,例如,可以是片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)、吸入剂、喷雾剂等形式。优选剂型取决于预期的给药方式和治疗用途。
本发明的融合多肽、融合蛋白、多聚体、复合物或药物组合物,可以通过本领域已知的任何合适的方法来施用,包括但不限于,口服、直肠、肠胃外或局部给药。
一种示例性施用途径是口服给药。用于口服给药的液体剂型包括药学上可接受的乳剂、微乳剂、溶液剂、悬浮剂、糖浆剂、酏剂等。除活性成分以外,液体剂型可含有本领域常用的惰性稀释剂,例如水或其它溶剂、增溶剂和乳化剂,例如乙醇、异丙醇、醋酸乙酯、乙酸乙酯、苯甲醇、苯甲酸苄酯、丙二醇、1,3-丁二醇、二甲基甲酰胺、油类(例如棉籽油、花生油、玉米油、胚芽油、橄榄油、蓖麻油和芝麻油)、甘油、四氢糠醇、聚乙二醇和脱水山梨糖醇的脂肪酸酯及其混合物。除惰性稀释剂以外,口服给药的液体剂型也可包括助剂,例如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和芳香剂等。用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、锭剂、粉剂、颗粒剂等。除活性成分以外,固体剂型可含有药学上可接受的惰性赋形剂或载体,例如填充剂(如乳糖、蔗糖、葡萄糖、甘露醇、淀粉、微晶纤维素、半乳糖、交联聚维酮和硫酸钙);粘合剂(如羧甲基纤维素、海藻酸盐、明胶、聚乙烯吡咯烷酮、蔗糖和阿拉伯胶);湿润剂(如鲸蜡醇和单硬脂酸甘油酯);崩解剂(如琼胶、碳酸钙、淀粉、褐藻酸、羧甲基纤维素钠、羧甲基淀粉钠);润滑剂(如滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、月桂基硫酸钠);及其混合物。
本发明的融合多肽、融合蛋白、多聚体、复合物或药物组合物也可通过非口服途径给药。
因此,另一种示例性的施用途径是肠胃外给药,例如,皮下注射、静脉注射、腹膜内注射、肌肉注射、胸骨内注射和注入。用于肠道外给药的剂型可以为注射制剂,包括注射液、注射用无菌粉末或注射用浓溶液。除活性成分以外,注射剂型可含有药学上可接受的载体例如无菌水、林格氏液和等渗氯化钠溶液,也可根据药物的性质加入适宜的附加剂例如抗氧化剂、缓冲剂和抑菌剂。
另一种示例性的施用途径是局部给药,例如经皮给药(如通过经皮贴剂或离子电渗装置给药)、眼内给药或者鼻内或吸入给药。用于经皮给药的剂型可以为局部凝胶剂、喷雾剂、软膏剂和霜剂。除活性成分以外,局部剂型可含有能够提高该活性成分通过皮肤或其它作用区域的吸收或渗透的成分。
另一种示例性的施用途径是直肠给药。用于直肠给药的剂型可以为栓剂。
此外,还可以使用药学领域已知的其它载体材料和给药方式。本发明的融合多肽、融合蛋白、多聚体、复合物或药物组合物可以通过任何公知的制药工艺制备,例如有效的制剂和给药方法。
在另一方面,本发明提供了一种试剂盒,其含有本发明的融合多肽、融合蛋白、多聚体、复合物、分离的核酸分子、载体或宿主细胞。在某些实施方案中,所述试剂盒进一步包括用于转染和/或胞内递送(intracellular delivery)的说明书。在某些实施方案中,所述试剂盒用于转染和/或胞内递送货物分子(例如,核酸、肽或蛋白、糖类、脂质、化学化合物以及其任意的混合物)。在某些实施方案中,所述细胞是哺乳动物细胞,例如人类细胞。
在另一方面,本发明还涉及本发明的融合多肽、融合蛋白、多聚体、复合物、分离的核酸分子、载体或宿主细胞,用作递送试剂(例如转染试剂或胞内递送试剂)的用途。在某些实施方案中,所述递送试剂用于胞内递送货物分子(例如,核酸、肽或蛋白、糖类、脂质、化学化合物以及其任意的混合物)。在某些实施方案中,所述细胞是哺乳动物细胞,例如人类细胞。
在另一方面,本发明提供了一种用于将货物分子递送入细胞的方法,其包括:使所述细胞与第五方面所述的复合物接触,其中所述货物分子是所述复合物中所包含的货物分子。
在某些实施方案中,所述细胞与所述复合物的接触在体内实施。
在某些实施方案中,所述细胞与所述复合物的接触离体实施。
在某些实施方案中,所述细胞与所述复合物的接触在体外实施。
在某些实施方案中,所述细胞是真核细胞,例如哺乳动物细胞,例如人类细胞。
术语定义
在本发明中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本文中所用的细胞培养、生物化学、核酸化学、免疫学实验室等操作步骤均为相应领域内广泛使用的常规步骤。同时,为了更好地理解本发明,下面提供相关术语的定义和解释。
如本文中所使用的,术语“载体(vector)”是指,可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时,载体称为表达载体。载体可以通过转化,转导或者转染导入宿主细胞,使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的,包括但不限于:质粒;噬菌粒;柯斯质粒;人工染色体,例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC);噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于,逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件,包括但不限于,启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外,载体还可含有复制起始位点。
如本文中所使用的,术语“宿主细胞”是指,可用于导入载体的细胞,其包括但不限于,如大肠杆菌或枯草菌等的原核细胞,如酵母细胞或曲霉菌等的真菌细胞,如S2果蝇细胞或Sf9等的昆虫细胞,或者如纤维原细胞,CHO细胞,COS细胞,NSO细胞,HeLa细胞,BHK细胞,HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的,术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如,两个DNA分子的每一个中的某个位置都被腺嘌呤占据,或两个多肽的每一个中的某个位置都被赖氨酸占据),那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如,如果两个序列的10个位置中有6个匹配,那么这两个序列具有60%的同一性。例如,DNA序列CTGACT和CAGGTT共有50%的同一性(总共6个位置中有3个位置匹配)。通常,在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用,例如,可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48:443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.ApplBiosci.,4:11-17(1988))的算法,使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外,可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoIBiol.48:444-453(1970))算法,使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如,Immunology-ASynthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991)),其以引用的方式并入本文中。在本发明中,术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中,氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如,丙氨酸可用A或Ala表示。
如本文中所使用的,术语“药学上可接受的载体和/或赋形剂”是指,在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂,其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19thed.Pennsylvania:Mack Publishing Company,1995),并且包括但不限于:pH调节剂,表面活性剂,离子强度增强剂,维持渗透压的试剂,延迟吸收的试剂,稀释剂,佐剂,防腐剂,稳定剂等。例如,pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子,阴离子或者非离子型表面活性剂,例如Tween-80。离子强度增强剂包括但不限于氯化钠。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水,水性缓冲液(如缓冲盐水),醇和多元醇(如甘油)等。佐剂包括但不限于铝佐剂(例如氢氧化铝),弗氏佐剂(例如完全弗氏佐剂)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂,例如硫柳汞,2-苯氧乙醇,对羟苯甲酸酯,三氯叔丁醇,苯酚,山梨酸等。稳定剂具有本领域技术人员通常理解的含义,其能够稳定药物中的活性成分的期望活性(例如对PSD-95泛素化的抑制活性),包括但不限于谷氨酸钠,明胶,SPGA,糖类(如山梨醇,甘露醇,淀粉,蔗糖,乳糖,葡聚糖,或葡萄糖),氨基酸(如谷氨酸,甘氨酸),蛋白质(如干燥乳清,白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文中所使用的,术语“治疗”是指,为了获得有益或所需临床结果而实施的方法。为了本发明的目的,有益或所需的临床结果包括(但不限于)减轻症状、缩小疾病的范围、稳定(即,不再恶化)疾病的状态,延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部),无论是可检测或是不可检测的。此外,“治疗”还可以指,与期望的存活期相比(如果未接受治疗),延长存活期。
如本文中使用的,术语“受试者”是指哺乳动物,例如灵长类哺乳动物,例如人。
发明的有益效果
本发明的多聚化递送系统能够显著提高货物分子的内吞效率以及小泡释放效率,从而显著提升货物分子的细胞质递送效率,使货物分子充分发挥其相应的生物学功能。特别地,本发明的多聚化递送系统能够显著提升血清耐受性,使得体内递送成为可能。因此,本发明的递送系统提供了用于影响细胞的生物学机制和途径的有效手段,可用于研究、治疗、诊断等诸多领域,具有广阔的应用前景及临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述,但是本领域技术人员将理解,下列附图和实施例仅用于说明本发明,而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述,本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了基于亮氨酸拉链的多聚化递送系统-eGFP克隆设计。
图2显示了亮氨酸拉链对于蛋白聚体状态的影响。
图3显示了亮氨酸结构域加入后递送系统内吞效率和小泡逃逸效率评价。
图4显示了亮氨酸结构域加入后递送系统蛋白在血清条件下内吞效率评价。
图5显示了基于多种多聚化结构域的递送系统-eGFP克隆设计。
图6显示了多种多聚化结构域对内吞效率影响荧光显微成像。
图7显示了多种多聚化结构域对内吞效率影响评价。
图8显示了TINNeL-eGFP/GFPβ1-10克隆设计。
图9显示了TINNeL递送系统内吞效率评价。
图10显示了TINNeL递送系统小泡逃逸效率评价。
图11显示了TINNeL递送系统在不同浓度血清条件下递送效率评价。
图12显示了本发明的多聚化递送系统运输机制示意图。
图13显示了TINNeL-Ppm1b递送系统蛋白及其他对照蛋白克隆构建设计图。
图14显示了TINNeL-Ppm1b抑制TNF-α引起的细胞坏死效果评价。
图15显示了TINNeL-Ppm1b抑制TNF-α导致的死亡小鼠存活率评价。
图16显示了TINNeL-Ppm1b抑制TNF-α导致的小鼠SIRS盲肠HE染色图。
图17显示了TINNeL-Ppm1b抑制TNF-α导致的小鼠SIRS盲肠损伤评分。
图18显示了TINNeL-Ppm1b治疗APAP导致的小鼠急性肝损伤后ALT/AST水平变化评价。
图19显示了TINNeL-Ppm1b治疗APAP导致的死亡小鼠存活率评价。
图20显示了TINNeL-ZFP9递送系统蛋白及其他对照蛋白克隆构建设计图。
图21显示了ZFP9递送用真核表达质粒结构示意图。
图22显示了TINNeL-ZFP9转导红色荧光蛋白表达质粒效率流式分析。
图23显示了TINNeL-ZFP9与X-tremeGNEN在血清条件下转导红色荧光蛋白表达质粒流式分析。
图24显示了TINNeL-ZFP9转导Luciferase质粒荧光表达情况小鼠成像图。
图25显示了递送系统转导GFPβ1-10的荧光显微镜观察结果。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1:序列的描述
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明,本发明中所使用的分子生物学实验方法和免疫检测法,基本上参照J.Sambrook等人,分子克隆:实验室手册,第2版,冷泉港实验室出版社,1989,以及F.M.Ausubel等人,精编分子生物学实验指南,第3版,John Wiley&Sons,Inc.,1995中所述的方法进行;限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓,实施例以举例方式描述本发明,且不意欲限制本发明所要求保护的范围。
以下实施例涉及的主要试剂的来源如下:
克隆构建相关试剂所需材料如下:DNA聚合酶(TaKaRa,R040A),DNA回收试剂盒(TianGen,DP214-03),质粒小提试剂盒(TianGen,DP103-03),质粒大提试剂盒(QIAGEN,12663),Gibson装配预混液5管(NEB,E2611L),DNA marker(ThmeroFisher,SM0331),琼脂糖(Biowest,BW-R0100),
蛋白大量表达所需材料如下:蛋白胨(BiSIGMA-ALDRICH,T7293-1KG),酵母粉(OXOID,LP0021B),氯化钠(西陇化工,10011012AR),IPTG(Inalco,1758-1400)
蛋白纯化所需介质如下:SP SEPHAROSE FAST FLOW(GE Healthcare,17-0729-01),NI SEPHAROSE(GE Healthcare,17-5268-02)
蛋白纯化及保存所需试剂如下:甘油/丙三醇/C3H8O3(SIGMA-ALDRICH,G5516),KCl(西陇化工,1002007),Na2HPO4·12H2O(西陇化工,1001067AR),KH2PO4(西陇化工,1002048AR500),咪唑(SIGMA-ALDRICH,V900153),Tris base(Seebio,183995),葡萄糖(西陇化工,1064008AR500),BCA蛋白浓度测定试剂盒(Thermo Scientific,23227);
细胞培养所需试剂:FBS(GIBCO,10099-133),DMEM(GIBCO,11965092),胰蛋白酶(AMRESCO,0458);
慢病毒包装及感染所需试剂:慢病毒包装质粒:pCMV-VSV-G(Addgene,8454),pRSV-Rev(Addgene,12253),pMDLg/pRRE(Addgene,12251);X-tremeGENE转染试剂(Roche,06366244001),Puromycin(InvivoGen,ant-pr-5),Blasticidin(InvivoGen,ant-bl-5b),polybrene(Santa Cruz,sc-134220);
实验中所用的GFPβ1-10,ZFP9,Ppm1b,dsRed,mCherry,Histone-H3相关质粒均由公司合成(生工生物),用于扩增Cas9序列的质粒pCasKP-hph(Addgene,117232);
其他试剂:TNF-α(Novoprotein,CF09),PI(ThmeroFisher,P3566)
细胞系:HEK-293T(人肾上皮细胞),L929(小鼠成纤维细胞)均购自ATCC。
实施例1:TL多聚化递送系统的建立及性能评价
本实施例利用亮氨酸拉链建立多聚化递送系统,通过递送绿色荧光蛋白eGFP的方式观察多聚化对内吞效率的影响,通过递送GFPβ1-10-NLS(简称GFPβ1-10)并基于Split-GFP评价方法观察多聚化对小泡逃逸效率的影响,从而综合评估多聚化方式对于递送系统的递送效率的影响。同时,通过在体系中加入血清(FBS)的方式,观察多聚化前后内吞效率的差别,从而评估血清耐受程度改变。
1.1多聚化递送系统(TAT-Leu Zipper)-货物分子复合物表达载体的构建
构建包含TAT、Leu-Zipper(亮氨酸拉链)、和货物分子的重组蛋白的表达载体,各重组蛋白的编码核酸的结构示意图如图1所示,从N端到C端各组分氨基酸序列如下表所示。所述构建方法如下:首先,通过PCR扩增得到编码递送系统中的TAT、亮氨酸拉链、货物分子(eGFP或GFPβ1-10)的核酸序列,并通过多轮PCR将各部分连接,并在最后一轮PCR过程中通过上游引在片段的5’端引入NdeI酶切位点及其与pET21b(+)对应的NdeI酶切位点上游的重叠序列,通过下游引在片段的3’端引入BamHI酶切位点及其与pET-21b(+)对应的BamHI酶切位点下游的重叠序列。pET-21b(+)质粒通过NdeI,BamHI双酶切处理。通过GIBSON组装将带有重叠序列的插入片段连接到酶切载体pET-21b(+)上。
表2:递送系统-货物分子复合物所包含的组分
1.2多聚化递送系统-货物分子复合物的表达及纯化:
将1.1中所述的表达质粒转化入表达菌株E.coli BL21(DE3);从转化的平板上挑选单菌落接种到含氨苄抗性的5ml LB液体培养基中培养过夜,然后取1ml过夜培养的菌液转接到含氨苄抗性的500ml LB液体培养基中,37℃,180rpm培养到菌液OD600在0.6左右,接着加入诱导剂IPTG至终浓度为0.2mM,25℃诱导8小时;诱导表达结束后,4℃,7000g离心10分钟后收集菌体;接着用10ml蛋白纯化平衡缓冲液(PBS,5%甘油)重悬菌体,进行超声破碎。接着离心取上清液,并上样到蛋白纯化系统的SP强阳离子层析柱上;然后利用蛋白纯化系统用蛋白洗脱缓冲液(PBS+0.2M NaCl~PBS+0.6M NaCl去除杂蛋白,PBS+1.6M NaCl收集洗脱产物)洗脱目的蛋白。蛋白浓度可以根据分光光度计或BCA蛋白浓度测定试剂盒进行测定。每个纯化后的融合蛋白分装后-20℃保存。
1.3亮氨酸拉链对递送系统-货物分子复合物的多聚化作用评价
将上述1.2获得的TAT-eGFP(T-eGFP)及带有亮氨酸拉链的TAT-Leu-eGFP(TL-eGFP),通过非还原性的聚丙烯酰胺凝胶电泳分析其聚体形成状态。结果如图2所示,单体状态的T-eGFP蛋白大小为35kD左右,而加入后的亮氨酸拉链的TL-eGFP的蛋白大小位置在70kD处,该实验说明亮氨酸拉链可促使递送系统蛋白形成二聚体。
1.4多聚化递送系统-eGFP复合物的内吞效率评价
将HEK-293T细胞接种到12孔的细胞培养板中,过夜培养,蛋白处理前保证每孔的细胞数约为2*106左右;用无血清DMEM培养基润洗三次细胞,然后在无血清培养基中分别加入5μM的TL-eGFP和T-eGFP,孵育3h后,用预冷的含有20U/mL肝素的无血清DMEM漂洗三次去除在细胞膜上吸附的未能进入细胞内的蛋白,收集细胞进行流式分析。结果如图3所示,二聚化蛋白TL-eGFP的胞内荧光强度为单体(T-eGFP)的4-5倍,证明多聚化过程提高了递送系统的内吞效率。
1.5多聚化递送系统-GFPβ1-10复合物的小泡逃逸效率评价
在Split-GFP系统中,GFP的11个β折叠被分拆成大片段(β1-10)和小片段(β11),二者均失去了其荧光活性,但如果相遇则可以自发缔合并恢复GFP的荧光性能。基于此,我们构建了稳定表达Histone-β11的HEK293T细胞,同时将带有入核信号(NLS)的GFPβ1-10作为Cargo与拟评价的多聚化递送系统融合表达,然后转导该稳定细胞系。当递送系统转导GFPβ1-10时,只有成功从内吞小泡逃逸并进入到细胞质或者细胞核后才能与GFPβ11结合生成完整的GFP,因此通过GFP的比例及相对荧光强度可对内吞小泡逃逸效率进行评估。
1.5.1 HEK293T-GFPβ11细胞系的构建
(1)GFPβ11细胞系慢病毒质粒的构建:
Histone-H3的编码序列(SEQ ID NO:27)通过PCR扩增得到,GFPβ11编码序列(SEQID NO:28)较短直接设计在上游引物中,通过多轮PCR将各组分连接,并在最后一轮PCR过程中通过上游引在片段的5’端引入Hind III酶切位点及其与Lenti载体对应的Hind III酶切位点上游的重叠序列,通过下游引在片段的3’端引入酶切位点BamHI及其与Lenti载体对应的BamHI酶切位点下游的重叠序列。Lenti质粒通过Hind III,BamH I双酶切处理。通过GIBSON组装将带有重叠序列的插入片段连接到酶切后的Lenti载体上。
(2)慢病毒包装、感染及抗性筛选细胞系:
将HEK-293T细胞接种到6孔板中,过夜培养,在质粒转染之前保证每孔的细胞数约为2*107/ml左右;转染之前,细胞更换为无血清DMEM培养基;在300μl无血清DMEM分别加入1.5μg Lenti重组质粒,0.75μg pMDL质粒,0.45μg pVSV-G质粒,0.3μg pREV(质量比为5:3:2:1),缓慢吹匀后,加入9μl(1:3)的X-tremeGENE转染试剂,缓慢吹匀,室温静置15min,滴加入细胞上清中,8h后更为换为含10%FBS的DMEM继续培养;60h后收取培养上清待感染。
将HEK-293T细胞接种到12孔板中,过夜培养,在慢病毒感染之前保证每孔的细胞数约为2*106/ml(50%密度)左右;弃去原有细胞培养上清,加入300μl慢病毒(moi=3)及700μl 10%FBS DMEM,按10μg/ml的浓度加入polybrene,细胞板无菌条件下2500rpm离心30min后继续培养。
慢病毒感染48h后按1/3的比例进行细胞传代,同时按2.5μg/ml的浓度加入puromycin进行抗性筛选;将筛选得到的阳性细胞进行克隆化,得到HEK-293T-Hitone-GFPβ11的单克隆细胞株。
1.5.2Split-GFP系统检测小泡逃逸效率
将上述HEK-293T Histone H3-β11细胞(简称HEK-293Tβ11)接种到12孔的细胞培养板中,过夜培养,蛋白处理前保证每孔的细胞数约为2*106左右;用无血清DMEM培养基润洗三次细胞,然后在无血清培养基中分别加入5μM的TL-GFPβ1-10和T-GFPβ1-10,孵育3h后,用预冷的含有20U/mL肝素的无血清DMEM漂洗三次去除在细胞膜上吸附的未能进入细胞内的蛋白,再换为有血清DMEM培养基再培养9h后,收集细胞进行流式分析。结果显示,二聚化蛋白TL-GFPβ1-10与单体状态的T-GFPβ1-10相比其小泡逃逸效率并未随之改变(图3)。
1.6多聚化递送系统对血清条件的耐受程度评价
将HEK-293T细胞接种到12孔的细胞培养板中,过夜培养,蛋白处理前保证每孔的细胞数约为2*106左右;用无血清DMEM培养基润洗三次细胞,分别在无血清,5%,10%,20%,50%,100%FBS DMEM中加入5μM的TL-eGFP和T-eGFP,孵育3h后,用预冷的含有20U/mL肝素的无血清DMEM漂洗三次去除在细胞膜上吸附的未能进入细胞内的蛋白,再换为无血清DMEM培养基,收集细胞进行流式分析。结果如图4所示,随着血清含量的增加TL-eGFP和T-eGFP的转导效率都随之降低,但TL-eGFP的转导效率受血清的影响会相对较小,在50%FBS条件下,T-eGFP转导细胞后在胞内几乎检测不到绿色荧光信号,而二聚化蛋白TL-eGFP仍有相比于不加血清时70%的转导效率。由此可见,多聚化不仅提高了蛋白的内吞效率,而且赋予了蛋白更高的血清耐受度。
1.7不同多聚化结构域对递送系统内吞效率的影响
本实施例进一步比较了不同多聚化结构域序列对递送系统内吞效率的影响,包含不同多聚化结构域的递送系统-货物分子复合物的各组分如下表所示。通过上述1.1-1.2中的方法制备表达上述复合物的表达载体、并表达纯化上述各复合物,构建的质粒示意图如图5所示。
表3:包含不同多聚化结构域的递送系统
将MA104细胞接种到12孔的细胞培养板中,过夜培养,蛋白处理前保证每孔的细胞数约为2*106左右;用无血清DMEM培养基润洗三次细胞,然后在无血清培养基中分别加入5μM的T-eGFP,TL-eGFP,TL2-eGFP,TN-eGFP,TG-eGFP和TD-eGFP,孵育3h后,用预冷的含有20U/mL肝素的无血清DMEM漂洗三次去除在细胞膜上吸附的未能进入细胞内的蛋白,更换为无血清培养基后荧光成像,结束后收集细胞进行流式分析。荧光成像结果如图6所示,T-eGFP组细胞内绿色荧光强度很低,而其他加入多聚化结构域的实验组胞内荧光强度明显增加。流式分析结果如图7所示,不同的多聚化结构域均会使TAT介导eGFP的平均荧光强度增强,即提升了TAT介导的内吞效率,而以其中亮氨酸拉链为基础的TL-eGFP及TL2-eGFP效果最为明显。
实施例2:TINNeL多聚化递送系统的建立及性能评价
本实施例在实施例1的多聚化递送系统基础上,进一步加入pH敏感肽(INF7)和内吞小泡蛋白酶特异性酶切位点(CTSL蛋白酶:N,Furin蛋白酶:Ne),以构建TINNEL递送系统,并评价其内吞效率和小泡逃逸效率。
2.1 TINNEL-货物分子复合物表达载体的构建
构建TINNEL-货物分子重组蛋白的表达载体,各重组蛋白从N端到C端所包含的各组分氨基酸序列如下表所示。所述构建方法如下:首先,通过PCR扩增得到编码递送系统中的TAT、INF7、亮氨酸拉链、N、Ne、货物分子(eGFP或GFPβ1-10)的核酸序列,并通过多轮PCR将各部分连接,并在最后一轮PCR过程中通过上游引在片段的5’端引入NdeI酶切位点及其与pET-21b(+)对应的NdeI酶切位点上游的重叠序列,通过下游引在片段的3’端引入BamHI酶切位点及其与pET-21b(+)对应的BamHI酶切位点下游的重叠序列。pET-21b(+)质粒通过NdeI,BamHI双酶切处理。通过GIBSON组装方式将带有重叠序列的插入片段连接到酶切载体pET-21b(+),质粒示意图如图8。
表4:递送系统-货物分子复合物所包含的组分
2.2 TINNeL-货物分子复合物的表达、纯化
首先,将2.1获得的表达质粒转化入表达菌株E.coli BL21(DE3);从转化的平板上挑选单菌落接种到含氨苄抗性的5ml LB液体培养基中培养过夜,然后取1ml过夜培养的菌液转接到含氨苄抗性的500mL LB液体培养基中,37℃,180rpm培养到菌液OD600在0.6左右,接着加入诱导剂IPTG至终浓度为0.2mM,25℃诱导8小时;诱导表达结束后,4℃,7000g离心10分钟后收集菌体;接着用10ml蛋白纯化平衡缓冲液(PBS,5%甘油)重悬菌体,进行超声破碎。接着离心取上清液,并上样到蛋白纯化系统的SP强阳离子层析柱上;然后利用蛋白纯化系统用蛋白洗脱缓冲液(PBS+0.2M NaCl~PBS+0.7M NaCl去除杂蛋白,PBS+1.8M NaCl收集洗脱产物)洗脱目的蛋白。蛋白浓度可以根据分光光度计或BCA蛋白浓度测定试剂盒进行测定。每个纯化后的融合蛋白分装后-20℃保存。
2.3 TINNeL递送系统的胞内递送效率评价
内吞效率评价:将HEK-293T接种到12孔的细胞培养板中,过夜培养,蛋白处理前保证每孔的细胞数约为2*106左右;用无血清DMEM培养基润洗三次细胞,然后在无血清培养基中分别加入5μM的递送系统蛋白TINNeL-eGFP和对照蛋白TINNe-eGFP,孵育3h后,用预冷的含有20U/mL肝素的无血清DMEM漂洗三次去除在细胞膜上吸附的未能进入细胞内的蛋白,然后收集细胞进行流式分析,我们发现二聚化后的TINNeL-eGFP的荧光强度是4倍,即亮氨酸拉链结构域加入后内吞效率明显提高(图9)。
小泡逃逸效率评价:我们利用Split-GFP体系通过观察其逃逸效率的改变,进一步评估TINNeL系统的递送效率。将HEK-293Tβ11接种到12孔的细胞培养板中,过夜培养,蛋白处理前保证每孔的细胞数约为2*106左右;用无血清DMEM培养基润洗三次细胞,然后在无血清培养基中分别加入1μM的递送系统蛋白TINNeL-GFPβ1-10和对照蛋白TINNe-GFPβ1-10,孵育3h后,用预冷的含有20U/mL肝素的无血清DMEM漂洗三次去除在细胞膜上吸附的未能进入细胞内的蛋白,更换为有血清培养基(10%FBS)继续培养9h后,收集细胞进行流式分析。结果如图10所示,TINNeL-GFPβ1-10有更高的荧光强度,说明TINNeL递送系统能够同时提高内吞效率和小泡逃逸效率,是一种更为高效的递送方法。
2.4 TINNeL递送系统在血清条件下的递送效率评价
将HEK-293T细胞接种到12孔的细胞培养板中,过夜培养,蛋白处理前保证每孔的细胞数约为2*106左右;用无血清DMEM培养基润洗三次细胞,分别在无血清,10%,20%,50%,100%FBS DMEM中加入5μM的TINNeL-GFPβ1-10和对照蛋白TINNe-GFPβ1-10,孵育3h后,用预冷的含有20U/mL肝素的无血清DMEM漂洗四次去除在细胞膜上吸附的未能进入细胞内的蛋白,再换为无血清DMEM培养基,继续培养9h后收集细胞进行流式分析。结果如图11所示,随着血清含量的增加TINNeL-GFPβ1-10和TINNe-GFPβ1-10组的荧光强度都随之降低,但TINNeL-GFPβ1-10的受到的影响会更小,而在100%FBS浓度条件下,TINNe-GFPβ1-10组几乎检测不到绿色荧光信号,而TINNeL-GFPβ1-10仍有相比于不加血清时70%的效率,因此TINNeL不仅拥有高效的递送能力,而且在血清存在的条件下其递送效果不会受到较大影响。图12显示了TINNeL递送系统的原理示意图。
此外,发明人还基于Split-GFP系统评价了包含蛋白酶识别序列N的递送系统(TIN-GFPβ1-10)、包含蛋白酶识别序列Ne的递送系统(TINe-GFPβ1-10)的小泡逃逸效率。上述递送系统与TINNe-GFPβ1-10的区别仅在于,蛋白酶识别序列被替换为单独的N或Ne。结果如图25所示,相比于未加入蛋白酶识别序列的TI-GFPβ1-10或TIN-GFPβ1-10,无论是包含单独蛋白酶识别序列N的递送系统(TIN-GFPβ1-10)、还是包含蛋白蛋白酶识别序列Ne的递送系统(TINe-GFPβ1-10),内吞小泡逃逸效率均明显提高,以上结果表明,单独的蛋白酶识别序列N或Ne均具备提高小泡逃逸效率的能力。
实施例3:TINNeL-Ppm1b在抑制TNF-α诱发的细胞凋亡中的应用
细胞死亡可以分为细胞凋亡和细胞坏死。其中细胞坏死则会导致细胞膜破裂,肿胀和内容物的泄漏,从而引起剧烈的炎症反应,其主要由受体相互作用蛋白激酶3(receptor-interaction kinase 3,RIP3)控制。在TNF-α刺激下,细胞内形成包含Rip1和Rip3坏死小体(necrosome),坏死小体中的Rip3募集并磷酸化Mlkl。磷酸化的Mlk1易位至细胞膜以执行坏死性凋亡,在这个过程中Rip3的磷酸化对于将Mlk1募集至坏死因子是必需的,因此,Rip3的磷酸化过程,很可能成为抑制细胞坏死并控制其引起的炎症反应重要靶点。已有研究表明,蛋白磷酸酶1B(Ppm1b)可以通过使Rip3去磷酸化而在培养细胞和小鼠体内抑制坏死性凋亡,Ppm1b蛋白成为控制程序性坏死的重要工具。鉴于目前已发现细胞程序性坏死与炎性疾病、缺血再灌注损伤、神经退行性疾病等多种疾病的发生密切相关,Ppm1b蛋白在治疗上述与细胞程序性坏死相关的疾病方面展现出巨大潜力。
3.1 TINNeL-Ppm1b复合物及其他对照蛋白表达载体构建
构建含有TAT、INF7、蛋白酶切割位点、多聚化结构域、货物分子Ppm1b(SEQ ID NO:29)的重组蛋白表达载体,各重组蛋白的结构示意图如图13所示,从N端到C端各组分氨基酸序列如下表所示。所述构建方法如下:通过PCR扩增得到TAT、INF7、N、Ne、Ppm1b的核酸序列,并通过多轮PCR将各部分连接,并在最后一轮PCR过程中通过上游引在片段的5’端引入NdeI酶切位点及其与pET-21b(+)对应的NdeI酶切位点上游的重叠序列,通过下游引在片段的3’端引入BamHI酶切位点及其与pET-21b(+)对应的BamHI酶切位点下游的重叠序列。pET-21b(+)质粒通过NdeI,BamHI双酶切处理。通过GIBSON组装方式将带有重叠序列的插入片段连接到酶切载体pET-21b(+)。
表5:递送系统-货物分子复合物所包含的组分
3.2 TINNeL-Ppm1b蛋白及其他对照蛋白的表达、纯化
首先,将3.1获得的表达质粒转化入表达菌株E.coli BL21(DE3);从转化的平板上挑选单菌落接种到含氨苄抗性的5ml LB液体培养基中培养过夜,然后取1ml过夜培养的菌液转接到含氨苄抗性的500ml LB液体培养基中,37℃,180rpm培养到菌液OD600在0.6左右,接着添加诱导IPTG至终浓度为0.2mM,25℃诱导8小时;诱导表达结束后,4℃,7000g离心10分钟后收集菌体,取部分菌体检测蛋白质诱导表达情况;接着用10ml蛋白纯化平衡缓冲液(50ml C3H8O3,3.6342g Tris(Hydroxymethyl)aminomethane溶于1L双蒸水中,调pH至8.0)重悬菌体,进行超声破碎。接着离心取上清液,并上样到AKTA蛋白纯化系统的Sulphopropyl(SP)阳离子交换柱上;然后利用平衡缓冲液与高盐洗脱液(50ml C3H8O3,116.88g NaCl,3.6342g Tris(Hydroxymethyl)aminomethane溶于1L双蒸水中,调pH至8.0)之间按不同比例来进行梯度洗脱得到目的蛋白。蛋白浓度可以根据分光光度计或BCA蛋白浓度测定试剂盒进行测定。每个纯化后的融合蛋白分装后-20℃保存。
3.3 TINNeL-Ppm1b对TNF-α诱发的L292细胞坏死的影响
将L929细胞接种到12孔的细胞培养板中,过夜培养,蛋白处理前保证每孔的细胞数约为2*106左右,用无血清DMEM培养基润洗三次细胞,然后在无血清培养基中分别加入递送系统蛋白TINNeL-Ppm1b和其他对照蛋白,孵育3h后,加入用10%FBS DMEM稀释好含20ng/ml TNF-α以及20mM z-VAD 1ml,孵育10h后,收集细胞进行PI染色,流式分析细胞观察细胞坏死的比率。同时,利用慢病毒转导Ppm1b(Lenti-Ppm1b)以及慢病毒(Lenti-vec)作为对照,在HEK-293T细胞上包装收集的带有Ppm1b表达序列的慢病毒和对照慢病毒,感染L929细胞24h后,以使Ppm1b在胞内充分表达,将感染后的L929细胞重新铺版待TNF-α刺激。结果如图14所示,TINNeL-Ppm1b处理后的L929,TNF-α导致的细胞坏死比例最低,仅有10%左右,其抑制效果甚至要好于作为阳性对照的慢病毒感染组(20%)。
同时,因为Ppm1b是通过抑制Rip3磷酸化的过程以实现抑制细胞坏死,因此,我们尝试通过蛋白免疫印迹验证不同处理组(处理方式同上)的L929细胞内Rip3和磷酸化的Rip3(p-Rip3)的含量,进一步确定TINNe-Ppm1b是通过进入细胞质化后,更为高效的抑制Rip3的磷酸化最终实现抑制TNF-α细胞坏死的。结果如图14所示,各处理组的L929细胞内Rip3的含量基本一致,而对于磷酸化状态的p-Rip3,TINNeL-Ppm1b组显著低于其他蛋白组,该结果表明TINNeL-Ppm1b可以更为高效的进入L929细胞质内,通过抑制Rip3的磷酸化过程,达到抑制TNF-α引起的细胞坏死。
3.4 TINNeL-Ppm1b对TNF-α诱发的周身性炎症反应综合症的抑制活性评价
在小鼠体内,TNF-α导致的细胞坏死会进一步引发小鼠的周身性炎症反应(systemic inflammatory response syndrome,SIRS)并最终导致小鼠死亡。而TINNeL递送系统不仅拥有较高的递送效率,而且在血清存在的条件下,仍应该能保持较高的递送效果,即其在体内仍能较好的发挥其递送作用,因此我们尝试基于TNF-α诱发的SIRS模型,观察TINNeL-Ppm1b在体内是否仍有较理想的递送效果。
将25mmol的TINNeL-Ppm1b及其他对照蛋白(每组12只)通过尾静脉途径注入6周龄的雌性BALB/C小鼠体内,2h后同样通过尾静脉途径向小鼠体内注入15μg TNF-α,并在此后每隔一个小时观察小鼠的存活情况(图15)。结果显示,在观察期(40h后存活率基本稳定)内,对照组小鼠在20h内全部因为TNF-α引起的炎症而死亡,而TINNeL-Ppm1b组小鼠在观察其结束仍然100%存活(图15)。;同时,TNF-α引起的SIRS可以从小鼠从盲肠的损伤程度上直接观察到,因此我们将25nmol的TINNeL-Ppm1b及其他对照蛋白(每组6只)通过尾静脉途径注入6周龄的雌性BALB/C小鼠体内,2h后同样通过尾静脉途径向小鼠体内给予15μg TNF-α,并在10h后安乐死处理小鼠,取出盲肠进行HE染色,观察不同处理组小鼠盲肠的损伤情况并对其进行损伤评分。结果显示,TINNeL-Ppm1b组小鼠盲肠几乎未收到损伤(图16),评分和Mock组(未接受TNF-α的对照组)相当,相对于其他蛋白可以更为高效抑制TNF-α引起的SIRS(图17)。因此,综合以上结果,我们在小鼠水平证实了TINNeL-Ppm1b可以有效抑制TNF-α引起SIRS,TINNeL的是一种更为有效的递送系统。
实施例4:TINNeL-Ppm1b在治疗APAP引起的急性肝损伤中的应用
乙酰氨基酚(Acetaminophen,APAP)是临床上常用的退热镇痛药物,但其过量使用会导致的急性肝损伤,存在死亡风险,近些年研究证实APAP引起的急性肝损伤是通过Rip3介导的细胞程序性坏死而引起的炎症反应导致,因此,通过Ppm1b去磷酸化Rip3很可能达到治疗APAP引起的急性肝损伤的效果。
首先,通过尾静脉途径向5-7周龄的小鼠(16-18g)体内注入500mg/kg(非致死剂量)的APAP,分别在之后的2h和6h尾静脉方式注射两针25nmol的TINNeL-Ppm1b及实施例3中制备的其他对照蛋白(每组7只),再过6h APAP注射后(12h)后眼眶采血,检测血清中ALT及AST水平,评估小鼠肝脏损伤情况。结果显示,TINNeL-Ppm1b组的ALT及AST水品均为最低,基本和APAP稀释缓冲液DMSO组一致(ALT/AST是上升是由于DMSO导致,这部分并非通过Rip3途径产生),因此,TINNeL-Ppm1b可以实现APAP引起的急性肝损伤的较为理想的治疗效果(图18)。
如果进一步提高APAP的使用剂量,会导致更为严重的肝损伤,进而诱发小鼠死亡,因此,我们尝试观察TINNeL-Ppm1b是否能够治疗APAP致死剂量下的小鼠肝损伤。同时,我们利用目前FDA批准的唯一的用于治疗APAP引起肝损伤的药物NAC作为对照。首先,通过尾静脉途径向5-7周龄的BALB/C小鼠(16-18g)体内注入800mg/kg(致死剂量)的APAP,并分别在之后的2h和6h尾静脉方式注射两次25mmol的TINNeL-Ppm1b及NAC,PBS(每组7只),之后每隔1h观察小鼠存活情况。结果如图19所示,PBS组的小鼠在18h内全部死亡,在72h的观察期内(24h之内为急性期),NAC组的小鼠存活率为20%,而TINNeL-Ppm1b组可以达到60%,因此,即便是APAP导致的更为严重的急性肝损伤,TINNeL-Ppm1b也表现出很好的治疗效果,甚至要略好于目前的临床用药NAC。上述结果充分显示了TINNeL在体内递送的优势。
实施例5:TINNeL-ZFP9的体内外核酸递送效率评价
5.1 TINNeL-ZFP9及其他对照蛋白表达载体的构建
构建含有TAT、INF7、蛋白酶切割位点、多聚化结构域、货物分子ZFP9(SEQ ID NO:31)的重组蛋白表达载体,各重组蛋白的结构示意图如图20所示,从N端到C端各组分氨基酸序列如下表所示。所述构建方法如下:通过PCR扩增得到编码TAT,INF7,蛋白酶切割位点,亮氨酸拉链,ZFP9的核酸序列,并通过多轮PCR将各部分连接,并在最后一轮PCR过程中通过上游引在片段的5’端引入NdeI酶切位点及其与pET21b(+)对应的NdeI酶切位点上游的重叠序列,通过下游引在片段的3’端引入BamHI酶切位点及其与pET21b(+)对应的BamHI酶切位点下游的重叠序列。pET-21b(+)质粒通过NdeI,BamHI双酶切处理。通过GIBSON组装将带有重叠序列的插入片段连接到酶切载体pET-21b(+)上。
表6:递送系统-货物分子复合物所包含的组分
5.2 TINNeL-ZFP9及对照蛋白的表达、纯化
首先,将5.1获得的表达质粒转化入表达菌株E.coli BL21(DE3);从转化的平板上挑选单菌落接种到含氨苄抗性的5ml LB液体培养基中培养过夜,然后取1ml过夜培养的菌液转接到含氨苄抗性的500ml LB液体培养基中,37℃,180rpm培养到菌液OD600在0.6左右,接着添加诱导IPTG至终浓度为0.2mM,25℃诱导8小时;诱导表达结束后,4℃,7000g离心10分钟后收集菌体,取部分菌体检测蛋白质诱导表达情况;接着用10ml蛋白纯化平衡缓冲液(50ml C3H8O3,3.6342g Tris(Hydroxymethyl)aminomethane溶于1L双蒸水中,调pH至8.0)重悬菌体,进行超声破碎。接着离心取上清液,并上样到AKTA蛋白纯化系统的Sulphopropyl(SP)阳离子交换柱上;然后利用平衡缓冲液与高盐洗脱液(50ml C3H8O3,116.88g NaCl,3.6342g Tris(Hydroxymethyl)aminomethane溶于1L双蒸水中,调pH至8.0)之间按不同比例来进行梯度洗脱得到目的蛋白。蛋白浓度可以根据分光光度计或BCA蛋白浓度测定试剂盒进行测定。每个纯化后的融合蛋白分装后-20℃保存。
5.3含有ZFP9结合位点的真核表达质粒的构建
构建含有tdTomato编码序列和ZFP9结合位点的表达载体(pTT5-tdTomato-6BS质粒),其结构示意图如图21所示。tdTomato编码序列(SEQ ID NO:33)及ZFP9结合位点6*binding sites(6个结合位点)(SEQ ID NO:34)序列通过PCR扩增得到,通过两轮PCR将二者连接,并在第二轮PCR过程中通过上游引在片段的5’端引入Hind III酶切位点及其与pTT5载体对应的Hind III酶切位点上游的重叠序列,通过下游引在片段的3’端引入酶切位点BamHI及其与pTT5载体对应的BamHI酶切位点下游的重叠序列。pTT5质粒通过Hind III,BamH I双酶切处理。通过GIBSON组装将带有重叠序列的插入片段连接到酶切后的pTT5载体上。
5.4 TINNeL-ZFP9递送质粒DNA的递送效率评价
首先,将HEK-293T细胞接种到12孔板中,过夜培养,蛋白处理前保证每孔的细胞数约为5*106左右;将5μM递送系统蛋白或其他对照蛋白和pTT5-tdTomato-6BS质粒5μg在37℃共孵育30min充分形成复合物;用无血清DMEM培养基润洗三次细胞,后加入复合物孵育3h;更换有10%FBS DMEM继续培养,分别在换液12h、24h、36h进行流式分析红色荧光蛋白表达情况。结果如图22所示,相比于其他对照蛋白,在三个检测时间点TINNeL-ZFP9组的红色荧光比率均是最高的,因此,TINNeL-ZFP9在结合pTT5-tdTomato-6BS更为有效的递送进入细胞核,实现质粒上红色荧光基因的表达。
5.5 TINNeL-ZFP9在血清条件下的递送效率评价
本实施例考察TINNeL-ZFP9在血清条件下的转染效果,以在血清条件下仍然可以工作的商品化转染试剂X-tremeGENE作为对照。
首先,将HEK-293T细胞接种到12孔板中,过夜培养,蛋白处理前保证每孔的细胞数约为5*106左右;将5μM的TINNeL-ZFP9和pTT5-tdTomato-6BS质粒5μg在37℃共孵育30min充分形成复合物;在100%FBS血清条件及无血清条件加入复合物孵育3h后,更换有10%FBSDMEM继续培养;对照的X-tremeGENE组,将5μg的pTT5-tdTomato-6BS质粒与X-tremeGENE按1μg DNA:3μl X-tremeGENE的比例混合后,室温静置30min,在100%FBS血清条件及无血清条件下孵育8h,然后后更换为10%FBS DMEM继续培养。以上处理组,分别在换液12h、24h、36h、48h、60h、72h、84h进行流式分析红色荧光蛋白表达情况。结果如图23所示,在无血清条件下,TINNeL-ZFP9组红色荧光细胞比例增长的更快,但达到平台期后与X-tremeGENE组的比例基本一致,但是在100%FBS条件下,在各个时间点,红色荧光细胞比例都有接近10%的差,TINNeL-ZFP9在血清条件下表现出更为高效的的转染能力。
5.5 TINNeL-ZFP9在小鼠体内的递送效率评价
构建带有Luciferase编码基因(SEQ ID NO:35)以及ZFP9结合位点(SEQ ID NO:34)的pTT5-Luc-6BS表达质粒,构建方法同上述5.3。将4nmol的TINNeL-ZFP9及其他对照蛋白TINNe-ZFP9,T-ZFP9与5μg pTT5-Luc-6BS表达质粒混合后30min,注射于裸鼠左腿肌肉处,24h后成像并分析荧光强度。结果如图24所示,T-ZFP9和无处理组基本检测不到荧光,TINNe-ZFP9有较弱荧光,而TINNeL-ZFP9组可以检测到较强的荧光,因此,在小鼠水平确认了TINNeL-ZFP9可以高效结合并递送DNA。
尽管本发明的具体实施方式已经得到详细的描述,但本领域技术人员将理解:根据已经公布的所有教导,可以对细节进行各种修改和变动,并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。
SEQUENCE LISTING
<110> 厦门大学
厦门万泰沧海生物技术有限公司
<120> 用于胞内递送分子的多聚化递送系统
<130> IDC200043
<150> CN201911414575.7
<151> 2019-12-31
<160> 50
<170> PatentIn version 3.5
<210> 1
<211> 35
<212> PRT
<213> 人工序列
<220>
<223> 亮氨酸拉链-1
<400> 1
Gly Asp Arg Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser
1 5 10 15
Lys Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile
20 25 30
Gly Glu Arg
35
<210> 2
<211> 24
<212> PRT
<213> 人工序列
<220>
<223> 亮氨酸拉链-2
<400> 2
Glu Ile Ala Ala Ile Lys Lys Glu Ile Ala Ala Ile Lys Gln Glu Ile
1 5 10 15
Ala Ala Ile Lys Gln Gly Tyr Gly
20
<210> 3
<211> 40
<212> PRT
<213> 人工序列
<220>
<223> NOE多聚化结构域
<400> 3
Ala Pro Val Pro Val Thr Lys Leu Val Cys Asp Gly Asp Thr Tyr Lys
1 5 10 15
Cys Thr Ala Tyr Leu Asp Tyr Gly Asp Gly Lys Trp Val Ala Gln Trp
20 25 30
Asp Thr Ala Val Phe His Thr Thr
35 40
<210> 4
<211> 33
<212> PRT
<213> 人工序列
<220>
<223> GCN4-P1多聚化结构域
<400> 4
Arg Met Lys Gln Leu Glu Asp Lys Val Glu Glu Leu Leu Ser Lys Asn
1 5 10 15
Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val Gly Glu
20 25 30
Arg
<210> 5
<211> 63
<212> PRT
<213> 人工序列
<220>
<223> Delta多聚化结构域
<400> 5
Leu Ala Ala Leu Thr Thr Arg Val Thr Thr Glu Asn Thr Arg Leu Asp
1 5 10 15
Gly Leu Ile Asn Ser Gly Gln Asn Ser Ile Gly Glu Leu Ser Thr Arg
20 25 30
Leu Ser Asn Val Glu Thr Ser Met Val Thr Thr Ala Gly Arg Gly Leu
35 40 45
Gln Lys Asn Gly Asn Thr Leu Asn Val Ile Val Gly Asn Gly Met
50 55 60
<210> 6
<211> 105
<212> DNA
<213> 人工序列
<220>
<223> 编码亮氨酸拉链-1的核酸序列
<400> 6
ggcgacagaa tgaagcagat cgaagacaag atcgaagaaa tcctgagcaa gatctaccac 60
atcgaaaacg aaatcgccag aatcaagaag ctgatcggcg aaaga 105
<210> 7
<211> 72
<212> DNA
<213> 人工序列
<220>
<223> 编码亮氨酸拉链-2的核酸序列
<400> 7
gaaattgcag caattaaaaa agaaattgca gcaattaaac aggaaattgc agcaattaaa 60
cagggttatg gt 72
<210> 8
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> 弗林蛋白酶识别序列Ne
<400> 8
Gln Ser Val Ala Ser Ser Arg Arg His Lys Arg Phe Ala Gly Val
1 5 10 15
<210> 9
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 编码Ne的核酸序列
<400> 9
cagagcgttg caagcagccg tcgtcataaa cgttttgcag gtgtt 45
<210> 10
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> CTSL识别序列N
<400> 10
Asn Asn Thr His Asp Leu Val Gly Asp Val Arg Leu Ala Gly Val
1 5 10 15
<210> 11
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 编码N的核酸序列
<400> 11
aacaacactc atgaccttgt cggtgatgtg agattagccg gagtt 45
<210> 12
<211> 23
<212> PRT
<213> 人工序列
<220>
<223> INF7
<400> 12
Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly
1 5 10 15
Met Ile Asp Gly Trp Tyr Gly
20
<210> 13
<211> 69
<212> DNA
<213> 人工序列
<220>
<223> 编码INF7的核酸序列
<400> 13
ggcctgttcg aagcaataga aggtttcata gaaaatggtt gggagggaat gatagacggt 60
tggtacggt 69
<210> 14
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> Tat(48-60)
<400> 14
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln
1 5 10
<210> 15
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 编码Tat(48-60)的核酸序列
<400> 15
ggccgtaaga agcggagaca gcgacgaaga ccgccgcag 39
<210> 16
<211> 100
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白TINL
<400> 16
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Gly Gly
1 5 10 15
Gly Ser Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp
20 25 30
Glu Gly Met Ile Asp Gly Trp Tyr Gly Gly Gly Gly Gly Ser Asn Asn
35 40 45
Thr His Asp Leu Val Gly Asp Val Arg Leu Ala Gly Val Gly Gly Gly
50 55 60
Gly Ser Gly Asp Arg Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile
65 70 75 80
Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys
85 90 95
Leu Ile Gly Glu
100
<210> 17
<211> 101
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白TINeL
<400> 17
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Gly Gly
1 5 10 15
Gly Ser Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp
20 25 30
Glu Gly Met Ile Asp Gly Trp Tyr Gly Gly Gly Gly Gly Ser Gln Ser
35 40 45
Val Ala Ser Ser Arg Arg His Lys Arg Phe Ala Gly Val Gly Gly Gly
50 55 60
Gly Ser Gly Asp Arg Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile
65 70 75 80
Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys
85 90 95
Leu Ile Gly Glu Arg
100
<210> 18
<211> 121
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白TINNeL
<400> 18
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Gly Gly
1 5 10 15
Gly Ser Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp
20 25 30
Glu Gly Met Ile Asp Gly Trp Tyr Gly Gly Gly Gly Gly Ser Asn Asn
35 40 45
Thr His Asp Leu Val Gly Asp Val Arg Leu Ala Gly Val Gly Gly Gly
50 55 60
Gly Ser Gln Ser Val Ala Ser Ser Arg Arg His Lys Arg Phe Ala Gly
65 70 75 80
Val Gly Gly Gly Gly Ser Gly Asp Arg Met Lys Gln Ile Glu Asp Lys
85 90 95
Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala
100 105 110
Arg Ile Lys Lys Leu Ile Gly Glu Arg
115 120
<210> 19
<211> 186
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白TINL-ZFP9
<400> 19
Glu Ile Ala Ala Ile Lys Lys Glu Ile Ala Ala Ile Lys Gln Glu Ile
1 5 10 15
Ala Ala Ile Lys Gln Gly Tyr Gly Gly Gly Gly Gly Ser Gly Arg Lys
20 25 30
Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Gly Gly Gly Ser Gly
35 40 45
Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly Met
50 55 60
Ile Asp Gly Trp Tyr Gly Gly Gly Gly Gly Ser Asn Asn Thr His Asp
65 70 75 80
Leu Val Gly Asp Val Arg Leu Ala Gly Val Gly Gly Gly Gly Ser Val
85 90 95
Ser Arg Pro Gly Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn
100 105 110
Phe Ser Asp Lys Thr Lys Leu Arg Val His Thr Arg Thr His Thr Gly
115 120 125
Glu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Val Arg
130 135 140
His Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Lys Pro Phe
145 150 155 160
Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser Thr Ser Leu Gln
165 170 175
Arg His Leu Lys Thr His Leu Arg Gly Ser
180 185
<210> 20
<211> 186
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白TINeL-ZFP9
<400> 20
Glu Ile Ala Ala Ile Lys Lys Glu Ile Ala Ala Ile Lys Gln Glu Ile
1 5 10 15
Ala Ala Ile Lys Gln Gly Tyr Gly Gly Gly Gly Gly Ser Gly Arg Lys
20 25 30
Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Gly Gly Gly Ser Gly
35 40 45
Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly Met
50 55 60
Ile Asp Gly Trp Tyr Gly Gly Gly Gly Gly Ser Gln Ser Val Ala Ser
65 70 75 80
Ser Arg Arg His Lys Arg Phe Ala Gly Val Gly Gly Gly Gly Ser Val
85 90 95
Ser Arg Pro Gly Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn
100 105 110
Phe Ser Asp Lys Thr Lys Leu Arg Val His Thr Arg Thr His Thr Gly
115 120 125
Glu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Val Arg
130 135 140
His Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Lys Pro Phe
145 150 155 160
Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser Thr Ser Leu Gln
165 170 175
Arg His Leu Lys Thr His Leu Arg Gly Ser
180 185
<210> 21
<211> 206
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白TINNeL-ZFP9
<400> 21
Glu Ile Ala Ala Ile Lys Lys Glu Ile Ala Ala Ile Lys Gln Glu Ile
1 5 10 15
Ala Ala Ile Lys Gln Gly Tyr Gly Gly Gly Gly Gly Ser Gly Arg Lys
20 25 30
Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Gly Gly Gly Ser Gly
35 40 45
Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly Met
50 55 60
Ile Asp Gly Trp Tyr Gly Gly Gly Gly Gly Ser Asn Asn Thr His Asp
65 70 75 80
Leu Val Gly Asp Val Arg Leu Ala Gly Val Gly Gly Gly Gly Ser Gln
85 90 95
Ser Val Ala Ser Ser Arg Arg His Lys Arg Phe Ala Gly Val Gly Gly
100 105 110
Gly Gly Ser Val Ser Arg Pro Gly Glu Arg Pro Phe Gln Cys Arg Ile
115 120 125
Cys Met Arg Asn Phe Ser Asp Lys Thr Lys Leu Arg Val His Thr Arg
130 135 140
Thr His Thr Gly Glu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn
145 150 155 160
Phe Ser Val Arg His Asn Leu Thr Arg His Leu Arg Thr His Thr Gly
165 170 175
Glu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser
180 185 190
Thr Ser Leu Gln Arg His Leu Lys Thr His Leu Arg Gly Ser
195 200 205
<210> 22
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> NLS
<400> 22
Pro Lys Lys Lys Arg Lys Val
1 5
<210> 23
<211> 239
<212> PRT
<213> 人工序列
<220>
<223> eGFP
<400> 23
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 24
<211> 720
<212> DNA
<213> 人工序列
<220>
<223> 编码eGFP的核酸序列
<400> 24
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
<210> 25
<211> 228
<212> PRT
<213> 人工序列
<220>
<223> GFPβ1-10-NLS
<400> 25
Met Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Ile Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Arg His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Ser Phe Lys Asp Asp Gly Lys Tyr Lys Thr Arg Ala Val
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Thr Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Ala Asn Phe Thr Val Arg His Asn Val Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Thr Val Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys His His His His His His Pro Lys Lys
210 215 220
Lys Arg Lys Val
225
<210> 26
<211> 687
<212> DNA
<213> 人工序列
<220>
<223> 编码GFPβ1-10-NLS的核酸序列
<400> 26
atgggcagca aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat 60
ggtgatgtta atgggcacaa attttctgtc agaggagagg gtgaaggtga tgctacaatc 120
ggaaaactca cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca 180
cttgtcacta ctctgaccta tggtgttcaa tgcttttccc gttatccgga tcacatgaaa 240
aggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaacg cactatatct 300
ttcaaagatg acgggaaata caagacgcgt gctgtagtca agtttgaagg tgataccctt 360
gttaatcgta tcgagttaaa gggtactgat tttaaagaag atggaaacat tctcggacac 420
aaactcgagt acaactttaa ctcacacaat gtatacatca cggcagacaa acaaaagaat 480
ggaatcaaag ctaacttcac agttcgccac aacgttgaag atggttccgt tcaactagca 540
gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat 600
tacctgtcga cacaaactgt cctttcgaaa gatcccaacg aaaagcacca ccaccaccac 660
caccccaaga agaagaggaa ggtgtaa 687
<210> 27
<211> 411
<212> DNA
<213> 人工序列
<220>
<223> 编码Histone-H3的核酸序列
<400> 27
atggctcgca ctaagcaaac tgctcggaag tctactggtg gcaaggcgcc acgcaaacag 60
ttggccacta aggcagcccg caaaagcgct ccggccaccg gcggcgtgaa aaagccccac 120
cgctaccggc cgggcaccgt ggctctgcgc gagatccgcc gttatcagaa gtccactgaa 180
ctgcttattc gtaaactacc tttccagcgc ctggtgcgcg agattgcgca ggactttaaa 240
acagacctgc gtttccagag ctccgctgtg atggctctgc aggaggcgtg cgaggcctac 300
ttggtagggc tatttgagga cactaacctg tgcgccatcc acgccaagcg cgtcactatc 360
atgcccaagg acatccagct cgcccgccgc atccgcggag agagggcgtg a 411
<210> 28
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> 编码GFPβ11的核酸序列
<400> 28
cgggaccaca tggtgctgca cgagtacgtg aacgccgccg gcatcac 47
<210> 29
<211> 378
<212> PRT
<213> 人工序列
<220>
<223> Ppm1b
<400> 29
Met Gly Ala Phe Leu Asp Lys Pro Lys Thr Glu Lys His Asn Ala His
1 5 10 15
Gly Ala Gly Asn Gly Leu Arg Tyr Gly Leu Ser Ser Met Gln Gly Trp
20 25 30
Arg Val Glu Met Glu Asp Ala His Thr Ala Val Val Gly Ile Pro His
35 40 45
Gly Leu Asp Asn Trp Ser Phe Phe Ala Val Tyr Asp Gly His Ala Gly
50 55 60
Ser Arg Val Ala Asn Tyr Cys Ser Thr His Leu Leu Glu His Ile Thr
65 70 75 80
Thr Asn Glu Asp Phe Arg Ala Ala Asp Lys Ser Gly Ser Ala Leu Glu
85 90 95
Pro Ser Val Glu Ser Val Lys Thr Gly Ile Arg Thr Gly Phe Leu Lys
100 105 110
Ile Asp Glu Tyr Met Arg Asn Phe Ser Asp Leu Arg Asn Gly Met Asp
115 120 125
Arg Ser Gly Ser Thr Ala Val Gly Val Met Val Ser Pro Thr His Met
130 135 140
Tyr Phe Ile Asn Cys Gly Asp Ser Arg Ala Val Leu Cys Arg Asn Gly
145 150 155 160
Gln Val Cys Phe Ser Thr Gln Asp His Lys Pro Cys Asn Pro Val Glu
165 170 175
Lys Glu Arg Ile Gln Asn Ala Gly Gly Ser Val Met Ile Gln Arg Val
180 185 190
Asn Gly Ser Leu Ala Val Ser Arg Ala Leu Gly Asp Tyr Asp Tyr Lys
195 200 205
Cys Val Asp Gly Lys Gly Pro Thr Glu Gln Leu Val Ser Pro Glu Pro
210 215 220
Glu Val Tyr Glu Ile Val Arg Ala Glu Glu Asp Glu Phe Val Val Leu
225 230 235 240
Ala Cys Asp Gly Ile Trp Asp Val Met Ser Asn Glu Glu Leu Cys Glu
245 250 255
Phe Val Lys Ser Arg Leu Glu Val Ser Asp Asp Leu Glu Asn Val Cys
260 265 270
Asn Trp Val Val Asp Thr Cys Leu His Lys Gly Ser Arg Asp Asn Met
275 280 285
Ser Val Val Leu Val Cys Phe Ser Asn Ala Pro Lys Val Ser Glu Glu
290 295 300
Ala Val Lys Arg Asp Ser Glu Leu Asp Lys His Leu Glu Ser Arg Val
305 310 315 320
Glu Glu Ile Met Gln Lys Ser Gly Glu Glu Gly Met Pro Asp Leu Ala
325 330 335
His Val Met Arg Ile Leu Ser Ala Glu Asn Ile Pro Asn Leu Pro Pro
340 345 350
Gly Gly Gly Leu Ala Gly Lys Arg His Val Ile Glu Ala Val Tyr Ser
355 360 365
Arg Leu Asn Pro His Lys Asp Asn Asp Gly
370 375
<210> 30
<211> 1134
<212> DNA
<213> 人工序列
<220>
<223> 编码Ppm1b的核酸序列
<400> 30
atgggtgcat ttttggataa acccaaaact gaaaagcaca atgctcacgg tgctgggaat 60
ggtctgcgtt atggcctgag cagtatgcaa ggatggagag tagaaatgga agatgcacac 120
acagctgttg tgggtattcc tcacggcttg gacaactggt cgttttttgc agtttatgac 180
ggtcatgctg gatcccgagt ggcaaattac tgttcaacac atctattaga acacatcacc 240
accaatgaag acttcagggc agctgacaaa tcaggctctg ctctcgagcc ttcagtagaa 300
agtgtgaaga ctggtatcag gactggcttt ttgaaaattg atgaatatat gcgtaacttc 360
tcagacctga ggaacgggat ggacaggagc ggctcgactg cagtgggcgt gatggtctcc 420
cctacacaca tgtacttcat caactgtggt gactctcgag ctgttctgtg taggaacgga 480
caggtctgct tttctaccca ggatcacaaa ccttgtaatc cagtggagaa ggagcgcatc 540
caaaacgcag gaggcagtgt gatgatccag cgtgtgaacg gctcactagc agtgtctcgg 600
gctctggggg actatgacta caagtgtgtg gatggcaagg gccctacaga gcagcttgtt 660
tctccagagc ctgaggttta tgagattgtg agagcagaag aggatgagtt tgtcgtcttg 720
gcctgtgatg ggatctggga tgtgatgagc aatgaggagc tctgtgagtt tgttaagtct 780
aggcttgagg tgtcggacga cctggagaat gtgtgcaatt gggtagtgga cacttgttta 840
cataagggaa gtcgagataa catgagtgtt gtattagttt gcttttcaaa tgcccccaag 900
gtctcagagg aagccgtgaa gagagattca gagttggata agcacttgga atcacgggtt 960
gaagaaatca tgcagaagtc tggggaggaa ggaatgcctg atcttgccca tgtgatgcgc 1020
attttgtctg cagaaaatat cccgaattta cctcccgggg gaggtctcgc tggcaagcgc 1080
catgttattg aagctgttta tagtagactt aatccacaca aagacaatga tggg 1134
<210> 31
<211> 98
<212> PRT
<213> 人工序列
<220>
<223> ZFP9-NLS
<400> 31
Val Ser Arg Pro Gly Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg
1 5 10 15
Asn Phe Ser Asp Lys Thr Lys Leu Arg Val His Thr Arg Thr His Thr
20 25 30
Gly Glu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Val
35 40 45
Arg His Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Lys Pro
50 55 60
Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser Thr Ser Leu
65 70 75 80
Gln Arg His Leu Lys Thr His Leu Arg Gly Ser Pro Lys Lys Lys Arg
85 90 95
Lys Val
<210> 32
<211> 294
<212> DNA
<213> 人工序列
<220>
<223> 编码ZFP9-NLS的核酸序列
<400> 32
gtctctagac ccggggagcg ccccttccag tgtcgcattt gcatgcggaa cttttcggat 60
aaaactaaat tgagagttca tacccgtact cataccggtg aaaaaccgtt tcagtgtcgg 120
atctgtatgc gaaatttctc cgttagacat aatttgacta gacatctacg tacgcacacc 180
ggcgagaagc cattccaatg ccgaatatgc atgcgcaact tcagtcaatc tacttctttg 240
caaagacacc taaaaaccca cctgagagga tcccccaaga agaagcgtaa ggtg 294
<210> 33
<211> 1431
<212> DNA
<213> 人工序列
<220>
<223> tdTomato编码序列
<400> 33
atggtgagca agggcgagga ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag 60
ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag 120
ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac 180
atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc 240
cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc 300
gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc 360
tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag 420
accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc 480
gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc 540
atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg 600
gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc 660
cgccaccacc tgttcctggg gcatggcacc ggcagcaccg gcagcggcag ctccggcacc 720
gcctcctccg aggacaacaa catggccgtc atcaaagagt tcatgcgctt caaggtgcgc 780
atggagggct ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc 840
tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc 900
tgggacatcc tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc 960
gacatccccg attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg 1020
aacttcgagg acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg 1080
ctgatctaca aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag 1140
aagaagacca tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg 1200
aagggcgaga tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc 1260
aagaccatct acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc 1320
aagctggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc 1380
gagggccgcc accacctgtt cctgtacggc atggacgagc tgtacaagta a 1431
<210> 34
<211> 11
<212> DNA
<213> 人工序列
<220>
<223> ZFP9结合位点
<400> 34
tgtagatgga g 11
<210> 35
<211> 1653
<212> DNA
<213> 人工序列
<220>
<223> Luciferase编码基因
<400> 35
atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatcctct tgaggatgga 60
accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 120
gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc 180
gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 240
tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 300
gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgaacatt 360
tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420
aaaaaattac caataatcca gaaaattatt atcatggatt ctaaaacgga ttaccaggga 480
tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 540
tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa ctcctctgga 600
tctactgggt tacctaaggg tgtggccctt ccgcatagaa ctgcctgcgt cagattctcg 660
catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 720
gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 780
cgagtcgtct taatgtatag atttgaagaa gagctgtttt tacgatccct tcaggattac 840
aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa aagcactctg 900
attgacaaat acgatttatc taatttacac gaaattgctt ctgggggcgc acctctttcg 960
aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc cagggatacg acaaggatat 1020
gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 1080
gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 1140
acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat tatgtccggt 1200
tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 1260
ggagacatag cttactggga cgaagacgaa cacttcttca tagttgaccg cttgaagtct 1320
ttaattaaat acaaaggata ccaggtggcc cccgctgaat tggagtcgat attgttacaa 1380
caccccaaca tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc cggtgaactt 1440
cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat 1500
tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 1560
gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata 1620
aaggccaaga agggcggaaa gtccaaattg taa 1653
<210> 36
<211> 23
<212> PRT
<213> 人工序列
<220>
<223> 流感病毒HA2
<400> 36
Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly
1 5 10 15
Met Ile Asp Gly Trp Tyr Gly
20
<210> 37
<211> 30
<212> PRT
<213> 人工序列
<220>
<223> KALA
<400> 37
Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Ala Lys His
1 5 10 15
Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Ala Cys Glu Ala
20 25 30
<210> 38
<211> 30
<212> PRT
<213> 人工序列
<220>
<223> GALA
<400> 38
Trp Glu Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu His
1 5 10 15
Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala
20 25 30
<210> 39
<211> 26
<212> PRT
<213> 人工序列
<220>
<223> Melittin
<400> 39
Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu
1 5 10 15
Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln
20 25
<210> 40
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> Penetratin
<400> 40
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys
1 5 10 15
<210> 41
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> HIV-TAT(47-57)
<400> 41
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10
<210> 42
<211> 8
<212> PRT
<213> 人工序列
<220>
<223> HIV-1 Rev(34-50)
<400> 42
Lys Gln Ala Ile Pro Val Ala Lys
1 5
<210> 43
<211> 34
<212> PRT
<213> 人工序列
<220>
<223> VP22
<400> 43
Asp Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr
1 5 10 15
Glu Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro
20 25 30
Val Glu
<210> 44
<211> 27
<212> PRT
<213> 人工序列
<220>
<223> Transportan
<400> 44
Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu
1 5 10 15
Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu
20 25
<210> 45
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> Pep-1
<220>
<221> MOD_RES
<222> (1)..(1)
<223> ACETYLATION
<220>
<221> MOD_RES
<222> (21)..(21)
<223> AMIDATION (-NH-CH2-CH2-SH)
<400> 45
Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys
1 5 10 15
Lys Lys Arg Lys Val
20
<210> 46
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> Pep-7
<400> 46
Ser Asp Leu Trp Glu Met Met Met Val Ser Leu Ala Cys Gln Tyr
1 5 10 15
<210> 47
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 弗林蛋白酶识别序列-1
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X=任意氨基酸
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> X=K或R
<400> 47
Arg Xaa Xaa Arg
1
<210> 48
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 弗林蛋白酶识别序列-2
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> X=任意氨基酸
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> X=K或R
<400> 48
Arg Arg Xaa Xaa Arg
1 5
<210> 49
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 弗林蛋白酶识别序列-3
<400> 49
Arg Arg His Lys Arg
1 5
<210> 50
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 肽接头
<400> 50
Gly Gly Gly Gly Ser
1 5
Claims (28)
1.融合多肽,其包含多聚化结构域序列、细胞穿膜肽、pH敏感融合肽以及蛋白酶识别序列。
2.权利要求1所述的融合多肽,其中,所述多聚化结构域是二聚化结构域、三聚化结构域、四聚化结构域或任何更高级的多聚化结构域;
优选地,所述多聚化结构域选自:亮氨酸拉链、NOE、GCN4-P1、Delta及其任何组合;
优选地,所述多聚化结构域选自亮氨酸拉链;
优选地,所述多聚化结构域序列包含SEQ ID NO:1或2所示的序列。
3.权利要求1或2所述的融合多肽,其中,所述细胞穿膜肽选自穿透素(Penetratin)、Tat衍生肽(例如,Tat(48-60)或Tat(47-57))、Rev(34-50)、VP22、转运肽(transportan)、Pep-1、Pep-7,及其任意组合;
优选地,所述细胞穿膜肽包含Tat衍生肽,例如Tat(48-60);
优选地,所述细胞穿膜肽包含如SEQ ID NO:14所示的序列。
4.权利要求1-3任一项所述的融合多肽,其中,所述pH敏感融合肽选自流感病毒HA2或其突变体(例如INF7,KALA或GALA)、蜂毒素(Melittin),及其任意组合;
优选地,所述pH敏感融合肽包含INF7;
优选地,所述pH敏感融合肽包含如SEQ ID NO:12所示的序列。
5.权利要求1-4任一项所述的融合多肽,其中,所述蛋白酶选自弗林蛋白酶和/或溶酶体半胱氨酸蛋白酶;
优选地,所述蛋白酶识别序列选自弗林蛋白酶识别序列、溶酶体半胱氨酸蛋白酶识别序列及其组合。
6.权利要求5所述的融合多肽,其中,所述弗林蛋白酶识别序列包含R-X1-X2-R(SEQ IDNO:47),其中,X1为任意氨基酸,X2为K或R;
优选地,所述弗林蛋白酶识别序列包含R-R-X1-X2-R(SEQ ID NO:48);
优选地,所述弗林蛋白酶识别序列包含SEQ ID NO:49所示的序列;
优选地,所述弗林蛋白酶识别序列包含SEQ ID NO:8所示的序列。
7.权利要求5或6所述的融合多肽,其中,所述溶酶体半胱氨酸蛋白酶选自组织蛋白酶B、组织蛋白酶C、组织蛋白酶X、组织蛋白酶S、组织蛋白酶L、组织蛋白酶D或组织蛋白酶H;
优选地,所述溶酶体半胱氨酸蛋白酶是组织蛋白酶L;
优选地,所述组织蛋白酶L识别序列包含SEQ ID NO:10所示的序列。
8.权利要求1-7任一项所述的融合多肽,其中,所述蛋白酶识别序列包含弗林蛋白酶识别序列和组织蛋白酶L识别序列;
优选地,所述蛋白酶识别序列包含SEQ ID NO:49和SEQ ID NO:10;
优选地,所述蛋白酶识别序列包含SEQ ID NO:8和SEQ ID NO:10。
9.权利要求1-8任一项所述的融合多肽,其中,所述融合多肽从N端至C端包含:所述细胞穿膜肽、pH敏感融合肽、蛋白酶识别序列,或者,从N端至C端包含:所述pH敏感融合肽、细胞穿膜肽、蛋白酶识别序列;并且,所述多聚化结构域序列位于所述融合多肽的N端或C端,或者位于上述任何两个相邻结构域之间;
优选地,所述蛋白酶识别序列从N端至C端包含所述弗林蛋白酶识别序列和组织蛋白酶L识别序列,或者从N端至C端包含所述组织蛋白酶L识别序列和弗林蛋白酶识别序列。
10.权利要求1-9任一项所述的融合多肽,其中,所述融合多肽中所包含的任何两个相邻结构域之间任选地通过肽接头连接;
优选地,所述肽接头是(GmS)n,其中m选自1-4的整数,n选自1-3的整数。
11.权利要求1-10任一项所述的融合多肽,其中,所述融合多肽包含SEQ ID NOs:16-18任一项所示的序列。
12.权利要求1-11任一项所述融合多肽的多聚体;
优选地,所述多聚体是二聚体、三聚体或四聚体;
优选地,所述多聚体是同源多聚体。
13.融合蛋白,其包含权利要求1-11任一项所述的融合多肽,以及另外的多肽;
优选地,所述另外的多肽包含可检测的标记;
优选地,所述另外的多肽包含表位标签、报告基因编码蛋白序列和/或核定位信号(NLS)序列。
14.权利要求13所述的融合蛋白,其中,所述另外的多肽是核酸结合结构域序列;
优选地,所述核酸结合结构域序列是锌指蛋白(例如ZFP9);
优选地,所述核酸结合结构域序列包含SEQ ID NO:31所示的序列;
优选地,所述融合蛋白包含SEQ ID NOs:19-21任一项所示的序列。
15.权利要求13或14所述的融合蛋白,其中,
(i)所述融合蛋白从N端至C端包含:所述细胞穿膜肽、pH敏感融合肽、蛋白酶识别序列和另外的多肽;并且,所述多聚化结构域序列位于所述融合蛋白的N端或C端,或者位于上述任何两个相邻结构域之间;优选地,所述蛋白酶识别序列从N端至C端包含所述弗林蛋白酶识别序列和组织蛋白酶L识别序列,或者从N端至C端包含所述组织蛋白酶L识别序列和弗林蛋白酶识别序列;或,
(ii)所述融合蛋白包含从N端至C端:所述pH敏感融合肽、细胞穿膜肽、蛋白酶识别序列和另外的多肽;并且,所述多聚化结构域序列位于所述融合蛋白的N端或C端,或者位于上述任何两个相邻结构域之间;优选地,所述蛋白酶识别序列从N端至C端包含所述弗林蛋白酶识别序列和组织蛋白酶L识别序列,或者从N端至C端包含所述组织蛋白酶L识别序列和弗林蛋白酶识别序列;或,
(iii)所述另外的多肽融合至所述融合多肽的C端。
16.权利要求13-15任一项所述的融合蛋白的多聚体;
优选地,所述多聚体是二聚体、三聚体或四聚体;
优选地,所述多聚体是同源多聚体。
17.分离的核酸分子,其包含编码权利要求1-11任一项所述的融合多肽,或权利要求13-15任一项所述的融合蛋白的核苷酸序列。
18.载体,其包含权利要求17所述的分离的核酸分子。
19.宿主细胞,其包含权利要求17所述的分离的核酸分子或权利要求18所述的载体。
20.制备权利要求1-11任一项所述的融合多肽,或权利要求13-15任一项所述的融合蛋白的方法,其包括,在合适的条件下培养权利要求19的宿主细胞,和从细胞培养物中回收所述融合多肽或融合蛋白,其中所述融合多肽或融合蛋白以多聚体形式存在。
21.复合物,其包含权利要求12所述的多聚体或权利要求16所述的多聚体,以及货物分子;
优选地,所述货物分子选自蛋白、核酸、糖类、脂质、化学化合物以及其任意的混合物;
优选地,所述货物分子与所述多聚体融合、化学偶联或非共价连接。
22.权利要求21所述的复合物,其中,所述货物分子是肽或蛋白;
优选地,所述货物分子与所述多聚体融合。
23.权利要求21所述的复合物,其中,所述货物分子是核酸;
优选地,所述核酸选自DNA分子、RNA分子、siRNA、反义寡核苷酸、核酶、适体(aptamer)及其任意组合;
优选地,所述多聚体是权利要求14所述的融合蛋白的多聚体。
24.药物组合物,其包含权利要求21-23任一项所述的复合物,以及药学上可接受的载体和/或赋形剂;
优选地,所述复合物包含的货物分子是药学活性剂或可检测的标记。
25.权利要求21-23任一项所述的复合物或权利要求24所述的药物组合物在制备用于治疗疾病的药物中的用途;其中,所述复合物中所包含的货物分子能够治疗所述疾病;
优选地,所述疾病是与细胞程序性坏死相关的疾病,并且,所述货物分子包含蛋白磷酸酶1B;优选地,所述与细胞程序性坏死相关的疾病包括肝损伤(例如药源性肝损伤)、炎性疾病、缺血再灌注损伤和/或神经退行性疾病。
26.试剂盒,其包含权利要求1-11任一项所述的融合多肽、权利要求12所述的多聚体、权利要求13-15任一项所述的融合蛋白、权利要求16所述的多聚体、权利要求17所述的分离的核酸分子、权利要求18所述的载体、权利要求19所述的宿主细胞、或权利要求21-23任一项所述的复合物;
优选地,所述试剂盒进一步包括用于转染和/或胞内递送的说明书。
27.权利要求1-11任一项所述的融合多肽、权利要求12所述的多聚体、权利要求13-15任一项所述的融合蛋白、权利要求16所述的多聚体、权利要求17所述的分离的核酸分子、权利要求18所述的载体、权利要求19所述的宿主细胞、或权利要求21-23任一项所述的复合物,用作递送试剂的用途。
28.用于将货物分子递送入细胞的方法,其包括:使所述细胞与权利要求21-23任一项所述的复合物接触,其中所述货物分子是所述复合物中所包含的货物分子;
优选地,所述细胞与所述复合物的接触在体外实施;
优选地,所述货物分子选自核酸、肽或蛋白、糖类、脂质、化学化合物以及其任意的混合物;优选地,所述核酸选自DNA分子、RNA分子、siRNA、反义寡核苷酸、核酶、适体(aptamer)及其任意组合。
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WO2009080816A1 (en) * | 2007-12-21 | 2009-07-02 | Altonabiotec Ag | Fusion polypeptides comprising a shbg dimerization component and uses thereof |
US20160272678A1 (en) * | 2013-03-15 | 2016-09-22 | Board Of Regents, The University Of Texas System | Inhibition of pulmonary fibrosis with nutlin-3a and peptides |
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