CN103298938A - 氧化应激指示物表达用核酸构建物及其应用 - Google Patents
氧化应激指示物表达用核酸构建物及其应用 Download PDFInfo
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Abstract
本发明提供氧化应激指示物表达用核酸构建物以及使用该核酸构建物的氧化应激测定方法及氧化应激抑制剂的筛选方法,该核酸构建物至少包含Nrf2蛋白中的Neh2结构域序列,并且包含:编码使Neh1结构域序列或Neh1-Neh3结构域序列实质上缺失或丧失功能而成的部分蛋白质的核酸序列,配置于该核酸序列的上游的应激诱导性启动子序列,配置于该核酸序列的下游的、编码能发出可检测信号的蛋白质的核酸序列。
Description
技术领域
本发明涉及氧化应激指示物表达用核酸构建物。具体而言,氧化应激是与Nrf2-Keap1通路有关的应激。
本发明也涉及使用上述核酸构建物来测定氧化应激的方法。
本发明还涉及使用上述核酸构建物来筛选氧化应激抑制用药剂的方法。
背景技术
氧化应激是指机体内的活性氧类的生成和清除系统的平衡紊乱、活性氧类过量的状态。如果该状态发展下去,则构成机体的核酸、蛋白质、脂类等被氧化,对机体产生损伤。细胞如果暴露于氧化应激或亲电子性物质,则会通过诱导表达谷胱甘肽合成酶、血红素加氧酶1(HO-1)等抗氧化蛋白质和II相解毒酶来参与机体防御(非专利文献1)。这种基于氧化应激的基因表达机制中,存在于基因上游的抗氧化剂应答元件ARE(Antioxidant Response Element)和与其结合的转录因子Nrf2起着重要的作用(非专利文献2和3)。
Nrf2是碱性亮氨酸拉链(bZip)型转录因子,作为针对氧化应激、亲电子性应激、毒性化合物及致癌物质等的细胞防御的重要控制因子起作用(非专利文献3、4、5和6)。在常规条件下,Nrf2通过由其与Keap1(Kelch样ECH联合蛋白1(Kelch-like ECH associating protein 1);其是Cul3系泛素E3连接酶复合物的底物衔接蛋白)的结合介导的泛素-蛋白酶体通路而迅速地分解(非专利文献7~10)。如果暴露于氧化应激或亲电子性应激,则Keap1中的多个反应性半胱氨酸残基被共价键修饰,通过由Keap1介导的分解而释放出Nrf2。其结果是,发生Nrf2的稳定化和随后向核中的转移,在核中,Nrf2与小Maf家族蛋白的成员形成二聚体。该复合物由已知作为氧化/亲电体应答元件(ARE/EpRE)的顺式作用DNA元件(cis-acting DNA element)介导,激活大范围的转录(非专利文献2和3)。
重要的是,体外和体内模型显示,Keap1-Nrf2系统与阿尔茨海默病、帕金森病、亨延顿舞蹈病、肌萎缩性侧索硬化症等神经变性疾病有关,或是减轻该疾病(非专利文献11)。此外,使用Nrf2基因敲除(Nrf2 KO)小鼠的最近的研究表明,Nrf2的机能障碍与人类疾病的病况有关,Nrf2在人类疾病中有可能起着重要的作用(非专利文献12~14)。还有,最近人们发现,Nrf2作为急性肺损伤和急性炎症的机体侧防御因子起着重要的作用(非专利文献15)。
迄今为止,氧化应激和针对氧化应激的细胞应答反应主要用培养细胞来进行研究,此外,关于监控氧化应激状态的报告物(reporter),也只知道低灵敏度的报告物。还有,氧化应激的检测一般通过测定某种蛋白质的表达量来进行。具体而言,通过以下方法进行:通过蛋白质印迹分析来测定氧化应激诱导性的转录因子Nrf2的表达量,或者通过RNA印迹分析来测定在其下游诱导的HO-1的表达水平。但是,这些方法由于存在将细胞裂解的过程,因此难以在个体水平上确定受到氧化应激的脏器。迄今为止,制作了由氧化应激依赖性地受Nrf2诱导的启动子来使碱性磷酸酶表达的报告物,但灵敏度不足,而且报告蛋白不适于个体观察,因此不能进行动物个体的分析,而只是停留在培养细胞水平的分析(非专利文献16)。
此外,迄今为止,已知将应激诱导性启动子和蛋白质分解控制位点组合而成的报告物(非专利文献17),包含荧光或发光蛋白、标记蛋白及调节蛋白的用于测定氧化应激的探针试剂(专利文献1)等。
综上所述,体内的氧化应激研究应该能提供在与氧化应激有关的神经变性疾病、心血管疾病、糖尿病、类风湿性关节炎等疾病的病理、发病和细胞生物学上有用的信息。
现有技术文献
专利文献
专利文献1:国际申请第WO2009/1078号小册子
非专利文献
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发明内容
如上所述,不仅已知氧化应激与各种疾病有关,而且对于能在细胞和机体内以高灵敏度监控或检测氧化应激状态的系统的构建的需求很高。
这次,为了使体内的氧化应激的分析变得容易,本发明人着眼于通过至少保持Nrf2中的Neh2结构域的与Keap1结合而泛素化的功能、使Neh1结构域或Neh1-Neh3结构域的DNA结合能力缺失、从而提高氧化应激时的应激反应的检测时的S/N(信号/噪声)比这一点,发现了即使在叫做OKD48 (Keap1依赖性氧化应激检测器(Keap1-dependent Oxidative stress Detector), No-48)(也称“AJISAI (ARE-Nrf2联合应激相关指示物(ARE-Nrf2 jointed stress associated indicator))”)的活细胞中也能使用的氧化应激指示物。本说明书中,对于OKD48这样的氧化应激指示物的特异性和感受性、以及表达氧化应激指示物的转基因非人动物的有用性进行了记载。
即,本发明包含以下特征。
(1)氧化应激指示物表达用核酸构建物,其包含:编码使Nrf2蛋白中至少包含Neh2结构域序列、并且使Neh1结构域序列或Neh1-Neh3结构域序列实质上缺失或丧失功能而成的部分蛋白质的核酸序列,配置于该核酸序列的上游的应激诱导性启动子序列,和配置于该核酸序列的下游的、编码能发出可检测信号的蛋白质(例如发光或荧光蛋白或者结合有可标记的标签序列的蛋白质)的核酸序列。
(2)上述(1)所述的核酸构建物,其中,部分蛋白质包含从Nrf2蛋白中使Neh1结构域序列或Neh1-Neh3结构域序列实质上缺失而成的氨基酸序列。
(3)上述(1)或(2)所述的核酸构建物,其中,Nrf2蛋白是来源于哺乳动物的Nrf2。
(4)上述(3)所述的核酸构建物,其中,来源于哺乳动物的Nrf2是人Nrf2或小鼠Nrf2。
(5)上述(1)~(4)中任一项所述的核酸构建物,其中,部分蛋白质包括:(a)由序列编号1(人)或序列编号2(小鼠)的Nrf2蛋白的氨基酸序列中的1位~93位氨基酸构成的Neh2结构域序列,(b)与该Neh2结构域序列具有80%以上的同一性的氨基酸序列,或者(c)在该Neh2结构域序列中包含1个或多个氨基酸的缺失、置换或添加的氨基酸序列。
(6)上述(1)~(4)中任一项所述的核酸构建物,其中,部分蛋白质包括:(d)由序列编号1(人)或序列编号2(小鼠)的Nrf2蛋白的氨基酸序列中的1位~433位氨基酸或1位~425位氨基酸各自构成的、包含Neh2-Neh6结构域序列的氨基酸序列,(e)与该包含Neh2-Neh6结构域序列的氨基酸序列具有80%以上的同一性的氨基酸序列,或者(f)在该包含Neh2-Neh6结构域序列的氨基酸序列中包含1个或多个氨基酸的缺失、置换或添加的氨基酸序列。
(7)上述(1)~(6)中任一项所述的核酸构建物,其中,应激诱导性启动子由抗氧化剂应答元件(ARE)或ARE与启动子的组合构成。
(8)上述(7)所述的核酸构建物,其中,ARE由多次重复构成。
(9)上述(1)~(8)中任一项所述的核酸构建物,其中,所述核酸构建物在其3'末端包含标签编码序列。
(10)上述(9)所述的核酸构建物,其中,标签编码序列是Flag标签编码序列。
(11)载体,其包含上述(1)~(10)中任一项所述的核酸构建物。
(12)细胞,其包含上述(1)~(10)中任一项所述的核酸构建物或上述(11)所述的载体。
(13)非人动物,其包含上述(1)~(10)中任一项所述的核酸构建物或上述(11)所述的载体。
(14)上述(13)所述的非人动物,其中,非人动物还包含内质网应激指示物表达用核酸构建物或低氧应激指示物表达用核酸构建物。
(15)用于测定氧化应激的方法,其包括在上述(12)所述的细胞或者上述(13)或(14)所述的非人动物中测定在提供氧化应激时增大的可检测信号(例如发光或荧光信号)的强度的步骤。
(16)筛选氧化应激抑制剂的方法,其包括如下步骤:在候选药剂的存在下对上述(12)所述的细胞或者上述(13)或(14)所述的非人动物提供某种特定的氧化应激,当可检测信号(例如发光或荧光信号)的强度与不存在该候选药剂的对照相比有所减小时,判定为该候选药剂具有氧化应激抑制能力。
本发明的指示物能特异性地检测细胞或动物中与Nrf2-Keap1通路有关的氧化应激,而且具有使氧化应激时的应激反应检测的S/N比提高的优点。
本说明书包含作为本申请优先权基础的日本国专利申请2011-010833号的说明书和/或附图中记载的内容。
附图说明
图1表示Nrf2的结构域结构(A)以及人Nrf2和小鼠Nrf2的各结构域的位置和同一性百分比(B)。
图2表示人Nrf2和小鼠Nrf2的氨基酸序列比对。
图3模式地表示由p(3xARE)TKbasal-hNrf2(1-433)-GL4-Flag构成的核酸构建物(OKD48)以及常规条件和氧化应激条件下的该核酸构建物的转录的抑制和诱导。作为OKD48构建物,制备了p(3xARE)TKbasal-hNrf2(1-433)GL4-Flag。人Nrf2中,Neh2结构域起到Keap1结合及泛素化区域的作用,Neh1结构域起到DNA结合区的作用。OKD48构建物具有3×ARE启动子、人Nrf2(1-433氨基酸)编码序列和Flag标签结合荧光素酶(GL4)编码序列。在常规条件下,OKD48构建物的转录不被诱导。此外,漏出的OKD48蛋白被Keap1系统分解。在氧化应激下,OKD48由3×ARE启动子诱导转录,所得的OKD48蛋白由Keap1系统稳定化。由此,只在受到氧化应激的细胞中检测到发光。
图4表示包含由p(3xARE)TKbasal-hNrf2(1-433)-GL4-Flag构成的核酸构建物(OKD48)的载体的结构。
图5表示体外的OKD48的特性确定。(A)相对于氧化应激的AJISA的特异性。将OKD48构建物转染至HeLa细胞中,在各种应激物质(亚砷酸钠(ASN)、马来酸二乙酯(DEM)、H2O2、衣霉素(Tun)、毒胡萝卜素(Tg)、二硫苏糖醇(DTT)、依托泊苷(Etp)或噻吩甲酰三氟丙酮(TTFA))的存在下或不存在下处理8小时或16小时后,进行荧光素酶试验。(B)各种应激下的OKD48构建物的蛋白质表达。将OKD48构建物转染至HEK293T细胞中,在各种应激物质的存在下或不存在下处理8小时。通过使用抗Luc抗体或抗GAPDH抗体的蛋白质印迹法来分析细胞的裂解液。(C)各种应激下的内源性Nrf2的蛋白质表达。将HeLa细胞在各种应激物质的存在下或不存在下处理8小时或16小时后,用抗Luc抗体或抗GAPDH抗体的蛋白质印迹法来分析细胞的裂解液。(D)Nrf2过表达对OKD48活性的影响。将OKD48构建物和Nrf2过表达载体转染至HeLa细胞中,在亚砷酸钠(ASN)的存在下或不存在下处理8小时后,进行荧光素酶试验。(E)Keap1过表达对OKD48活性的影响。将OKD48构建物和Keap1过表达载体(全长或1-314切割型氨基酸)转染至HeLa细胞中,在亚砷酸钠(ASN)的存在下或不存在下处理8小时后,进行荧光素酶试验。
图6表示荧光素酶融合Nrf2片段的优化。(A)试验构建物的示意图。将人Nrf2的部分片段(1-93氨基酸或1-433氨基酸)或全长编码序列与Flag标签结合荧光素酶(GL4)编码序列融合。这些融合基因由3×ARE启动子或作为阴性对照的HSV-TK(单纯疱疹病毒胸苷激酶 (Herpes simplex virus thymidine kinase))启动子驱动。(B)使用试验构建物的荧光素酶试验。将各试验构建物转染至HeLa细胞中,在亚砷酸钠(ASN)、马来酸二乙酯、H2O2的存在下或不存在下处理8小时或16小时后,进行荧光素酶试验。
图7表示体外的来源于OKD48-Venus的荧光的检测。(A)来源于OKD48-Venus构建物的荧光图像。将OKD48-Venus构建物转染至HEK293T细胞中,然后在亚砷酸钠(ASN)或马来酸二乙酯的存在下或不存在下处理8小时。和相位差图像一起获得它们的荧光图像。(B)各种应激下的来源于OKD48-Venus构建物的荧光强度。将OKD48-Venus构建物转染至HEK293T细胞中,然后将细胞在亚砷酸钠(ASN)、马来酸二乙酯(DEM)、H2O2、衣霉素(Tun)、毒胡萝卜素(Tg)、二硫苏糖醇(DTT)、依托泊苷(Etp)或噻吩甲酰三氟丙酮(TTFA))的存在下或不存在下处理8小时。测定来源于这些细胞的裂解液的荧光,相对于同时转染的荧光素酶进行标准化。图中,n.d.表示未检出。(C)各种应激下的OKD48-Venus构建物的蛋白质表达。将OKD48-Venus构建物转染至HEK293T细胞中,在各种应激物质的存在下或不存在下处理8小时。通过使用抗GFP抗体或抗GAPDH抗体的蛋白质印迹法来分析这些细胞裂解液。
图8表示给予作为氧化应激物质的ASN试剂,经过6小时后剖开小鼠的腹部,观察发光信号的结果。
图9表示小鼠OKD48在应激条件下(应激物质:DEM或ASN)的发光特性。
具体实施方式
以下,对本发明进行详细说明。
1.氧化应激指示物表达用核酸构建物
本发明的氧化应激指示物表达用核酸构建物的特征在于,包含:编码使Nrf2蛋白中至少包含Neh2结构域序列、并且使Neh1结构域序列或Neh1-Neh3结构域序列实质上缺失或丧失功能而成的部分蛋白质的核酸序列,配置于该核酸序列的上游的应激诱导性启动子序列,和配置于该核酸序列的下游的、编码发光或荧光蛋白或者结合有(在各种试剂的作用下)可标记的标签序列的蛋白质的核酸序列。
本说明书中的“核酸”这一用语的使用含义包括基因、DNA、cDNA、RNA、mRNA、它们的化学修饰体等。
本说明书中的“氧化应激指示物”这一用语是指细胞或机体内产生的氧化应激的指示物(指示物质或指示剂),在本发明中是指使Nrf2蛋白中至少在功能上保持Neh2结构域、而使Neh1结构域或Neh1-Neh3结构域的功能丧失的物质。
关于Neh2结构域而使用的“在功能上保持”这一用语是指一种Neh2结构域,该Neh2结构域在保持着与Keap1结合而泛素化的功能的基础上,还具有与序列编号1(人Nrf2蛋白)或序列编号2(小鼠Nrf2蛋白)的氨基酸序列中的1位~93位氨基酸的序列有着80%以上、优选90%以上、更优选95%以上的同一性的氨基酸序列。从在没有氧化应激的平常时候氧化应激指示物在Nrf2的作用下受到分解这一方面来看,Neh2结构域的功能是必需的。
还有,“使Neh1结构域或Neh1-Neh3结构域实质上缺失或丧失功能”这一用语是指只要丧失Neh1结构域的功能即“DNA结合能力”即可的状态,因此未必需要整个Neh1结构域都缺失,即,意味着Neh1结构域的一部分、例如其1~50个、优选1~20个氨基酸可以不缺失而残存下来。此外,Neh3优选不存在。无论如何,由于Neh1结构域的存在,即使在没有氧化应激的情况下也会显示出氧化应激反应,因此必须使Neh1结构域没有功能。
“氧化应激”这一用语是指机体内的活性氧类的生成和清除系统的平衡紊乱、活性氧类过量的状态,本发明中特别是与Keap1-Nrf2通路有关。已知由氧化应激诱导的血红素加氧酶1(HO-1)被紫外线(UV-A)照射诱导,Nrf2蛋白参与该诱导(Allanson M 和Reeve VE, J. Invest. Dermatol. 2004;122:1030-1036;Zhong JL等. Photochem. Photobiol. Sci. 2010;9:18-24)。因此,例如通过本发明,即使是由紫外线照射引起的与生理条件接近的低水平的氧化应激也能够检出。紫外线照射的强度例如为1~100mW/cm2、优选为2~50mW/cm2、更优选为5~30mW/cm2。氧化应激与动脉粥样硬化症等心血管疾病,阿尔茨海默病、帕金森病、亨延顿舞蹈病、肌萎缩性侧索硬化症等神经变性疾病,以肝病为代表的各脏器的变性疾病,糖尿病、类风湿性关节炎等多种疾病有关。此外,也报道了与急性炎症和慢性炎症等的炎症反应的相关性。本发明中,可以使用上述氧化应激指示物在体外或体内测定氧化应激。
“Nrf2蛋白”这一用语是指作为碱性亮氨酸拉链(bZip)型转录因子起作用的蛋白质,该蛋白质在动物细胞内对氧化应激作出应答,诱导担负针对该氧化应激的防御功能的抗氧化蛋白质的表达。在细胞没有氧化应激的状态下,Nrf2以与Keap1结合的形态存在于细胞质中。此时,Keap1进一步与泛素连接酶复合物结合,促进Nrf2的泛素化。泛素化的Nrf2在作为蛋白质分解酶复合物的蛋白酶体的作用下受到分解,因此其功能被抑制。另一方面,如果细胞暴露于氧化应激,则活性氧与Keap1反应,使其结构发生变化。由于该结构变化,Keap1从Nrf2解离,因而Nrf2的泛素化被抑制。分解由此得到了抑制的Nrf2稳定化,在细胞内的量增加,转移至核内,发挥出作为转录因子的功能。此时,Nrf2与抗氧化剂应答元件(antioxidant responsive element;ARE)结合,表达氧化应激防御基因。关于Nrf2-Keap1通路介导的氧化应激防御机制,在伊东健、生物化学(Biochemistry) 78(2) pp.79-92 (2006)(日本)中有记载。
Nrf2如图1A所示,由自N末端侧起的依次包含Neh2结构域、Neh4域、Neh5域、Neh6结构域、Neh1结构域及Neh3结构域的6个Neh(Nrf2-ECH homology)结构域构成,在物种间高度保守(伊东健 2006,见上文)。ECH是鸡Nrf2的别名。这里,Neh2是Nrf2活性的抑制性的控制结构域,具有与Keap1结合的位点和泛素化位点。Neh4和Neh5均为转录活化结构域(TAD;Trans-Activation Domain),与作为辅助因子的转录辅激活因子(CBP)结合。Neh6是在核中固定地被分解的结构域(降解决定子(Degron))。Neh1由碱性-CNC特异性区域(Basic)和亮氨酸拉链区域(L-Zip)构成,Basic是与DNA特异性结合以及参与向核中的转移的区域,L-Zip对与小Maf群因子形成异源二聚体而言是重要的区域。Neh3是与转录活化有关的区域(TAD)(Nioi P等. Mol Cell Biol 2005;25:10895-10906)。
本说明书中使用的“Neh2-Neh6结构域”这一用语是指从Nrf2蛋白的Neh2结构域的N末端氨基酸残基到Neh1结构域的N末端氨基酸的前一个氨基酸残基为止。此外,“Neh1-Neh3结构域”是指从Neh1结构域的N末端氨基酸残基到Nrf2蛋白的C末端氨基酸残基为止。
本说明书中使用的“Nrf2”这一用语包括来源于脊椎动物、无脊椎动物、温血动物、哺乳动物、鸟类等动物的Nrf2,其同系物(同源体(homolog)或直系同源体(ortholog))及其类似物(analog)(例如突变体(虽然保持Nrf2的生物活性,但部分地包含氨基酸残基的缺失、置换、插入或添加)或化学修饰体)。来源于动物的Nrf2的氨基酸序列或碱基序列可通过访问NCBI(GenBank)、EMBL、DDBJ等序列数据库而获得。
例如图2表示来源于哺乳动物的Nrf2中、特别是人Nrf2和小鼠Nrf2的氨基酸序列(分别为序列编号1、序列编号2)的比对。人和小鼠的Nrf2之间的序列同一性约为80%。此外,图1B表示人和小鼠的Nrf2蛋白的Neh2、Neh4、Neh5、Neh6、Neh1、Neh3的各结构域的位置和同一性百分比。序列同一性可以用BLAST(基础局部比对搜寻工具(Basic Local Alignment Search Tool))、FASTA等公知的算法来确定(Altschul SF等, J Mol Biol 215(3):403-10, 1990),此外,同一性百分比是指将2条氨基酸序列按照一致度最大的方式导入缺口、或者不导入缺口来进行比对时,相同的氨基酸残基数相对于总氨基酸残基数(也包括缺口的数目)的百分比。该定义也同样适用于DNA的碱基序列的同一性百分比。此时,分别以碱基序列来代替上述氨基酸序列作为对象,以核苷酸(或碱基)来代替上述氨基酸作为对象。
如上所述,本发明中,Nrf2优选来源于哺乳动物的Nrf2,例如更优选来源于人的Nrf2和来源于小鼠的Nrf2,但在保守性高的结构域域Neh2以外的Nrf2区域内,可以有全部成熟氨基酸残基数的约20%以下、优选约10%以下、更优选约5%以下、例如约4%以下、约3%以下、约2%以下或约1%以下的氨基酸突变(缺失、置换、插入或添加)。即,人Nrf2是包含序列编号1所示的氨基酸序列或与该氨基酸序列具有80%以上、优选90%以上、更优选95%以上、例如96%、97%、98%或99%以上的同一性的氨基酸序列的蛋白质,另一方面,小鼠Nrf2是包含序列编号2所示的氨基酸序列或与该氨基酸序列具有80%以上、优选90%以上、更优选95%以上、例如96%、97%、98%或99%以上的同一性的氨基酸序列的蛋白质。
本发明的核酸构建物至少包含编码上述指示物的核酸序列和应激诱导性启动子序列。
“应激诱导性启动子”这一用语是指控制应激防御基因的基因表达的启动子。这种启动子不限定于以下的启动子,例如为醌还原酶(QR)启动子、Nrf2的目标基因启动子、作为启动子活化位点的抗氧化剂应答元件(ARE)或Maf识别元件(MARE;Maf-recognition element)等。或者也可以是这些启动子或启动子活化位点与其它启动子或增强子的组合。这里,其它启动子或增强子包括来源于病毒的启动子、来源于哺乳动物的启动子、组织特异性启动子等,例如有HSV-TK基础启动子(Exp Cell Res. 2009 315(15):2496-504)、HSV-TK启动子(Cell 1981; 25:385-398, Arch. Biol. Sci. Belgrade 2006; 58(4):197-203, phRL-TK (Promega))、CMV(巨细胞病毒)启动子/增强子(J. Vitol. 1995; 69:2194-2207, pKM2L-pvCMV (RDB No. 5551; RIKEN, pRc/CMV (Invitrogen), pCMVTNT (Promega))、SV-40病毒启动子/增强子(Cell 1981; 27:299-308, J. Virol. 1991; 65(12):6900-6912, pKM2L-pvSV40 (RDB No. 5550; RIKEN), pCAT(注册商标)3-增强子(Promega), pCAT(注册商标)3-启动子(Promega), pGL3-对照(Promega))、延伸因子1(EF-1)启动子(Nucleic Acids Res.1999; 27(24);4775-4782)等。此外,增强子的例子有Nrf2的来源于目标基因的增强子、例如血红素加氧酶-1(HO-1)增强子(药学杂志2007; 127(4): 757-764(日本), Am. J. Physiol. Renal Physiol. 2003; 285:F515-F523)、NAD(P)H脱氢酶-1(NQO1)增强子(Proc Natl Acad Sci U S A. 1996;93:14960-5)等。上述组合例如有ARE和HSV-TK基础启动子(basal promoter)的组合、ARE和HO-1增强子的组合等,优选ARE和HSV-TK基础启动子的组合。
上述启动子/增强子的序列的具体例如下所示。
HSV-TK基础启动子(序列编号3):
HSV-TK启动子(phRL-TK (Promega); 543-758部分)(序列编号4):
SV40启动子/增强子(pGL3-对照(Promega); 48-250部分)(序列编号5):
巨细胞病毒极早期基因增强子/启动子(Cytomegalovirus immediate-early enhancer/promoter)(pCMVTNT (Promega); 1-795部分)(序列编号6):
小鼠HO-1增强子-TK基础启动子(序列编号7):
(粗体部分表示限制酶位点,标有“HO-1增强子”的部分表示包含HO-1增强子的序列,标有“TK基础启动子”的部分表示TK基础启动子序列。)。
ARE是对作为氧化应激原因物质的亲电子性物质(例如马来酸二乙酯(DEM)、亚砷酸钠(ASN)、叔丁基氢醌(t-BHQ)等)作出应答、介导机体防御基因的诱导表达的基因表达控制序列(或顺式作用增强子区(cis-acting enhancer region))。根据本发明的实施方式,ARE为多次重复,优选为3~5次重复,更优选为3次重复。多次重复的情况下,优选在ARE序列和ARE序列之间存在由例如1~20个、例如1~10个的任意的碱基构成的序列作为间隔物。ARE的碱基序列是TGA(G/C)NNNGC(这里,N是G、C、A或T)(Trends Mol Med. 2004;10(11):549-57),例如是TGACATTGC(序列编号8)和/或TGACAAAGC(序列编号9)。下述实施例中使用的ARE的3次重复序列(3×ARE)如下所述(序列编号10)。
(粗体部分表示限制酶位点,标有“ARE”的部分表示包含ARE的序列。)。
此外,下述实施例中使用的3xARE-TK基础启动子序列如下所述(序列编号11)。
(粗体部分表示限制酶位点,标有“ARE”的部分表示包含ARE的序列,标有“TK基础启动子”的部分表示TK基础启动子序列。)。
本发明的核酸构建物还可以包含终止子、Poly A序列、核糖体结合序列等调节序列、根据需要使用的筛选标记序列。还有,该核酸构建物还可以在上游和下游包含Nrf2基因的5'-非翻译区序列和3'-非翻译区序列,这是意欲整合到细胞基因组中而进行的。3'-非翻译区序列或5'-非翻译区序列的大小没有限定,例如为1kb以上,优选为1~7kb。
本发明的氧化应激指示物是Nrf2蛋白中至少包含Neh2结构域、并且Neh1结构域或Neh1-Neh3结构域、优选Neh1-Neh3结构域被发光或荧光蛋白或者结合有可标记的标签序列的蛋白质置换而得的改性Nrf2蛋白。本发明的核酸构建物可以通过改性Nrf2蛋白的表达来进行发光或荧光的观察。
筛选标记序列可以用于筛选整合了本发明的核酸构建物的细胞,可以包括例如抗药性基因(例如卡那霉素抗性基因、氨苄西林抗性基因、潮霉素抗性基因等)等。此外,整合到细胞基因组中后除去筛选标记序列时,可以在核酸构建物中在筛选标记序列的两端配置loxP序列、FRT序列等重组酶识别序列,利用Cre-loxP系统、FLP-FRT系统等重组酶-重组酶识别序列系统进行除去。如果需要,可以将HSV-TK基因或者白喉毒素(DT)基因或其片段等负向筛选标记序列插入核酸构建物中。
本发明中,氧化应激的检测可以使用能发出可检测信号的任意的蛋白质。此外,例如也可以通过使用细胞死亡诱导分子等来特异性地杀灭受到氧化应激的细胞或组织。优选使用发光或荧光蛋白或者结合有可标记的标签序列的蛋白质。本说明书中关于上述内容对本发明进行说明,但本发明不限定于此。
本发明中使用的发光或荧光蛋白无特别限制,可以使用任意的蛋白。作为荧光蛋白,可以使用来源于维多利亚多管发光水母的荧光蛋白及其衍生物、例如GFP、YFP、EBFP、ECFP、EGFP、EYFP、Venus等,或者来源于珊瑚的荧光蛋白及其衍生物、例如DsRed、HcRed、mCherry等。作为发光蛋白的代表性的例子,包括荧光素酶,其是萤火虫、叩头虫等甲虫类、海萤、细菌等生物所具有的催化生物发光反应的酶。关于荧光蛋白,可例举例如Shimomura O等(1962) J Cell Comp Physiol 59: 223–39; Phillips G (2001) FEMS Microbiol Lett 204 (1): 9–18; Shaner N等(2005) Nat Methods 2: 905–909; Heim R等(1995) Nature 373: 663–664等文献。此外,关于荧光素酶,可例举例如de Wet JR等(1985) Proc Natl Acad Sci USA 82: 7870-7873、日本特开2008-289475号公报、日本特开2005-245457号公报等文献。这种荧光或发光蛋白也由Clontech、Roche Diagnostics、CHISSO(日本)等在市场上销售。
结合有可标记的标签序列的蛋白质也称为标签蛋白,例如包括四半胱氨酸标签(标记试剂:FlAsH标记试剂或ReAsH标记试剂;Invitrogen)、HaloTag(注册商标)(标记试剂:有多种;Promega)、SNAP-tag、CLIP-tag、ACP-tag和MCP-tag(以上,标记:有多种;New Englnad Biolabs)、荧光发生素激活蛋白(Fluorogen activating proteins)(FAPs)(Nat. Biotechnol. 2008; 26(2):235-240)等,但不限定于此。这些标签蛋白是将蛋白质与荧光标签序列结合来对其进行标记的蛋白质。
本发明的核酸构建物可以如下所述制备,但以下是示例,并不对该方法进行限制。
将来源于人、小鼠等动物种类的Nrf2基因进行cDNA克隆,进行PCR扩增,整合到载体中后,将Nrf2的Neh1编码区或Neh1-Neh3编码区的两末端实质上用限制酶切割、除去,将预先制备好的、编码发光或荧光蛋白或标签蛋白或者连接有报告序列(例如Flag标签等)的发光或荧光蛋白或标签蛋白的核酸插入该切割位点。例如为人Nrf2时,Neh1编码区和Neh3编码区分别相当于编码序列编号1的氨基酸序列的434位~561位、562位~605位的序列的区域,另一方面,为小鼠Nrf2时,Neh1编码区和Neh3编码区分别相当于编码序列编号2的氨基酸序列的426位~553位、554位~597位的序列的区域(参照图1B)。Neh1编码区或Neh1-Neh3编码区的置换中,如果该区域的功能有障碍或功能丧失,则可以不将该区域全部置换,而是将一部分用编码发光或荧光蛋白或标签蛋白的核酸置换。此时,编码从Nrf2蛋白上缺失Neh1或Neh1-Neh3而残留的蛋白质部分的核酸由编码至少包含Neh2的氨基酸序列的核酸构成。通过PCR来扩增编码如上所述得到的改性Nrf2蛋白的核酸,回收扩增产物。Neh3编码区不是必需的,因此在PCR扩增时,可以按照不扩增该区域的方式来设计引物,从而制成不含Neh3编码区的扩增产物。下述实施例中,作为扩增产物得到编码hNrf2(1-433)-Luc、小鼠Nrf2(1-426)-Luc(这里,Luc表示编码荧光素酶的DNA)的核酸。这些核酸构建物与未改性Nrf2-Luc核酸构建物相比,可实现更高的S/N比。
下述实施例中实际使用的序列(3xARE-TKbasal-OKD48-LUC)如下所示(序列编号12)。
(粗体部分表示限制酶位点,标有“ARE”的部分表示包含ARE的序列,标有“TK基础启动子”的部分表示包含TK基础启动子的序列,加框部分表示Nrf2部分(aa1-433),斜体部分表示Flag标签序列,剩余部分表示荧光素酶序列(GL4)。)。
下述实施例中实际使用的序列(3xARE-TKbasal-OKD48-Venus)如下所示(序列编号13)。
(粗体部分表示限制酶位点,标有“ARE”的部分表示包含ARE的序列,标有“TK基础启动子”的部分表示包含TK基础启动子的序列,加框部分表示Nrf2部分(aa1-433),斜体部分表示Flag标签序列,剩余部分表示Venus序列(GL4)。)。
PCR扩增用的引物可以根据目标扩增产物的碱基序列来设计,此外,PCR条件可以采用惯用的条件。
一般来说,PCR反应包括:以使双链DNA变性而分离成单链DNA的步骤、将单链DNA与引物退火的步骤、以及以单链DNA为模板使引物延伸的步骤作为一个循环,将其重复约20~45个循环。变性步骤例如由94~98℃、约10秒~约5分钟的处理构成,退火步骤例如由约50~68℃、约10~60秒的处理构成,延伸步骤例如由72℃、约20秒~约10分钟的处理构成。在所有循环开始前,可以进行94~98℃、约30秒~约5分钟的处理,此外,在所有循环结束后,可以进行72℃、约1~10分钟的处理。引物由正向引物和反向引物构成,根据模板DNA的碱基序列来设计,此外,引物的大小一般为15~30个碱基,优选为20~25个碱基。PCR反应液由模板DNA、耐热性聚合酶、包含Mg2+、dNTP(N=A、T、C、G)等的PCR缓冲液构成。如果使用热循环器等PCR装置,则能方便地实施PCR反应。关于PCR的方法的具体例,可参照FM Ausubel等、Short Protocols in Molecular Biology (2002), John Wiley & Sons、RA Siki等, Science 1985, 230:1350-1354、HA Erlich等, Science 1991, 252:1643-1651等。
另一方面,关于核酸构建物的另一要素、即应激诱导性启动子,合成该启动子,或者由包含该启动子的文库、例如来源于动物组织的基因组文库来制备,整合到含有单一限制位点或多克隆位点的载体中。该载体中,在应激诱导性启动子的3’侧的限制位点或多克隆位点处插入编码改性Nrf2蛋白的核酸。根据需要将制成的核酸构建物从载体上切下,用电泳等纯化方法回收。这里,应激诱导性启动子是以上例举的启动子,优选氧化应激诱导性启动子,本发明中优选ARE、ARE和启动子(例如HSV-TK基础启动子等)的组合。ARE如上所述,优选ARE的多次重复,更优选例如ARE的3次重复(3×ARE)。
如上所述,可以制成图3所示的本发明的核酸构建物。如果欲将该核酸构建物整合到基因组中,则可以在核酸构建物的5'侧和3'侧分别连接Nrf2基因的约1~7kb的5'-非翻译区序列、约1~7kb的3'-非翻译区序列。包含这种核酸构建物的载体与细胞基因组上的Nrf2基因(或Nrf2基因座)发生同源重组,由此将核酸构建物整合到该基因组中。该方法特别适合用于制备包含该核酸构建物的转基因非人动物、例如转基因小鼠。
2.载体
本发明还提供包含上述核酸构建物的载体。
载体可以是质粒(例如pBruescript系、pUC系等)、噬菌体、粘粒、病毒、BAC、PAC等中的任一种,但不限定于此。质粒是动物细胞用质粒、例如哺乳动物用质粒,为了方便可以使用市售的质粒。此外,病毒是腺病毒、腺相关病毒、逆转录病毒、仙台病毒、慢病毒等病毒载体。BAC和PAC是人工染色体。
本发明的载体构建物的例子示于图4。
3.细胞
本发明还提供包含上述核酸构建物或上述载体的细胞。
细胞一般是动物细胞,优选温血动物细胞,更优选哺乳动物细胞,进一步更优选人细胞或小鼠细胞。此外,细胞非限定地包含培养细胞、原代细胞、传代细胞、细胞系、转化细胞、转染细胞、体细胞、生殖细胞、胚胎干(ES)细胞、组织干细胞、通过基因操作等而赋予了分化多能性的细胞(例如包括人工多能性(iPS)细胞)以及由该细胞分化而成的细胞等。哺乳动物细胞的例子包括HEK293细胞、CHO细胞、BHK细胞、COS细胞、HeLa细胞等。
上述细胞被本发明的核酸构建物或载体转化或转染。转化或转染的方法通常是动物细胞的转化方法,例如包括病毒感染法、电穿孔、显微注射、脂质体法、磷酸钙法等公知的方法。
病毒感染法是使用逆转录病毒、慢病毒、腺病毒、腺相关病毒、仙台病毒等公知的病毒载体的方法。该方法利用了病毒容易感染动物细胞的特性。
脂质体法是使用阳离子脂质体、例如胆固醇系阳离子脂质体等脂质体的方法,也称脂质体转化法或脂质体转染。该方法利用了细胞表面具有阴离子性的电性质这一点。此外,也可以使用在脂质体表面结合有细胞膜穿透肽(例如HIV-1 Tat肽、穿膜肽(penetratin)、寡聚精氨酸肽等)的脂质体。
4.非人动物
本发明还提供包含上述核酸构建物或上述载体的非人动物,即转基因动物(人除外)或其子孙动物。
转基因动物(人除外)的制备例如可以采用核移植法、ES细胞、iPS细胞等分化多能性细胞。
本说明书中,“动物”包括除人以外的动物,优选鸟类、哺乳类、例如猴、黑猩猩等灵长目、小鼠、大鼠、仓鼠等啮齿目、作为家畜动物有用的牛、山羊、绵羊、猪等偶蹄目、狗、猫等食肉目、鸡等鸟类等。
核移植法例如包括:在成纤维细胞等体细胞的基因组中导入本发明的核酸构建物或载体后,将从该细胞取出的核显微注射至已除核的受精卵或未受精卵中(为已除核的未受精卵时在施加电刺激后),将其移植到养母的子宫或输卵管,使其发育、出生,得到嵌合体动物。
使用ES细胞的方法包括:在从动物(人除外)的卵子的胚泡中取出的内细胞团中通过显微注射、微细胞融合法、电穿孔等方法导入本发明的核酸构建物或载体后,将ES细胞注入另一胚胎的胚泡中,得到移植胚,将该胚移植到养母的子宫中,使其出生,得到嵌合体动物(例如Evans MJ 和Kaufman MH 1981, Nature 292:154-156、押村光雄等编、染色质·染色体实验方案(クロマチン?染色体実験プロトコール)(2004年)羊土社)。
如果要例举使用iPS细胞的方法的一例,则可以在来源于动物的体细胞中以能表达编码改性Nrf2蛋白的核酸的状态导入上述核酸构建物或载体,制成转化细胞后,通过文献记载的方法(例如Takahashi K 和Yamanaka S 2006, Cell 126:663-676)在该细胞中导入转录因子(例如Oct3/4、Sox2、Klf4、c-Myc等)或编码其的核酸(包括载体),通过培养得到ES细胞样的分化多能性细胞,将该iPS细胞注入胚泡,得到移植胚,然后将该胚胎移植到养母的子宫中,使其出生,得到嵌合体动物。嵌合体动物的筛选中,可以从动物组织中取出基因组DNA,通过Southern杂交、原位杂交、PCR等方法考察导入核酸的存在,或者也可以通过小鼠等动物的毛色变化来判定。
如上所述制成的嵌合体动物可以再与同种的野生型交配,进而重复进行所得杂合性的动物个体之间的交配,从而得到纯合性的子孙动物(人除外)。
此外,也可以使上述杂合性或纯合性的子孙动物与任意的同种动物交配,在赋予氧化应激指示物的同时还赋予其它特征。作为该任意的动物,可例举包含内质网应激指示物表达用核酸构建物的同种动物或包含低氧应激指示物表达用核酸构建物的同种动物,但不限定于此。此外,只要在持续进行交配的子孙动物中也能显示出本发明的氧化应激指示物的功能,就可以理解为本发明的子孙动物。
例如,与包含内质网应激指示物表达用核酸构建物的同种动物交配时,可以制成包含氧化应激指示物表达用核酸构建物和内质网应激指示物表达用核酸构建物的非人动物,与之同样地,与包含低氧应激指示物表达用核酸构建物的同种动物交配时,可以制成包含氧化应激指示物表达用核酸构建物和低氧应激指示物表达用核酸构建物的非人动物。
内质网应激与IRE1a-XBP1通路、ATF6通路、PERK-ATF4通路等有关,与阿尔茨海默病、帕金森病、多聚谷氨酰胺病、肌萎缩性侧索硬化症等神经变性疾病、糖尿病、高脂血症、肥胖等代谢性疾病、癌症等疾病有关。内质网应激由内质网膜蛋白ATF6、IRE1、PERK等感知,例如IRE1诱导XBP1的剪接,因此利用该反应,开发出了能在体内检查内质网应激的系统(ERAI系统),还有,ATF6被切割而转化成活性型ATF6蛋白,另一方面,PERK将eIF-2α磷酸化,降低其功能,促进ATF4的合成,因此这些诱导物被用于内质网应激的分析(日本特开2005-204516号公报;Yoshida H., FEBS J. 2007 Feb;274(3):630-58. ER应激与疾病(ER stress and diseases.))。
低氧应激是指当暴露在与细胞正常发育的氧浓度相比更低的氧浓度条件下时引起的应激反应,已知与HIF-1α参与的通路等有关。
包含内质网应激指示物表达用核酸构建物的非人动物是具有内质网应激指示物表达用核酸构建物或包含该构建物的载体的非人动物,该构建物中,在启动子(并且根据情况可以与增强子组合)的下游连接并包含XBP1基因(包括内含子),在其3'侧连接并包含发光或荧光蛋白或标签蛋白质编码序列。启动子等的例子没有限定,有来源于CMV(巨细胞病毒)、SV-40病毒等病毒的启动子和/或增强子,β-肌动蛋白启动子、延伸因子1(EF-1)启动子、它们的组合等。XBP1基因上例如可以在其5'端连接编码Flag等标签的序列。
内质网应激指示物表达用核酸构建物的例子有:在包含CMV增强子和β-肌动蛋白启动子的序列的下游依次包含标签编码序列(标签的例子:Flag)、XBP1基因、发光或荧光蛋白或标签蛋白质编码序列的构建物。
XBP1(X-Box结合蛋白1(X-Box Binding Protein 1))的碱基序列和氨基酸序列可以从GenBank(美国NCBI)等获得,例如登录号的例子有NM_013842(小鼠)、NM_00107953(人)、NM_005080(人)等。
本发明的非人动物也可以与具有上述氧化应激相关疾病的非人动物、例如该疾病的模型动物(例如小鼠)交配,制成包含氧化应激指示物表达用核酸构建物以及根据情况使用的内质网应激指示物表达用核酸构建物、低氧应激指示物表达用核酸构建物等其它应激指示物表达用核酸构建物的具有上述疾病的非人动物。
例如与作为具有氧化应激相关疾病的非人动物的、包含能产生人型Aβ的嵌合APP基因的AD模型非人动物交配时,可制成包含氧化应激指示物表达用核酸构建物的AD模型非人动物。其它疾病模型非人动物也一样。
此外,包含氧化应激指示物表达用核酸构建物和内质网应激指示物表达用核酸构建物的非人动物与包含能产生人型Aβ的嵌合APP基因的AD模型非人动物交配时,可制成包含氧化应激指示物表达用核酸构建物和内质网应激指示物表达用核酸构建物的AD模型非人动物。氧化应激指示物表达用核酸构建物以外的其它应激指示物表达用核酸构建物可以像上述制造方法那样任意地选择,交配的其它疾病模型非人动物也一样。
如上所述,只要保持氧化应激指示物表达用核酸构建物,就能任意地制造具有氧化应激的指示物的特征并包含其它特征的同种动物。
具有氧化应激相关疾病的非人动物包括:包含能产生人型Aβ的嵌合APP基因的AD模型非人动物(日本特开2008-000027号公报)、包含人早老素2基因的突变体的AD模型非人动物(日本特开平11-146743号公报)、包含α-突触核蛋白(synuclein)基因的帕金森病模型非人动物(日本再表2005/041649号公报)、包含Parkin基因的突变体的帕金森病模型非人动物(日本特开2003-018992号公报)、糖尿病模型非人动物(Diabetes 1997 46:887-894)、肥胖症模型非人动物(Nature 372 425-432, 1994)、慢性炎症模型非人动物(Cell Metab 2009 10 178-88)等,但不限定于此。
5.氧化应激测定方法
本发明还提供测定氧化应激水平的方法,其包括在本发明的上述细胞或上述非人动物中测定在提供氧化应激时增大的可检测信号(例如发光或荧光信号)的强度的步骤。
本发明的测定方法中,作为测定氧化应激时的对照,例如可以使用不含氧化应激指示物表达用核酸构建物的细胞或非人动物、未施加氧化应激的细胞或非人动物、施加了氧化应激的阳性或阴性对照的细胞或非人动物等。
使用细胞时,例如可以在培养细胞的培养基中添加氧化应激物质,在细胞中诱导氧化应激,采用荧光显微镜和图像化装置、或者使用细胞裂解液(lysate)的荧光素酶试验或荧光测定法等来测定细胞内的发光或荧光强度的增大。此外,可以实时地观察氧化应激物质怎样对细胞造成影响。
作为培养基,可以将常规的动物细胞培养基、例如DMEM、BME、Ham's F12、RPMI1640、Fisher培养基、ES培养基、灵长类ES培养基等作为基础培养基,在其中适当添加抗生素(例如青霉素、链霉素等)、血清(例如胎牛血清等)、蛋白因子(例如碱性成纤维细胞生长因子(bFGF)、转铁蛋白、胰岛素、白血病抑制因子(LIF)等)、非必需氨基酸、巯基乙醇等。
培养可以是固体培养、液体培养等中的任一种,例如可以在35~40℃范围内的温度下、在含CO2气体的空气气氛下、根据需要使用成纤维细胞等饲养细胞来进行。
使用非人动物时,通过使用可以在个体水平上对动物进行分析的例如Xengen公司的IVIS成像系统(IVIS Imaging System),可以在氧化应激的存在下定量地测定活体动物个体中的发光或荧光信号。如果是该方法,则可以在组织水平上考察受到氧化应激的细胞。使用具有氧化应激相关疾病的动物作为非人动物时,既可以通过视觉考察氧化应激与疾病的关系,另一方面,也可以将上述动物用于抗氧化应激药、即该疾病的治疗药的开发。另外,此时,为了得到发光信号,例如只要是核酸构建物中具有荧光素酶基因的非人动物,则仅通过对该动物注射荧光素酶底物,就能与氧化应激相对应地检测出发光。荧光素酶底物例如是荧光素,荧光素在荧光素酶的作用下被氧化,转化成发光物质。
使用非人动物来实施本发明的氧化应激测定方法时,可以将非人动物反复供于试验。即,可以将非人动物暴露于氧化应激,在一定时间内使该动物从氧化应激的影响下恢复,再次将非人动物暴露于氧化应激时,可以使用该非人动物来测定氧化应激。藉此,可以应用于需要随时间进行评价的事例、长期发展的疾病等。上述时间例如为1天以上、优选2天以上、更优选3天以上,并且例如为10天以下、优选7天以下、更优选5天以下、最优选约4天。
6.筛选方法
本发明还提供筛选氧化应激抑制剂的方法,其包括如下步骤:在候选药剂的存在下对本发明的上述细胞或者上述非人动物提供某种特定的氧化应激,当可检测信号(例如发光或荧光信号等)的强度与不含药剂的对照相比有所减小时,判定为该候选药剂具有氧化应激抑制能力。
候选药剂可以从天然物、非天然物(或合成物)、有机物质、无机物质、低分子化合物、蛋白质、糖蛋白、脂蛋白、肽、糖类、脂类、核酸、核苷酸、寡核苷酸、多核苷酸、核苷等、或者公知的治疗用药剂或生物活性物质等中选择。特别地,只要是公知的治疗用药剂或生物活性物质,则有可能作为未知具有氧化应激抑制效果的物质的新效果而被筛选出来,具有氧化应激抑制效果的物质还有可能成为动脉粥样硬化症等心血管疾病,阿尔茨海默病、帕金森病、亨延顿舞蹈病等神经变性疾病,以肝病为代表的各脏器的变性疾病,糖尿病、类风湿性关节炎等氧化应激相关疾病的治疗药。
此外,例如本发明的非人动物是包含氧化应激指示物表达用核酸构建物和内质网应激指示物表达用核酸构建物的动物时,可以将该动物用于抑制氧化应激和/或内质网应激的药剂的筛选。内质网应激如上所述,与神经变性疾病、糖尿病、高脂血症、肥胖等代谢性疾病、慢性炎症、癌症等疾病有关,如果使用该动物,则可以发现针对各应激的应激抑制剂。对于像这样在获得氧化应激指示物的同时还获得其它功能的动物,可以适当利用其功能,和氧化应激状态一起分析机体内的其它反应等。
筛选方法可以通过“5.氧化应激测定方法”中记载的方法或实验系统来实施。
即,如果是细胞,则在对培养细胞施加应激前、或在施加应激的同时、或在施加应激后在培养基中添加候选药剂来培养细胞,测定细胞内的例如发光或荧光信号等可检测信号,选择与对照(无候选药剂)相比能减小该信号的物质。
如果是非人动物,则可以在施加氧化应激前、或在施加应激的同时、或在施加应激后通过口服给药、静脉内给药、直肠内给药、皮下给药、肌肉内给药、经粘膜给药等给药途径给予该动物候选药剂,或者和饲料一起摄取,测定发光或荧光信号等可检测信号,选择能减小该信号的药剂。
只要本发明的非人动物是通过与具有氧化应激相关疾病的动物的交配而制成的动物,则也可以同时判定疾病的治疗效果,因此认为,通过使用这种动物,可以容易地发现该疾病的治疗剂。
通过上述方法选出的药剂可以与赋形剂、溶剂或载体组合,制剂化成医药品的形态。医药品可以配制成固体制剂(例如片剂、颗粒剂、粉末剂、胶囊剂等)、液体制剂(例如溶液剂、悬浮剂等)、气溶胶制剂、缓释制剂(例如肠溶剂、多层制剂、包衣制剂等)等多种制剂。制剂化过程中可以适当添加各种添加剂,例如崩解剂、粘合剂、稳定剂、润滑剂、乳化剂、香味剂、着色剂等制剂。
(实施例)
进一步通过以下实施例对本发明进行具体说明,但本发明的范围不受这些实施例的限制。
实施例1
<方法>
[质粒的构建]
为了制备p(3xARE)TKbasal,将通过PCR扩增的ARE片段(来源于小鼠GSTYa启动子; ACTAGTACTAGTGGAAATGACATTGCTAATGGTGACAAAGCAACTTTTCTAGA(序列编号14);结合的限制位点以粗体表示)用SpeI-XbaI消化,自连,形成3次重复片段,然后插入具有XbaI-SpeI位点的pTKbasal中。XbaI-SpeI位点位于TK基础启动子的5'部位。将编码人Nrf2(图1的1-93、1-433、全长(full-length))的cDNA用PCR扩增,插入具有KpnI-XhoI位点的p(3xARE)TKbasal(或图6的pTKX)中。将编码荧光素酶(GL4)的cDNA在其3'末端处用1×Flag标签进行PCR扩增,插入具有XhoI-NheI位点的p(3xARE)TKbasal-hNrf2(1-433)中。将所得的p(3xARE)TKbasal-hNrf2(1-433)GL4-Flag作为OKD48-Luc质粒使用。通过同样的方法制备GFP型、即p(3xARE)TKbasal-hNrf2(1-433)-Venus-Flag,作为OKD48-Venus质粒使用。
在具有KpnI-XhoI的pCAX中插入PCR扩增人Nrf2片段,从而构建人Nrf2的过表达载体(pCAX-hNrf2)。此外,在具有HindIII-XhoI位点的pCAX中插入PCR扩增人Keap1片段,从而构建人Keap1的过表达载体(pCAX-hKeap1)。通过同样的方法构建末端切割型的pCAX-hKeap1(1-314)。
[细胞培养、转染及处理]
将HeLa细胞和HEK293T细胞在补充了100U/ml青霉素、100μg/ml链霉素和10%胎牛血清的DMEM培养基中、在含5%CO2的气氛下、在37℃下培养。使用磷酸钙-DNA沉淀法将质粒DNA插入细胞内。为了测试细胞对药剂的应答,以各种时间用10μM ASN(亚砷酸钠)、100μM DEM(马来酸二乙酯)、200μM H2O2、2.5μg/ml衣霉素、1μM毒胡萝卜素、1mM DTT、100μM依托泊苷或100μg/ml TTFA (噻吩甲酰三氟丙酮)处理细胞。
[荧光素酶试验]
在使用OKD48-Luc报告物的二连荧光素酶试验中,使用phRL-TK(Promega)作为内部对照。将HeLa细胞接种于24孔板,然后用质粒DNA进行转染。转染后第24小时,将细胞裂解,用于荧光素酶试验。各报告物活性的测定用荧光素酶试验系统(Promega)和发光计(Berthold)来进行。其结果是以3次实验的平均±SEM表示。各值以诱导倍数表示,该倍数分别相对于未处理(NT)的倍数(图5A)、无Nrf2过表达的未处理(NT)的倍数(图5D)或无Keap1过表达的未处理(NT)的倍数(图5E)进行了标准化。此时,将各NT的倍数设为1.0。
[荧光图像化及荧光强度测定]
为了获取细胞的图像,将HEK293T细胞接种于6孔板,然后用质粒DNA进行转染。在转染后第24小时,用FSX100 (Olympus)获得荧光图像。为了测定细胞裂解液的荧光,将细胞在毛地黄皂苷(digitonin)缓冲液(50mM Tris-HCl(pH7.5), 1mM EDTA, 10mM EGTA和10μM毛地黄皂苷)中以37℃、30分钟裂解。通过16500g的离心分离使裂解液变得透明。用荧光光度计(ARVO MX-2, PerkinElmer)测定上清的荧光(发光波长535nm; 激发波长485nm)。使用pCAG-GL3作为内部对照。各样品的荧光强度相对于同时转染的荧光素酶活性进行标准化。数值以3次实验的平均±SEM表示。
[蛋白质印迹分析]
将细胞在SDS样品缓冲液(50mM Tris-HCl(pH 6.8), 2% SDS, 50mM DTT, 10%甘油和1μg/ml溴酚蓝)中裂解。将裂解液在98℃下加热10分钟,用SDS-PAGE分析裂解液中的蛋白质。电泳后,将蛋白质电转移至聚偏氟乙烯多孔膜,通过标准方法用针对荧光素酶的单克隆抗体(Promega)、针对GFP的单克隆抗体(Nacalai tesque)、针对GAPDH的单克隆抗体(Cell Signaling Technology)或针对Nrf2的多克隆抗体(Santa Cruz)进行免疫学检测。
[转基因小鼠]
将p(3xARE)TKbasal-hNrf2(1-433)-GL4-Flag的4.5-kb SpeI-SfiI片段作为转基因,显微注射至C57BL/6小鼠受精卵中,通过使用5'-ATC ACC AGA ACA CTC AGT GG-3'(序列编号15)和5'-ACT CGG CGT AGG TAA TGT CC-3'(序列编号16)的引物的PCR来筛选转基因子孙。将所得的小鼠用于体内的图像化试验。腹腔内注射D-荧光素(4.5mg)后,按照标准方案用体内图像化系统IVIS (Xenogen)对小鼠进行分析。包含动物的实验方案由RIKEN(日本)动物研究委员会认可。
<结果>
[新型的氧化应激指示物(OKD48)的设计和构建]
这次,本发明人利用Keap1-Nrf2通路来进行新型的氧化应激指示物OKD48的制备。将与应激依赖性稳定化有关的Nrf2的部分片段与荧光素酶(Luc)基因融合,使其从应激诱导性的启动子开始表达。如图3所示,在平常时候,因为核内不存在内源性的Nrf2,所以不会诱导报告物的转录水平的表达,而且漏出的报告物蛋白也因Keap1而受到分解抑制,无法检出信号。但是,在氧化应激时,随着内源性Nrf2量的增加和向核内的转移,报告物基因的转录被诱导,而且基于Keap1的分解抑制也被解除,因此报告物蛋白的表达增强,能检出信号。
如图6中的其一例所示,对于所使用的Nrf2的部分片段和启动子的组合,制备了多种变异体,寻找显示出最好的应答性的变异体。关于Nrf2片段,对氨基酸(a.a.)1~93、1~433或全长(full length)进行了考察。对于启动子,除了作为阴性对照使用的HSV-TK启动子外,也研究了HO-1增强子、3×ARE启动子。其结果是,Nrf2的a.a.1~433区域和3×ARE启动子的组合对于氧化应激诱导剂ASN和DEM显示出最好的应答性。
[体外的OKD48的特性确定]
为了测试如上所述构建的OKD48(ARE-Nrf2联合应激相关指示物(ARE-Nrf2 jointed stress associated indicator))-Luc在实际的哺乳动物细胞中是否起到氧化应激指示物的功能,将融合基因的表达载体导入培养细胞,进行试验(图5)。用HeLa细胞通过荧光素酶试验来评价对于各种药剂的应答性,结果OKD48-Luc对ASN(亚砷酸钠)和DEM(马来酸二乙酯)等氧化应激诱导剂作出特异性的应答,而在Tun (衣霉素)和Tg (毒胡萝卜素)等内质网应激诱导剂、作为还原剂的DTT (dithiothreitol)、通过拓扑异构酶Ⅱ的抑制剂来诱导凋亡的Etp (依托泊苷)、作为化学缺氧诱导剂的TTFA (噻吩甲酰三氟丙酮)的作用下几乎未被活化(图5A)。与该报告物试验的结果一致的是,通过使用Luc抗体的蛋白质印迹,也能确认到对ASN和DEM有特异性的、蛋白质水平上的OKD48-Luc的升高(图5B)。如图5C所示,这些药剂中,显著地诱导内源性Nrf2的只有ASN和DEM。这些结果表明,制得的报告物的特异性与内源性Nrf2一致,意味着OKD48-Luc作为氧化应激指示物的有用性。
接着,本发明人考察了相关因子的过表达对OKD48-Luc的影响。如图5D所示,由于Nrf2的过表达,即使在常规条件(normal condition)下,OKD48-Luc的活性也升高100倍以上,而且其活性通过ASN处理有一定升高。另一方面,由于Keap1的过表达,无论有无应激,OKD48-Luc的活性都降低,在与Nrf2的结合区域缺失的Keap1(1-314)的过表达条件下,OKD48-Luc的活性反而升高(图5E)。这些结果支持假定OKD48-Luc被Nrf2诱导、受到基于Keap1的分解/稳定化的控制的实施模型。
除了这种融合了荧光素酶的报告物,还制备了融合了GFP的报告物,进行了使用培养细胞的检验(图7)。与Luc型的报告物一致的是,在显微镜观察中,OKD48-Venus随着ASN和DEM处理而发出荧光(图7A)。此外,通过使用细胞裂解液(Lysate)的荧光测定(图7B)和使用GFP抗体的蛋白质印迹(图7C)也确认到同样的结果。
[OKD48小鼠的制备及体内的氧化应激的监控]
为了在体内监控氧化应激,制备了具有3×ARE驱动的Nrf2(1-433)-Luc表达基因的OKD48-转基因小鼠。为了确认在体内可以有效地使用氧化应激下的细胞,对转基因小鼠腹腔内注射(12.5mg/kg)在各种组织和器官中诱导氧化应激的ASN,6小时后,在剖开腹部的条件下观察发光信号。注射了ASN的转基因小鼠的肝脏中显示出强发光(图8)。在其它组织和器官中也检出了一定的发光(例如胃和肾脏;数据未示出)。与之相对,注射了PBS的对照转基因小鼠几乎未显示出发光。该结果表明,制成的转基因小鼠可以用于动物个体内的氧化应激的检测。
实施例2
[小鼠OKD48的制备及其特性]
将导入有通过与实施例1同样的方法制备的小鼠OKD48-Luc(3xARE+TK基础启动子+小鼠Nrf2(1-426)+Luc)的HeLa细胞用氧化应激物质(DEM或ASN)处理,通过荧光素酶试验法测定发光强度。其结果是,对于DEM和ASN这两者,小鼠Nrf2(1-426)-Luc与小鼠Nrf2(1-93)和小鼠Nrf2(full)相比,显示出最高的S/N比(图9)。
产业实用性
本发明的指示物能特异性地检出细胞或动物中与Nrf2-Keap1通路有关的氧化应激,因此可以在机体水平上(体内)通过视觉捕捉受到氧化应激的细胞和组织,或者通过使用能表达上述指示物的非人动物,可有助于各种氧化应激相关疾病的病理研究和治疗用药剂的开发。
序列表自由内容
序列编号3:HSV-TK基础(basal)启动子
序列编号7:小鼠HO-1增强子-TK基础(basal)启动子
序列编号10:3×ARE
序列编号11:3xARE-TK基础(basal)启动子
序列编号12:3xARE-TKbasal-OKD48-Luc
序列编号13:3xARE-TKbasal-OKD48-Venus
序列编号15:引物
序列编号16:引物。
本说明书中引用的全部发行物、专利和专利申请都直接作为参考在本说明书中引用。
序列表
<110> RIKEN
<120> 氧化应激指示物表达用核酸构建物及其应用
<130> PH-5127-PCT
<150> JP 2011-010833
<151> 2011-01-21
<160> 16
<170> PatentIn 版本 3.4
<210> 1
<211> 604
<212> PRT
<213> 智人(Homo sapiens)
<400> 1
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<220>
<223> HSV-TK基础启动子
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aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag 180
gcctaggctt ttgcaaaaag ctt 203
<210> 6
<211> 795
<212> DNA
<213> 巨细胞病毒(cytomegalovirus)
<400> 6
tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60
ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120
aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180
gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240
gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300
agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360
ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420
cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480
gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 660
cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 720
tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat 780
tgctaacgca gtcag 795
<210> 7
<211> 432
<212> DNA
<213> 人工的
<220>
<223> 小鼠HO-1增强子-TK基础启动子
<400> 7
actagtgagc tccaccccca cccaggattc cagcccccac aggagctgaa ctttgttttt 60
cccgcagcgg ctggaatgct gagttgtgat ttcctcactg ctcatttcct cagctgcttt 120
tatgctgtgt catggttggg aggggtgatt agcagacaaa gggaagacag attttgctga 180
gtcaccctct gttccctctg cctcagctag gaatagttgg taaaaggttc cggaacggct 240
ttaacttcag gcagaaggaa gtgaaagttc tagaggcccc gcccagcgtc ttgtcattgg 300
cgaattcgaa cacgcagatg cagtcggggc ggcgcggtcc gaggtccact tcgcatatta 360
aggtgacgcg tgtggcctcg aacaccgagc gaccctgcag cgacccgctt aacagcgtca 420
acagcgaagc tt 432
<210> 8
<211> 9
<212> DNA
<213> 小鼠(Mus musculus)
<400> 8
tgacattgc 9
<210> 9
<211> 9
<212> DNA
<213> 小鼠(Mus musculus)
<400> 9
tgacaaagc 9
<210> 10
<211> 129
<212> DNA
<213> 人工的
<220>
<223> 3xARE
<400> 10
actagtggaa atgacattgc taatggtgac aaagcaactt ttctagtgga aatgacattg 60
ctaatggtga caaagcaact tttctagtgg aaatgacatt gctaatggtg acaaagcaac 120
ttttctaga 129
<210> 11
<211> 287
<212> DNA
<213> 人工的
<220>
<223> 3xARE-TK基础启动子
<400> 11
actagtggaa atgacattgc taatggtgac aaagcaactt ttctagtgga aatgacattg 60
ctaatggtga caaagcaact tttctagtgg aaatgacatt gctaatggtg acaaagcaac 120
ttttctagag gccccgccca gcgtcttgtc attggcgaat tcgaacacgc agatgcagtc 180
ggggcggcgc ggtccgaggt ccacttcgca tattaaggtg acgcgtgtgg cctcgaacac 240
cgagcgaccc tgcagcgacc cgcttaacag cgtcaacagc gaagctt 287
<210> 12
<211> 3283
<212> DNA
<213> 人工的
<220>
<223> 3xARE-TKbasal-OKD48-Luc
<400> 12
actagtggaa atgacattgc taatggtgac aaagcaactt ttctagtgga aatgacattg 60
ctaatggtga caaagcaact tttctagtgg aaatgacatt gctaatggtg acaaagcaac 120
ttttctagag gccccgccca gcgtcttgtc attggcgaat tcgaacacgc agatgcagtc 180
ggggcggcgc ggtccgaggt ccacttcgca tattaaggtg acgcgtgtgg cctcgaacac 240
cgagcgaccc tgcagcgacc cgcttaacag cgtcaacagc gaagcttcta ggtaccatga 300
tggacttgga gctgccgccg ccgggactcc cgtcccagca ggacatggat ttgattgaca 360
tactttggag gcaagatata gatcttggag taagtcgaga agtatttgac ttcagtcagc 420
gacggaaaga gtatgagctg gaaaaacaga aaaaacttga aaaggaaaga caagaacaac 480
tccaaaagga gcaagagaaa gcctttttcg ctcagttaca actagatgaa gagacaggtg 540
aatttctccc aattcagcca gcccagcaca tccagtcaga aaccagtgga tctgccaact 600
actcccaggt tgcccacatt cccaaatcag atgctttgta ctttgatgac tgcatgcagc 660
ttttggcgca gacattcccg tttgtagatg acaatgaggt ttcttcggct acgtttcagt 720
cacttgttcc tgatattccc ggtcacatcg agagcccagt cttcattgct actaatcagg 780
ctcagtcacc tgaaacttct gttgctcagg tagcccctgt tgatttagac ggtatgcaac 840
aggacattga gcaagtttgg gaggagctat tatccattcc tgagttacag tgtcttaata 900
ttgaaaatga caagctggtt gagactacca tggttccaag tccagaagcc aaactgacag 960
aagttgacaa ttatcatttt tactcatcta taccctcaat ggaaaaagaa gtaggtaact 1020
gtagtccaca ttttcttaat gcttttgagg attccttcag cagcatcctc tccacagaag 1080
accccaacca gttgacagtg aactcattaa attcagatgc cacagtcaac acagattttg 1140
gtgatgaatt ttattctgct ttcatagctg agcccagtat cagcaacagc atgccctcac 1200
ctgctacttt aagccattca ctctctgaac ttctaaatgg gcccattgat gtttctgatc 1260
tatcactttg caaagctttc aaccaaaacc accctgaaag cacagcagaa ttcaatgatt 1320
ctgactccgg catttcacta aacacaagtc ccagtgtggc atcaccagaa cactcagtgg 1380
aatcttccag ctatggagac acactacttg gcctcagtga ttctgaagtg gaagagctag 1440
atagtgcccc tggaagtgtc aaacagaatg gtcctaaaac accagtacat tcttctgggg 1500
atatggtaca acccttgtca ccatctcagg ggcagagcac tcacgtgcat gatgcccaat 1560
gtgagaacac accagagaaa gaattgcctg taagtctcga ggaagatgcc aaaaacatta 1620
agaagggccc agcgccattc tacccactcg aagacgggac cgccggcgag cagctgcaca 1680
aagccatgaa gcgctacgcc ctggtgcccg gcaccatcgc ctttaccgac gcacatatcg 1740
aggtggacat tacctacgcc gagtacttcg agatgagcgt tcggctggca gaagctatga 1800
agcgctatgg gctgaataca aaccatcgga tcgtggtgtg cagcgagaat agcttgcagt 1860
tcttcatgcc cgtgttgggt gccctgttca tcggtgtggc tgtggcccca gctaacgaca 1920
tctacaacga gcgcgagctg ctgaacagca tgggcatcag ccagcccacc gtcgtattcg 1980
tgagcaagaa agggctgcaa aagatcctca acgtgcaaaa gaagctaccg atcatacaaa 2040
agatcatcat catggatagc aagaccgact accagggctt ccaaagcatg tacaccttcg 2100
tgacttccca tttgccaccc ggcttcaacg agtacgactt cgtgcccgag agcttcgacc 2160
gggacaaaac catcgccctg atcatgaaca gtagtggcag taccggattg cccaagggcg 2220
tagccctacc gcaccgcacc gcttgtgtcc gattcagtca tgcccgcgac cccatcttcg 2280
gcaaccagat catccccgac accgctatcc tcagcgtggt gccatttcac cacggcttcg 2340
gcatgttcac cacgctgggc tacttgatct gcggctttcg ggtcgtgctc atgtaccgct 2400
tcgaggagga gctattcttg cgcagcttgc aagactataa gattcaatct gccctgctgg 2460
tgcccacact atttagcttc ttcgctaaga gcactctcat cgacaagtac gacctaagca 2520
acttgcacga gatcgccagc ggcggggcgc cgctcagcaa ggaggtaggt gaggccgtgg 2580
ccaaacgctt ccacctacca ggcatccgcc agggctacgg cctgacagaa acaaccagcg 2640
ccattctgat cacccccgaa ggggacgaca agcctggcgc agtaggcaag gtggtgccct 2700
tcttcgaggc taaggtggtg gacttggaca ccggtaagac actgggtgtg aaccagcgcg 2760
gcgagctgtg cgtccgtggc cccatgatca tgagcggcta cgttaacaac cccgaggcta 2820
caaacgctct catcgacaag gacggctggc tgcacagcgg cgacatcgcc tactgggacg 2880
aggacgagca cttcttcatc gtggaccggc tgaagagcct gatcaaatac aagggctacc 2940
aggtagcccc agccgaactg gagagcatcc tgctgcaaca ccccaacatc ttcgacgccg 3000
gggtcgccgg cctgcccgac gacgatgccg gcgagctgcc cgccgcagtc gtcgtgctgg 3060
aacacggtaa aaccatgacc gagaaggaga tcgtggacta tgtggccagc caggttacaa 3120
ccgccaagaa gctgcgcggt ggtgttgtgt tcgtggacga ggtgcctaaa ggactgaccg 3180
gcaagttgga cgcccgcaag atccgcgaga ttctcattaa ggccaagaag ggcggcaaga 3240
tcgccgtgga ctacaaggat gacgatgaca agtaatagct agc 3283
<210> 13
<211> 2350
<212> DNA
<213> 人工的
<220>
<223> 3xARE-TKbasal-OKD48-Venus
<400> 13
actagtggaa atgacattgc taatggtgac aaagcaactt ttctagtgga aatgacattg 60
ctaatggtga caaagcaact tttctagtgg aaatgacatt gctaatggtg acaaagcaac 120
ttttctagag gccccgccca gcgtcttgtc attggcgaat tcgaacacgc agatgcagtc 180
ggggcggcgc ggtccgaggt ccacttcgca tattaaggtg acgcgtgtgg cctcgaacac 240
cgagcgaccc tgcagcgacc cgcttaacag cgtcaacagc gaagcttcta ggtaccatga 300
tggacttgga gctgccgccg ccgggactcc cgtcccagca ggacatggat ttgattgaca 360
tactttggag gcaagatata gatcttggag taagtcgaga agtatttgac ttcagtcagc 420
gacggaaaga gtatgagctg gaaaaacaga aaaaacttga aaaggaaaga caagaacaac 480
tccaaaagga gcaagagaaa gcctttttcg ctcagttaca actagatgaa gagacaggtg 540
aatttctccc aattcagcca gcccagcaca tccagtcaga aaccagtgga tctgccaact 600
actcccaggt tgcccacatt cccaaatcag atgctttgta ctttgatgac tgcatgcagc 660
ttttggcgca gacattcccg tttgtagatg acaatgaggt ttcttcggct acgtttcagt 720
cacttgttcc tgatattccc ggtcacatcg agagcccagt cttcattgct actaatcagg 780
ctcagtcacc tgaaacttct gttgctcagg tagcccctgt tgatttagac ggtatgcaac 840
aggacattga gcaagtttgg gaggagctat tatccattcc tgagttacag tgtcttaata 900
ttgaaaatga caagctggtt gagactacca tggttccaag tccagaagcc aaactgacag 960
aagttgacaa ttatcatttt tactcatcta taccctcaat ggaaaaagaa gtaggtaact 1020
gtagtccaca ttttcttaat gcttttgagg attccttcag cagcatcctc tccacagaag 1080
accccaacca gttgacagtg aactcattaa attcagatgc cacagtcaac acagattttg 1140
gtgatgaatt ttattctgct ttcatagctg agcccagtat cagcaacagc atgccctcac 1200
ctgctacttt aagccattca ctctctgaac ttctaaatgg gcccattgat gtttctgatc 1260
tatcactttg caaagctttc aaccaaaacc accctgaaag cacagcagaa ttcaatgatt 1320
ctgactccgg catttcacta aacacaagtc ccagtgtggc atcaccagaa cactcagtgg 1380
aatcttccag ctatggagac acactacttg gcctcagtga ttctgaagtg gaagagctag 1440
atagtgcccc tggaagtgtc aaacagaatg gtcctaaaac accagtacat tcttctgggg 1500
atatggtaca acccttgtca ccatctcagg ggcagagcac tcacgtgcat gatgcccaat 1560
gtgagaacac accagagaaa gaattgcctg taagtctcga ggtgagcaag ggcgaggagc 1620
tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt 1680
tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagctga 1740
tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgggctacg 1800
gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg 1860
ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca 1920
agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg 1980
gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca 2040
gccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc aacttcaaga 2100
tccgccacaa catcgaggac ggcggcgtgc agctcgccga ccactaccag cagaacaccc 2160
ccatcggcga cggccccgtg ttgctgcccg acaaccacta cctgagctac cagtccgccc 2220
tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg 2280
ccgggatcac tctcggcatg gacgagctgt acaaggacta caaggatgac gatgacaagt 2340
aatagctagc 2350
<210> 14
<211> 53
<212> DNA
<213> 小鼠(Mus musculus)
<400> 14
actagtacta gtggaaatga cattgctaat ggtgacaaag caacttttct aga 53
<210> 15
<211> 20
<212> DNA
<213> 人工的
<220>
<223> 引物
<400> 15
atcaccagaa cactcagtgg 20
<210> 16
<211> 20
<212> DNA
<213> 人工的
<220>
<223> 引物
<400> 16
actcggcgta ggtaatgtcc 20
Claims (16)
1. 氧化应激指示物表达用核酸构建物,其包含:编码使Nrf2蛋白中至少包含Neh2结构域序列、并且使Neh1结构域序列或Neh1-Neh3结构域序列实质上缺失或丧失功能而成的部分蛋白质的核酸序列,配置于该核酸序列的上游的应激诱导性启动子序列,和配置于该核酸序列的下游的、编码能发出可检测信号的蛋白质的核酸序列。
2. 权利要求1所述的核酸构建物,其中,部分蛋白质包含从Nrf2蛋白中使Neh1结构域序列或Neh1-Neh3结构域序列实质上缺失而成的氨基酸序列。
3. 权利要求1或2所述的核酸构建物,其中,Nrf2蛋白是来源于哺乳动物的Nrf2。
4. 权利要求3所述的核酸构建物,其中,来源于哺乳动物的Nrf2是人Nrf2或小鼠Nrf2。
5. 权利要求1~4中任一项所述的核酸构建物,其中,部分蛋白质包括:(a)由序列编号1(人)或序列编号2(小鼠)的Nrf2蛋白的氨基酸序列中的1位~93位氨基酸构成的Neh2结构域序列,(b)与该Neh2结构域序列具有80%以上的同一性的氨基酸序列,或者(c)在该Neh2结构域序列中包含1个或多个氨基酸的缺失、置换或添加的氨基酸序列。
6. 权利要求1~4中任一项所述的核酸构建物,其中,部分蛋白质包括:(d)由序列编号1(人)或序列编号2(小鼠)的Nrf2蛋白的氨基酸序列中的1位~433位氨基酸或1位~425位氨基酸各自构成的、包含Neh2-Neh6结构域序列的氨基酸序列,(e)与该包含Neh2-Neh6结构域序列的氨基酸序列具有80%以上的同一性的氨基酸序列,或者(f)在该包含Neh2-Neh6结构域序列的氨基酸序列中包含1个或多个氨基酸的缺失、置换或添加的氨基酸序列。
7. 权利要求1~6中任一项所述的核酸构建物,其中,应激诱导性启动子由抗氧化剂应答元件(ARE)、或ARE与启动子的组合构成。
8. 权利要求7所述的核酸构建物,其中,ARE由多次重复构成。
9. 权利要求1~8中任一项所述的核酸构建物,其中,所述核酸构建物在其3'末端包含标签编码序列。
10. 权利要求9所述的核酸构建物,其中,标签编码序列是Flag标签编码序列。
11. 载体,其包含权利要求1~10中任一项所述的核酸构建物。
12. 细胞,其包含权利要求1~10中任一项所述的核酸构建物或权利要求11所述的载体。
13. 非人动物,其包含权利要求1~10中任一项所述的核酸构建物或权利要求11所述的载体。
14. 权利要求13所述的非人动物,其中,非人动物还包含内质网应激指示物表达用核酸构建物或低氧应激指示物表达用核酸构建物。
15. 用于测定氧化应激的方法,其包括在权利要求12所述的细胞或者权利要求13或14所述的非人动物中测定在提供氧化应激时增大的可检测信号的强度的步骤。
16. 筛选氧化应激抑制剂的方法,其包括如下步骤:在候选药剂的存在下对权利要求12所述的细胞或者权利要求13或14所述的非人动物提供某种特定的氧化应激,当可检测信号的强度与不存在该候选药剂的对照相比有所减小时,判定为该候选药剂具有氧化应激抑制能力。
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