CN1279689A - 新的维生素d受体相关多肽、编码其的核苷酸序列及其用途 - Google Patents
新的维生素d受体相关多肽、编码其的核苷酸序列及其用途 Download PDFInfo
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Abstract
本发明涉及新的维生素D受体相关(VDRR)多肽和包含同一多肽的制剂。还公开了编码VDRR多肽的核酸序列、包含此序列的表达载体和经此表达载体转化的宿主细胞,以及用以表达本发明的新的VDRR多肽的方法。本发明进一步涉及了用作药物的VDRR多肽,以及影响VDRR信号转导的物质用于制造治疗代谢性、增生性或炎性疾病的药物的用途。本发明还涉及了鉴定编码VDRR多肽的克隆的方法、鉴定VDRR配体的方法和鉴定用于治疗受VDRR多肽影响的疾病的物质的方法。更确切地讲,这种新的VDRR多肽可以是称作VDRRγ的多肽,其可以由任何在结构上相似于已知核受体之配体的小化学分子调节。
Description
发明领域
本发明涉及了新的维生素D受体相关(VDRR)多肽。并且公开了编码同一多肽的核酸序列、包含此种序列的表达载体及经此种表达载体转化宿主细胞,本发明还公开了这种新的VDRR多肽的表达方法,及其用途。
发明背景
核激素受体是很大一组条件控制的转录因子。结合了各种小的化学分子(配体),包括类固醇、维生素D3、类视色素、二十烷类(前列腺素类)、甲状腺激素和胆固醇衍生物后,这些受体应答性地得到激活,并调控目标基因表达。
已有越来越多的结构相关性受体得到识别,却尚未鉴别出它们的配体。这群受体被称为孤核受体(ONR)。在Enmark等,分子内分泌学(Mol.Endo.),卷10,第11(1996)pp.1293-1307中能够发现关于ONR的综述,因此在此引入作为参考。大量的ONR对各种过程,如代谢平衡、细胞分化和发育的关键作用已由生化和基因技术得到阐明。此外,已显示几种ONR也在如糖尿病、肥胖症、炎性疾病和增生性疾病等多种常见病和失调中作为关键因子。
基于这些发现,普遍认为新的ONR将会成为常见病治疗用途发明的潜在药物靶点。因此,识别这些受体是非常重要的。
发明概述
本发明涉及新的维生素D受体相关(VDRR)多肽和含有其的制剂。并且公开了编码此VDRR多肽的核酸序列、包含此种序列的表达载体和经此载体转化的宿主细胞,以及本发明的新的VDRR多肽的表达方法。本发明进一步涉及了利用VDRR多肽作药物,及利用影响VDRR信号转导的物质制造治疗代谢性、增生性或炎症性疾病的药物的用途。本发明还涉及了鉴别编码VDRR多肽的克隆的方法、鉴别VDRR配体的方法和鉴别治疗受VDRR多肽影响的疾病的物质的方法。更确切地讲,此种新的VDRR多肽可以是命名为VDRRγ的多肽,任何在结构上与核受体的已知配体相似的小化学分子都可能调节此VDRRγ多肽。
附图概述
图1-显示编码此种新的核受体多肽维生素D受体相关γ(VDRRg)的cDNA序列。
图2-如DBD-HMM排列所示的VDRRg进化邻近点树图。
图3-如LBD-HMM排列所示的VDRRg进化邻近点树图。
图4-显示推断的VDRRg氨基酸序列。
图5-成人组织中VDRRg的表达。右侧的数字指的是mRNA以千碱基对为单位的长度。
图6-维生素D3在CV-1细胞的瞬时转染过程中激活GAL4-DBD/VDR-LBD融合蛋白,而不是GAL4-DBD/VDRRγ-LBD融合蛋白,左侧的数字指的是GAL4-萤光素酶报道基因的相对萤光素酶活性。
图7-显示与VDRRg相比具有备择剪接的5′端的VDRRg-2的cDNA序列。
图8-显示推断的VDRR-2氨基酸序列。
图9-显示类视色素X受体(RXR)与VDRRg的异二聚体。
图10-显示作为VDRRg激活物的孕烯醇酮衍生物的作用。
图11-显示作为VDRRg激活物的孕烯醇酮16α-腈(PCN)、地塞米松和抗孕酮(RU486)的作用。
图12-新基因VDRRg-1和VDRRg-2以及已知基因XOR-6、HVDR、CAR-1和CAR-2之间的相似性百分比。
图13-新基因VDRRg-1和VDRRg-2以及已知基因XOR-6、HVDR、CAR-1和CAR-2之间的同一性百分比。
发明详述
上述目的均在本发明得到实现,这涉及哺乳动物,优选人类的分离的或重组的核酸,其包含编码维生素D受体相关(VDRR)多肽的连续核酸序列。此VDRR多肽有适当的起源。
在本发明的优选实施方案中,编码VDRR多肽的核酸包含约77个氨基酸的DNA结合结构域(DBD),其中具有9个半胱氨酸残基。相对于人维生素D受体(hVDR)的DBD和从爪蟾(Xenopus laevis)分离来的孤核受体1(xDNR1=XOR-6)的DBD,此DBD进一步具有如下氨基酸序列相似性的特征,分别为:(ⅰ)与hVDR的DBD具有至少约60%氨基酸序列相似性;和(ⅱ)与xONR1的DBD具有至少约65%氨基酸序列相似性。
更加确切地说,相对于hVDR和xONR1的DBD,此氨基酸序列的相似性分别是:(ⅰ)与hDVR的DBD具有约65%氨基酸序列相似性;和(ⅱ)与xONR1的DBD具有约71%氨基酸序列相似性。
在本发明的优选实施方案中,相对于hVDR和xONR1的LBD,编码包含配体结合结构域(LBD)的VDRR多肽的核酸特征在于分别有如下氨基酸序列相似性:(ⅰ)与hVDR的LBD具有至少约30%氨基酸序列相似性,恰当地,与hVDR的LBD具有至少35%氨基酸序列相似性;和(ⅱ)与xONR1的LBD具有至少约40%氨基酸序列相似性,恰当地,与xONR1的LBD具有至少45%氨基酸序列相似性。
更确切地说,相对于hVDR和xONR1的LBD,氨基酸序列相似性分别为:(ⅰ)与hVDR的LBD约42%氨基酸序列相似性;和(ⅱ)与xONR1的LBD约54%的氨基酸序列相似性。
“氨基酸序列相似性”指的是:100x一致性长度除以(一致性长度+错配+缺口)。
也可应用术语氨基酸序列同一性。通过比较绝对氨基酸序列残基同一性计算氨基酸序列同一性。图13显示了新基因VDRRg-1和VDRRg-2与已知基因之间的氨基酸序列同一性。
在特别优选的实施方案中,本发明的核酸序列与图1或图7中所示的序列及其相同或等位基因基本相同。
本发明还涉及用于在样本中探测编码VDRR多肽的核酸序列的核酸探针。此探针适宜地包含图1或图7所示的核酸序列中的至少14个连续核苷酸,优选至少28个连续核苷酸。此核酸探针能用于鉴别编码VDRR多肽的克隆的方法中,此方法包括在低严格度杂交条件下用此探针筛选基因组或cDNA文库,并且鉴别与所述探针表现实质性杂交程度的克隆。
本发明进一步涉及了分离的或重组的VDRR多肽。此多肽可以是全长的,在全长上此氨基酸序列与一般哺乳动物,尤其是人类中发现的相应序列是一致的。在本发明中,此多肽也可以是此全长多肽的截短的、延长的或突变形式。截短的和延长的形式指的是在VDRR多肽链的N端分别丢失或加上一个或多个氨基酸。突变形式指的是VDRR多肽上的一个或多个氨基酸被另一个氨基酸序列替代。此分离的或重组的VDRR多肽恰当地显示了图4或图8中所示的氨基酸序列。
同本VDRR多肽的氨基酸序列一样,编码VDRR多肽的本核酸的N端序列也可以改变。因此,设想了各种各样的N端异构体,例如转录因子3:核受体、蛋白质手册,卷2,11期(1995),pp 1173-1235图7B中所示的α1、α2、β1、β2、β3、β4、γ1或γ2中的任何一个。因此,这篇关于核受体的评述全部引入供作参考。更特别地,维生素D受体及相关的孤受体,如ONR1,在p1191-1992更专门地得到讨论。
本发明进一步涉及包含分离的或重组的VDRR多肽及一个或多个治疗上可接受的赋形剂之药物制剂。可以应用的赋形剂的例子有:糖类,例如单糖、二糖和糖醇,如蔗糖和山梨醇。其它的例子包括氨基酸,例如组氨酸和精氨酸,表面活性剂,如聚氧乙烯脱水山梨糖醇脂肪酸酯,无机盐,如氯化钠和氯化钙,和鳌合剂,如EDTA和枸橼酸。
本制剂可以是易用的水溶液形式或干的尤其是冻干的形式。在后一个情况中,应用前此制剂用液体,如无菌水或盐水重构。
本发明进一步涉及含有分离的或重组的核酸的表达载体,该核酸包含编码维生素D受体相关(VDRR)多肽的连续核酸序列。本发明还涉及含有此表达载体的细胞。
本发明还涉及含有此要求保护的核酸的细胞,该核酸含有编码维生素D受体相关(VDRR)多肽的连续核酸序列。
本发明进一步涉及在适宜的宿主细胞中,优选在真核生物细胞中,通过表达编码维生素D受体相关(VDRR)多肽的要求保护的分离的或重组的连续核酸序列,重组生产VDRR多肽的方法。
本发明进一步涉及了鉴别VDRR配体的方法,例如通过基于细胞的报道分子测定、转基因动物报道分子测定或体外结合测定。本发明还涉及了鉴定治疗受VDRR多肽影响的疾病的物质的方法,包括筛选VDRR多肽信号转导的激动剂或抑制剂,以用于治疗代谢性、增生性或炎性疾病。
本发明还涉及了将VDRR多肽用作药物,以及影响VDRR信号转导的物质用于制造治疗代谢性、增生性或炎性疾病的药物的用途。更确切地说,本发明能用于制备治疗肥胖症、糖尿病、厌食症、脂蛋白缺乏、高脂血症、高胆固醇血症或高脂蛋白血症的药物。本发明还能用于制备治疗骨质疏松症、类风温性关节炎、良性和恶性肿瘤、过度增生性皮肤病或甲状旁腺功能亢进的药物。
本发明进一步涉及通过将编码表达VDRR多肽的核酸载体引入哺乳动物,以治疗代谢性、增生性或炎性疾病的方法。核酸载体能在体内转化细胞,并且在所述的转化细胞中表达所述的多肽。
本发明还涉及了一种治疗代谢性、增生性或炎性疾病的方法,其是通过给予治疗有效量的影响VDRR信号转导的物质,尤其是VDRR多肽。
在本发明中,术语“分离的”结合VDRR多肽或编码同一VDRR多肽的核酸,涉及从天然来源,如人类的肝、小肠或结肠分离出的核酸或多肽。本发明的分离的VDRR多肽或核酸在自然界中还没有发现纯的或分离的形式,从这种意义上讲,它们是独一无二的。术语“分离的”的应用表示已经将天然存在的序列从它的正常的细胞环境中移出。因此,此序列可能处于无细胞的环境或处于不同的细胞的环境。该术语并不意味着此序列是存在的唯一核酸或氨基酸序列,而是意味着它是占存在的绝大多数的核酸或氨基酸序列。另外,此核酸或多肽应该基本上没有与各自产物天然结合的非氨基酸或非核酸物质。在本文中,基本上没有指的是纯度大于80%,适宜的纯度大于90%,优选的纯度大于95%。
当提及图1或图7中核酸序列和图4或图8中氨基酸序列时,术语“基本上相同”意思是它们来源于图中所示的序列,并且有和这些序列相同的功能。
本发明的发明人已令人吃惊地分离出了新的核酸序列和由所述的核酸序列编码的多肽。因此,编码命名为VDRRγ的多肽的新的cDNA已得到克隆和表征。基于氨基酸序列相似性,此多肽是核(激素)受体超基因家族的新成员。Hidden Markov模型(HMM)与种系发生分析,如邻近点相连树图方法,和其它的统计算法相结合,表明VDRRγ属于维生素D受体(VDR)亚家族和命名为xONR1(见Smith等,核酸研究(Nucl.Acids.Res)22(1994),No.1,pp.61-71)或如WO 96/22390中所示的XOR-6来自爪蟾的VDR样受体。因此,此VDRRγ是维生素D受体相关(VDRR)多肽家族中的一员。
与最密切相关的受体XOR-6、hVDR和CAR(见WO 93/17041)相比较,VDRRg的DBD和LBD的氨基酸相似性程度近似于其它不同但相关的受体之间的关系(见图12)。甲状腺素(TRb)和视黄酸受体(RARb)在DBD和LBD氨基酸水平上分别有约60%和40%的相同性。经比较,密切相关但独立的编码人类RARa和RARb核受体的基因在DBD和LBD上分别有97%和82%的相同性。
正如核受体领域的普通技术人员所认识的那样,在不同核受体之间(共生同源的)和来自于不同种属的相同受体(直向同源的)之间DBD均显示了最高的保守程度(氨基酸一致性)。DBD中的两个“锌指”由两个进化保守的氨基酸基元产生:Cys-X2-Cys-X13-Cys-X2-Cys(氨基端或第一锌指)和Cys-Xn-Cys-X9-Cys-X2-Cys(羧基端或第二锌指),其中两个半胱氨酸鳌合在锌离子上。大多数的核受体在第二锌指(Cys-Xs-Cys-X9-Cys-X2-Cys)的前二个半胱氨酸残基之间有五个氨基酸残基,见Gronemeyer和Laudet(蛋白质手册(Protein Profile)1995,2,卷11)。关于此作用,现在知道的例外仅有PPAR,其有三个氨基酸(Cys-X3-Cys-X9-Cys-X2-Cys)残基;和TLL受体群,其有七个(Cys-X7-Cys-X9-Cys-X2-Cys)。因此,具有在这里所述的新的VDRRg多肽的特性的另一个特征是DBD部分的氨基酸残基数是6个(Cys-X6-Cys-X9-Cys-X2-Cys),如图4和图8所示。如今,在TREMBLE数据库中发现的,在两个半胱氨酸残基之间具有相同数目的氨基酸残基的唯一另外的核受体样序列是来自于线虫C.elegans(Q20097和Q18155)的两个序列(Q20097和Q18155)。然而,这些推定的C.elegans核受体的整个DBD远缘地与VDRRg的DBD相关。综上,比较此处所述的核受体VDRRg的DBD和LBD(见图12),清楚地表明了此受体是核受体超基因家族的新成员,它不同于其它已知的与包括ONR-1(在Smith等,1994,核酸研究),22,pp 66-71中)或XOR-6(WO 96/22390)、hVDR和CAR(WO93/17041)在内的VDRRg最密切相关的核受体。
这个发现,结合我们从人类VDRRγ(肝、小肠和结肠粘膜)观察到的高度限制性表达模式,并类比其它展示组织特异性表达模式的核受体,如微体增殖激活受体(PPPA),提示VDRRγ在肝、小肠和结肠中履行重要的生理功能。因此,VDRRγ可能是影响脂类、糖类或氨基酸代谢/平衡的关键代谢路径的重要传感器。另外,高度选择的组织特异性表达模式提示VDRRγ可能参与了这些组织的细胞分化和发育。
具有备择剪接的5′端的一个另外的人类VDRRγ cDNA已经得到鉴别(见图7)。此VDRRγ cDNA因而可编码除图4所示的VDRRγ多肽外至少一种备择性N端变体。类比于核受体超基因家族的其它成员,如RORα和RARα,这些VDRRγ的N-端异构体可以确定不同的功能,包括DNA结合特异性和/或启动子特异性激活(Gronemeyer和Laudet,1995)。
在本说明书中,术语VDRRγ指的是与不同剪接的VDRRγcDNA相对应的各种多肽(包括VDRRγ-1和VDRRγ-2)。然而,就图1和图4来说,VDRRγcDNA和VDRRγ分别特指VDRRγ-1cDNA和VDRRγ-1。同样,就图7和图8来讲,VDRRγcDNA和VDRRγ分别特指VDRRγ-2cDNA和VDRRγ-2。
与VDRRγ-2cDNA相反,VDRRγ-1cDNA不包含经典的AUG起始密码子,但可能代之以在另外的CUG密码子处起始。此推断的非AUG起始位点定位在用于从另外的起始位点有效启动的有利的序列位置中,处于整个开放阅读结构的读框中,且位于终止密码子之前。
总的来说,此VDRR,尤其是VDRRγ,通常在以下方面可表现出重要性:
1)代谢性疾病,如肥胖症、糖尿病(Ⅰ型和Ⅱ型),脂蛋白失调,
2)增生性疾病,如小肠和结肠的肿瘤(良性和恶性),
3)小肠和结肠的溃疡性-炎性疾病,如局限性回肠炎和溃疡性结肠炎,及
4)小肠和结肠的先天异常。
在DNA结合结构域(DBD)和配体结合结构域(LBD)中VDRRγ与VDR的高度氨基酸序列一致性表明,这两个受体也许还有重叠但独特的功能特性。在类比中,类似于VDR和VDRRγ,视黄酸受体(RAR)和类视色素X受体(RXR)在DBD和LBD区域中具有相似的氨基酸序列一致性。RAR和RXR已显示有不同的功能相似性,使得两受体都结合9-顺视黄酸,并且有重叠的DNA结合特异性,因而调控重叠的基因网络。基于这些发现,VDRRγ可被在结构上类似于核受体的已知配体,但不一定等同于1α,25-二羟维生素D3受体的配体的小化学分子所调控。另外,VDRRγ也可通过结合于维生素D应答元件(VDRE)样DNA序列调控维生素D3应答性基因网络。在本申请中,1α,25-二羟维生素D3受体缩写为维生素D受体(VDR)。
在本发明中,影响VDRR信号转导的物质能够是任何天然或合成来源的小化学分子,例如,诸如芳香化合物之类的糖类。这些小分子的分子量可以在大约100到约500Da范围内。适当的,小化学分子的分子量范围在200到400Da之间。优选的分子量为300Da左右。
人类VDRRγ多肽,包括VDRRγ-1和VDRRγ-2,已显示可出被如孕烯醇酮和雌二醇(微弱地)激活,但不能被诸如皮质醇、醛固酮、孕酮和雌激素之类的其它特定类固醇激素激活,并且最不可能被孕激素和糖皮质激素激活。因此,人VDRRγ不能被糖皮质激素拮抗剂孕烯醇酮16α-腈(PCN)激活。由于此原因,人VDRRγ也能称做人孕烯醇酮激活(核)受体(hPAR)。例如在默克手册(Merck Index),第11版,Merck和Co.,Inc.Rahuay,N.J.,美国,p.7735,1983中,可以发现关于孕烯醇酮的资料。
人VDRRγ多肽,包括VDRRγ-1和VDRRγ-2,(分别为hPAR-1和hPAR-2)的激活物包括但不限于诸如孕烷酮、孕烷二酮、孕烷三酮和孕烷二醇之类的孕烯醇酮,以及诸如雄烷醇和雄烷二醇之类的雄烷。恰当地,孕烯醇酮,尤其是5β-孕烷是非平面的。
以下化合物是人VDRRγ多肽,包括VDRRγ-1和VDRRγ-2的激活物和可能的配体的具体实例,它们由瑞典的Sigma-Aldrich公司销售:ⅰ)5β-孕烷-3,20-二酮ⅱ)3α-羟基-5β-孕烷-11,20-二酮甲磺酸酯ⅲ)5β-孕烷-3α,20β-二醇ⅳ)孕烯醇酮ⅴ)孕-4-烯醇[16,17-δ][2]异噁唑啉-3,20-二酮,6α-甲基-3′-苯基乙基醚溶剂合物ⅵ)孕-1,4,9(11)-三烯-3,20-二酮,21-[4-[6-甲氧基-2-(4-吗啉基)-4-嘧啶基]-1-哌嗪基]-16-甲基-,(16α)-ⅶ)雌烷-3-醇,17-[[[3-(三氟甲基)苯基]甲基]氨基]-,(E)-2-丁烯二酸(1∶1)(盐)ⅷ)9α-氟-5α-雄烷-11β,17β-二醇ⅸ)螺[5α-雄烷-3,2′-苯并噻唑]-11-酮,17β-羟基-17-甲基-ⅹ)螺[孕烷-3,2′-噻唑烷]-4′-羧酸,11α-羟基-20-氧代-,钠盐ⅹⅰ)17β-二甲氨基-17-乙炔基-5α-雄烷-11β-醇ⅹⅱ)6β-羟基-3,5-环-5α-孕烷-20-酮亚硝酸酯ⅹⅲ)3α-羟基-5β-孕烷-11,20-二酮乙酸酯,20-O-甲磺酰-肟ⅹⅳ)17α-甲基-5α-雄烷-11β,17-二-醇ⅹⅴ)5β-孕烷-3,11,20-三酮,三肟ⅹⅵ)3α-羟基-5β-孕烷-11,20-二酮,1-(羧甲基)氯化吡啶鎓酰肼的20位腙
VDRRg拮抗剂的可能用途可以是协同共施用,此VDRRg拮抗剂与其它药物,诸如但不限于同HIV蛋白酶抑制剂和环孢菌素一起服用,抑制CYP3A4的表达,因此提高了由于CYP3A4代谢使药物动力学不良的药物的生物利用率。通过将编码此多肽的DNA片段掺入重组DNA运载体,如载体,可以克隆编码诸如人类维生素D受体相关γ(hVDRg)之类的多肽的基因,并转化适当的原核或真核宿主细胞。这些重组DNA技术已公知,且在如酶学方法(Methods in Enzymology),Academic Press,San Diego,CA,美国(1994),65和68卷(1979),及100和101卷(1983)中有所描述。
用于本发明的宿主细胞可以是原核或真核生物的,优选真核生物细胞。恰当的真核生物宿主细胞包括但不限于诸如酵母属(Saccharomyces)的来源于酵母菌的细胞、昆虫细胞、诸如中国仓鼠卵巢(CHO)、幼仓鼠肾(BHK)、COS细胞等的哺乳动物细胞。合适的原核生物宿主细胞包括但不限于来自肠杆菌科(Enterobacteriacea)的细胞,例如大肠杆菌(E.coli)、芽孢杆菌属(Bacillus)和链霉菌属(Streptomyces)。
实施例
提供以下的实施例只是用于例证的目的,不能理解成以任何方式限制本发明的范围,这一点由附加的权利要求书规定。
实施例1人VDRRgcDNA的鉴定和分离
用核受体DNA结合结构域(DBD)曲线就核受体相关序列筛选表达序列标记(DEST)资料库。核受体亚结构域的选定组之多重比较后依统计学计算创建此检索曲线,以得到核受体超基因家族不同亚族成员的所谓的Hidden Markov模型(HMM)。通过测序详细分析一个已识别的核受体相关EST序列(Incyte克隆第2211526号)的cDNA。在整个IncytecDNA克隆(大约2200碱基对)DNA测序后,发现此克隆编码一个与xONR-1和维生素D受体(VDR)分别具有54%和44%相似性的推定的配体结合结构域(LBD)。此Incyte克隆的cDNA不是全长的,并且不编码相应于完整DBD的序列。
来自人肝RNA(InVitrogen)的经随机引物处理的cDNA的5′-RACE(cDNA末端的迅速扩增)后经克隆和序列测定显示,相应于Incyte克隆的cDNA的5′部分编码核受体特有的DBD,而且与xONR-1和VDR分别有71%和65%的序列相似性。与进化邻近点相连树图分析法相结合的多重比较法把由cDNA(图1中说明)编码的多肽归入VDR组(图2和3),并命名为人类维生素D受体相关γ(VDRRg)。图4给出VDRRg的推断的氨基酸序列。
实施例2人组织中VDRRgmDNA的表达
用多组织Northern印迹法(Clontech)来确定成年人类组织中VDRRg的表达模式。如图5所示,VDRRg在小肠、结肠粘膜表层和肝中大量表达,但在其它几种组织,包括脾、胸腺、前列腺、睾丸、卵巢、外周血白细胞、心脏、脑、胎盘、肺、骨骼肌、肾和胰腺中,不大量表达。为调查VDRRγ是否在任何一个其它组织中低水平表达,在延长的时间(相对于过夜延长至一周)内曝光滤膜。即使在这种延长的曝光后(资料未给出),仍只能在相同组织中探测到VDRRγ的表达,在任何其它检查的组织中未检出。VDRRg受限制的表达提示此受体可能在肝脏和肠中具有重要的调节功能。
实施例3应用维生素D3的GAL4-DBD/VDRRγ-LBD融合蛋白质的瞬时转染
施行瞬时转染以分析是否维生素D3激活VDRRγ多肽。为了这个目的,用表达质粒和报道分子质粒瞬时共转染CV-1细胞,其中表达质粒编码与VDR或者VDRR的LBD融合的GAL-4-DBD融合蛋白质,报道分子质粒含有在萤光素酶基因上游的5个GAL4应答元件。转染之后,单独用溶剂(DMSO)处理细胞,或者用维生素D3处理细胞48小时,接着采集细胞并测量细胞提取物中的萤光素酶活性。如图6所示,维生素D3(1μM)反式激活GAL4-DBD/VDR-LBD,但在这些条件下,不激活相应的GAL4-DBD/DRRγ-LBD多肽。这说明这两个受体可能具有不同的配体结合特异性。
实施例4编码多重N-端异构体的人类VDRRγcDNA的鉴定和分离
来自人肝RNA的cDNA经5′-RACE(见实施例1)后经克隆和DNA测序,鉴定人VDRRγcDNA,其带有另外剪接的5′端(见图7)。因而除编码图4所示的VDRRγ多肽外,此VDRRγcDNA还能编码至少一种另外的N-端变异体(图8)。图4和图8显示的相应于不同剪接的VDRRγcDNA的多肽分别称作VDRRγ-1和VDRRγ-2。
实施例5具有RXR并与由三或四个核苷酸分隔开的同向重复(DR)结合的VDRRγ异二聚体
用T7多聚酶转录包含VDRRγ或RXRβcDNA的表达质粒,并在TNT网织红细胞溶解产物(Promega公司,Madison,WI,美国)中体外翻译。为调查VDRRγ的DNA结合特异性;应用一种天然凝胶泳动度测定法,基本如Berkenstam等,细胞(Cell),69,401-402,1992中所述,其中在有或没有图9所示的具有由不同的32P标记的同向重复(DR-1到DR-5)的体外翻译的RXRβ的情况下,孵育体外翻译的VDRRγ。这些同向重复衍生自RAR-β2启动子中的DR-5元件(de Thé等,自然(Nature),343,177-180,1990),并得到修改以被一到五个核苷酸分隔(Pettersson等,发育机制(Mechanisms of Dev.),54,1-13,1995)。在天然的5%聚丙烯酰胺/0.25X TBE凝胶上分离蛋白质-DNA复合物,接着进行放射自显影。如图9所示,在这五个检测的DR中,只有用由三或四个核苷酸分隔的DR可检测到有效的VDRRγ结合,而且必须在RXR存在的情况下。然而,对DR-2和DR-1元件也能观察到较弱的RXR依赖性结合。这些结果说明,VDRRγ需要RXR异二聚体化,以便把DNA高效地结合到DR的特定亚组。然而,这些结果并不排除VDRRγ作为单体、双体或异二聚体结合到独立但相关的DNA序列上的可能性。重要的是,我们的结果说明了VDRRγ和其它核受体,包括VDR(例如Markose,E.R.等,美国国家科学院院报(Proc.Natl.Acad.Sci.NSA.),87,1701-1705,1990)、THR(例如Gronemeyer,H.和Moras,D.,自然,375,190-191,1995)、LXR(例如Willv,P.J.等,基因进展(Genes.Dev.),9,1033-1045,1995),具有不同的但重叠的DNA序列,因此可以调控重叠的基因网络。
有趣的是,据报道,称作ONR-1(在Smith等,1994,核酸研究,22,pp 66-71中)或XOR-6(在WO 96/22390中)的最密切相关的核受体“很好地结合到视黄酸应答元件,bRARE”上(WO 96/22390 11页30行)。
尽管此处报道这种新的核受体VDRRg与XOR-6(图12)比较在DBD有71%的氨基酸相似性,VDRRg却好象不结合于相同的bRARE序列(图9中D1-5)。实施例6孕烯醇酮衍生物作为VDRRγ的激活剂
为鉴定VDRRγ的激活剂或配体,检测了对不同种类的核受体激活剂和配体有结构偏向的物质文库。在短暂Caco-2(TC7)细胞中以报道基因测定法分析VDRRγ的激活(Carriere等,1994)。在此初期的筛选中,发现具有激活VDRRγ能力的合成物质结构上相似于孕烯醇酮(资料未给出)。基于这些结果,检测天然存在的孕烯醇酮衍生物对VDRRγ的激活。结果显示于图10中。从图10可以明显看到,孕烯醇酮、5β-孕烷-3,20-二酮、5β-孕烷-3α,20β-二醇和3α-羟-5β-孕烷-11,20-二酮甲磺酸酯大约5到12倍激活VDRRγ。对比于观察到的由5β-孕烷-3,20-二酮的高效激活,相应的平面类固醇衍生物5α-孕烷-3,20-二酮不激活此受体。相对于所有检测的平面孕烯醇酮衍生物,其它5β-孕烷也高效激活VDRRγ,这从图10中也可明显看出。
实施例7孕烯醇酮-16α-腈(PCN)、地塞米松和抗孕酮(RU486)作为VDRRγ的激活剂
施行进一步的实验,以明确孕烯醇酮-16α-腈(PCN)、糖皮质激素拮抗剂或地塞米松是否为VDRRγ的激活剂。为了达到效果,如前面一样用VDRRγ转染Caco-2细胞,并且用10μM PCN或地塞米松处理细胞后分析VDRRγ的激活。结果显示于图11中。从图11可明显看出这些物质不激活VDRRγ,说明VDRRγ不是人类PCN受体。这种提示与如下观察相一致:即抗孕酮也仅仅引起VDRRγ介导受体基因活性的略有增加(两倍),这从图11可以明显看出。
在WO 96/22390中所使用的相似的报道分子测定中,诸如4-胺苯甲酸丁酯之类的XOR-6(在WO 96/22390的图3中)的激活剂不激活VDRRγ(资料未给出)。
序列表<110>Pharmacia和Upjohn AB<120>新的维生素D受体相关多肽、编码其的核酸序列及其用途<130>1788序列表<140><141><150>9703745-1<151>1997-10-14<150>9801148-9<151>1998-03-31<160>4<170>PatentIn Ver.2.0<210>1<211>2910<212>DNA<213>人<400>1cctctgaagg ttctagaatc gatagtgaat tcgtgggacg ggaagaggaa gcactgcctt 60tacttcagtg ggaatctcgg cctcagcctg caagccaagt gttcacagtg aaaaaagcaa 120gagaataagc taatactcct gtcctgaaca aggcagcggc tccttggtaa agctactcct 180tgatcgatcc tttgcaccgg attgttcaaa gtggacccca ggggagaagt cggagcaaag 240aacttaccac caagcagtcc aagaggccca gaagcaaacc tggaggtgag acccaaagaa 300agctggaacc atgctgactt tgtacactgt gaggacacag agtctgttcc tggaaagccc 360agtgtcaacg cagatgagga agtcggaggt ccccaaatct gccgtgtatg tggggacaag 420gccactggct atcacttcaa tgtcatgaca tgtgaaggat gcaagggctt tttcaggagg 480gccatgaaac gcaacgcccg gctgaggtgc cccttccgga agggcgcctg cgagatcacc 540cggaagaccc ggcgacagtg ccaggcctgc cgcctgcgca agtgcctgga gagcggcatg 600aagaaggaga tgatcatgtc cgacgaggcc gtggaggaga ggcgggcctt gatcaagcgg 660aagaaaagtg aacggacagg gactcagcca ctgggagtgc aggggctgac agaggagcag 720cggatgatga tcagggagct gatggacgct cagatgaaaa cctttgacac taccttctcc 780catttcaaga atttccggct gccaggggtg cttagcagtg gctgcgagtt gccagagtct 840ctgcaggccc catcgaggga agaagctgcc aagtggagcc aggtccggaa agatctgtgc 900tctttgaagg tctctctgca gctgcggggg gaggatggca gtgtctggaa ctacaaaccc 960ccagccgaca gtggcgggaa agagatcttc tccctgctgc cccacatggc tgacatgtca 1020acctacatgt tcaaaggcat catcagcttt gccaaagtca tctcctactt cagggacttg 1080cccatcgagg accagatctc cctgctgaag ggggccgctt tcgagctgtg tcaactgaga 1140ttcaacacag tgttcaacgc ggagactgga acctgggagt gtggccggct gtcctactgc 1200ttggaagaca ctgcaggtgg cttccagcaa cttctactgg agcccatgct gaaattccac 1260tacatgctga agaagctgca gctgcatgag gaggagtatg tgctgatgca ggccatctcc 1320ctcttctccc cagaccgccc aggtgtgctg cagcaccgcg tggtggacca gctgcaggag 1380caattcgcca ttactctgaa gtcctacatt gaatgcaatc ggccccagcc tgctcatagg 1440ttcttgttcc gtgaagatca tggctatgct caccgagctc cgcagcatca atgctcagca 1500cacccagcgg ctgctgcgca tccaggacat acaccccttt gctacgcccc tcatgcagga 1560gttgttcggc atcacaggta gctgagcggc tgcccttggg tgacacctcc gagaggcagc 1620cagacccaga gccctctgag ccgccactcc cgggccaaga cagatggaca ctgccaagag 1680ccgacaatgc cctgctggcc tgtctcccta gggaattcct gctatgacag ctggctagca 1740ttcctcagga aggacatggg tgccccccac ccccagttca gtctgtaggg agtgaagcca 1800cagactctta cgtggagagt gcactgacct gtaggtcagg accatcagag aggcaaggtt 1860gccctttcct tttaaaaggc cctgtggtct ggggagaaat ccctcagatc ccactaaagt 1920gtcaaggtgt ggaagggacc aagcgaccaa ggataggcca tctggggtct atgcccacat 1980acccacgttt gttcgcttcc tgagtctttt cattgctacc tctaatagtc ctgtctccca 2040cttcccactc gttcccctcc tcttccgagc tgctttgtgg gctcaaggcc tgtactcatc 2100ggcaggtgca tgagtatctg tgggagtcct ctagagagat gagaagccag gaggcctgca 2160ccaaatgtca gaagcttggc atgacctcat tccggccaca tcattctgtg tctctgcatc 2220catttgaaca cattattaag cactgataat aggtagcctg ctgtggggta tacagcattg 2280actcagatat agatcctgag ctcacagagt ttatagttaa aaaaacaaac agaaacacaa 2340acaatttgga tcaaaaggag aaaatgataa gtgacaaaag cagcacaagg aatttccctg 2400tgtggatgct gagctgtgat ggcaggcact gggtacccaa gtgaaggttc ccgaggacat 2460gagtctgtag gagcaagggc acaaactgca gctgtgagtg cgtgtgtgtg atttggtgta 2520ggtaggtctg tttgccactt gatggggcct gggtttgttc ctggggctgg aatgctgggt 2580atgctctgtg acaaggctac gctgacaatc agttaaacac accggagaag aaccatttac 2640atgcacctta tatttctgtg tacacatcta ttctcaaagc taaagggtat gaaagtgcct 2700gccttgttta tagccacttg tgagtaaaaa tttttttgca ttttcacaaa ttatacttta 2760tataaggcat tccacaccta agaactagtt ttgggaaatg tagccctggg tttaatgtca 2820aatcaaggca aaaggaatta aataatgtac ttttggctaa aaaaaaaaaa aaaaaaaaaa 2880aaaaaaaaaa aaaaaaaaaa aaaaaaagcnt 2910<210>2<211>437<212>PRT<213>人<400>2Met Glu Val Arg Pro Lys Glu Ser Trp Asn His Ala Asp Phe Val His1 5 10 15Cys Glu Asp Thr Glu Ser Val Pro Gly Lys Pro Ser Val Asn Ala Asp
20 25 30Glu Glu Val Gly Gly Pro Gln Ile Cys Arg Val Cys Gly Asp Lys Ala
35 40 45Thr Gly Tyr His Phe Asn Val Met Thr Cys Glu Gly Cys Lys Gly Phe
50 55 60Phe Arg Arg Ala Met Lys Arg Asn Ala Arg Leu Arg Cys Pro Phe Arg65 70 75 80Lys Gly Ala Cys Glu Ile Thr Arg Lys Thr Arg Arg Gln Cys Gln Ala
85 90 95Cys Arg Leu Arg Lys Cys Leu Glu Ser Gly Met Lys Lys Glu Met Ile
100 105 110Met Ser Asp Glu Ala Val Glu Glu Arg Arg Ala Leu Ile Lys Arg Lys
115 120 125Lys Ser Glu Arg Thr Gly Thr Gln Pro Leu Gly Val Gln Gly Leu Thr130 135 140Glu Glu Gln Arg Met Met Ile Arg Glu Leu Met Asp Ala Gln Met Lys145 150 155 160Thr Phe Asp Thr Thr Phe Ser His Phe Lys Asn Phe Arg Leu Pro Gly
165 170 175Val Leu Ser Ser Gly Cys Glu Leu Pro Glu Ser Leu Gln Ala Pro Ser
180 185 190Arg Glu Glu Ala Ala Lys Trp Ser Gln Val Arg Lys Asp Leu Cys Ser
195 200 205Leu Lys Val Ser Leu Gln Leu Arg Gly Glu Asp Gly Ser Val Trp Asn210 215 220Tyr Lys Pro Pro Ala Asp Ser Gly Gly Lys Glu Ile Phe Ser Leu Leu225 230 235 240Pro His Met Ala Asp Met Ser Thr Tyr Met Phe Lys Gly Ile Ile Ser
245 250 255Phe Ala Lys Val Ile Ser Tyr Phe Arg Asp Leu Pro Ile Glu Asp Gln
260 265 270Ile Ser Leu Leu Lys Gly Ala Ala Phe Glu Leu Cys Gln Leu Arg Phe
275 280 285Asn Thr Val Phe Asn Ala Glu Thr Gly Thr Trp Glu Cys Gly Arg Leu290 295 300Ser Tyr Cys Leu Glu Asp Thr Ala Gly Gly Phe Gln Gln Leu Leu Leu305 310 315 320Glu Pro Met Leu Lys Phe His Tyr Met Leu Lys Lys Leu Gln Leu His
325 330 335Glu Glu Glu Tyr Val Leu Met Gln Ala Ile Ser Leu Phe Ser Pro Asp
340 345 350Arg Pro Gly Val Leu Gln His Arg Val Val Asp Gln Leu Gln Glu Gln
355 360 365Phe Ala Ile Thr Leu Lys Ser Tyr Ile Glu Cys Asn Arg Pro Gln Pro370 375 380Ala His Arg Phe Leu Phe Leu Lys Ile Met Ala Met Leu Thr Glu Leu385 390 395 400Arg Ser Ile Asn Ala Gln His Thr Gln Arg Leu Leu Arg Ile Gln Asp
405 410 415Ile His Pro Phe Ala Thr Pro Leu Met Gln Glu Leu Phe Gly Ile Thr
420 425 430Gly Ser Phe Ile Gly
435<210>3<211>2802<212>DNA<213>人<400>3tgaattcgtg ggcctgctgg gttagtgctg gcagcccccc tgaggccaag gacagcagca 60tgacagtcac caggactcac cacttcaagg aggggtccct cagagcacct gccatacccc 120tgcacagtgc tgcggctgag ttggcttcaa accatccaag aggcccagaa gcaaacctgg 180aggtgagacc caaagaaagc tggaaccatg ctgactttgt acactgtgag gacacagagt 240ctgttcctgg aaagcccagt gtcaacgcag atgaggaagt cggaggtccc caaatctgcc 300gtgtatgtgg ggacaaggcc actggctatc acttcaatgt catgacatgt gaaggatgca 360agggcttttt caggagggcc atgaaacgca acgcccggct gaggtgcccc ttccggaagg 420gcgcctgcga gatcacccgg aagacccggc gacagtgcca ggcctgccgc ctgcgcaagt 480gcctggagag cggcatgaag aaggagatga tcatgtccga cgaggccgtg gaggagaggc 540gggccttgat caagcggaag aaaagtgaac ggacagggac tcagccactg ggagtgcagg 600ggctgacaga ggagcagcgg atgatgatca gggagctgat ggacgctcag atgaaaacct 660ttgacactac cttctcccat ttcaagaatt tccggctgcc aggggtgctt agcagtggct 720gcgagttgcc agagtctctg caggccccat cgagggaaga agctgccaag tggagccagg 780tccggaaaga tctgtgctct ttgaaggtct ctctgcagct gcggggggag gatggcagtg 840tctggaacta caaaccccca gccgacagtg gcgggaaaga gatcttctcc ctgctgcccc 900acatggctga catgtcaacc tacatgttca aaggcatcat cagctttgcc aaagtcatct 960cctacttcag ggacttgccc atcgaggacc agatctccct gctgaagggg gccgctttcg 1020agctgtgtca actgagattc aacacagtgt tcaacgcgga gactggaacc tgggagtgtg 1080gccggctgtc ctactgcttg gaagacactg caggtggctt ccagcaactt ctactggagc 1140ccatgctgaa attccactac atgctgaaga agctgcagct gcatgaggag gagtatgtgc 1200tgatgcaggc catctccctc ttctccccag accgcccagg tgtgctgcag caccgcgtgg 1260tggaccagct gcaggagcaa ttcgccatta ctctgaagtc ctacattgaa tgcaatcggc 1320cccagcctgc tcataggttc ttgttcctga agatcatggc tatgctcacc gagctccgca 1380gcatcaatgc tcagcacacc cagcggctgc tgcgcatcca ggacatacac ccctttgcta 1440cgcccctcat gcaggagttg ttcggcatca caggtagctg agcggctgcc cttgggtgac 1500acctccgaga ggcagccaga cccagagccc tctgagccgc cactcccggg ccaagacaga 1560tggacactgc caagagccga caatgccctg ctggcctgtc tccctaggga attcctgcta 1620tgacagctgg ctagcattcc tcaggaagga catgggtgcc ccccaccccc agttcagtct 1680gtagggagtg aagccacaga ctcttacgtg gagagtgcac tgacctgtag gtcaggacca 1740tcagagaggc aaggttgccc tttcctttta aaaggccctg tggtctgggg agaaatccct 1800cagatcccac taaagtgtca aggtgtggaa gggaccaagc gaccaaggat aggccatctg 1860gggtctatgc ccacataccc acgtttgttc gcttcctgag tcttttcatt gctacctcta 1920atagtcctgt ctcccacttc ccactcgttc ccctcctctt ccgagctgct ttgtgggctc 1980aaggcctgta ctcatcggca ggtgcatgag tatctgtggg agtcctctag agagatgaga 2040agccaggagg cctgcaccaa atgtcagaag cttggcatga cctcattccg gccacatcat 2100tctgtgtctc tgcatccatt tgaacacatt attaagcact gataataggt agcctgctgt 2160ggggtataca gcattgactc agatatagat cctgagctca cagagtttat agttaaaaaa 2220acaaacagaa acacaaacaa tttggatcaa aaggagaaaa tgataagtga caaaagcagc 2280acaaggaatt tccctgtgtg gatgctgagc tgtgatggca ggcactgggt acccaagtga 2340aggttcccga ggacatgagt ctgtaggagc aagggcacaa actgcagctg tgagtgcgtg 2400tgtgtgattt ggtgtaggta ggtctgtttg ccacttgatg gggcctgggt ttgttcctgg 2460ggctggaatg ctgggtatgc tctgtgacaa ggctacgctg acaatcagtt aaacacaccg 2520gagaagaacc atttacatgc accttatatt tctgtgtaca catctattct caaagctaaa 2580gggtatgaaa gtgcctgcct tgtttatagc cacttgtgag taaaaatttt tttgcatttt 2640cacaaattat actttatata aggcattcca cacctaagaa ctagttttgg gaaatgtagc 2700cctgggttta atgtcaaatc aaggcaaaag gaattaaata atgtactttt ggctaaaaaa 2760aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 2802<210>4<211>473<212>PRT<213>人<400>4Met Thr Val Thr Arg Thr His His Phe Lys Glu Gly Ser Leu Arg Ala1 5 10 15Pro Ala Ile Pro Leu His Ser Ala Ala Ala Glu Leu Ala Ser Asn His
20 25 30Pro Arg Gly Pro Glu Ala Ash Leu Glu Val Arg Pro Lys Glu Ser Trp
35 40 45Asn His Ala Asp Phe Val His Cys Glu Asp Thr Glu Ser Val Pro Gly
50 55 60Lys Pro Ser Val Asn Ala Asp Gtu Glu Val Gly Gly Pro Gln Ile Cys65 70 75 80Arg Val Cys Gly Asp Lys Ala Thr Gly Tyr His Phe Asn Val Met Thr
85 90 95Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ala Met Lys Arg Asn Ala
100 105 110Arg Leu Arg Cys Pro Phe Arg Lys Gly Ala Cys Glu Ile Thr Arg Lys
115 120 125Thr Arg Arg Gln Cys Gln Ala Cys Arg Leu Arg Lys Cys Leu Glu Ser130 135 140Gly Met Lys Lys Glu Met Ile Met Ser Asp Glu Ala Val Glu Glu Arg145 150 155 160Arg Ala Leu Ile Lys Arg Lys Lys Set Glu Arg Thr Gly Thr Gln Pro
165 170 175Leu Gly Val Gln Gly Leu Thr Glu Glu Gln Arg Met Met Ile Arg Glu
180 185 190Leu Met Asp Ala Gln Met Lys Thr Phe Asp Thr Thr Phe Ser His Phe
195 200 205Lys Asn Phe Arg Leu Pro Gly Val Leu Ser Ser Gly Cys Glu Leu Pro210 215 220Glu Set Leu Gln Ala Pro Set Arg Glu Glu Ala Ala Lys Trp Set Gln225 230 235 240Val Arg Lys Asp Leu Cys Set Leu Lys Val Ser Leu Gln Leu Arg Gly
245 250 255Glu Asp Gly Set Val Trp Asn Tyr Lys Pro Pro Ala Asp Set Gly Gly
260 265 270Lys Glu Ile Phe Ser Leu Leu Pro His Met Ala Asp Met Ser Thr Tyr
275 280 285Met Phe Lys Gly Ile Ile Ser Phe Ala Lys Val Ile Ser Tyr Phe Arg290 295 300Asp Leu Pro Ile Glu Asp Gln Ile Ser Leu Leu Lys Gly Ala Ala Phe305 310 315 320Glu Leu Cys Gln Leu Arg Phe Asn Thr Val Phe Asn Ala Glu Thr Gly
325 330 335Thr Trp Glu Cys Gly Arg Leu Set Tyr Cys Leu Glu Asp Thr Ala Gly
340 345 350Gly Phe Gln Gln Leu Leu Leu Glu Pro Met Leu Lys Phe His Tyr Met
355 360 365Leu Lys Lys Leu Gln Leu His Glu Glu Glu Tyr Val Leu Met Gln Ala370 375 380Ile Ser Leu Phe Ser Pro Asp Arg Pro Gly Val Leu Gln His Arg Val385 390 395 400Val Asp Gln Leu Gln Glu Gln Phe Ala Ile Thr Leu Lys Ser Tyr Ile
405 410 415Glu Cys Asn Arg Pro Gln Pro Ala His Arg Phe Leu Phe Leu Lys Ile
420 425 430Met Ala Met Leu Thr Glu Leu Arg Ser Ile Asn Ala Gln His Thr Gln
435 440 445Arg Leu Leu Arg Ile Gln Asp Ile His Pro Phe Ala Thr Pro Leu Met450 455 460Gln Glu Leu Phe Gly Ile Thr Gly Ser465 470
Claims (31)
1.一种哺乳动物优选人的分离的或重组核酸,其包含编码维生素D受体相关(VDRR)多肽的连续核酸序列。
2.一种根据图1或图7所述的分离的或重组的DNA/核酸或其等位基因,其编码一种新的VDRR多肽。
3.根据权利要求1或权利要求2所述的编码VDRR多肽的核酸,其含有包含大约77个的氨基酸的DNA结合结构域(DBD),其中具有9个半胱氨酸残基,其中所述的DBD以下列氨基酸序列相似性为特征:(ⅰ)同hVDR的DBD具有至少60%的氨基酸序列相似性;以及(ⅱ)同xONR1的DBD具有至少65%的氨基酸序列相似性。
4.根据权利要求3所述的核酸,其中所述的DBD以下列氨基酸序列相似性为特征:(ⅰ)同hVDR的DBD具有大约65%的氨基酸序列相似性;以及(ⅱ)同xONR1的DBD具有大约71%的氨基酸序列相似性。
5.根据任一先前的权利要求所述的编码VDRR多肽的核酸,其中所述多肽的配体结合结构域(LBD)以下列氨基酸序列相似性为特征,其分别相对于hVDR和xONR1的LBD:(ⅰ)同hVDR的LBD具有至少大约30%的氨基酸序列相似性;以及(ⅱ)同xONR1的LBD具有至少大约40%的氨基酸序列相似性。
6.根据权利要求5所述的核酸,其中所述的LBD以下列氨基酸序列相似性为特征:(ⅰ)同hVDR的LBD具有至少35%的氨基酸序列相似性;以及(ⅱ)同xONR1的LBD具有至少45%的氨基酸序列相似性。
7.根据权利要求6所述的核酸,其中所述的LBD以下列氨基酸序列相似性为特征:(ⅰ)同hVDR的LBD具有至少大约42%的氨基酸序列相似性;以及(ⅱ)同xONR1的LBD具有至少大约54%的氨基酸序列相似性。
8.根据任一先前的权利要求所述的核酸,其中所述的核酸序列如图1或图7中所示或其等位基因。
9.根据权利要求8所述的核酸,其中所述的核酸序列同图1或图7中所示的相同或基本相同。
10.一种用于检测样本中编码VDRR多肽的核酸序列的核酸探针。
11.根据权利要求10所述的核酸探针,其中所述的探针包含图1或图7中所示核酸序列的至少15个连续核苷酸。
12.一种鉴定编码VDRR多肽的克隆的方法,所述的方法包括在低严格杂交条件下用根据权利要求10或11所述的核酸探针筛选基因组文库或cDNA文库,以及鉴定显示与所述探针实质程度杂交的那些克隆。
13.一种表达载体,其包含根据权利要求1至9中任何一个所述的核酸。
14.一种包含根据权利要求1至9中任何一个所述的核酸的细胞。
15.一种包含根据权利要求14所述的表达载体的细胞。
16.一种用于VDRR多肽的重组生产的方法,所述的方法包括在合适的宿主细胞中表达权利要求1至9的核酸。
17.根据权利要求16所述的方法,其中宿主细胞是真核生物细胞。
18.一种分离的或重组的哺乳动物优选为人的VDRR多肽。
19.根据权利要求18所述的分离的或重组的VDRR多肽,其包含与图4或图8中所示的相同或基本相同的氨基酸序列。
20.一种产生针对根据权利要求18和19中任何一个所述的多肽的特异性单克隆抗体和多克隆抗体的方法,包括向哺乳动物注射该蛋白质。
21.一种药物制剂,其包含根据权利要求18或19所述的分离或重组的VDRR多肽,和一种或多种治疗上可接受的赋形剂。
22.一种通过基于细胞的报道分子测定、转基因动物报道分子测定或体外结合测定用于鉴定VDRR配体的方法。
23.一种鉴定用于治疗受VDRR多肽影响的疾病的物质的方法,其包括筛选用于治疗代谢性、增生性或炎性疾病的VDRR多肽信号转导的激动剂或拮抗剂。
24.一种根据权利要求18和19中任一项所述的哺乳动物优选为人的VDRR多肽,其用作药物。
25.影响VDRR信号转导的物质的用途,诸如VDRR多肽信号转导的激动剂或拮抗剂用于制造治疗代谢性、增生性或炎性疾病的药物。
26.根据权利要求25所述的影响VDRR信号转导的物质的用途,其用于制造治疗肥胖症、糖尿病、厌食、脂蛋白缺乏症、高脂血症、高胆固醇血症或高脂蛋白血症的药物。
27.影响VDRR信号转导的物质的用途,其用于制造治疗骨质疏松症、类风湿性关节炎、良性和恶性肿瘤、过度增生性皮肤疾病或甲状旁腺功能亢进的药物。
28.根据权利要求25至27中任何一个所述的用途,其中影响VDRR信号转导的物质是天然或合成来源的化学分子,其分子量在从大约100到大约500Da的范围内,优选分子量为大约300Da。
29.一种用于治疗代谢性、增生性或炎性疾病的方法,其包括将根据权利要求13所述的编码表达VDRR多肽的核酸载体引入哺乳动物,而且其中所述的核酸载体能在体内转化细胞,并在所述的转化细胞中表达所述的多肽。
30.一种通过给予治疗有效剂量的影响VDRR信号转导的物质治疗代谢性、增生性或炎性疾病的方法。
31.根据权利要求30所述的方法,其中影响VDRR信号转导的物质是天然或合成来源的化学分子,其分子量在从大约100到大约500Da的范围内,优选分子量为大约300Da。
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SE9703745A SE9703745D0 (sv) | 1997-10-14 | 1997-10-14 | New receptors |
SE9703745-1 | 1997-10-14 | ||
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SE9801148A SE9801148D0 (sv) | 1997-10-14 | 1998-03-31 | New receptors |
SE9801148-9 | 1998-03-31 | ||
SE98011489 | 1998-03-31 |
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JP (1) | JP2001519441A (zh) |
KR (1) | KR20010031120A (zh) |
CN (1) | CN1134452C (zh) |
AU (1) | AU732079B2 (zh) |
CA (1) | CA2306453A1 (zh) |
NZ (1) | NZ504025A (zh) |
SE (1) | SE9801148D0 (zh) |
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EP1044216A4 (en) * | 1997-12-12 | 2001-10-31 | Merck & Co Inc | DNA MOLECULES ENCODING HUMAN NUCLEAR RECEPTOR PROTEINS, nNR7 AND nNR7-1 |
AU2003200641B2 (en) * | 1998-01-09 | 2008-04-03 | The Salk Institute For Biological Studies | Novel Steroid-activated Nuclear Receptors and Uses therefor |
US6984773B1 (en) | 1998-01-09 | 2006-01-10 | The Salk Institute For Biological Studies | Transgenic mice expressing a human SXR receptor polypeptide |
US6756491B2 (en) | 1998-01-09 | 2004-06-29 | The Salk Institute For Biological Studies | Steroid-activated nuclear receptors and uses therefor |
US6911537B2 (en) * | 1998-01-09 | 2005-06-28 | The Salk Institute For Biological Studies | Xenobiotic compound modulated expression systems and uses therefor |
US7238491B1 (en) | 1998-03-27 | 2007-07-03 | Smithkline Beecham Corporation | Pregnane X receptor method |
EP1066320A4 (en) * | 1998-03-27 | 2005-03-16 | Glaxo Group Ltd | ORPHAN NUCLEAR RECEPTOR |
FR2801311B1 (fr) * | 1999-11-22 | 2005-08-26 | Centre Nat Rech Scient | Polypeptides derives du recepteur nucleaire de la vitamine d, et leurs utilisations notamment dans le cadre du criblage d'analogues de la vitamine d |
AU2006200258B2 (en) * | 1999-12-09 | 2009-04-09 | The Salk Institute For Biological Studies | Novel steroid-activated nuclear receptors and uses therefor |
US6514941B1 (en) | 1999-12-10 | 2003-02-04 | Campina Melkunie B.V. | Method of preparing a casein hydrolysate enriched in anti-hypertensive peptides |
US20040053866A1 (en) * | 2002-08-21 | 2004-03-18 | The Regents Of The University Of California | Tumor suppressor genes and their uses |
SE0400489D0 (sv) * | 2004-02-27 | 2004-02-27 | Biovitrum Ab | Therapeutic proteins |
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EP0609240B1 (en) * | 1991-09-17 | 2002-04-03 | The Salk Institute For Biological Studies | Receptors of the thyroid/steroid hormone receptor superfamily |
US5756448A (en) * | 1992-02-26 | 1998-05-26 | The General Hospital Corporation | Constitute activator of retinoid (CAR) receptor polypeptides |
US6391847B1 (en) * | 1995-01-17 | 2002-05-21 | The Salk Institute For Biological Studies | Method, polypeptides, nucleotide sequence of XOR-6, a vitamin D-like receptor from xenopus |
AU5426596A (en) * | 1995-05-16 | 1996-11-29 | Salk Institute For Biological Studies, The | Modulators for new members of the steroid/thyroid superfamily of receptors |
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NZ504025A (en) | 2003-04-29 |
KR20010031120A (ko) | 2001-04-16 |
CN1134452C (zh) | 2004-01-14 |
AU732079B2 (en) | 2001-04-12 |
JP2001519441A (ja) | 2001-10-23 |
CA2306453A1 (en) | 1999-04-22 |
AU9013198A (en) | 1999-05-03 |
SE9801148D0 (sv) | 1998-03-31 |
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