CN113474013B - 肌肉生长抑制素剪接变体衍生蛋白对肌肉生长抑制素信号的抑制及其利用 - Google Patents
肌肉生长抑制素剪接变体衍生蛋白对肌肉生长抑制素信号的抑制及其利用 Download PDFInfo
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- CN113474013B CN113474013B CN202080018143.9A CN202080018143A CN113474013B CN 113474013 B CN113474013 B CN 113474013B CN 202080018143 A CN202080018143 A CN 202080018143A CN 113474013 B CN113474013 B CN 113474013B
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Abstract
本发明提供抑制肌肉生长抑制素信号的方法。以下的(a)或(b)的蛋白:(a)蛋白,该蛋白由下述的氨基酸序列构成;(b)蛋白,该蛋白由与下述的氨基酸序列具有至少70%以上的序列同一性的氨基酸序列构成,并且可抑制肌肉生长抑制素信号。
Description
技术领域
本发明涉及肌肉生长抑制素(myostatin)剪接变体衍生蛋白对肌肉生长抑制素信号的抑制及其利用。
背景技术
肌肉生长抑制素在细胞内以前体的形式产生,被蛋白酶切割而转化为成熟肌肉生长抑制素。该成熟肌肉生长抑制素若与细胞表层的受体结合,则Smad2/3被磷酸化,磷酸化Smad2/3转移至核内。磷酸化Smad2/3与存在于靶基因的启动子内的Smad结合元件结合,从而诱导靶基因的表达(肌肉生长抑制素信号)。肌肉生长抑制素信号的激活会诱导基因表达,被诱导的因子对肌肉形成起抑制作用。相对于此,肌肉生长抑制素信号的抑制会促进肌肉形成。促进肌肉形成可用于治疗肌肉萎缩症等肌肉萎缩性疾病,因此认为肌肉生长抑制素信号的抑制可成为针对肌肉萎缩的治疗方法(非专利文献1)。另外,通过降低肌肉生长抑制素的表达量,癌细胞的生长被抑制(非专利文献2)。这暗示了:在癌症治疗中肌肉生长抑制素量的降低以及肌肉生长抑制素信号的抑制是有效的。而且,2型糖尿病患者的肌肉生长抑制素表达量增加,由此认为:肌肉生长抑制素与糖尿病之间存在某种关系(非专利文献3)。由这些情况认为:抑制肌肉生长抑制素信号对抑制糖尿病或抑制其进展有效。
作为肌肉生长抑制素信号的抑制方法,正在验证与肌肉生长抑制素结合的肽的利用。该肽来自肌肉生长抑制素自身所具有的序列,实际上能够抑制肌肉生长抑制素信号。然而,在对生物体内给药时存在稳定性低的问题(非专利文献4、专利文献1)。
另外,还公开了利用羊(sheep,绵羊)的肌肉生长抑制素剪接变体抑制肌肉生长抑制素信号的方法(非专利文献5、专利文献2)。然而,在人体内还未发现与该羊中发现的肌肉生长抑制素剪接变体同样的物质。
现有技术文献
非专利文献
非专利文献1:Bogdanovich等人,Nature,2002,420;418-421;
非专利文献2:Han等人,RedoxBiol.2018,19;412-4128;
非专利文献3:Palsgaard等人.2009,4;e6575;
非专利文献4:Ohsawa等人,PlosOne,2015,10;e0133713;
非专利文献5:Jeanplong等人,PlosOne,2013,8;e81713;
专利文献
专利文献1:WO2014/119753A1;
专利文献2:WO2006/036074A1。
发明内容
发明所要解决的课题
本发明的目的在于:提供抑制肌肉生长抑制素信号的方法。
用于解决课题的手段
本发明人刻苦努力的结果,发现了由肌肉生长抑制素剪接变体(由于剪接方式(splicing pattern)的变化而产生的多样化变体)翻译的蛋白抑制肌肉生长抑制素信号。由肌肉生长抑制素剪接变体的mRNA翻译的蛋白虽不具有成熟肌肉生长抑制素所需的活性区,但保持前域的90%以上(图3)。通过使肌肉生长抑制素变体在培养细胞中过度表达,可抑制肌肉生长抑制素信号(图6)。由于由肌肉生长抑制素剪接变体翻译的蛋白是在生物体内天然产生的蛋白,所以认为其稳定性好于外源性蛋白。通过抑制肌肉生长抑制素信号,可通过促进肌肉形成来治疗肌肉萎缩性疾病。另外,可通过抑制肌肉生长抑制素信号来抑制癌细胞生长抑制。还暗示了肌肉生长抑制素增加所伴随的肌肉生长抑制素信号的激活可能与糖尿病有关,由此认为:抑制肌肉生长抑制素信号对预防糖尿病、或抑制其进展有效。因此,该蛋白以及蛋白表达***可用于肌肉生长抑制素相关疾病的治疗方法。
本发明的宗旨如下。
(1)以下的(a)或(b)的蛋白:
(a)蛋白,该蛋白由SEQ ID NO:1的氨基酸序列构成;
(b)蛋白,该蛋白由与SEQ ID NO:1的氨基酸序列具有至少70%以上的序列同一性的氨基酸序列构成,并且可抑制肌肉生长抑制素信号。
(2)多核苷酸,该多核苷酸包含编码(1)所述的蛋白的核苷酸序列或与其互补的序列。
(3)载体,该载体包含(2)所述的多核苷酸。
(4)细胞,该细胞包含(3)所述的载体。
(5)制作以下的(a)或(b)的蛋白的方法,该方法包括:培养(4)所述的细胞,
(a)蛋白,该蛋白由SEQ ID NO:1的氨基酸序列构成;
(b)蛋白,该蛋白由与SEQ ID NO:1的氨基酸序列具有至少70%以上的序列同一性的氨基酸序列构成,并且可抑制肌肉生长抑制素信号;
(6)用于抑制肌肉生长抑制素信号的组合物,该组合物包含:选自(1)所述的蛋白、(2)所述的多核苷酸、(3)所述的载体和(4)所述的细胞的至少一种。
(7)用于促进肌肉形成的组合物,该组合物包含:选自(1)所述的蛋白、(2)所述的多核苷酸、(3)所述的载体和(4)所述的细胞的至少一种。
(8)用于预防和/或治疗肌肉生长抑制素相关疾病的组合物,该组合物包含:选自(1)所述的蛋白、(2)所述的多核苷酸、(3)所述的载体和(4)所述的细胞的至少一种。
(9)药物,该药物包含:选自(1)所述的蛋白、(2)所述的多核苷酸、(3)所述的载体和(4)所述的细胞的至少一种。
(11)饲料,该饲料包含:选自(1)所述的蛋白、(2)所述的多核苷酸、(3)所述的载体和(4)所述的细胞的至少一种。
(12)预防和/或治疗肌肉生长抑制素相关疾病的方法,该方法包括:以药学上的有效量对受试者给予选自(1)所述的蛋白、(2)所述的多核苷酸、(3)所述的载体和(4)所述的细胞的至少一种。
(13)用于在预防和/或治疗肌肉生长抑制素相关疾病的方法中使用的、选自(1)所述的蛋白、(2)所述的多核苷酸、(3)所述的载体和(4)所述的细胞的至少一种。
发明效果
可利用由肌肉生长抑制素剪接变体翻译的蛋白来抑制肌肉生长抑制素信号。
本说明书包含作为本申请的优先权基础的日本专利申请、日本特愿2019-37915的说明书和/或附图中记载的内容。
附图说明
[图1]肌肉生长抑制素V的PCR扩增例。
显示人横纹肌肉瘤细胞的肌肉生长抑制素(MSTN)基因产物的PCR扩增结果。通过扩增得到了2个扩增产物(MSTN、MSTN-V)(左)。分析各产物的核苷酸序列,将结果得到的外显子的结构作成示意图(右)。在MSTN-V中,MSTN的碱基编号第881位~第1843号的碱基缺失。显示各产物的外显子2与外显子3的结合部的序列和第1844位碱基部分的核苷酸序列的一部分(右下)。
[图2]肌肉生长抑制素基因的剪接。
MSTN是通过剪接从肌肉生长抑制素基因的pre-mRNA中去除了内含子1和2,由外显子1、外显子2、外显子3构成(实线)。内含子2是在5’和3’的两端具有GT和AG的序列的最一般的内含子。另一方面,在MSTN-V中,内含子2的剪接方式不同,作为潜在性剪接受***点的外显子3内的TG被激活而形成GT-TG内含子(虚线)。其结果,在肌肉生长抑制素V中,外显子3的第881位~第1843位的963个碱基缺失。
[图3]肌肉生长抑制素V蛋白。
将肌肉生长抑制素、肌肉生长抑制素V的蛋白结构作成示意图。肌肉生长抑制素从N末端起由信号肽(第1位~第18位)、前域(第19位~第266位)、成熟肌肉生长抑制素(第267位~第375位)构成。在肌肉生长抑制素和肌肉生长抑制素V的核酸序列中,外显子1、外显子2相同,到氨基酸249为止均共通。在肌肉生长抑制素V中,由于外显子3的核苷酸序列不同,所以从N末端起第250位的氨基酸成为天冬酰胺(N)、第251位成为缬氨酸(V)、第252位成为终止密码子(*)(SEQ ID NO:1)。因此,肌肉生长抑制素V不具有成熟肌肉生长抑制素的结构域。
[图4]肌肉生长抑制素信号。
肌肉生长抑制素通过使前体产生的成熟肌肉生长抑制素与细胞表层的受体结合而激活下游的信号传递。若成熟肌肉生长抑制素与受体结合,则Smad2/3被磷酸化,磷酸化Smad2/3转移至核内。磷酸化Smad2/3与存在于靶基因的启动子中的Smad结合元件(bindingelement)结合,从而诱导靶基因的表达。
[图5]肌肉生长抑制素V的表达载体和表达蛋白。
在对SEQ ID NO:2进行密码子使用频率最优化而得的序列(SEQ ID NO:3)的5’侧添加Nhe I识别序列(GCTTGC)、在3’侧添加BamH I识别序列(GGATCC),将所得的人工合成核酸***到pcDNATM3.1(+)载体的Nhe I、BamH I识别位点,制作了MSTN-V表达载体(左)。将所制作的载体导入到肌细胞中,对表达的蛋白进行蛋白质印迹分析。在由导入了MSTN-V表达载体的细胞提取的样品中,在分子量35kDa附近检测到特异性的谱带(箭头),确认了肌肉生长抑制素V的表达(右)。左起依次显示分子量标志、模拟品(Mock)(来自导入了空载体的细胞的样品)、来自导入了MSTN-V表达载体的细胞的样品。
[图6]肌肉生长抑制素V对肌肉生长抑制素信号的抑制。
显示肌肉生长抑制素V和肌肉生长抑制素对体外肌肉生长抑制素转录活性测定***的作用的示意图(左)。在肌肉生长抑制素信号分析中,将空载体、MSTN-N表达载体、MSTN-V表达载体分别导入到肌细胞中,根据由Smad2/3诱导表达的荧光素酶的活性进行评价。
以来自导入了空载体的细胞的提取液的测定结果为1,用相对值表示荧光素酶活性(中央、右)。在人横纹肌肉瘤细胞、人骨骼肌成肌细胞中,若使肌肉生长抑制素表达,则均可见荧光素酶活性的升高。另一方面,在使肌肉生长抑制素V表达的情况下,可见荧光素酶活性的降低,暗示了肌肉生长抑制素V抑制肌肉生长抑制素信号传递。
具体实施方式
以下,更详细地对本发明的实施方式进行说明。
本发明提供以下的(a)或(b)的蛋白:
(a)蛋白,该蛋白由SEQ ID NO:1的氨基酸序列构成;
(b)蛋白,该蛋白由与SEQ ID NO:1的氨基酸序列具有至少70%以上的序列同一性的氨基酸序列构成,并且可抑制肌肉生长抑制素信号。
(a)的蛋白是由SEQ ID NO:1的氨基酸序列构成的蛋白,是由人的肌肉生长抑制素的剪接变体翻译的蛋白。如图3所示,肌肉生长抑制素(375aa、43kDa)由信号肽(1-18)、前域(19-266)和成熟肌肉生长抑制素(267-375)的区域构成,而肌肉生长抑制素变体由信号肽(1-18)和一部分前域(19-251)构成,C末端氨基酸(251)由脯氨酸取代成缬氨酸。因此,在肌肉生长抑制素变体中没有形成成熟肌肉生长抑制素。
(a)的蛋白可抑制肌肉生长抑制素信号(图4)。
(a)的蛋白可如下制造:由人横纹肌肉瘤细胞(CRL-2061、ATCC)提取RNA,使用逆转录酶和随机引物合成cDNA,通过PCR进行扩增,之后进行序列分析来确定序列,然后在将可读框的密码子使用频率最优化而得的序列的5’侧和3’侧添加限制酶识别序列,之后***到适当的载体中,再导入到适当的宿主细胞中,以重组蛋白的形式生产,从而可制造(a)的蛋白。
(b)的蛋白是由与SEQ ID NO:1的氨基酸序列具有至少70%以上的序列同一性的氨基酸序列构成、并且可抑制肌肉生长抑制素信号的蛋白。如后述的实施例中所述,若通过肌肉生长抑制素将信号传递激活,则转录因子(Smad蛋白)与Smad结合序列结合而诱导转录,因此利用该现象,将在Smad结合序列的下游配置有荧光素酶基因的报道基因和表达(b)的蛋白的载体一同导入到细胞中,测定荧光素酶的发光,从而可评价是否有肌肉生长抑制素信号的抑制。
(b)的蛋白与(a)的蛋白的氨基酸序列的序列同一性至少为70以上,依次更优选为80%以上、90%以上、95%以上、98%以上。(b)的蛋白可以是由在SEQ ID NO:1的氨基酸序列中有1个或多个(2~76个中的任一个、依次优选为2个、3个、4个、5个、6个、7个、8个、9个、10个、20个、30个、40个、50个、60个、70个、75个、76个。)氨基酸缺失、取代或添加而得的氨基酸序列构成、并且可抑制肌肉生长抑制素信号的蛋白。
(b)的蛋白可利用位点特异性诱变法将(a)的蛋白中的任意的氨基酸用其他的氨基酸取代来制作。
本发明提供多核苷酸,该多核苷酸包含:编码(a)或(b)的蛋白的核苷酸序列或与其互补的序列。
本发明的多核苷酸可以是单链也可以是双链。在双链的情况下,由包含编码(a)或(b)的蛋白的核苷酸序列的多核苷酸及其互补链构成。
多核苷酸可以是DNA、RNA、DNA与RNA的嵌合中的任一种,构成多核苷酸的核苷酸可被修饰。作为修饰核苷酸,可示例:糖被修饰的核苷酸(例如,D-呋喃核糖被2’-O-烷基化而得的核苷酸、D-呋喃核糖被2’-O,4’-C-亚烷基化而得的核苷酸)、磷酸二酯键被修饰(例如,硫代化)而得的核苷酸、碱基被修饰而得的核苷酸、将它们组合而得的核苷酸等。
作为编码(a)的蛋白的核苷酸序列的一例,可列举:SEQ ID NO:2的核苷酸序列。SEQ ID NO:2的核苷酸序列是由人横纹肌肉瘤细胞(CRL-2061、ATCC)提取的肌肉生长抑制素变体的mRNA的序列。SEQ ID NO:2的核苷酸序列中包含5’非翻译区、可读框(起始密码子(atg)与终止密码子(tga)之间的序列)、3’非翻译区、poly(A)。编码(a)的蛋白的核苷酸序列可以是SEQ ID NO:2的核苷酸序列中的起始密码子(atg)与终止密码子(tga)之间的序列或包含该序列的序列。对可读框的核苷酸序列可进行密码子使用频率的最优化,作为其一例,将SEQ ID NO:2中的可读框的密码子使用频率最优化而得的核苷酸序列见SEQ ID NO:3。
包含编码(a)的蛋白的核苷酸序列的多核苷酸例如可通过后述的实施例1中所记载的方法进行调制。
包含与编码(a)的蛋白的核苷酸序列互补的序列的多核苷酸可使用逆转录酶和寡dT引物由包含编码(a)的蛋白的核苷酸序列的肌肉生长抑制素变体的mRNA(在3’末端具有聚(A)链)来合成。可通过碱处理降解mRNA,之后以产生的单链DNA为模板,通过逆转录酶或DNA聚合酶形成双链。
编码(b)的蛋白的核苷酸序列和与其互补的序列例如可通过位点特异性诱变法向编码(a)的蛋白的核苷酸序列和与其互补的序列中导入碱基取代突变而获得。
将编码(a)或(b)的蛋白的DNA***到载体中制作重组载体,将其导入到宿主细胞中进行转化,培养该转化细胞,使其生产(a)或(b)的蛋白,从而可制作(a)或(b)的蛋白。因此,本发明提供制作(a)或(b)的蛋白的方法,该方法包括:培养包含载体的细胞,该载体包含多核苷酸,该多核苷酸包含编码(a)或(b)的蛋白的核苷酸序列或与其互补的序列。本发明还提供包含多核苷酸的载体(重组载体),该多核苷酸包含编码(a)或(b)的蛋白的核苷酸序列或与其互补的序列。另外,本发明还提供包含载体的细胞,该载体包含多核苷酸,该多核苷酸包含编码(a)或(b)的蛋白的核苷酸序列或与其互补的序列。
本发明的重组载体可通过将包含编码(a)或(b)的蛋白的核苷酸序列和与其互补的序列的多核苷酸***到适当的载体中而获得。
作为载体,可使用来自大肠杆菌的质粒(例如,pBR322、pBR325、pUC12、pUC13、pUC19、pET-44、pBlueScriptII)、来自枯草杆菌的质粒(例如,YEp13、pYES2、YRp7、YIp5、pYAC2、pUB110、pTP5、pC194)、来自酵母的质粒(例如,pSH19、pSH15)、λ噬菌体(λphage)等噬菌体(bacteriophages)、逆转录病毒、腺病毒、慢病毒、腺相关病毒、痘苗病毒等动物病毒、杆状病毒等昆虫病原病毒等。
在表达载体中可添加启动子、增强子、终止子、剪接信号、添加了Poly A的信号、选择标志、SV40复制起点等。
另外,表达载体可以是融合蛋白表达载体。各种融合蛋白表达载体有市售,可示例:pGEX系列(GE Healthcare公司)、Novagen’s(注册商标)pET Systems(Merck公司)、Clontech荧光蛋白载体系列(Takara公司)、用于添加His6、HaloTag的表达载体(Promega公司)、FLAG标签融合蛋白表达***(Sigma-Aldrich公司)、pCruzTM哺乳动物细胞用表达载体系列(Santa Cruz Biotechnology公司)等。
通过将本发明的重组载体导入到宿主细胞中,可得到转化细胞。本发明还提供导入了重组载体的细胞(宿主细胞)。
作为宿主,可示例:细菌细胞(例如,埃希氏菌属菌、芽孢杆菌属菌、枯草杆菌等)、真菌细胞(例如,酵母、曲霉等)、昆虫细胞(例如,S2细胞、Sf细胞等)、动物细胞(例如,CHO细胞、COS细胞、HeLa细胞、C127细胞、3T3细胞、BHK细胞、HEK293细胞等)、植物细胞等。
将重组载体导入到宿主中可通过Molecular Cloning第2版,J.Sambrook等人,Cold Spring Harbor Lab.Press,1989中记载的方法(例如,磷酸钙法、DEAE-葡聚糖法、转染法、显微注射法、脂质转染法、电穿孔法、转导法、scrape-loading法、鸟枪法等)或感染来进行。
可将转化细胞在培养基中进行培养,由培养物采集(a)或(b)的蛋白。在(a)或(b)的蛋白被分泌到培养基中的情况下,只要回收培养基,由该培养基分离(a)或(b)的蛋白并进行纯化即可。在(a)或(b)的蛋白于所转化的细胞内产生的情况下,只要溶解该细胞,由该溶解物分离(a)或(b)的蛋白并进行纯化即可。
在(a)或(b)的蛋白以与其他蛋白(作为标签起作用)的融合蛋白的形式表达的情况下,可在分离和纯化融合蛋白后进行因子Xa或酶(肠激酶)处理,从而切割其他蛋白,得到目标的(a)或(b)的蛋白。
(a)或(b)的蛋白的分离和纯化可通过已知的方法进行。作为已知的分离、纯化方法,可采用:盐析或溶剂沉淀法等利用溶解度的方法、透析法、超滤法、凝胶过滤法、以及SDS-聚丙烯酰胺凝胶电泳法等利用分子量之差的方法、离子交换层析等利用电荷之差的方法、亲和层析等利用特异的亲和性的方法、反相高效液相色谱法等利用疏水性之差的方法、等电点电泳法等利用等电点之差的方法等。
通过使用本发明的蛋白、包含编码本发明的蛋白的核苷酸序列或与其互补的序列的多核苷酸、包含本发明的核苷酸和/或本发明的细胞的载体抑制肌肉生长抑制素信号,可促进肌肉形成。因此,本发明提供用于抑制肌肉生长抑制素信号的组合物,该组合物包含:选自(a)和/或(b)的蛋白、包含编码该蛋白的核苷酸序列或与其互补的序列的多核苷酸、包含该多核苷酸的载体以及包含该载体的细胞的至少一种。另外,本发明还提供用于促进肌肉形成的组合物,该组合物包含:选自(a)和/或(b)的蛋白、包含编码该蛋白的核苷酸序列或与其互补的序列的多核苷酸、包含该多核苷酸的载体以及包含该载体的细胞的至少一种。在载体中含有包含编码(a)和/或(b)的蛋白的核苷酸序列或与其互补的序列的多核苷酸的情况下,载体只要是可将包含编码(a)和/或(b)的蛋白的核苷酸序列或与其互补的序列的多核苷酸导入到细胞中的载体即可,例如可列举:腺病毒、逆转录病毒、慢病毒、腺相关病毒、仙台病毒、脂质体、质粒等基因治疗用载体。另外,还可将本发明的多核苷酸或载体导入到细胞(自身、同种)中而得的产物用于细胞治疗。向载体中导入目标基因的方法、向细胞中导入重组载体的方法、对人给予导入了重组载体或基因的细胞的方法或给予部位均已知,也可直接或修饰后适用于本发明。在基因治疗或细胞治疗中可利用基因组编辑技术。在基因组编辑中可使用ZFN(zinc-finger nuclease:锌指核酸酶)、TALEN(transcriptionactivator-like effector nuclease:转录激活因子样效应物核酸酶)、CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associatedprotein 9:成簇的规律间隔的短回文重复序列/CRISPR-相关蛋白9)等人工核酸酶。
本发明的组合物可用于药物、实验用试剂、饲料等。
作为药物,可用于肌肉生长抑制素相关疾病(肌肉生长抑制素的相关可以是直接的也可以是间接的)、具体而言是肌肉萎缩性疾病(例如,肌肉萎缩症、脊髄性肌肉萎缩症、肌肉减少症、废用性肌肉萎缩)、循环***疾病(例如,心功能不全、动脉硬化等)、肾病(例如,慢性肾功能不全等)、骨病(例如,炎症性关节炎等)、癌症或糖尿病的预防和/或治疗。肌肉生长抑制素相关疾病可以是肌肉生长抑制素量的降低或肌肉生长抑制素信号的抑制有效的疾病。本发明提供预防和/或治疗肌肉生长抑制素相关疾病的方法,该方法包括:以药学上的有效量对受试者给予选自(a)和/或(b)的蛋白、包含编码该蛋白的核苷酸序列或与其互补的序列的多核苷酸、包含该多核苷酸的载体以及包含该载体的细胞的至少一种。另外,本发明还提供:用于在预防和/或治疗肌肉生长抑制素相关疾病的方法中使用的、选自(a)和/或(b)的蛋白、包含编码该蛋白的核苷酸序列或与其互补的序列的多核苷酸、包含该多核苷酸的载体以及包含该载体的细胞的至少一种。
由于肌肉生长抑制素的抑制还会带来骨骼肌量的增加,所以不管肌肉萎缩的原因如何,可用于所有的呈肌肉萎缩的疾病的治疗。骨骼肌量的增加可谋求运动量的增加,也有助于全身代谢的改善。另外,还可期待对心肌起作用以恢复其功能。
另一方面,肌肉生长抑制素抑制还被期待:作用于破骨细胞而抑制骨破坏、激活血管内皮细胞的稳态维持能力、诱导细胞凋亡、提高胰岛素的敏感性等。
选自(a)和/或(b)的蛋白、包含编码该蛋白的核苷酸序列或与其互补的序列的多核苷酸、包含该多核苷酸的载体以及包含该载体的细胞的至少一种(以下,记作“有效成分(活性成分)”。)可单独、或者与药理学上可接受的载体、稀释剂或赋形剂一起,以适当的剂型的药物组合物的形式对哺乳动物(例如,人、兔、狗、猫、大鼠、小鼠)进行口服或胃肠外给药。给药量还根据给药对象、对象疾病、症状、给药途径等而不同,例如,在用于预防/治疗肌肉萎缩性疾病(例如,肌肉萎缩症)的情况下,以有效成分的1次剂量计,在有效成分为蛋白的情况下,通常为0.1μg~100mg/kg体重左右、优选0.5mg~100mg体重左右,在有效成分为多核苷酸的情况下,通常为0.1~50mg/kg体重左右、优选0.5mg/kg体重左右,在有效成分为包含多核苷酸的载体的情况下,通常为1×1014~9×1014基因组拷贝/kg体重左右、优选1×1012基因组拷贝/kg体重左右,以每周1次~每月1次左右或每年1次左右的频率、优选每年1次左右的频率,通过口服/肌肉内注射/皮下注射/静脉注射进行给药(优选连续或隔日给药)即可。在有效成分为包含载体的细胞的情况下,以有效成分的1次剂量计为10,000-100,000个细胞,以每周1次~每月1次左右或每年1次左右的频率、优选每年1次左右的频率,通过肌肉内注射/皮下注射/静脉注射、优选通过静脉注射进行给药即可。
在其他的胃肠外给药和口服给药的情况下,也可给予基于此的量。在症状特别严重的情况下,根据其症状可增量。
作为用于口服给药的组合物,可列举:固体或液体的剂型、具体而言是片剂(包括糖衣片、薄膜包衣片)、丸剂、颗粒剂、散剂、胶囊剂(包括软胶囊剂)、糖浆剂、乳剂、悬浮剂等。这样的组合物可通过常规方法来制造,可含有制剂领域通常使用的载体、稀释剂或赋形剂。例如,作为片剂用载体、赋形剂,可列举:乳糖、淀粉、蔗糖、硬脂酸镁等。
作为用于胃肠外给药的组合物,例如可列举:注射剂、栓剂等,注射剂可以是静脉注射剂、皮下注射剂、皮内注射剂、肌肉注射剂、点滴注射剂等剂型。这样的注射剂通过常规方法、即将有效成分溶解、悬浮或乳化于通常用于注射剂的无菌的水性或油性液中来调制。作为注射用水性液,可列举:生理盐水、含有葡萄糖或其他辅药的等渗液等,可并用适当的助溶剂、例如醇(例如,乙醇)、多元醇(例如,丙二醇、聚乙二醇)、非离子表面活性剂(例如,聚山梨酯80、HCO-50(polyoxyethylene(50mol)adduct of hydrogenated castor oil,氢化蓖麻油的聚氧乙烯(50mol)加成物))等。作为油性液,可列举:芝麻油、大豆油等,可并用苯甲酸苄酯、苄醇等作为助溶剂。所调制的注射液通常填充在适当的安瓿中。用于直肠给药的栓剂可通过将有效成分混合于通常的栓剂用基质中来调制。
上述的口服用或胃肠外用药物组合物可调制成适合于有效成分的给药量这样的给药单位(剂量单位)的剂型。作为这样的给药单位的剂型,可列举:片剂、丸剂、胶囊剂、注射剂(安瓿)、栓剂等,各自的每给药单位剂型通常优选含有0.1~1.000mg的有效成分。
作为饲料,可用于促进动物的肌肉形成。动物只要是表达肌肉生长抑制素的动物即可,可示例:猫、狗、羊、猪、牛、鸡、火鸡等哺乳动物;鲑鱼、鳟鱼、鳕鱼、金枪鱼、黄尾鱼等鱼类等役用、食用的饲养动物。
在本发明的饲料中可添加蛋白质、脂质、碳水化合物、钠等一般成分、钾、钙、镁、磷等矿物质类、铁、锌、铜、硒、铬等微量元素、维生素A、β-胡萝卜素、维生素B1、维生素B2、维生素B6、维生素B12、维生素C、烟酸、叶酸、维生素D3、维生素E、生物素、泛酸等维生素类、辅酶Q10、α-硫辛酸、半乳寡糖、食物纤维、赋形剂(水、羧甲基纤维素、乳糖等)、甜味剂、矫味剂(苹果酸、枸橼酸、氨基酸等)、香料等。在将本发明的饲料制成液体制剂的情况下,可使用水、生理盐水、汤、牛乳、果汁等作为分散或溶解食品或饲料成分的液体。本发明的饲料可制成粉末、颗粒、片剂、液体制剂等形状。
饲料的摄取可在确认所期望的效果这样的摄取量和频率、摄取期间进行。
实施例
以下,根据实施例详细地对本发明进行说明,但本发明并不受这些实施例的限定。
[实施例1]肌肉生长抑制素变体的克隆和鉴定
在RNaseOUTTM重组核糖核酸酶抑制剂(#10777-019,Thermo Fisher Scientific)的存在下,使用随机引物(#48190011、Thermo Fisher Scientific),通过M-MLV逆转录酶(#28025013,Thermo Fisher Scientific)将使用高纯度RNA分离试剂盒(#11828665001、Roche Life Science)由人横纹肌肉瘤细胞(CRL-2061,ATCC)提取的500ng RNA逆转录成cDNA。使用引物MSTN Ex1_F1;5’-agattcactggtgtggcaag-3’(SEQ ID NO:6)、MSTN R2;5’-tgcatgacatgtctttgtgc-3’(SEQ ID NO:7)、TaKaRa Ex Taq(注册商标)DNA聚合酶(#RR001A,Takara)对所得的cDNA进行PCR。对PCR产物进行琼脂糖凝胶电泳。通过电泳(泳動)在DNA尺寸标志的2kbp与3kbp的中间、1kbp与2kbp的中间分别得到了扩增谱带,作为2.5kbp和1.5kbp的PCR片段(图1左)。使用MinElute(注册商标)凝胶提取试剂盒(#28604、Qiagen)由各片段区域提取DNA,将提取片段通过DNA连接试剂盒2.1版(#6022、Takara)亚克隆到pT7Blue(#69820、Novagen)中。通过使用了引物MSTN Ex1_F1、MSTN R2、TaKaRa Ex Taq(注册商标)DNA聚合酶(#RR001A、Takara)的PCR扩增已亚克隆的序列,用MinElute(注册商标)PCR纯化试剂盒(#28006、QIAGEN)纯化后,通过桑格法(Sanger method)进行测序分析。
测序的结果,约2.5kbp的PCR片段是由肌肉生长抑制素(MSTN)基因的全部的外显子1(Ex1)、外显子2(Ex2)、外显子3(Ex3)构成的正常剪接产物(MSTN)。该MSTN的示意图见图1右,外显子2与3的结合部的序列和外显子内的第1844位碱基部分的序列的各自的一部分见图1右下。另一方面,在作为肌肉生长抑制素变体(MSTN-V)的约1.5kbp的片段的核苷酸序列(SEQ ID NO:2)中,截止到肌肉生长抑制素基因的外显子1、外显子2为止与MSTN完全相同。然而,在相当于外显子3的序列中与MSTN完全不同。以aat ccg ttt开始的序列成为以aat gtc tga开始的序列。该aat gtc tga以下的序列与MSTN的外显子3内的第1844位碱基下游的序列完全匹配。这显示了外显子3的与核酸序列第881位~第1843位相当的区域缺失。
MSTN的内含子2是在5’和3’的两端具有GT和AG的序列的最一般的内含子。另一方面,在MSTN-V中内含子2的剪接方式不同,作为潜在性剪接受***点的外显子3内的TG被激活,形成GT-TG内含子。其结果,在肌肉生长抑制素V中,外显子3的第881位~第1843位的963个碱基缺失(图2)。
已鉴定的MSTN-V mRNA具有起始密码子和终止密码子,暗示了被翻译成251个氨基酸的蛋白。肌肉生长抑制素V的从N末端侧到第250位的氨基酸是与肌肉生长抑制素同一的序列,但第251位是缬氨酸、第252位是终止密码子。由于成熟肌肉生长抑制素由肌肉生长抑制素的第267位~第375位的氨基酸构成,所以不会由MSTN-V mRNA产生成熟肌肉生长抑制素(SEQ ID NO:1、图3)。
肌肉生长抑制素经由肌肉生长抑制素信号发挥作用。即,由前体产生的成熟肌肉生长抑制素若与存在于细胞膜的受体结合,则Smad2/3被磷酸化。被磷酸化的Smad2/3转移到核内,与位于靶基因的上游的Smad结合元件结合,使基因的表达亢进(图4)。肌肉生长抑制素前体的氨基酸序列见SEQ ID NO:5、mRNA的序列见SEQ ID NO:4。
[实施例2]培养细胞中的肌肉生长抑制素变体的表达
为了在培养细胞中表达肌肉生长抑制素V,制作了MSTN-V表达载体。在株式会社Fasmac人工合成将SEQ ID NO:2的可读框的密码子使用频率最优化(SEQ ID NO:3)、并在5’侧添加Nhe I识别序列(GCTTGC)、在3’侧添加BamH I识别序列(GGATCC)而得的核酸,之后***到cDNATM3.1(+)载体(#V79020、Thermo Fisher Scientific)的Nhe I、BamH I位点(图5左)。
来自MSTN-V表达载体的蛋白表达通过蛋白质印迹进行确认。使用Lipofectamin(注册商标)2000(11668019、Thermo Fisher Scientific)向人横纹肌肉瘤细胞(CRL-2061、ATCC)中导入MSTN-V表达载体和比较对象的空载体(pcDNATM3.1(+))。在载体导入的24小时后使用细胞裂解缓冲液(#9803、Cell Signaling)(添加1mM PMSF(#8553、CellSignaling))破碎细胞,得到可溶性组分作为样品。所得样品的蛋白定量使用Qubit(注册商标)蛋白测定试剂盒(#Q33211、Thermo Fisher Scientific)来进行。SDS-PAGE用样品是将样品与4×Laemmli样品缓冲液(#1610747、Bio-Rad)(添加2-巯基乙醇(#1610710、Bio-Rad))混合后通过热处理而调制的。在SDS-PAGE中,使用Mini-PROTEAN(注册商标)TGXTMPrecast Gels 4-20%Gel(#456-1094、BIO-RAD)进行电泳。作为分子量标志,将PrecisionPlus ProteinTM双色标准品(#1610374、BIO-RAD)进行电泳。向膜上的转录使用Trans-BlotTurboTM转录***(Bio-Rad)。将蛋白转录后的膜在室温下用2%ECLTM Prime封闭剂(#RPN418、Amersham)封闭1小时后,使用作为一次抗体的识别肌肉生长抑制素的N末侧的抗体(抗-GDF8/肌肉生长抑制素抗体、#ab71808、abcam)或识别肌动蛋白的抗体(β-肌动蛋白抗体(C4)、#sc-47778、Santa Cruz Biotechnology)在4℃下处理一夜。二次抗体使用HRP标记抗兔IgG抗体(#NA934,GE)、HRP标记抗小鼠IgG抗体(#NA931,GE),在室温下处理1小时。检测使用AmershamTM ECL SelectTM蛋白质印迹测定试剂(#RPN2235、GE),通过ChemiDocTM XRS+***(Bio-Rad)来进行。其结果,在35kDa附近检测到肌肉生长抑制素V的谱带(图5右)。同时还对肌动蛋白进行分析,得到了谱带。
[实施例3]肌肉生长抑制素变体对肌肉生长抑制素信号的抑制利用体外肌肉生长抑制素转录活性测定***评价了肌肉生长抑制素V对肌肉生长抑制素信号的抑制活性。在该评价***中,将在Smad结合序列的下游配置有荧光素酶基因的报道基因(SBE4-Luc质粒、#16495、Addgene)导入到细胞中,测定所表达诱导的荧光素酶的发光,从而对肌肉生长抑制素信号进行评价(图6左)。除MSTN-V表达载体以外,还使用肌肉生长抑制素(MSTN-N)表达载体进行研究。MSTN-N表达载体是在株式会社Fasmac人工合成在肌肉生长抑制素cDNA(SEQ ID NO:4)的5’侧添加Nhe I识别序列(GCTTGC)、在3’侧添加BamH I识别序列(GGATCC)而得的核酸、并***到pcDNATM3.1(+)载体(V79020、Thermo Fisher Scientific)的Nhe I、BamH I位点而制作的。来自MSTN-N表达载体的肌肉生长抑制素(SEQ ID NO:5)的表达与肌肉生长抑制素V同样地通过蛋白质印迹进行确认。
使用Lipofectamin(注册商标)2000(#11668019、Thermo Fisher Scientific)向人横纹肌肉瘤细胞(CRL-2061、ATCC)、人骨骼肌成肌细胞(Human Skeletal Myoblast)中同时导入2种载体。1种是SBE4-Luc质粒,另一种是MSTN-V表达载体或MSTN-N表达载体或空载体(pcDNATM3.1(+))的组合。在载体导入的24小时后使用带有报道基因裂解缓冲液的荧光素酶测定***(E4030、Promega)的报道基因裂解缓冲液破碎细胞,得到可溶性组分作为样品。所得样品的蛋白定量使用Qubit(注册商标)蛋白测定试剂盒(#Q33211、Thermo FisherScientific)来进行。荧光素酶活性通过以带有报道基因裂解缓冲液的荧光素酶测定***(#E4030、Promega)的荧光素酶测定***为底物,使用多标记读板仪(multi-label platereader)ARVOTM3(PerkinElmer)测定荧光素酶发光信号来进行评价。
以来自导入了空载体的细胞的提取液的测定结果为1,用相对值表示荧光素酶活性(图6中央、右)。在人横纹肌肉瘤细胞、人骨骼肌成肌细胞中,在使肌肉生长抑制素表达的情况下均可见荧光素酶比活性的升高。另一方面,在使肌肉生长抑制素V表达的情况下,可见荧光素酶比活性的下降。本实验***的荧光素酶活性与肌肉生长抑制素信号相关,荧光素酶活性的升高显示肌肉生长抑制素信号的亢进,荧光素酶活性的下降显示肌肉生长抑制素信号的抑制。由此可知:通过肌肉生长抑制素V的表达,肌肉生长抑制素信号被抑制。
本说明书中引用的所有出版物、专利和专利申请均直接作为参考而纳入本说明书。
产业实用性
本发明可用于促进人或动物的肌肉形成。
序列表自由文本
<SEQ ID NO:1>
显示肌肉生长抑制素变体蛋白的氨基酸序列(全部的251个氨基酸)。
<SEQ ID NO:2>
显示肌肉生长抑制素变体的核苷酸序列(全部的1860个碱基。起始密码子(atg)和终止密码子(tga)见方框内)。
<SEQ ID NO:3>
***到表达载体中的肌肉生长抑制素V(MSTN-V)核苷酸序列在5’侧添加Nhe I位点、在3’侧添加BamH I位点,并***到载体中。
(全部的1860个碱基。起始密码子(atg)和终止密码子(tga)见方框内)
<SEQ ID NO:4>
***到表达载体中的肌肉生长抑制素(MSTN-N)核苷酸序列。
在5’侧添加Nhe I位点、在3’侧添加BamH I位点,并***到载体中(全部的2823个碱基。起始密码子(atg)和终止密码子(tga)见方框内)
<SEQ ID NO:5>
肌肉生长抑制素的氨基酸序列信息。
氨基酸序列(全部的375个氨基酸)
<SEQ ID NO:6>
引物MSTN Ex1_F1的序列。5’-agattcactggtgtggcaag-3’<SEQ ID NO:7>
引物MSTN R2的序列。5’-tgcatgacatgtctttgtgc-3’。
<110> 神户天然物化学株式会社
学校法人神户学院
<120> 肌肉生长抑制素剪接变体衍生蛋白对肌肉生长抑制素信号的抑制及其利用
<130> FP-277PCT
<150> JP2019-37915
<151> 2019-03-01
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 251
<212> PRT
<213> Homo sapiens
<400> 1
Met Gln Lys Leu Gln Leu Cys Val Tyr Ile Tyr Leu Phe Met Leu Ile
1 5 10 15
Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn
20 25 30
Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr
35 40 45
Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu
50 55 60
Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Val Ile Arg Gln Leu
65 70 75 80
Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val
85 90 95
Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His
100 105 110
Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu
115 120 125
Met Gln Val Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser
130 135 140
Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu
145 150 155 160
Arg Pro Val Glu Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu
165 170 175
Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu
180 185 190
Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val
195 200 205
Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly
210 215 220
Ile Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr
225 230 235 240
Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Val
245 250
<210> 2
<211> 1860
<212> DNA
<213> Homo sapiens
<400> 2
agattcactg gtgtggcaag ttgtctctca gactgtacat gcattaaaat tttgcttggc 60
attactcaaa agcaaaagaa aagtaaaagg aagaaacaag aacaagaaaa aagattatat 120
tgattttaaa atcatgcaaa aactgcaact ctgtgtttat atttacctgt ttatgctgat 180
tgttgctggt ccagtggatc taaatgagaa cagtgagcaa aaagaaaatg tggaaaaaga 240
ggggctgtgt aatgcatgta cttggagaca aaacactaaa tcttcaagaa tagaagccat 300
taagatacaa atcctcagta aacttcgtct ggaaacagct cctaacatca gcaaagatgt 360
tataagacaa cttttaccca aagctcctcc actccgggaa ctgattgatc agtatgatgt 420
ccagagggat gacagcagcg atggctcttt ggaagatgac gattatcacg ctacaacgga 480
aacaatcatt accatgccta cagagtctga ttttctaatg caagtggatg gaaaacccaa 540
atgttgcttc tttaaattta gctctaaaat acaatacaat aaagtagtaa aggcccaact 600
atggatatat ttgagacccg tcgagactcc tacaacagtg tttgtgcaaa tcctgagact 660
catcaaacct atgaaagacg gtacaaggta tactggaatc cgatctctga aacttgacat 720
gaacccaggc actggtattt ggcagagcat tgatgtgaag acagtgttgc aaaattggct 780
caaacaacct gaatccaact taggcattga aataaaagct ttagatgaga atggtcatga 840
tcttgctgta accttcccag gaccaggaga agatgggctg aatgtctgag gctaccaggt 900
ttatcacata aaaaacattc agtaaaatag taagtttctc ttttcttcag gtgcattttc 960
ctacacctcc aaatgaggaa tggattttct ttaatgtaag aagaatcatt tttctagagg 1020
ttggctttca attctgtagc atacttggag aaactgcatt atcttaaaag gcagtcaaat 1080
ggtgtttgtt tttatcaaaa tgtcaaaata acatacttgg agaagtatgt aattttgtct 1140
ttggaaaatt acaacactgc ctttgcaaca ctgcagtttt tatggtaaaa taatagaaat 1200
gatcgactct atcaatattg tataaaaaga ctgaaacaat gcatttatat aatatgtata 1260
caatattgtt ttgtaaataa gtgtctcctt ttttatttac tttggtatat ttttacacta 1320
aggacatttc aaattaagta ctaaggcaca aagacatgtc atgcatcaca gaaaagcaac 1380
tacttatatt tcagagcaaa ttagcagatt aaatagtggt cttaaaactc catatgttaa 1440
tgattagatg gttatattac aatcatttta tattttttta catgattaac attcacttat 1500
ggattcatga tggctgtata aagtgaattt gaaatttcaa tggtttactg tcattgtgtt 1560
taaatctcaa cgttccatta ttttaatact tgcaaaaaca ttactaagta taccaaaata 1620
attgactcta ttatctgaaa tgaagaataa actgatgcta tctcaacaat aactgttact 1680
tttattttat aatttgataa tgaatatatt tctgcattta tttacttctg ttttgtaaat 1740
tgggattttg ttaatcaaat ttattgtact atgactaaat gaaattattt cttacatcta 1800
atttgtagaa acagtataag ttatattaaa gtgttttcac atttttttga aagacaaaaa 1860
<210> 3
<211> 1860
<212> DNA
<213> 人工序列
<220>
<223> 密码子优化序列
<400> 3
agattcactg gtgtggcaag ttgtctctca gactgtacat gcattaaaat tttgcttggc 60
attactcaaa agcaaaagaa aagtaaaagg aagaaacaag aacaagaaaa aagattatat 120
tgattttaaa atcatgcaga agctccagct ttgcgtgtac atctacctgt tcatgctgat 180
agttgcaggc ccagtggatc tgaatgagaa cagcgaacag aaggagaacg tagagaagga 240
aggcttgtgc aatgcctgta cttggcggca gaatacgaaa tcttcccgta ttgaggccat 300
caagatccag attctcagca aactgcgcct tgaaactgca cctaacatca gcaaggacgt 360
aatcagacag cttctgccca aagctcctcc actgagagag ctcattgacc agtacgacgt 420
ccaacgagat gacagttcag atggctcact tgaggatgac gactatcatg ccactaccga 480
aaccatcatt acaatgccga ccgaaagcga tttcctgatg caagtggatg ggaaaccaaa 540
gtgttgcttc ttcaagtttt cctccaagat ccagtacaac aaagtcgtca aggcgcaact 600
gtggatatat ctgaggcccg ttgagactcc aacaaccgtg tttgtgcaga ttttgaggct 660
gatcaagccc atgaaagacg gaacacgcta taccggaata cggagtctga aactggacat 720
gaatcccggt acagggattt ggcagtctat cgacgtcaaa acggttctcc agaactggct 780
gaaacaaccg gagtctaatc tcgggattga gatcaaggcc ttggacgaaa atggccacga 840
tctggctgtg acctttcctg gtcctggaga agatggcctg aacgtgtgag gctaccaggt 900
ttatcacata aaaaacattc agtaaaatag taagtttctc ttttcttcag gtgcattttc 960
ctacacctcc aaatgaggaa tggattttct ttaatgtaag aagaatcatt tttctagagg 1020
ttggctttca attctgtagc atacttggag aaactgcatt atcttaaaag gcagtcaaat 1080
ggtgtttgtt tttatcaaaa tgtcaaaata acatacttgg agaagtatgt aattttgtct 1140
ttggaaaatt acaacactgc ctttgcaaca ctgcagtttt tatggtaaaa taatagaaat 1200
gatcgactct atcaatattg tataaaaaga ctgaaacaat gcatttatat aatatgtata 1260
caatattgtt ttgtaaataa gtgtctcctt ttttatttac tttggtatat ttttacacta 1320
aggacatttc aaattaagta ctaaggcaca aagacatgtc atgcatcaca gaaaagcaac 1380
tacttatatt tcagagcaaa ttagcagatt aaatagtggt cttaaaactc catatgttaa 1440
tgattagatg gttatattac aatcatttta tattttttta catgattaac attcacttat 1500
ggattcatga tggctgtata aagtgaattt gaaatttcaa tggtttactg tcattgtgtt 1560
taaatctcaa cgttccatta ttttaatact tgcaaaaaca ttactaagta taccaaaata 1620
attgactcta ttatctgaaa tgaagaataa actgatgcta tctcaacaat aactgttact 1680
tttattttat aatttgataa tgaatatatt tctgcattta tttacttctg ttttgtaaat 1740
tgggattttg ttaatcaaat ttattgtact atgactaaat gaaattattt cttacatcta 1800
atttgtagaa acagtataag ttatattaaa gtgttttcac atttttttga aagacaaaaa 1860
<210> 4
<211> 2823
<212> DNA
<213> Homo sapiens
<400> 4
agattcactg gtgtggcaag ttgtctctca gactgtacat gcattaaaat tttgcttggc 60
attactcaaa agcaaaagaa aagtaaaagg aagaaacaag aacaagaaaa aagattatat 120
tgattttaaa atcatgcaaa agttgcagct gtgtgtgtac atctacctgt tcatgctgat 180
tgtcgccggt cctgttgatc tgaacgagaa ctctgagcag aaggagaacg tggagaaaga 240
aggcctgtgc aatgcttgca catggagaca gaataccaag agtagccgga tagaagccat 300
taagatccag atactgagca agctccgctt ggagacagcc cctaacattt ccaaggatgt 360
gatacggcaa cttctgccaa aggcaccacc acttagggaa ctcatcgacc agtacgacgt 420
tcagagggac gatagctccg atggctctct cgaggacgat gattaccacg ctactaccga 480
gactatcatt acaatgccta ctgagagcga ctttctgatg caagtagacg ggaaacccaa 540
gtgctgcttc ttcaaattct cctccaagat tcagtacaat aaggtcgtga aagcccaact 600
ctggatctat ctccgtccgg tggaaactcc tacgaccgta ttcgtccaga ttcttaggct 660
gattaagccc atgaaagatg gaacgcggta taccggcatc agaagtttga aactggacat 720
gaatccaggt accggaatct ggcagagtat cgacgtcaaa actgtgctgc agaattggct 780
gaaacagcct gagtcaaacc tggggatcga gataaaagcg ctggatgaaa atgggcatga 840
tctggctgtc acctttccgg gtcctggcga agatggcctg aatcccttcc tggaagtgaa 900
agtgaccgac acacccaaac gatccagaag ggactttggc ttggattgcg acgaacactc 960
aaccgagtct cgctgttgcc gctatcctct cactgttgac tttgaggcct ttggatggga 1020
ttggatcatt gctcccaagc ggtacaaagc gaactactgt tcaggggaat gcgagtttgt 1080
gttcctccag aagtatccgc atacacacct tgttcatcaa gccaatccaa gagggtctgc 1140
aggaccctgt tgtacaccca cgaagatgag ccccatcaac atgctgtatt tcaacggaaa 1200
ggaacagata atctatggca agattccagc aatggtggta gaccgatgtg gttgcagctg 1260
agatttatat taagcgttca taacttccta aaacatggaa ggttttcccc tcaacaattt 1320
tgaagctgtg aaattaagta ccacaggcta taggcctaga gtatgctaca gtcacttaag 1380
cataagctac agtatgtaaa ctaaaagggg gaatatatgc aatggttggc atttaaccat 1440
ccaaacaaat catacaagaa agttttatga tttccagagt ttttgagcta gaaggagatc 1500
aaattacatt tatgttccta tatattacaa catcggcgag gaaatgaaag cgattctcct 1560
tgagttctga tgaattaaag gagtatgctt taaagtctat ttctttaaag ttttgtttaa 1620
tatttacaga aaaatccaca tacagtattg gtaaaatgca ggattgttat ataccatcat 1680
tcgaatcatc cttaaacact tgaatttata ttgtatggta gtatacttgg taagataaaa 1740
ttccacaaaa atagggatgg tgcagcatat gcaatttcca ttcctattat aattgacaca 1800
gtacattaac aatccatgcc aacggtgcta atacgatagg ctgaatgtct gaggctacca 1860
ggtttatcac ataaaaaaca ttcagtaaaa tagtaagttt ctcttttctt caggtgcatt 1920
ttcctacacc tccaaatgag gaatggattt tctttaatgt aagaagaatc atttttctag 1980
aggttggctt tcaattctgt agcatacttg gagaaactgc attatcttaa aaggcagtca 2040
aatggtgttt gtttttatca aaatgtcaaa ataacatact tggagaagta tgtaattttg 2100
tctttggaaa attacaacac tgcctttgca acactgcagt ttttatggta aaataataga 2160
aatgatcgac tctatcaata ttgtataaaa agactgaaac aatgcattta tataatatgt 2220
atacaatatt gttttgtaaa taagtgtctc cttttttatt tactttggta tatttttaca 2280
ctaaggacat ttcaaattaa gtactaaggc acaaagacat gtcatgcatc acagaaaagc 2340
aactacttat atttcagagc aaattagcag attaaatagt ggtcttaaaa ctccatatgt 2400
taatgattag atggttatat tacaatcatt ttatattttt ttacatgatt aacattcact 2460
tatggattca tgatggctgt ataaagtgaa tttgaaattt caatggttta ctgtcattgt 2520
gtttaaatct caacgttcca ttattttaat acttgcaaaa acattactaa gtataccaaa 2580
ataattgact ctattatctg aaatgaagaa taaactgatg ctatctcaac aataactgtt 2640
acttttattt tataatttga taatgaatat atttctgcat ttatttactt ctgttttgta 2700
aattgggatt ttgttaatca aatttattgt actatgacta aatgaaatta tttcttacat 2760
ctaatttgta gaaacagtat aagttatatt aaagtgtttt cacatttttt tgaaagacaa 2820
aaa 2823
<210> 5
<211> 375
<212> PRT
<213> Homo sapiens
<400> 5
Met Gln Lys Leu Gln Leu Cys Val Tyr Ile Tyr Leu Phe Met Leu Ile
1 5 10 15
Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn
20 25 30
Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr
35 40 45
Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu
50 55 60
Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Val Ile Arg Gln Leu
65 70 75 80
Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val
85 90 95
Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His
100 105 110
Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu
115 120 125
Met Gln Val Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser
130 135 140
Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu
145 150 155 160
Arg Pro Val Glu Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu
165 170 175
Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu
180 185 190
Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val
195 200 205
Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly
210 215 220
Ile Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr
225 230 235 240
Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Lys
245 250 255
Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys
260 265 270
Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val
275 280 285
Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr
290 295 300
Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys
305 310 315 320
Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala
325 330 335
Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr
340 345 350
Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val
355 360 365
Val Asp Arg Cys Gly Cys Ser
370 375
<210> 6
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 6
agattcactg gtgtggcaag 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 7
tgcatgacat gtctttgtgc 20
Claims (10)
1.由SEQ ID NO:1的氨基酸序列构成的蛋白。
2.多核苷酸,该多核苷酸包含:编码权利要求1所述的蛋白的核苷酸序列或与其互补的序列。
3.载体,该载体包含权利要求2所述的多核苷酸。
4.细胞,该细胞包含权利要求3所述的载体,且该细胞为细菌细胞、真菌细胞、昆虫细胞、动物细胞。
5.制作由SEQ ID NO:1的氨基酸序列构成的蛋白的方法,该方法包括:培养权利要求4所述的细胞。
6.用于抑制肌肉生长抑制素信号的组合物,该组合物包含:选自权利要求1所述的蛋白、权利要求2所述的多核苷酸、权利要求3所述的载体和权利要求4所述的细胞的至少一种。
7.用于促进肌肉形成的组合物,该组合物包含:选自权利要求1所述的蛋白、权利要求2所述的多核苷酸、权利要求3所述的载体和权利要求4所述的细胞的至少一种。
8.用于预防和/或治疗肌肉生长抑制素相关疾病的组合物,该组合物包含:选自权利要求1所述的蛋白、权利要求2所述的多核苷酸、权利要求3所述的载体和权利要求4所述的细胞的至少一种。
9.药物,该药物包含:选自权利要求1所述的蛋白、权利要求2所述的多核苷酸、权利要求3所述的载体和权利要求4所述的细胞的至少一种。
10.饲料,该饲料包含:选自权利要求1所述的蛋白、权利要求2所述的多核苷酸、权利要求3所述的载体和权利要求4所述的细胞的至少一种。
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CA2538208A1 (en) * | 2005-05-04 | 2006-11-04 | Universite Laval | Modulation of myostatin and use thereof in cell transplantation-based treatment of muscle disease |
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US6369201B1 (en) * | 1998-02-19 | 2002-04-09 | Metamorphix International, Inc. | Myostatin multimers |
US20080118487A1 (en) | 2004-09-30 | 2008-05-22 | Orico Limited | Myostatin Isoform |
WO2010134686A2 (ko) * | 2009-05-20 | 2010-11-25 | 강릉원주대학교산학협력단 | 마이오스타틴 억제 활성을 갖는 가용성 마이오스타틴 프로도메인 재조합 단백질 및 그의 용도 |
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JP2017226651A (ja) * | 2016-06-15 | 2017-12-28 | 興和株式会社 | 水性なる組成物 |
BR112018073628A2 (pt) * | 2016-06-17 | 2019-02-26 | Chugai Seiyaku Kabushiki Kaisha | anticorpos antimiostatina e métodos de utilização |
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