CN111484550A - Dkk-3蛋白功能性片段及其应用 - Google Patents
Dkk-3蛋白功能性片段及其应用 Download PDFInfo
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- CN111484550A CN111484550A CN202010335115.1A CN202010335115A CN111484550A CN 111484550 A CN111484550 A CN 111484550A CN 202010335115 A CN202010335115 A CN 202010335115A CN 111484550 A CN111484550 A CN 111484550A
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Abstract
本公开提供了一种Dkk‑3蛋白功能性片段及其应用。具体来说，本公开揭示了一种由氨基酸编码的Dkk‑3蛋白的不同片段在肌肉萎缩中的功能。其中，Dkk‑3蛋白的N端片段具有诱导肌肉萎缩的完整功能，Dkk‑3蛋白的L‑CDR2‑C片段具有拮抗N端片段诱导肌肉萎缩的功能。本公开还提供了以Dkk‑3蛋白的特定位点为靶点治疗肌肉萎缩的药物，Dkk‑3蛋白的L‑CDR2‑C片段本身或其融合蛋白可以作为拮抗Dkk‑3诱导肌肉萎缩功能的靶向药物。
Description
技术领域
本公开涉及生物技术领域，具体涉及肌肉萎缩相关的蛋白质分子标记Dkk-3蛋白的功能性片段、前述功能性片段的筛选及其应用。
背景技术
Dkk蛋白家族(Dickkopf family protein)属于分泌型的糖蛋白，包括五个家族成员，分别是Dkk-1、Dkk-2、Dkk-3、Dkk-4和DkkL1(Niehrs，2006)。
Dkk蛋白家族成员的蛋白结构包括N端的信号肽(Signal peptide)，两个半胱氨酸富集区(Cysteine-rich domain，CRD)(DkkL1除外)以及连接两个半胱氨酸富集区的连接肽段(Linker)(见图2)。每一个CRD区域都存在十个保守的半胱氨酸位点。靠近N端的CRD1为Dkk蛋白家族所特有(Krupnik et al.，1999)。另一个靠近C端(碳端)的半胱氨酸富集区CRD2在蛋白质序列上与共脂肪酶折叠区(Colipase fold)的功能域相似(Aravind andKoonin，1998)。
与功能研究相似，在Dkk蛋白家族中，结构研究较多家族成员是Dkk-1和Dkk-2。将Dkk-1和Dkk-2从连接肽段处分为N段和C段，二者的C段均能够与LRP6受体结合。Dkk-1的N段能够抑制Dkk-1的C段与LRP6的结合，Dkk-2的N段则没有类似功能(Brott and Sokol，2002)。这可能解释了Dkk-1和Dkk-2在爪蟾发育中的不同功能(Brott and Sokol，2002)。这些结果提示，Dkk蛋白家族中，不同成员的N段结构域的功能可能不相同，甚至存在很大差异，部分结构域与全长蛋白的功能有可能完全相反。
与Dkk-1及Dkk-2相比，目前针对Dkk-3结构域功能的研究还比较少。2008年，Abarzua等人研究发现Dkk-3 N端78个氨基酸组成的结构域是Dkk-3诱导人前列腺癌细胞发生内质网应激反应和细胞凋亡的最主要区域(Abarzua et al.，2008)。2015年，Kinoshita等人发现，Dkk-3诱导单核细胞向树突状细胞分化，激活细胞毒性T淋巴细胞，引起抗肿瘤免疫反应的重要功能结构域为CRD1和CRD2区域(Kinoshita et al.，2015)。这说明，在不同类型细胞中，行使不同生物学功能时，Dkk-3起主要作用的结构域可能并不相同。
Dkk-1能够通过抑制Wnt信号通路从而诱导非洲爪蟾头部发育的起始(Glinka etal.，1998)。
Dkk-2的功能也与Wnt信号通路紧密相连。但与Dkk-1不同，Dkk-2在不同细胞中，对Wnt信号通路的调控方向不同。其对Wnt信号通路的影响却既有激活又有抑制(Wu et al.，2000)。
Dkk-4的功能与Dkk-1相似，也能通过结合LRP5/6受体，对Wnt信号通路进行负调控(Krupnik et al.，1999)。
DkkL1到目前为止，功能研究几乎处于空白状态。
与其它Dkk家族成员不同，Dkk-3不能与LRP5/6和Kremen受体结合(Mao andNiehrs，2003；Li et al.，2002；Brott and Sokol，2002)，在Wnt信号通路中的作用及机制仍不清楚。很多报道表明Dkk-3不影响Wnt信号通路(Abarzua et al.，2005)。但是，也有报道认为Dkk-3能够抑制Wnt信号通路(Caricasole et al.，2003；Hoang et al.，2004)。相反，也有文章认为Dkk-3能增强Wnt信号通路(Nakamura et al.，2007)。上述研究表明，在不同类型的细胞，不同细胞状态等不同背景下，Dkk-3的功能可能都不相同。
Dkk-3是一个公认的肿瘤抑制因子(Veeck and Dahl，2012)。非肿瘤疾病中，Dkk-3的研究较少。已知Dkk-3的表达水平与动脉粥样硬化(Yu et al.，2017)，肾纤维化(Federico et al.，2016)，阿尔兹海默症(Zhang et al.，2017)及肌肉萎缩(Yin et al.，2018)密切相关。
目前针对Dkk-3在骨骼肌中的相关研究还很少。现有研究表明，在不同类型的小鼠骨骼肌中，Dkk-3的表达量存在差异。在股四头肌中表达水平较高，在腓肠肌和比目鱼肌中表达水平相对较低，但并没有深入研究具体是哪一类型肌肉中Dkk-3表达量较高(de Wildeet al.，2010)。另外，在斑马鱼胚胎肌肉发育过程中，Dkk-3能够正调控成肌因子Myf5的表达，从而促进肌肉发育(Fu et al.，2012)。上述研究揭示了Dkk-3在肌肉的发育和分化过程中有非常重要的调控作用。
Dkk-3不仅在肌肉的发育和分化中起重要的作用，而且与由衰老引起的肌肉萎缩密切相关。Dkk-3的表达水平在衰老肌肉中显著提高。同时，在肌肉萎缩患者的外周血中，Dkk-3的蛋白水平显著升高，提示Dkk-3可以成为肌肉萎缩的血液诊断标志物(Yin et al.，2018)。利用腺病毒在年轻小鼠胫骨前肌中过表达Dkk-3，胫骨前肌肌肉质量下降，肌纤维横截面积明显变小，肌肉力量下降，肌肉发生了肌肉萎缩现象。相反，在老年小鼠胫骨前肌中敲低Dkk-3表达水平，胫骨前肌的肌纤维横截面积增大，肌肉质量显著增加，更重要的是肌肉收缩力量恢复，这提示Dkk-3是引起肌肉萎缩的重要因素，Dkk-3可能成为新的肌肉萎缩的治疗靶点(Yin et al.，2018)。
肌少症(sarcopenia)或称“肌肉减少症”，源于希腊语，“sarx”意为肌肉，“penia”意为减少或丢失，这是个新名词，于1989年由Rosenberg首次命名。2010年欧洲老年肌少症工作组(European Working Group on Sarcopenia in Older People，EWGSOP)发表了肌肉萎缩共识。此后，国际肌少症工作组(International Working Group on Sarcopenia，IWGS)也公布了新共识，将肌少症定义为：“与增龄相关的进行性、全身肌量减少和/或肌强度下降或肌肉生理功能减退”。
肌少症与活动障碍、跌倒、低骨密度及代谢紊乱密切相关，是老年人生理功能逐渐减退的重要原因和表现之一。肌肉少症会增加老年人的住院率及医疗花费，严重影响老年人的生活质量，甚至缩短老年人的寿命。
目前还没有以老年肌肉萎缩为适应症的药物，扩展使用的药物副作用大而效果有限。运动疗法和营养疗法的效果更有限。
发明内容
发明要解决的问题
基于现有技术中存在的问题，本公开提供一种治疗肌肉萎缩，特别是老年肌肉萎缩的药物。
用于解决问题的方案
(1)一种蛋白，所述蛋白由氨基酸序列编码，其中，所述氨基酸序列选自以下序列：
(a)所述氨基酸序列与SEQ ID NO：3所示的序列相比，具有80％以上且90％以下的同源性；优选的，具有82％以上且86％以下的同源性；更优选的，具有84％以上且85％以下的同源性。
(2)如(1)所述的蛋白，其特征在于，所述核苷酸的序列为如SEQ ID NO：4所示的序列。
(3)一种蛋白，所述蛋白由氨基酸序列编码，其中，所述氨基酸序列选自以下序列：
(b)所述氨基酸序列与SEQ ID NO：5所示的序列相比，具有75％以上且84％以下的同源性；优选的，具有77％以上且82％以下的同源性；更优选的，具有79％以上且80％以下的同源性；
可选的，所述氨基酸序列中氨基酸的数量比SEQ ID NO：5所示的氨基酸数量多1个。
(4)如(3)所述的蛋白，其特征在于，所述氨基酸序列为如SEQ ID NO：6所示的序列。
(5)一种核酸，所述核酸编码如(1)-(4)任一项所述的蛋白。
(6)一种遗传修饰的载体，其特征在于，所述载体包含核苷酸序列，其中，所述核苷酸序列编码如(1)-(4)任一项所述的蛋白；可选的，所述核苷酸序列包含如SEQ ID NO：8所示的序列或如SEQ ID NO：10所示的序列。
(7)一种表达蛋白的细胞，其含有如(6)所述的载体。
(8)一种抗体或其片段，所述抗体或其片段竞争性地结合(1)-(2)任一项所述的蛋白；优选的，所述抗体为单克隆抗体。
(9)一种制备蛋白的方法，所述方法包括以下步骤：
(s1)将(6)所述的载体转染表达蛋白的细胞；
(s2)收集步骤(s1)中的所述细胞表达的所述蛋白。
(10)一种制备单克隆抗体的方法，所述方法包括以下步骤(s1)-(s3)或以下步骤(s1)-(s2)、(s4)：
(s1)将(6)所述的载体转染表达蛋白的细胞；
(s2)收集步骤(s1)中的所述细胞表达的蛋白；
(s3)用步骤(s2)获得的蛋白免疫动物，从被免疫动物的淋巴细胞中分离特异性识别所述蛋白的所述单克隆抗体；或者
(s4)用步骤(s2)获得的蛋白筛选单克隆抗体噬菌体展示文库，得到特异性识别所述蛋白的单克隆抗体。
(11)一种制备融合蛋白的方法，所述蛋白由核苷酸编码，所述核苷酸至少包含第一核苷酸和第二核苷酸，所述方法包括以下步骤：
(i)将编码所述第一核苷酸和第二核苷酸的基因进行连接，形成融合蛋白基因；
(ii)将所述融合蛋白基因转染能够表达蛋白的细胞；
(iii)收集步骤(ii)中的所述细胞表达的所述蛋白；
其中，所述第一核苷酸的序列选自如SEQ ID NO：8所示的序列或如SEQ ID NO：10所示的序列；
所述第二核苷酸编码其他蛋白的基因；
可选的，所述核苷酸还包含连接序列。
(12)一种融合蛋白的抗体或其抗体片段，其中，所述融合蛋白由(10)所述的方法制备得到。
(13)一种融合蛋白，其中，所述融合蛋白由权利要求11所述的方法制备得到。
(14)(3)-(4)任一项所述的蛋白、(8)所述的抗体或其片段、(12)所述的融合蛋白的抗体或其抗体片段或(13)所述的融合蛋白在制备用于治疗肌肉萎缩的药物中的用途。
(15)一种药物组合物，所述药物组合物包含如(3)-(4)任一项所述的蛋白、(8)所述的抗体或其片段、(12)所述的融合蛋白的抗体或其抗体片段或(13)所述的融合蛋白。
(16)一种治疗肌肉萎缩的方法，所述方法包括施用如(3)-(4)任一项所述的蛋白、(8)所述的抗体或其片段、(12)所述的融合蛋白的抗体或其抗体片段或(13)所述的融合蛋白。
(17)一种筛选治疗肌肉萎缩的候选药物的方法，其中，所述方法包含如下步骤：
(s1)监测患有肌肉萎缩的实验动物中，如(1)-(4)任一项所述的蛋白的表达水平；
(s2)根据预定频率和用量及给药时间给予所述患有肌肉萎缩的实验动物候选药物；
(s3)再次监测患有肌肉萎缩的实验动物中，如(1)-(4)任一项所述的蛋白的表达水平；其中，
如果步骤(s3)中监测得到的如(1)-(2)任一项所述蛋白的表达水平低于在步骤(s1)中所监测的蛋白的表达水平；或者
如果步骤(s3)中监测得到的如(3)-(4)任一项所述蛋白的表达水平高于在步骤(s1)中所监测的蛋白的表达水平；则
所述候选药物为目标药物。
在一种具体的实施方式中，本公开提供一种Dkk-3蛋白片段、其基因、其抗体、其融合蛋白的用途，用于制备治疗肌肉萎缩的药物。
在一种具体的实施方式中，本公开提供一种制备Dkk-3蛋白片段的方法，所述方法包括以下步骤：
(1)将编码Dkk-3片段的基因装入表达载体，转染哺乳动物细胞株，制备表达Dkk-3蛋白片段。
步骤(1)获得的Dkk-3蛋白片段可能直接作为肌肉萎缩的候选药物，也可能作为工具进一步开发肌肉萎缩药物。
在一种具体的实施方式中，本公开提供一种利用Dkk-3蛋白片段制备特异性单克隆抗体的方法，所述方法包括以下步骤：
(1)将编码Dkk-3片段的基因装入表达载体，转染哺乳动物细胞株，制备表达Dkk-3蛋白片段。
(2)用步骤(1)获得的Dkk-3蛋白片段免疫动物，从被免疫动物的淋巴细胞中分离特异性识别Dkk-3蛋白片段的单克隆抗体；
(3)或者用步骤(1)获得的Dkk-3蛋白片段筛选单克隆抗体噬菌体展示文库，得到特异性识别Dkk-3蛋白片段的单克隆抗体。
(4)步骤(2)和步骤(3)获得的单克隆抗体是治疗肌肉萎缩的候选药物。
在一种具体的实施方式中，本公开提供一种利用Dkk-3蛋白片段制备融合蛋白的方法，所述方法包括以下步骤：
(1)将编码Dkk-3片段的基因与编码其他蛋白的基因进行体外连接，形成融合蛋白基因。在融合蛋白基因中，其他蛋白的基因可能位于Dkk-3片段基因的上游，也可能位于Dkk-3片段基因的下游。
(2)将步骤(1)得到的融合蛋白基因装入表达载体，转染哺乳动物细胞株，制备表达包含Dkk-3蛋白片段的融合蛋白。
(3)步骤(2)获得的融合蛋白是治疗肌肉萎缩的候选药物。
发明的效果
在一个实施方式中，本公开首次揭示了Dkk-3蛋白的不同片段在肌肉萎缩中的功能。其中，Dkk-3蛋白的N端片段(23-146)具有诱导肌肉萎缩的完整功能。Dkk-3蛋白的L-CDR2-C片段(196-350)具有拮抗N端片段诱导肌肉萎缩的功能。
在另一个实施方式中，本公开提供了一种以Dkk-3蛋白的特定位点为靶点治疗肌肉萎缩的药物，其能够特异性识别Dkk-3蛋白的N端片段(23-146)。
在另一个实施方式中，本公开还提供了另一种以Dkk-3蛋白的特定位点为靶点治疗肌肉萎缩的药物，Dkk-3蛋白的L-CDR2-C片段(196-350)本身或其融合蛋白可以作为拮抗Dkk-3诱导肌肉萎缩功能的靶向药物。
附图说明
图1所示的是人Dkk-3蛋白序列和小鼠Dkk-3蛋白序列比对。
图2所示的是Dkk家族成员结构示意图。其中，Sp代表信号肽；CRD1表示半胱氨酸富集区1；CRD2表示半胱氨酸富集区2；圆圈中N及数字表示在这一个位置的天冬酰胺发生了糖基化修饰。
图3所示的是用于结构-功能研究的Dkk-3蛋白及其片段的载体结构示意图。其中，N表示N端片段；CRD1表示半胱氨酸富集区1；L区域表示连接CRD1及CRD2的连接肽；CRD2表示半胱氨酸富集区2；C表示C端片段。
图4所示的是在体外培养的小鼠肌小管中过表达Dkk-3及一组片段。其中，图4中的A所示的是过表达Dkk-3及其片段后肌小管的直径分布情况；图4中的B所示的是统计学分析显示，过表达带有FLAG标签的Dkk-3及N、N-DRD1、N-CRD1-CRD2片段后肌小管的直径显著减小；图4中的C所示的是肌小管中Dkk-3及其片段的过表达；图4中的D所示的是过表达Dkk-3及其片段的肌小管中肌肉萎缩特异性基因MAFbx的表达情况。
图5所示的是在体外培养的小鼠肌小管中过表达Dkk-3及另一组片段。其中，图5中的A所示的是过表达Dkk-3及片段后肌小管的直径分布情况；图5中的B所示的是统计学分析显示，过表达带有FLAG标签的CRD1-L-CDR2-C和L-CDR2-C片段后肌小管的直径没有显著变化；图5中的C所示的是肌小管中Dkk-3及其片段的过表达；图5中的D所示的是过表达Dkk-3及其片段的肌小管中肌肉萎缩特异性基因MAFbx的表达情况。
图6所示的是重组表达的N片段、L-CDR2-C片段及二者的混合液处理体外培养的小鼠肌小管细胞。
图7所示的用1nM小鼠Dkk-3和人Dkk-3处理肌管细胞的处理结果。
具体实施方式
当在权利要求和/或说明书中与术语“包含”联用时，词语“一(a)”或“一(an)”可以指“一个”，但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。
如在权利要求和说明书中所使用的，词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。
在整个申请文件中，术语“约”表示：一个值包括测定该值所使用的装置或方法的误差的标准偏差。
虽然所公开的内容支持术语“或”的定义仅为替代物以及“和/或”，但除非明确表示仅为替代物或替代物之间相互排斥外，权利要求中的术语“或”是指“和/或”。
当用于权利要求书或说明书时，选择/可选/优选的“数值范围”既包括范围两端的数值端点，也包括相对于前述数值端点而言，所述数值端点中间所覆盖的所有自然数。
本公开中的术语“取代、重复、缺失或添加一个或多个氨基酸”包括“保守突变”。本公开中的术语“保守突变”是指可正常维持蛋白质的功能的保守突变。保守突变的代表性例子为保守置换。保守置换是指，例如，在置换部位为芳香族氨基酸的情况下，在Phe、Trp、Tyr间相互置换的突变；在置换部位为疏水性氨基酸的情况下，在Leu、Ile、Val间相互置换的突变；在为极性氨基酸的情况下，在Gln、Asn间相互置换的突变；在为碱性氨基酸的情况下，在Lys、Arg、His间相互置换的突变；在为酸性氨基酸的情况下，在Asp、Glu间相互置换的突变；在为具有羟基的氨基酸的情况下，在Ser、Thr间相互置换的突变。作为被视作保守置换的置换，具体而言，可以举出Ala向Ser或Thr的置换、Arg向Gln、His或Lys的置换、Asn向Glu、Gln、Lys、His或Asp的置换、Asp向Asn、Glu或Gln的置换、Cys向Ser或Ala的置换、Gln向Asn、Glu、Lys、His、Asp或Arg的置换、Glu向Gly、Asn、Gln、Lys或Asp的置换、Gly向Pro的置换、His向Asn、Lys、Gln、Arg或Tyr的置换、Ile向Leu、Met、Val或Phe的置换、Leu向Ile、Met、Val或Phe的置换、Lys向Asn、Glu、Gln、His或Arg的置换、Met向Ile、Leu、Val或Phe的置换、Phe向Trp、Tyr、Met、Ile或Leu的置换、Ser向Thr或Ala的置换、Thr向Ser或Ala的置换、Trp向Phe或Tyr的置换、Tyr向His、Phe或Trp的置换、及Val向Met、Ile或Leu的置换。此外，保守突变还包括起因于基因所来源的个体差异、株、种的差异等天然产生的突变。
在一种实施方式中，本公开采用筛选单克隆抗体噬菌体展示文库技术，得到特异性识别所述蛋白的单克隆抗体。
在一种具体的实施方式中，将抗体编码基因插入噬菌体外壳蛋白结构基因的适当位置，在阅读框正确且不影响外壳蛋白正常功能的情况下，使抗体与外壳蛋白融合表达，融合蛋白随子代噬菌体的重新组装而展示在噬菌体表面。噬菌体文库与靶蛋白分子经过一定时间孵育后，洗去未结合的游离噬菌体，然后洗脱下与靶分子结合吸附的噬菌体，洗脱的噬菌体感染宿主细胞后经繁殖扩增，进行下一轮孵育吸附，经过3轮～5轮的“吸附-洗脱-扩增”后，从高度富集的噬菌体中获得与靶分子特异结合的单克隆抗体的基因。
本公开中的“本领域的常规生物学方法”，可以参见“最新分子生物学实验方法汇编(Current Protocols in Molecular Biology，Wiley出版)”，“分子克隆实验指南(Molecular Cloning：A Laboratory Manual，冷泉港实验室出版)”等公开出版物中记载的相应方法。
本公开中涉及的核苷酸或氨基酸序列含义如下。
SEQ ID NO：1所示的是小鼠Dkk-3蛋白的氨基酸序列；
SEQ ID NO：2所示的是人Dkk-3蛋白的氨基酸序列；
SEQ ID NO：3所示的是小鼠Dkk-3蛋白的N端片段(23-146)的氨基酸序列；
SEQ ID NO：4所示的是人Dkk-3蛋白的N端片段(23-146)的氨基酸序列；
SEQ ID NO：5所示的是小鼠Dkk-3蛋白的L-CDR2-C端片段(196-349)的氨基酸序列；
SEQ ID NO：6所示的是人Dkk-3蛋白的L-CDR2-C端片段(196-350)的氨基酸序列；
SEQ ID NO：7所示的是小鼠Dkk-3蛋白的N端片段(67-438)的核苷酸序列；
SEQ ID NO：8所示的是人Dkk-3蛋白的N端片段(67-438)的核苷酸序列；
SEQ ID NO：9所示的是小鼠Dkk-3蛋白的L-CDR2-C端片段(586-1047)的核苷酸序列；
SEQ ID NO：10所示的是人Dkk-3蛋白的L-CDR2-C端片段(586-1050)的核苷酸序列；
SEQ ID NO：11所示的是人Dkk-3蛋白的CRD1片段的氨基酸序列；
SEQ ID NO：12所示的是人Dkk-3蛋白的L片段的氨基酸序列；
SEQ ID NO：13所示的是人Dkk-3蛋白的CRD2片段的氨基酸序列；
SEQ ID NO：14所示的是人Dkk-3蛋白的C端片段的氨基酸序列；
SEQ ID NO：15所示的是小鼠Dkk-3蛋白的CRD1片段的氨基酸序列；
SEQ ID NO：16所示的是小鼠Dkk-3蛋白的L片段的氨基酸序列；
SEQ ID NO：17所示的是小鼠Dkk-3蛋白的CRD2片段的氨基酸序列；
SEQ ID NO：18所示的是小鼠Dkk-3蛋白的C端片段的氨基酸序列。
实施例
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是，应当理解的是，详细描述和具体实施例(虽然表示本公开的具体实施方式)仅为解释性目的而给出，因为在阅读该详细说明后，在本公开的精神和范围内所作出的各种改变和修饰，对于本领域技术人员来说将变得显而易见。
除非有特别说明，否则本公开中采用的所有试剂和原料均可以通过商业渠道购买。
实施例1：小鼠Dkk-3蛋白及其片段的功能检测
具体实验步骤：
C2C12细胞系购于ATCC，按1x 105密度接种于6孔板，每孔加2ml含10％胎牛血清(Hyclone)的DMEM培养基(Hyclone)，于37℃，5％CO2培养2-3天，待细胞密度约100％时，更换含2％马血清(Hyclone)的DMEM培养基，再培养3～4天，可形成肌管细胞。
向肌管细胞中分别加入腺病毒。所有病毒滴度均调整为1x 106pfu/μl，每孔中加入1μl病毒感染细胞。感染病毒后的肌管细胞培养三天后显微镜下观察并计数。利用Image-Pro Plus软件统计肌管细胞直径大小。
从肌管细胞中提取总蛋白。将肌管细胞用胰酶消化，3000rpm，离心5min，收集在1.5ml微量离心管中。细胞沉淀用D100裂解液重悬，D100配方为HEPES(pH：7.9)20mM，EDTA(pH：8.0)0.5mM，KCl 100mM，甘油20％，Tween-20 0.5％。将细胞悬液放置在冰上，每隔5min用胰岛素针头吹吸重悬5-8次，重复5-6遍。非接触式超声破碎细胞，30s on30s off，处理5min。破碎的细胞13000rpm离心20min，取上清为细胞内总蛋白。
免疫印迹(Western blot)测定小鼠Dkk-3蛋白片段和肌肉萎缩标记蛋白MAFbx的表达水平。配制8～12％SDS-PAGE胶。根据实验要求制备所需蛋白样品，加入Loadingbuffer，100℃变性10分钟，上样到SDS-PAGE胶上样孔中。接通电源，浓缩胶80V 30～40分钟，分离胶120V 1～2小时。待电泳结束，电转膜，恒压100V，1～2小时。待转膜完成，在含有5％脱脂奶粉的TPBS中，室温封闭1小时。TBST洗一次，加一抗(Rabbit anti Atrogin1(ECMBioscience)；Mouse anti-GAPDH(Protein tech,60004)；Rabbit anti-Dkk3(苏州神肌赢生物医药科技有限公司))，4℃孵育过夜。弃一抗，TBST洗三次，每次10分钟。加二抗(HRPGoat anti-rabbit(Sigma)；HRP Goat anti-mouse(Sigma))，室温孵育1小时，弃二抗，TBST洗三次，每次10分钟。显色和拍照。
实验结果：通过细胞模型对Dkk-3蛋白及其片段进行了功能检测。用表达Dkk-3蛋白或其不同片段的病毒感染肌管细胞，结果发现N端片段(23-146)具有诱导肌肉萎缩的完整功能(图4)，而去掉N端后的蛋白片段(CDR1-L-CDR2-C)不能诱导肌肉萎缩(图5)。
实施例2：小鼠Dkk-3蛋白及其片段的功能确认
具体实验步骤：
肌管的培养和分化同实施例1。
用纯化的小鼠Dkk-3蛋白片段处理肌管细胞，设定实验组为三组，两种蛋白以不同比例处理肌管细胞，分别为N:L-CRD2-C＝1nM：1nM、N:L-CRD2-C＝1nM：5nM和N:L-CRD2-C＝1nM：10nM。平行设置两个蛋白对照组(N端蛋白浓度1nM和L-CRD2-C蛋白1nM)和一个空白对照组。两天后观察细胞形态变化并计数。利用Image-Pro Plus软件统计肌管细胞直径大小。
实验结果：重组表达并纯化了Dkk-3 N端片段(23-146)和L-CDR2-C片段(196-349)，将两种片段按不同比例混合后处理肌管细胞，发现L-CDR2-C片段(196-349)可以拮抗N端片段(23-146)诱导肌肉萎缩的功能，并且这种拮抗效应表现出对L-CDR2-C片段的浓度依赖(图6)。
实施例3：人Dkk-3蛋白和小鼠Dkk-3蛋白的功能比较
通过BLAST分析，人Dkk-3蛋白(350个氨基酸编码)和小鼠Dkk-3蛋白(349个氨基酸编码)的同源性为83％。
肌管的培养和分化同实施例1。
具体来说，用纯化的小鼠Dkk-3蛋白和人Dkk-3蛋白处理肌管细胞，设定实验组为两组，分别用1nM小鼠Dkk-3和人Dkk-3处理肌管细胞。平行设置一个空白对照组。两天后观察细胞形态变化并计数。利用Image-Pro Plus软件统计肌管细胞直径大小。
实验结果：如图7所示，重组表达并纯化了全长小鼠Dkk-3和全长人Dkk-3，使用相同浓度的上述蛋白处理肌管细胞，发现两个物种的Dkk-3诱导肌肉萎缩的功能高度相似。
本公开并不旨在限于具体公开的实施方案的范围，提供所述实施方案例如来说明本发明的各方面。从本文的描述和教导，对所述组合物和方法的各种修改将变得明显。可以在不脱离本公开的真正范围和精神的情况下实践这类变化，并且这类变化旨在落入本公开的范围内。
序列表
<110> 苏州神肌赢生物医药科技有限公司
<120> Dkk-3蛋白功能性片段及其应用
<130> 6923-190374I-SU
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Met Gln Arg Leu Gly Gly Ile Leu Leu Cys Thr Leu Leu Ala Ala Ala
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Val Pro Thr Ala Pro Ala Pro Ser Pro Thr Val Thr Trp Thr Pro Ala
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Glu Pro Gly Pro Ala Leu Asn Tyr Pro Gln Glu Glu Ala Thr Leu Asn
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Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gln His Lys
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Leu Arg Ser Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys
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Thr Ser Ser Glu Val Asn Leu Ala Ser Leu Pro Pro Asn Tyr His Asn
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Glu Thr Ser Thr Glu Thr Arg Val Gly Asn Asn Thr Val His Val His
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Gln Glu Val His Lys Ile Thr Asn Asn Gln Ser Gly Gln Val Val Phe
115 120 125
Ser Glu Thr Val Ile Thr Ser Val Gly Asp Glu Glu Gly Lys Arg Ser
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His Glu Cys Ile Ile Asp Glu Asp Cys Gly Pro Thr Arg Tyr Cys Gln
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Phe Ser Ser Phe Lys Tyr Thr Cys Gln Pro Cys Arg Asp Gln Gln Met
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Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Ala Trp
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Gly His Cys Thr Gln Lys Ala Thr Lys Gly Gly Asn Gly Thr Ile Cys
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Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln Arg
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Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu
225 230 235 240
Cys His Asp Pro Thr Ser Gln Leu Leu Asp Leu Ile Thr Trp Glu Leu
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Glu Pro Glu Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu
260 265 270
Cys Gln Pro His Ser His Ser Leu Val Tyr Met Cys Lys Pro Ala Phe
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Val Gly Ser His Asp His Ser Glu Glu Ser Gln Leu Pro Arg Glu Ala
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Pro Asp Glu Tyr Glu Asp Val Gly Phe Ile Gly Glu Val Arg Gln Glu
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Leu Glu Asp Leu Glu Arg Ser Leu Ala Gln Glu Met Ala Phe Glu Gly
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Pro Ala Pro Val Glu Ser Leu Gly Gly Glu Glu Glu Ile
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Met Gln Arg Leu Gly Ala Thr Leu Leu Cys Leu Leu Leu Ala Ala Ala
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Val Pro Thr Ala Pro Ala Pro Ala Pro Thr Ala Thr Ser Ala Pro Val
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Lys Pro Gly Pro Ala Leu Ser Tyr Pro Gln Glu Glu Ala Thr Leu Asn
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Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gln His Lys
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Leu Arg Ser Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys
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Ala Ser Ser Glu Val Asn Leu Ala Asn Leu Pro Pro Ser Tyr His Asn
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Glu Thr Asn Thr Asp Thr Lys Val Gly Asn Asn Thr Ile His Val His
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Arg Glu Ile His Lys Ile Thr Asn Asn Gln Thr Gly Gln Met Val Phe
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Ser Glu Thr Val Ile Thr Ser Val Gly Asp Glu Glu Gly Arg Arg Ser
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His Glu Cys Ile Ile Asp Glu Asp Cys Gly Pro Ser Met Tyr Cys Gln
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Phe Ala Ser Phe Gln Tyr Thr Cys Gln Pro Cys Arg Gly Gln Arg Met
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Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Val Trp
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Gly His Cys Thr Lys Met Ala Thr Arg Gly Ser Asn Gly Thr Ile Cys
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Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln Arg
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Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu
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Cys His Asp Pro Ala Ser Arg Leu Leu Asp Leu Ile Thr Trp Glu Leu
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Glu Pro Asp Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu
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Val Gly Ser Arg Asp Gln Asp Gly Glu Ile Leu Leu Pro Arg Glu Val
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Glu Glu Leu Met Glu Asp Thr Gln His Lys Leu Arg Ser Ala Val Glu
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ccttccccga cggtcacttg gactccggcg gagccgggcc cagctctcaa ctaccctcag 60
gaggaagcta cgctcaatga gatgtttcga gaggtggagg agctgatgga agacactcag 120
cacaaactgc gcagtgccgt ggaggagatg gaggcggaag aagcagctgc taaaacgtcc 180
tctgaggtga acctggcaag cttacctccc aactatcaca atgagaccag cacggagacc 240
agggtgggaa ataacacagt ccatgtgcac caggaagttc acaagataac caacaaccag 300
agtggacagg tggtcttttc tgagacagtc attacatctg taggggatga agaaggcaag 360
aggagccatg aa 372
<210> 8
<211> 372
<212> DNA
<213> Homo sapiens
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cccgctccga cggcgacctc ggctccagtc aagcccggcc cggctctcag ctacccgcag 60
gaggaggcca ccctcaatga gatgttccgc gaggttgagg aactgatgga ggacacgcag 120
cacaaattgc gcagcgcggt ggaagagatg gaggcagaag aagctgctgc taaagcatca 180
tcagaagtga acctggcaaa cttacctccc agctatcaca atgagaccaa cacagacacg 240
aaggttggaa ataataccat ccatgtgcac cgagaaattc acaagataac caacaaccag 300
actggacaaa tggtcttttc agagacagtt atcacatctg tgggagacga agaaggcaga 360
aggagccacg ag 372
<210> 9
<211> 462
<212> DNA
<213> Mus musculus
<400> 9
acccaaaagg ccaccaaagg tggcaatggg accatctgtg acaaccagag ggattgccag 60
cctggcctgt gttgtgcctt ccaaagaggc ctgctgttcc ccgtgtgcac acccctgccc 120
gtggagggag agctctgcca tgaccccacc agccagctgc tggatctcat cacctgggaa 180
ctggagcctg aaggagcttt ggaccgatgc ccctgcgcca gtggcctcct atgccagcca 240
cacagccaca gtctggtgta catgtgcaag ccagccttcg tgggcagcca tgaccacagt 300
gaggagagcc agctgcccag ggaggccccg gatgagtacg aagatgttgg cttcataggg 360
gaagtgcgcc aggagctgga agacctggag cggagcctag cccaggagat ggcatttgag 420
gggcctgccc ctgtggagtc actaggcgga gaggaggaga tt 462
<210> 10
<211> 465
<212> DNA
<213> Homo sapiens
<400> 10
accaaaatgg ccaccagggg cagcaatggg accatctgtg acaaccagag ggactgccag 60
ccggggctgt gctgtgcctt ccagagaggc ctgctgttcc ctgtgtgcac acccctgccc 120
gtggagggcg agctttgcca tgaccccgcc agccggcttc tggacctcat cacctgggag 180
ctagagcctg atggagcctt ggaccgatgc ccttgtgcca gtggcctcct ctgccagccc 240
cacagccaca gcctggtgta tgtgtgcaag ccgaccttcg tggggagccg tgaccaagat 300
ggggagatcc tgctgcccag agaggtcccc gatgagtatg aagttggcag cttcatggag 360
gaggtgcgcc aggagctgga ggacctggag aggagcctga ctgaagagat ggcgctgagg 420
gagcctgcgg ctgccgccgc tgcactgctg ggaggggaag agatt 465
<210> 11
<211> 49
<212> PRT
<213> Homo sapiens
<400> 11
Cys Ile Ile Asp Glu Asp Cys Gly Pro Ser Met Tyr Cys Gln Phe Ala
1 5 10 15
Ser Phe Gln Tyr Thr Cys Gln Pro Cys Arg Gly Gln Arg Met Leu Cys
20 25 30
Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Val Trp Gly His
35 40 45
Cys
<210> 12
<211> 12
<212> PRT
<213> Homo sapiens
<400> 12
Thr Lys Met Ala Thr Arg Gly Ser Asn Gly Thr Ile
1 5 10
<210> 13
<211> 77
<212> PRT
<213> Homo sapiens
<400> 13
Cys Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln
1 5 10 15
Arg Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu
20 25 30
Leu Cys His Asp Pro Ala Ser Arg Leu Leu Asp Leu Ile Thr Trp Glu
35 40 45
Leu Glu Pro Asp Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu
50 55 60
Leu Cys Gln Pro His Ser His Ser Leu Val Tyr Val Cys
65 70 75
<210> 14
<211> 66
<212> PRT
<213> Homo sapiens
<400> 14
Lys Pro Thr Phe Val Gly Ser Arg Asp Gln Asp Gly Glu Ile Leu Leu
1 5 10 15
Pro Arg Glu Val Pro Asp Glu Tyr Glu Val Gly Ser Phe Met Glu Glu
20 25 30
Val Arg Gln Glu Leu Glu Asp Leu Glu Arg Ser Leu Thr Glu Glu Met
35 40 45
Ala Leu Arg Glu Pro Ala Ala Ala Ala Ala Ala Leu Leu Gly Gly Glu
50 55 60
Glu Ile
65
<210> 15
<211> 49
<212> PRT
<213> Mus musculus
<400> 15
Cys Ile Ile Asp Glu Asp Cys Gly Pro Thr Arg Tyr Cys Gln Phe Ser
1 5 10 15
Ser Phe Lys Tyr Thr Cys Gln Pro Cys Arg Asp Gln Gln Met Leu Cys
20 25 30
Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Ala Trp Gly His
35 40 45
Cys
<210> 16
<211> 12
<212> PRT
<213> Mus musculus
<400> 16
Thr Gln Lys Ala Thr Lys Gly Gly Asn Gly Thr Ile
1 5 10
<210> 17
<211> 77
<212> PRT
<213> Mus musculus
<400> 17
Cys Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln
1 5 10 15
Arg Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu
20 25 30
Leu Cys His Asp Pro Thr Ser Gln Leu Leu Asp Leu Ile Thr Trp Glu
35 40 45
Leu Glu Pro Glu Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu
50 55 60
Leu Cys Gln Pro His Ser His Ser Leu Val Tyr Met Cys
65 70 75
<210> 18
<211> 65
<212> PRT
<213> Mus musculus
<400> 18
Lys Pro Ala Phe Val Gly Ser His Asp His Ser Glu Glu Ser Gln Leu
1 5 10 15
Pro Arg Glu Ala Pro Asp Glu Tyr Glu Asp Val Gly Phe Ile Gly Glu
20 25 30
Val Arg Gln Glu Leu Glu Asp Leu Glu Arg Ser Leu Ala Gln Glu Met
35 40 45
Ala Phe Glu Gly Pro Ala Pro Val Glu Ser Leu Gly Gly Glu Glu Glu
50 55 60
Ile
65
Claims (17)
1.一种蛋白，所述蛋白由氨基酸序列编码，其中，所述氨基酸序列选自以下序列：
(a)所述氨基酸序列与SEQ ID NO：3所示的序列相比，具有80％以上且90％以下的同源性；优选的，具有82％以上且86％以下的同源性；更优选的，具有84％以上且85％以下的同源性。
2.如权利要求1所述的蛋白，其特征在于，所述核苷酸的序列为如SEQ ID NO：4所示的序列。
3.一种蛋白，所述蛋白由氨基酸序列编码，其中，所述氨基酸序列选自以下序列：
(b)所述氨基酸序列与SEQ ID NO：5所示的序列相比，具有75％以上且84％以下的同源性；优选的，具有77％以上且82％以下的同源性；更优选的，具有79％以上且80％以下的同源性；
可选的，所述氨基酸序列中氨基酸的数量比SEQ ID NO：5所示的氨基酸数量多1个。
4.如权利要求3所述的蛋白，其特征在于，所述氨基酸序列为如SEQ ID NO：6所示的序列。
5.一种核酸，所述核酸编码如权利要求1-4任一项所述的蛋白。
6.一种遗传修饰的载体，其特征在于，所述载体包含核苷酸序列，其中，所述核苷酸序列编码如权利要求1-4任一项所述的蛋白；可选的，所述核苷酸序列包含如SEQ ID NO：8所示的序列或如SEQ ID NO：10所示的序列。
7.一种表达蛋白的细胞，其含有如权利要求6所述的载体。
8.一种抗体或其片段，所述抗体或其片段竞争性地结合权利要求1-2任一项所述的蛋白；优选的，所述抗体为单克隆抗体。
9.一种制备蛋白的方法，所述方法包括以下步骤：
(s1)将权利要求6所述的载体转染表达蛋白的细胞；
(s2)收集步骤(s1)中的所述细胞表达的所述蛋白。
10.一种制备单克隆抗体的方法，所述方法包括以下步骤(s1)-(s3)或以下步骤(s1)-(s2)、(s4)：
(s1)将权利要求6所述的载体转染表达蛋白的细胞；
(s2)收集步骤(s1)中的所述细胞表达的蛋白；
(s3)用步骤(s2)获得的蛋白免疫动物，从被免疫动物的淋巴细胞中分离特异性识别所述蛋白的所述单克隆抗体；或者
(s4)用步骤(s2)获得的蛋白筛选单克隆抗体噬菌体展示文库，得到特异性识别所述蛋白的单克隆抗体。
11.一种制备融合蛋白的方法，所述蛋白由核苷酸编码，所述核苷酸至少包含第一核苷酸和第二核苷酸，所述方法包括以下步骤：
(i)将编码所述第一核苷酸和第二核苷酸的基因进行连接，形成融合蛋白基因；
(ii)将所述融合蛋白基因转染能够表达蛋白的细胞；
(iii)收集步骤(ii)中的所述细胞表达的所述蛋白；
其中，所述第一核苷酸的序列选自如SEQ ID NO：8所示的序列或如SEQ ID NO：10所示的序列；
所述第二核苷酸编码其他蛋白的基因；
可选的，所述核苷酸还包含连接序列。
12.一种融合蛋白的抗体或其抗体片段，其中，所述融合蛋白由权利要求10所述的方法制备得到。
13.一种融合蛋白，其中，所述融合蛋白由权利要求11所述的方法制备得到。
14.权利要求3-4任一项所述的蛋白、权利要求8所述的抗体或其片段、权利要求12所述的融合蛋白的抗体或其抗体片段或权利要求13所述的融合蛋白在制备用于治疗肌肉萎缩的药物中的用途。
15.一种药物组合物，所述药物组合物包含如权利要求3-4任一项所述的蛋白、权利要求8所述的抗体或其片段、权利要求12所述的融合蛋白的抗体或其抗体片段或权利要求13所述的融合蛋白。
16.一种治疗肌肉萎缩的方法，所述方法包括施用如权利要求3-4任一项所述的蛋白、权利要求8所述的抗体或其片段、权利要求12所述的融合蛋白的抗体或其抗体片段或权利要求13所述的融合蛋白。
17.一种筛选治疗肌肉萎缩的候选药物的方法，其中，所述方法包含如下步骤：
(s1)监测患有肌肉萎缩的实验动物中，如权利要求1-4任一项所述的蛋白的表达水平；
(s2)根据预定频率和用量及给药时间给予所述患有肌肉萎缩的实验动物候选药物；
(s3)再次监测患有肌肉萎缩的实验动物中，如权利要求1-4任一项所述的蛋白的表达水平；其中，
如果步骤(s3)中监测得到的如权利要求1-2任一项所述蛋白的表达水平低于在步骤(s1)中所监测的蛋白的表达水平；或者
如果步骤(s3)中监测得到的如权利要求3-4任一项所述蛋白的表达水平高于在步骤(s1)中所监测的蛋白的表达水平；则
所述候选药物为目标药物。
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