CN110028570A - 一种肌肉素的表达方法及其在代谢疾病中的应用 - Google Patents
一种肌肉素的表达方法及其在代谢疾病中的应用 Download PDFInfo
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Abstract
本发明公开了一种肌肉素蛋白及一段基于其序列的多肽的表达及其在代谢疾病中的应用。本发明通过肌肉素基因的克隆、重组质粒的构建、重组蛋白的表达制备、重组蛋白的纯化等一系列步骤获得了全长肌肉素重组蛋白(Mus‑F)。同时构建了截短的肌肉素核心多肽—Musclin 33(Mus 33)肽。本发明对肌肉素Mus‑F多肽与Mus33核心多肽在代谢疾病中的作用进行了研究,发现肌肉素Mus‑F及Mus 33核心多肽能够显著地减轻高脂喂养C57小鼠的体重,同时能抑制高脂引起的肝脏及脂肪组织中脂质积累,明显降低小鼠血清中甘油三酯水平。本发明的肌肉素及核心多肽可以用于制备治疗肥胖、2型糖尿病、非酒精性脂肪肝等代谢疾病的药物。
Description
技术领域
本发明属于生物药学领域,更具体地,涉及一种重组蛋白的表达纯化及其在糖脂代谢治疗中的应用。
背景技术
糖脂代谢是为细胞提供能量及其所需生物大分子中间体的重要生命过程。糖脂代谢不但响应生物及环境刺激,还可充当传感器,在各种生理条件下起到平衡机体代谢系统的作用。糖脂代谢失衡会导致肥胖、2型糖尿病、非酒精性脂肪肝等多种代谢性疾病,严重威胁人类健康。
肌肉的分泌作用被发现后,肌肉因子在代谢疾病中的多种作用相继被揭示。肌肉素是2004年发现的一种肌肉分泌因子,因其mRNA仅在肌肉中表达,故命名为肌肉素。肌肉素由130个氨基酸组成,其N端含25个氨基酸组成的信号肽,中间含有一段与利尿钠肽(natriuretic peptide,NP)高度同源的区域和假定的丝氨酸蛋白酶切割位点—KKKR位点,紧接着该位点之后的一段33氨基酸序列,在大、小鼠及人类中高度同源,被认为是肌肉素核心序列,即Musclin 33肽。
肌肉素是一种运动依赖型分泌的肌肉因子,血清肌肉素水平在运动后可升至正常水平的2倍,发挥促进骨骼肌线粒体合成、增强运动耐受性的功能[1]。体外研究发现肌肉素可通过降低PPARγ和LXRα的mRNA水平从而降低细胞的葡萄糖摄取,提示肌肉素很可能参与糖脂代谢[2,3]。在STZ诱导的1型糖尿病小鼠中,肌肉素低表达[2];在肥胖的胰岛素抵抗KKAy小鼠中,肌肉素显著性高表达[2];2型糖尿病患者的肌肉素表达水平升高[2,4];由此推测肌肉素与代谢疾病密切相关。
目前对肥胖、2型糖尿病、非酒精性脂肪肝等多种代谢性疾病的治疗尚缺乏有效的药物,尽管研究人员对于肌肉素能否作为代谢疾病的治疗靶点尚缺乏了解,对其深入研究有望为相关代谢性疾病的发病机制和临床防治提供一定的理论依据,开发其作为治疗代谢疾病的药物显得非常有前景。参考文献
1.Subbotina,E.,A.Sierra,Z.Zhu,Z.Gao,S.R.Koganti,S.Reyes,E.Stepniak,S.A.Walsh,M.R.Acevedo,C.M.Perez-Terzic,D.M.Hodgson-Zingman,and L.V.Zingman,Musclin is an activity-stimulated myokine that enhances physicalendurance.Proc Natl Acad Sci U S A,2015.112(52):p.16042-7.
2.Nishizawa,H.,M.Matsuda,Y.Yamada,K.Kawai,E.Suzuki,M.Makishima,T.Kitamura,and I.Shimomura,Musclin,a novel skeletal muscle-derived secretoryfactor.J Biol Chem,2004.279(19):p.19391-5.
3.Liu,Y.,X.Huo,X.F.Pang,Z.H.Zong,X.Meng,and G.L.Liu,Musclin inhibitsinsulin activation of Akt/protein kinase B in rat skeletal muscle.J Int MedRes,2008.36(3):p.496-504.
4.Chen,W.J.,Y.Liu,Y.B.Sui,B.Zhang,X.H.Zhang,and X.H.Yin,Increasedcirculating levels of musclin in newly diagnosed type 2diabetic patients.DiabVasc Dis Res,2017.14(2):p.116-121.
发明内容
为实现上述目的,本发明提供了一种融合蛋白及其在糖脂代谢中的应用。实现本发明的方法是:
本发明提供的这种具有治疗肥胖、2型糖尿病或非酒精性脂肪肝代谢疾病功效的蛋白,其氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示
SEQ ID NO:1所示氨基酸序列的重组蛋白的表达方法,包括以下步骤:
(1)重组载体质粒的构建:将与SEQ ID NO:1所示氨基酸序列对应的cDNA序列插入原核表达载体中,构建重组质粒,所述的cDNA序列如SEQ ID NO:3所示;
(2)目的蛋白的诱导:将鉴定正确的重组表达载体质粒转入宿主细胞,然后诱导表达重组蛋白;
(3)蛋白的纯化及浓缩:宿主细胞采用超声破碎的方式获取蛋白,而后将蛋白上清进行纯化。
SEQ ID NO:1所示的氨基酸序列是添加了蛋氨酸的肌肉素26-130AA序列。需要强调的是,由于载体构建过程中,需要插入起始密码子,故所表达氨基酸序列,即SEQ ID NO:1为蛋氨酸+肌肉素26-130AA序列。
步骤(1)中所述的表达载体可以为pet15b。
步骤(2)中所述的宿主细胞可以为E.coli,所述的诱导表达的诱导剂可以为IPTG(异丙基-β-D-硫代半乳糖苷)。
优选地,步骤(3)中的蛋白纯化可以是通过镍柱亲和层析进行纯化。
进一步地,蛋白进行浓缩及溶液置换是通过超滤管装置。
本发明所述的这种肌肉素重组蛋白的表达方法制备的蛋白在制备用于治疗肥胖、2型糖尿病或非酒精性脂肪肝代谢疾病药物中的应用。
本发明所述的这种肌肉素重组蛋白的表达方法制备的蛋白在制备用于治疗肥胖的保健产品中的应用。
SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的蛋白在制备用于治疗肥胖、2型糖尿病或非酒精性脂肪肝代谢疾病药物中的应用。
SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的蛋白在治疗肥胖的保健产品中的应用。
本发明的有益效果是:
1、本发明首次提供了全长无信号肽肌肉素的表达体系。采用大肠杆菌表达系统,利用简单快捷的方式,具有表达效率高、成本低、简单易操作等特点。为各种蛋白表达的研发工作提供了一定的基础。
2、本研究发现全长肌肉素Mus-F蛋白及其截短多肽Mus 33肽能够显著地减轻高脂喂养C57小鼠的体重,同时能抑制高脂引起的肝脏及脂肪组织中脂质积累,其中Mus-F效果更好,并能明显降低小鼠血清中甘油三酯水平。本发明的肌肉素为制备治疗肥胖、2型糖尿病、非酒精性脂肪肝等多种代谢性疾病的药物奠定了理论基础,为开发出更加有效的治疗药物提供了新思路。
附图说明
图1是全长肌肉素基因PCR琼脂糖凝胶图;
图2是全长肌肉素蛋白表达制备及纯化图;
图3是动物实验设计图;
图4是两种肌肉素给药后各组小鼠的体重变化(图4A)及摄食量统计(图4B);
图5是两种肌肉素给药后各组小鼠的组织解剖重量统计;
图6是两种肌肉素给药后各组小鼠的血清甘油三酯及总胆固醇的测试:
图6(A)为对注射两种肌肉素小鼠的血清进行TG(甘油三脂)含量检测;图6(B)为TC(总胆固醇)含量检测;
图7是两种肌肉素给药后各组小鼠的肝脏甘油三酯及总胆固醇的测试:
图7(A)为两种肌肉素给药后各组小鼠的肝脏甘油三酯测试;
图7(B)为两种肌肉素给药后各组小鼠的肝脏总胆固醇的测试;
图8是两种肌肉素给药后各组小鼠的肝脏油红O染色测试;
图9是两种肌肉素给药后各组小鼠的白色脂肪组织苏木精-伊红染色测试。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。此外,下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
1、全长无信号肽肌肉素基因的克隆
从GenBank中查找到肌肉素基因的cDNA序列,从中挑取26AA-130AA氨基酸序列对应的cDNA序列。从肌肉组织中获取肌肉cDNA,以其为模板,利用PCR技术克隆扩增出肌肉素26AA-130AA氨基酸序列对应的cDNA序列。
正向引物序列为:5′attcatatgttctctgtggacttagcat3′;
反向引物序列为:5′attggatcctcagcctctggaactggagag3′。
分别引入酶切位点Nde 1、BamH 1。PCR反应条件为:94℃预变性5min,94℃变性30s,60℃退火30s,72℃延伸30s,进行35个循环,72℃延伸5min。PCR扩增获得目的片段,并进行胶回收。
2、原核表达载体的构建
(1)PCR产物及原核表达载体pet15b分别用Nde 1、BamH 1进行双酶切3h。
(2)胶回收,而后用T4连接酶连接目的片段及载体。
(3)将连接产物转化于感受态细胞DH5α中,利用氨苄平板筛选重组子,12-16h后挑取单克隆。
(4)单克隆扩增,提取质粒,进行酶切鉴定,酶切鉴定成功者进行测序。测序分析获得正确的重组质粒pet15b Musclin。
3、重组蛋白的表达
(1)将待表达的质粒用BL21感受态进行转化,接种于氨苄培养基过夜增菌。37℃摇床培养。
(2)过夜培养物接种于大体积新鲜培养基中,当用紫外分光光度计测量OD600nm=0.6时,加入IPTG=0.2mM,而后继续诱导8h。
(3)取1ml培养物用100μl PBS/灭菌水超声,8%功率,超5s,停5s,共超声5次。实验组1ml培养物超声后作为全菌液;另1ml培养物超声后12000rpm,室温离心1min,上清为可溶性蛋白,沉淀为包涵体蛋白。包涵体蛋白再用100μl PBS/灭菌水超声一次。
(4)超声后的蛋白样品分别加入100μl 2*SDS loading buffer,95℃变性10min,收蛋白样品。
(5)进行SDS-PAGE电泳,考马斯亮蓝染色鉴定目的蛋白的分布情况。鉴定表明该重组蛋白在上清及包涵体中均有表达。
4、重组蛋白的制备纯化
(1)6000rpm,78min,4℃离心收菌。
(2)用0.1体积的重悬液(20mM TrisHcl,pH=8.0,5mM咪唑、500mM Nacl)重悬。
(3)冰浴下超声破菌,超声30min,功率25%(总功率960W)。破菌后加入蛋白酶抑制剂PMSF(50X,终浓度为1mM)
(4)10000g,10min,4℃,收集上清,弃沉淀。取上清,用0.22um滤头过滤。
(5)使用重悬液洗镍柱,洗2次。后加入已过滤的蛋白上清,4℃结合2h。去上清,用蛋白洗涤液(20mM TrisHcl,pH=8.0,20mM咪唑、500mM Nacl)洗涤3次镍柱。
(6)用蛋白洗脱液(20mM TrisHcl,pH=8.0,500mM咪唑、500mM Nacl)洗脱下目的蛋白。
(7)将洗脱下的蛋白用超滤管置换浓缩,置换溶液为PBS。
5、动物实验设计
本发明实施例中使用的C57BL/6小鼠购买于湖北省实验动物中心。在正式实验进行前我们进行了本发明的两种多肽注射剂量预实验,不同剂量两种多肽注射后小鼠各方面生理指标均正常,认为这两种多肽是安全的。
小鼠高脂食物喂养是一个经典的模拟2型糖尿病的小鼠模型。在注射肌肉素多肽前,将年龄在4周大的C57小鼠分为两组:正常食物喂养组(NC组)8只、高脂食物喂养组(HFD组)18只。连续高脂食物喂养8周后,检测到HFD组小鼠相较于NC组小鼠体重出现显著性差异。
将HFD组18只小鼠按体重平均分成3组:对照组注射生理盐水(HFD+Veh组)、实验组分别注射两种多肽:全长肌肉素(HFD+Mus-F组)及截短肌肉素33肽(HFD+Mus33组);NC组8只小鼠注射生理盐水(NC+Veh组)。正式注射Mus-F及Mus33多肽,选取皮下注射方式,每次按照25ug剂量注射,每天两次,共注射7天,每天对小鼠的体重及摄食量进行监测。注射7天后,解剖小鼠,收集小鼠血液,用于后续生化指标检测;称重并收取小鼠的肝脏、白色脂肪、棕色脂肪、骨骼肌等,用于后续病理检测分析。动物实验设计如图3。
6、两种肌肉素给药一周后各组小鼠的体重变化及摄食量统计
在注射两种肌肉素一周后,对小鼠体重相较于注射前的体重变化(△BW)进行统计计算:NC+Veh组、HFD+Veh组小鼠△BW都是升高的,HFD+Mus33组及HFD+Mus-F组小鼠△BW相较于HFD+Veh组有显著性下降(图4A);同时对各组小鼠的摄食量(Food intake)进行统计,HFD各组小鼠摄食量无显著性差异(图4B),说明短期内注射两种肌肉素可以显著抑制高脂喂养的C57小鼠的体重增长,且抑制效果不来自饮食摄取量改变。
7、两种肌肉素给药一周后各组小鼠的组织解剖重量统计
在注射两种肌肉素一周后,解剖小鼠,通过对小鼠的肝脏、白色脂肪、棕色脂肪、骨骼肌等组织进行称重统计:相较HFD+Veh组,HFD+Mus-F组小鼠白色脂肪(eWAT、iWAT)重量有显著性降低,HFD+Mus33组小鼠白色脂肪重量也有一定程度的降低,但各组小鼠的肝脏(Liver)、棕色脂肪(BAT)、骨骼肌各个部分(Gastroc、Soleus、TA、EDL)重量都无显著性差异(图5)。说明短期内注射两种肌肉素可以降低高脂喂养的C57小鼠脂肪积累。
8、两种肌肉素给药一周后各组小鼠的血清甘油三酯及总胆固醇的测试
对注射两种肌肉素小鼠的血清进行TG(甘油三脂)、TC(总胆固醇)含量检测:小鼠在注射两种肽前的血清TG和TC含量提示HFD喂养小鼠已处于高血脂状态;值得注意的是,全长肌肉素能够明显降低血清TG含量,但肌肉素33肽却无此作用(图6A);HFD下,相较于对照组,注射两种肽对血清TC含量无影响(图6B)。提示着肌肉素与其高度保守的33肽区域有着对肝脏和白色脂肪相同的作用,但对血脂的降低有不同的影响。
9、两种肌肉素给药后各组小鼠的肝脏甘油三酯及总胆固醇的测试
对注射两种肌肉素小鼠的肝脏进行TG、TC含量检测:相较HFD+Veh组,注射两种肽能够显著性降低HFD组小鼠肝脏中脂滴积累(图7A),但对肝脏中TC含量无影响(图7B)。
10、两种肌肉素给药后各组小鼠的肝脏油红O染色测试
肝脏切片Oil red O染色显示,在HFD喂养条件下,两种多肽注射组小鼠肝脏中的脂质积累显著降低(图8)。提示两种多肽能够显著抑制高脂引起的肝脏中脂质积累。
11、两种肌肉素多肽给药后各组小鼠的白色脂肪组织苏木精-伊红染色测试
白色脂肪石蜡切片的苏木精-伊红(H&E)染色显示,在HFD喂养条件下,两种多肽注射组小鼠eWAT及iWAT细胞面积显著减小(图9)。提示两种多肽能够显著抑制高脂引起的脂肪组织中脂质积累。
本领域的技术人员容易理解,以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
序列表
<120> 肌肉素的表达方法及其在代谢疾病中的应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 106
<212> PRT
<213> Mus musculus
<400> 1
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Gln Ser Pro Pro Thr Ala Arg Glu Glu Lys Ser Ala Thr Glu Leu Ser
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Ala Lys Leu Leu Arg Leu Asp Asp Leu Val Ser Leu Glu Asn Asp Val
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Phe Glu Thr Lys Lys Lys Arg Ser Phe Ser Gly Phe Gly Ser Pro Leu
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Asp Arg Leu Ser Ala Gly Ser Val Glu His Arg Gly Lys Gln Arg Lys
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Ala Val Asp His Ser Lys Lys Arg Phe Gly Ile Pro Met Asp Arg Ile
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Gly Arg Asn Arg Leu Ser Ser Ser Arg Gly
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<210> 2
<211> 33
<212> PRT
<213> Mus musculus
<400> 2
Ser Phe Ser Gly Phe Gly Ser Pro Leu Asp Arg Leu Ser Ala Gly Ser
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Val Glu His Arg Gly Lys Gln Arg Lys Ala Val Asp His Ser Lys Lys
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Arg
<210> 3
<211> 321
<212> DNA
<213> Mus musculus
<400> 3
atgttctctg tggacttagc atcacaggag tttggaacag caagcttgca gtctccaccc 60
acagccagag aagagaagtc agccactgag ctttcggcta agctcctgcg tcttgatgat 120
ctggtgtcct tagagaatga cgtatttgag accaagaaaa agagaagctt ctctggcttt 180
gggtctcccc ttgacagact ctcagctggg tctgtagagc atagagggaa acaaaggaaa 240
gcagtagatc attcaaaaaa gcggtttggt attcccatgg atcggattgg tagaaaccgg 300
ctctccagtt ccagaggctg a 321
Claims (9)
1.具有治疗肥胖、2型糖尿病或非酒精性脂肪肝代谢疾病功效的蛋白,其氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
2.如SEQ ID NO:1所示氨基酸序列的重组蛋白的表达方法,包括以下步骤:
(1)重组载体质粒的构建:将与SEQ ID NO:1所示氨基酸序列对应的cDNA序列插入原核表达载体中,构建重组质粒,所述的cDNA序列如SEQ ID NO:3所示;
(2)目的蛋白的诱导:将鉴定正确的重组表达载体质粒转入宿主细胞,然后诱导表达重组蛋白;
(3)蛋白的纯化及浓缩:宿主细胞采用超声破碎的方式获取蛋白,而后将蛋白上清进行纯化。
3.根据权利要求2所述的方法,其特征在于,步骤(1)中所述的表达载体为pet15b。
4.根据权利要求2所述的方法,其特征在于,步骤(2)中所述的宿主细胞为E.coli,所述的诱导表达的诱导剂为IPTG(异丙基-β-D-硫代半乳糖苷)。
5.根据权利要求2所述的方法,其特征在于,步骤(3)中的蛋白纯化是通过镍柱亲和层析进行纯化。
6.权利要求2-5中任意一项所述方法制备的蛋白在制备用于治疗肥胖、2型糖尿病或非酒精性脂肪肝代谢疾病药物中的应用。
7.权利要求2-5中任意一项所述方法制备的蛋白在制备用于治疗肥胖的保健产品中的应用。
8.SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的蛋白在制备用于治疗肥胖、2型糖尿病或非酒精性脂肪肝代谢疾病药物中的应用。
9.SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的蛋白在治疗肥胖的保健产品中的应用。
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