CN104560994A - 抑制酪氨酸酶活性的植物miRNA及其应用 - Google Patents
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Abstract
抑制酪氨酸酶活性的植物miRNA及其应用,序列如SEQ?ID?No.1所示。该miRNA可用于制备抑制酪氨酸酶活性的药物。本发明是天然植物中特异性分离提取的微小核苷酸,简单易行,成本相对低。该物质水解溶液可低温长期保存,无其他任何有毒物质残留,安全无污染。
Description
技术领域
本发明涉及微小核苷酸领域,具体来说是一种抑制酪氨酸酶活性的植物miRNA及其应用。
背景技术
微小核糖核酸(miRNA)是一类长约22个核苷酸的单链非编码RNA小分子。成熟的miRNA会选择与RNA诱导沉默复合体(RISC)结合,形成miRISC复合物来识别mRNA。这些miRNA通常靶向一个或多个基因的mRNA,并且与mRNA 3’非编码区域(3’-UTR)以碱基互补配对的方式特异性结合。从而以转录后水平抑制或者降解目标基因mRNA的方式实现对基因表达的调控,在多种生理和病理过程中发挥重要作用。在已发现的3000多个miRNA中,大部分在基因表达的调控中扮演关键角色,据估计动植物体内约2/3的基因受到某一个或多种miRNA的调控。miRNA区别于普通RNA的一个重要特点就是它的高度稳定性,在高温、长期保存以及RNA酶存在的环境中都不易被降解。这种稳定性是miRNA可以用作药物或者化妆品成分的一个重要前提。从miRNA被发现以来,内源性miRNA的研究一直占miRNA研究领域主导地位,其中血清中miRNA以及分泌型miRNA(secreted miRNA)是近年研究的热点。而最近已有研究表明植物miRNA可以通过饮食的方式进入到动物体内,并且跨界调控动物的基因表达。这是外源性miRNA可以调控动物体生理过程的重要证据。这一跨时代研究成果,也是我们本次研究直接的理论基础。
酪氨酸酶(Tyrosinase,TYR),广泛存在于生物界中,是黑色素生物合成的关键酶和限速酶,也是目前唯一明确的黑色素代谢相关酶。研究表明酪氨酸酶是一种铜结合蛋白,TYR的突变可能会导致其断裂与铜的结合而丧失催化活性。在酶促反应中的酪氨酸酶有三种形态:氧化态、还原态和脱氧态三种形式存在。其中氧化态酶可以与单酚或二酚结合作用底物,得到醌,H2O和脱氧态酶,脱氧态酶会再结合氧形成氧化态酶;而还原态的酶只能与二酚结合,不能结合单酚。在人体黑色素形成的过程中,酪氨酸酶首先催化L-酪氨酸羟基转变成L-多巴,并同时氧化L-多巴形成多巴醌。而醌类物质易于多聚化,通过与细胞内蛋白的结合最终形成黑色或棕色的黑色素。人皮肤颜色的深浅主要由黑色素细胞的数量以及细胞合成黑色素的能力决定,而酪氨酸酶的蛋白表达水平和酶活性与细胞黑色素的合成能力密切相关。降低皮肤细胞中酪氨酸酶的含量,将对减少黑色素形成、美白皮肤等产生直接的作用。
传统的酪氨酸酶抑制剂(Tyrosinase Inhibitor,TI),一般是从动植物当中提取的多肽类,糖苷类,黄铜,多酚类和醛类等。这些抑制剂一般认为可以与L-酪氨酸羟基或L-酪氨酸多巴竞争性地结合,达到抑制酪氨酸酶活性的目的。但是这些抑制剂不仅来源单一,而且由于提 取工艺的水平有限,成品中会一定量的有害杂质。再加上提取的成本相对较高,使得这些抑制剂的批量生产和推广受到限制。
发明内容
解决的技术问题:本发明提供一种抑制酪氨酸酶活性的植物miRNA及其应用。具体的说,所涉及的抑制酪氨酸酶表达的微小核糖核酸,可从多种天然植物中特异性分离提取,也可以用人工合成的方式得到,纯度高、得率高、成本低,抑制效果好。
技术方案:抑制酪氨酸酶活性的植物miRNA,序列如SEQ ID No.1所示。
序列如SEQ ID No.1所示的miRNA在抑制酪氨酸酶活性中的应用。
序列如SEQ ID No.1所示的miRNA在制备抑制酪氨酸酶活性药物中的应用。
抑制酪氨酸酶活性药物,有效成分为序列如SEQ ID No.1所示的miRNA。
序列如SEQ ID No.1所示的miRNA在抑制酪氨酸酶活性中的应用。
有益效果:1)本发明是在常见植物中特异性分离提取的天然微小核苷酸,提取纯度高,适于批量生产,成本相对低。2)本发明的固态粉末或水解溶液均可在低温较长期保存,无任何有害物质残留,适合在药物和护肤产品中应用。3)本发明可明显降低细胞中酪氨酸酶的含量,其效果优于传统的酪氨酸酶活性抑制剂。
附图说明
图1为生物信息学软件RNAhybrid计算得出的上述miR394a与酪氨酸酶的mRNA 3’-UTR区域相互作用关系示意图,以及产生转录后抑制的作用机制;
图2为载体图谱:pMIR-REPORTTM miRNA表达报告基因载体;
图3为miR394a与野生型(WT)TYR 3’-UTR以及突变体(Mut)TYR 3’-UTR相互作用关系验证的荧光素酶活性检测结果图;
图4为miR394a对酪氨酸酶蛋白表达水平调控效果的蛋白印记(western blotting)结果图。
具体实施方式
本发明是一种酪氨酸酶抑制剂,该抑制剂中含有微小核苷酸miR394a。此处,所述“miR394a”是指下面的核酸序列:UUGGCAUUCUGUCCACCUCC。
以下,通过实施例进一步详细说明本发明,但本发明并不限于这些实施例。
实施例1:
1、序列比对
对所述miR394a不特别限定植物物种,在对其进行物种间序列保守性对比发现miR394a在种植物中高度保守,序列比对结果如表1。
2、序列合成及提取方法
关于获得miR394a的方法没有特别限制,可以通过从植物中提取得到,也可以通过人工合成的方式得到。其中,在从植物中提取的方法,对作为原料的植物没有具体要求。另外,可以只使用一种,或者同时用两种或两种以上的植物来提取miR394a。对于大豆这种植物,优选原料组织为叶和种子;对于马铃薯,则建议用块茎和叶子。
2.1从植物中提取miR394a方法步骤
1).匀浆处理
取50-100mg植物组织于液氮中研磨,保持研钵中有液氮(如果没有液氮也可以直接加1mL的裂解液PL后匀浆。组织样品容积不能超过PL容积的10%)。
2).转移样品至1.5mL RNase free离心管中,加入1mL的Trizol颠倒混匀,在65℃条件下孵育10分钟(期间颠倒混匀几次)以使核蛋白体完全分解。
3).室温条件下12,000rpm离心5分钟,取上清转入一个新的2mL RNase free离心管中。
4).加入等体积事先预冷的异丙醇,颠倒混匀,4℃12,000rpm离心5分钟。
5).去除上清,加入100μL RNase-free H2O重新溶解沉淀。
6).加入200μL氯仿,涡旋混匀,放置2-3分钟。
7).于4℃12,000rpm离心10分钟,样品会分成三层:下层有机相,中间层和上层无色的水相,RNA存在于水相中。把水相(约700μL)转移到新的1.5mL RNase free离心管中,进行下一步操作。
8).加入0.6倍体积70wt.%乙醇,颠倒混匀。
9).于4℃13,000rpm离心5分钟。
10).弃上清将沉淀晾干直至变透明。
11).用50μL DEPC H2O溶解沉淀。
12).测定OD值确定总RNA浓度。
13).用过滤杂交方法亲和纯化总RNA中的miR394a。
3、生物信息学预测靶位点相互作用
图1显示用生物信息学软件RNAhybrid计算得出的上述miR394a与所述酪氨酸酶(TYR)的mRNA3’-非编码区(3’-UTR)相互作用从而产生转录后抑制作用机制关系。
4、细胞培养
将MCF-7细胞(中国科学院上海生命科学研究院细胞资源中心)在含有10wt.%胎牛血清(FBS)的DMEM中,于37℃下培养。
5、萤光素酶分析
我们利用人工合成的方式得到一段包含miR394a靶位点的野生型TYR 3’-UTR片段序列,以及靶位点突变后的TYR 3’-UTR片段序列,然后此产物被嵌入一个荧光素酶的报告基因p-MIR-report(Ambion公司)的3’-UTR端(图3)。测序结果显示质粒构建是成功的。
具体步骤为:
(1)构建荧光素酶报告质粒
合成一段miR394a靶位点序列,然后通过DNA酶连接反应将其插入到荧光素酶报告基因的3’非编码区,之后把构建得到的荧光素酶报告质粒转化进入感受态大肠杆菌中,再挑选单克隆菌落进行测序验证构建得到的荧光素酶报告质粒的正确性。具体操作为:
I)利用HindIII和SpelI内切酶酶切(37℃,4小时)荧光素酶报告质粒的3’非编码区,再从琼脂糖中回收目的酶切产物;
II)通过T4DNA连接酶分别催化miR394a靶位点与酶切产物的连接(16℃,过夜)。miR394a靶位点序列是由invitrogen公司合成提供,其140个核苷酸的片段为:5’-CTGTAAAGACCATTTGCAAAATTGTAACCTAATACAAAGTGTAGCCTTCTTCCAACTCAGGTAGAACACACCTGTCTTTGTCTTGCTGTTTTCACTCAGCCCTTTTAACATTTTCCCCTAAGCCCATATGTCTAAGGAAA-3’;
III)把构建好的荧光素酶报告质粒转化进入感受态大肠杆菌中然后铺在含有100μg/mL抗生素(Amp)固体培养基板上37℃过夜培养。
IV)挑选单克隆菌落,扩大培养,再从中提取重组质粒进行测序验证。
实验中所用质粒用Endo-free Midi Kit(OMEGA BIO-TEK)试剂盒提取,提取步骤按照说明书步骤进行,最后一步以相同体积三蒸水代替缓冲液洗下质粒。
(2)质粒转染
利用lipofectamine2000(invitrogen公司)及按照厂商提供的标准试验流程将荧光素酶报告质粒转染到细胞膜内。具体操作为:
分别将MCF-7细胞种入24孔板中,以含有10%(v/v)牛血清和1%(v/v)的双抗培育。在其生长密度达到70%时,给细胞换液,将正常细胞培养液换为Opti-MEM I培养基(每孔400μL),再加入100μL的miR394a-荧光素酶报告质粒(0.5μg),β-gal(0.3μg)质粒和lipofectamine2000(2μL)混合液;往对照孔内加入等量的miR394a荧光素酶报告质粒,β-gal质粒和lipofectamine2000混合液。
(3)收集细胞及荧光素酶活性检测
主要分为两个部分:(1)目的蛋白-荧光素酶活性检测(2)内参-β-gal的测定。具体检测方法为:
(a)荧光素酶活性检测:每孔加入100μL 1X Cell CμLture Lysis Reagent(由5×稀释而来),剧烈摇动10min。用枪头或刮板尽可能多地将细胞残余物与溶液一起收集到2mL离心管中,液氮冻融两次。取20μL细胞溶液到1.5mL离心管中,加入100μL荧光底物(luciferase assay system中溶剂与荧光物质混合而成),涡旋振荡数秒后,放入检测仪ModμLus single tube mμLtimode reader检测,读数记录。
(b)β-gal测定:溶液体系组成为1.5μL 100×Mg2+溶液,33μL 1×ONPG,100.5μL磷酸钠溶液,15μL细胞溶液。将混合溶液在37℃放置30min,至黄色出现,加入250μL 1M碳酸钠溶液终止反应。吸取100μL溶液加入96孔细胞培养板,在420nm处测定吸光度。
(4)数据分析
数据分析为相对比较方法,β-gal为内参。实验结果证明miR394a直接识别并作用于TYR的3’-UTR:为证明miR394a通过直接识别TYR的3’-UTR使蛋白的表达受抑制,我们将TYR的3’-UTR构建到荧光素酶报告基因的3’端,然后在细胞系中过表达miR394a,若荧光素酶活性受抑制,说明miRNA是直接作用于TYR的3’-UTR的。由图3可知,过表达miR-164可以显著抑制荧光素酶活性,而过表达ncRNA没什么作用,因此miR-164可以特异性地识别TYR的3’-UTR并调控TYR的表达。
6、蛋白印记法(Western blotting)检测miR394a对酪氨酸酶(TYR)蛋白表达水平的影响
实验步骤为:(1)总蛋白的提取。按照6孔板每孔加入150-250微升裂解液的比例分别向转染了miR394a和ncRNA的MCF-7细胞(中国科学院上海生命科学研究院细胞资源中心) 中加入细胞裂解液(50mM Tris-Cl,150mM NaCl,0.02wt.%NaN3,1wt.%NP-40,使用前加入0.5μg/mL蛋白酶抑制剂),充分吹打并转移至Eppendorf管中,离心去除不溶物,把上清分装成小份保存于-80℃备用。注意细胞裂解液加入量不宜过多,否则造成蛋白浓度过低,一般细胞加入100μL,组织大于200μL,线粒体50μL。(2)用BCA方法测定总蛋白或线粒体蛋白的浓度。(3)取100μg总蛋白进行常规Western blotting分析。堆积胶配方:H2O 2.92mL,0.5M Tris-HCl(pH 6.8)1.25mL,10wt.%SDS 50μL,Acryl/Bis 0.8mL,10wt.%APS 25μL,TEMED 2.5μL。10%分离胶配方:H2O 4mL,1.5M Tris-HCl(pH 8.8)2.5mL,10wt.%SDS 100μL,Acryl/Bis 3.3mL,10wt.%APS 66.7μL,TEMED 5μL。样品煮沸变性后上样,20mA电泳至loading buffer的染料前沿到达底部,小心剥胶,蛋白条带电转移至PVDF膜上,5%牛奶中室温封闭一小时,按体积比1:1000的一抗在4℃孵育过夜,第二天按1:2000在室温孵育二抗2小时,再加入荧光底物,置暗房内用胶片显影、定影。这里使用的一抗为小鼠抗TYR单克隆抗体(Santa Cruz公司);二抗为羊抗小鼠IgG(Santa Cruz公司)。
图4为miR394a调控TYR表达的蛋白印记(western blotting)结果,用ImageJ软件分析测定转染ncRNA灰度值为22319;过表达miR394a灰度值为4204,即TYR表达量降低了81%。而在加入了miR394a的抑制剂后,TYR的表达量上升了35%左右。这表明miR394a的确可以在动物细胞中调控TYR的表达水平。
7.制成潜在药物剂型说明
可将从植物中纯化的miR394a或者人工合成的miR394a或者或者miR394a的过表达质粒与不含RNA水解酶高压灭菌后的甘油混合,辅以其他芳香剂,制成液剂、乳剂、乳膏剂、固融体油膏剂及固融体棒型剂等。
上述具体实施方式不以任何形式限制本发明的技术方案,凡是采用等同替换或等效变换的方式所获得的技术方案均落在本发明的保护范围。
SEQUENCE LISTING
<110> 南京大学
<120> 抑制酪氨酸酶活性的植物miRNA及其应用
<130> F1404856
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> RNA
<213> 人工序列
<400> 1
uuggcauucu guccaccucc 20
<210> 2
<211> 140
<212> DNA
<213> 人工序列
<400> 2
ctgtaaagac catttgcaaa attgtaacct aatacaaagt gtagccttct tccaactcag 60
gtagaacaca cctgtctttg tcttgctgtt ttcactcagc ccttttaaca ttttccccta 120
agcccatatg tctaaggaaa 140
Claims (5)
1.抑制酪氨酸酶活性的植物miRNA,其特征在于序列如SEQ ID No.1所示。
2.序列如SEQ ID No.1所示的miRNA在抑制酪氨酸酶活性中的应用。
3.序列如SEQ ID No.1所示的miRNA在制备抑制酪氨酸酶活性药物中的应用。
4.抑制酪氨酸酶活性药物,其特征在于有效成分为序列如SEQ ID No.1所示的miRNA。
5.序列如SEQ ID No.1所示的miRNA在抑制酪氨酸酶活性中的应用。
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