CN113881666B - 干扰p300蛋白表达的siRNA及应用 - Google Patents
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Abstract
本发明涉及神经系统药物领域，公开了干扰p300蛋白表达的siRNA及应用，所述siRNA的序列如SEQ ID No.1所示。本发明首次揭示了β‑羟基丁酰化修饰是一种新的调控CaMKII活性的机制，于体外实验中发现酰基转移酶p300可催化CaMKII发生Kbhb导致其活性降低，本发明由此研发的siRNA可通过干预p300的表达而导致CaMKII的Kbhb减少，进而增强CaMKII的活性，起到改善认知和记忆功能障碍的作用，为糖尿病及与CaMKII活性下降相关的疾病的治疗提供了新的方向。
Description
技术领域
本发明涉及神经系统药物领域，具体涉及干扰p300蛋白表达的siRNA及应用。
背景技术
CBP和p300(也叫EP300)是哺乳动物里最主要的乙酰转移酶，因为其结构相似，功能冗余，所以常常被一起称为CBP/p300，编码p300蛋白的基因是EP300。CaMKII是一个Ser/Thr特异性的蛋白激酶，主要包括催化域、调节域和连接域三个结构域，由Ca2+/CaM激活。在正常生理状态下，由于缺少Ca2+/CaM，CaMKII通过催化域与调节域的相互作用，使其处于自身抑制状态；当在突触可塑性诱导过程中，神经元突触后膜内Ca2+浓度升高后会激活CaMKII，Ca2+/CaM与CaMKII调节域的结合会打破CaMKII的自身抑制状态，使其变为一种开放式的结构，导致CaMKII的活性显著升高。若Ca2+浓度回落到基础水平，CaMKII的活性则会恢复到正常水平，因此CaMKII的活性可作为观察钙离子信号的探测器，以及标记突触可塑性中“记忆分子”的作用，近几十年来大量研究已证实CaMKII的活性在突触可塑性和记忆的形成过程中发挥着重要作用。
酰化修饰(Acylation)是蛋白质翻译后修饰的一种，通过对蛋白质在翻译后的酰化修饰，从而调节蛋白质的活性、定位、折叠、以及蛋白与其他生物大分子间(包括蛋白、核酸、脂质等)的相互作用。在众多酰化修饰中，赖氨酸3-羟基丁酰化(lysineβ-hydroxybutyrylation,Kbhb)修饰的供体分子是β-羟基丁酰辅酶A，其前体物质为β-羟基丁酸，也是酮体的主要成份之一。酮体生成是以脂肪酸β-氧化生成的乙酰CoA为原料，在肝线粒体的酮体合成酶中生成。经典的生化理论认为，酮体的主要功能是机体在饥饿时的替代能量，经由肝细胞释放入血后，为脑、心脏等其他组织所利用。但在病理状态下如糖尿病引起的糖类物质利用受阻时，也会诱导酮体的产生量增多，持续的高酮血症会导致包括神经系统及消化道在内的多脏器损伤。组蛋白的Kbhb是由芝加哥大学赵英明教授课题组于2016年首次报道发现的，该修饰广泛存在于细胞的组蛋白赖氨酸上，因此目前对其研究主要集中在组蛋白上，但关于非组蛋白，特别是激酶的β-羟基丁酰化修饰还知之甚少。
发明内容
基于以上问题，本发明提供干扰p300蛋白表达的siRNA及应用，本发明首次揭示β-羟基丁酰化修饰是一种新的调控CaMKII活性的机制，由此研发的siRNA及其shRNA为提高CaMKII活性的药物提供了新的选择。
为解决以上问题，本发明提供了干扰p300蛋白表达的siRNA，所述siRNA的序列如SEQ ID No.1所示。
进一步的，所述siRNA可用于构建shRNA，所述shRNA可用于构建含有该shRNA的重组载体，所述shRNA的序列见SEQ ID No.2所示，所述重组载体含有BamHI和Mlul双酶切位点，shRNA可在细胞内被剪切成干扰p300蛋白表达的siRNA。
进一步的，所述重组载体为含有shRNA序列且连接入BamHI和Mlul双酶切线性化的RNA干扰载体pLent-U6-shRNA-CMV-RFP-P2A-Blasticidin。
为解决以上问题，本发明还提供了siRNA在制备提高CaMKII活性的药物中的应用，其特征在于，所述药物通过干预p300的表达而导致CaMKII的Kbhb减少，进而提高CaMKII的活性。
与现有技术相比，本发明的有益效果是：本发明首次揭示了β-羟基丁酰化修饰是一种新的调控CaMKII活性的机制，于体外实验中发现酰基转移酶p300可催化CaMKII发生Kbhb导致其活性降低，本发明由此研发的siRNA可通过干预p300的表达而导致CaMKII的Kbhb减少，进而增强CaMKII的活性，起到改善认知和记忆功能障碍的作用，为糖尿病及与CaMKII相关的疾病的治疗提供了新的方向。
附图说明
图1为本发明的实施例的载体图谱；
图2为本发明的实施例的293T细胞中RFP荧光检测图；
图3为本发明的实施例的p300的蛋白免疫印迹检测图；
图4为本发明的实施例的Kbhb CaMKII的蛋白免疫印迹检测图；
图5为本发明的实施例的CaMKII活性检测结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例和附图，对本发明作进一步的详细说明，本发明的示意性实施方式及其说明仅用于解释本发明，并不作为对本发明的限定。
实施例：
本实施例根据mouse p300基因的转录本设计siRNA靶点，安排引物合成，并将单链的引物退火成双链oligo(寡核苷酸)序列(shRNA)，再将shRNA序列连接入含有BamHI和Mlul双酶切位点的表达载体中形成RNA干扰载体pLent-U6-shRNA-CMV-RFP-P2A-Blasticidin，RNA干扰载体pLent-U6-shRNA-CMV-RFP-P2A-Blasticidin的图谱如附图1所示。之后本实施例通过菌落PCR筛选转化子，对筛选的阳性克隆进行测序验证，对测序验证正确的克隆进行高纯度质粒抽提，具体见下文。
本实施例根据shRNA设计的一般原则以及经验，设计3到4个siRNA靶点，并安排单链引物合成，经筛选后选择如表1所示的靶点siRNA，靶点siRNA的序列为GCAATGGACAAGGGATAATT，具体如SEQ ID No.1所示；本实施例由该siRNA构建的shRNA的序列为GCAATGGACAAGGGATAATTTCAAGAGAATTATCCCTTGTCCATTGCTTTTT T，具体见SEQ ID No.2。
表1siRNA靶点序列
载体酶切：
将克隆所需载体pLent-U6-GFP-Puro进行酶切，酶切体系如下：
加样混匀后置于37℃酶切1-2h(勿加AP)，反应结束后用1％琼脂糖凝胶电泳检测酶切，并用胶回收试剂盒回收载体。
退火：收到引物后先瞬时离心，将引物干粉中加入(nmol数*10)μLH2O，稀释成100μM的母液；退火反应体系如下：
反应程序：
连接：将退火产物稀释100倍，与酶切好的载体连接，连接体系如下：
混匀后瞬时离心，22℃连接2h.
转化：连接产物转化大肠杆菌DH5α感受态细胞，涂布于相应抗性的LB平板上进行筛选；转化的具体步骤：
(1)从-80℃取出提前制备好的DH5a感受态置于冰浴中
(2)待DH5a感受态细胞融化后，取1μL连接产物于20μL DH5a感受态细胞中，充分混匀，冰浴中静置30分钟。
(3)将离心管放入42℃水浴锅中40秒(期间不要摇动离心管)，然后快速移至冰浴中，静置2分钟。
(4)向离心管中加入200μL的无菌的LB培养基(不加抗生素)，混匀后置于摇床中37℃，200rpm，振摇1小时。目的是使质粒上相关的抗性标记基因表达，使菌体复苏。
(5)涂布到相应抗性的固体培养基平皿中
(6)37℃培养箱中培养过夜。
测序：挑取单菌落培养后提取质粒测序验证。对鉴定得到的阳性克隆进行测序验证，并对测序结果进行分析，经测序验证正确的阳性克隆，使用AxyPrep质粒DNA小量试剂盒提取质粒，并与293T细胞进行病毒包装，病毒包装结果见附图2，为干扰Tau蛋白表达的重组慢病毒(简称“sh-p300慢病毒”)转染293T细胞72h病变情况(200×视野)，可见慢病毒成功入侵细胞，并大量复制。
如表3所示，本实施例得到的sh-p300慢病毒滴度很高，最高可达6×108。
表3病毒滴度检测
本实施例还研究了上述制备的Sh-p300慢病毒与CaMKII的Kbhb的关系，本实施例因此进行了细胞培养、慢病毒感染目的细胞、蛋白提取、蛋白免疫印迹、CaMKII酶活性检测等实验。见附图3，其中LV+β-OHB为对照组，LV-Sh-p300+β-OHB为含有sh-p300慢病毒的实验组，p300代表目标蛋白，GAPDH代表内参；Kbhb CaMKII蛋白免疫印迹实验结果显示，向HT22细胞(小鼠海马神经元细胞)中转染sh-p300后，细胞内源性p300蛋白的表达显著下降。如附图4和附图5所示，其中LV+β-OHB为对照组，LV-Sh-p300+β-OHB为含有sh-p300慢病毒的实验组，Kbhb-CaMKII代表CaMKII的赖氨酸3-羟基丁酰化，GAPDH代表内参；结果显示，sh-p300慢病毒感染的细胞出现Kbhb CaMKII的表达显著降低，同时CaMKII的活性显著上升，结果表明干扰p300蛋白的表达后，CaMKII的Kbhb显著降低，同时CaMKII的活性显著上升，说明本实施例的siRNA和shRNA1可用于制备提高CaMKII活性的药物中。
如上即为本发明的实施例。上述实施例以及实施例中的具体参数仅是为了清楚表述发明验证过程，并非用以限制本发明的专利保护范围，本发明的专利保护范围仍然以其权利要求书为准，凡是运用本发明的说明书及附图内容所作的等同结构变化，同理均应包含在本发明的保护范围内。
序列表
<110> 四川大学华西医院
<120> 干扰p300蛋白表达的siRNA及应用
<130> 2021.08.23
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工合成(Artificial synthesis)
<400> 1
gcaatggaca agggataatt 20
<210> 2
<211> 53
<212> DNA
<213> 人工合成(Artificial synthesis)
<400> 2
gcaatggaca agggataatt tcaagagaat tatcccttgt ccattgcttt ttt 53
Claims (1)
1.干扰p300蛋白表达的siRNA在制备提高CaMKII活性的药物中的应用，其特征在于，所述siRNA的序列如SEQ ID No.1所示，所述药物通过干扰p300的表达而导致CaMKII的Kbhb减少，进而提高CaMKII的活性。
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