CN116904469B - 一种p300蛋白表达的抑制剂和制备方法及其用途 - Google Patents
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Abstract
本发明涉及代谢性药物领域,具体涉及一种p300蛋白表达的抑制剂和制备方法及其用途。本发明提供的一种p300蛋白表达的抑制剂,通过选择如SEQ ID NO.1所示核苷酸序列作为靶序列,依据该靶序列设计的shRNA特异性高,能够显著抑制p300蛋白的表达,从而可以缓解或治疗由p300蛋白引起的疾病,在代谢性疾病方面,可以调节脂质代谢紊乱,抑制脂肪组织脂肪累积,从而可以起到缓解肥胖的作用。
Description
技术领域
本发明涉及代谢性药物领域,具体涉及一种p300蛋白表达的抑制剂和制备方法及其用途。
背景技术
肥胖是目前全球发病率最高的代谢性疾病,主要特点是体脂率和体重的异常升高。肥胖是多种因素共同作用的结果。近年来随着表观遗传学的发展,多项研究从表观遗传学角度进一步解释环境和生活方式的改变在肥胖的发病机制中的重要作用。蛋白质的翻译后修饰是表观遗传过程中的重要环节,其中组蛋白修饰的变化可以改变染色质的可及性,影响基因转录及表达,调控细胞功能。p300是一种乙酰化修饰转移酶,催化乙酰辅酶A与蛋白质的赖氨酸残基的结合,具有转录共激活作用。已有研究在肥胖的疾病模型中发现组蛋白乙酰化修饰通过上调代谢相关基因的转录活性调节糖酵解和脂肪代谢等代谢相关途径(Morigny, P., Boucher, J., Arner, P.,&Langin, D. (2021). Lipid and glucosemetabolism in white adipocytes: pathways, dysfunction and therapeutics.Nat Rev Endocrinol, 17(5), 276-295. doi:10.1038/s41574-021-00471-8)。除了乙酰化修饰,P300还可以催化多种类型的组蛋白修饰,也有研究发现p300也可以通过新型酰化修饰上调细胞糖酵解过程。因此通过靶向p300调控酰化修饰可以对细胞代谢产生影响,并进一步影响全身代谢水平的变化。
近年来p300的抑制剂主要为小分子化合物,靶向结合其溴结构域或/和乙酰转移酶区域的来抑制P300的催化活性,并主要应用于肿瘤的治疗( Lasko, L. M., Jakob, C.G., Edalji, R. P., Qiu, W., Montgomery, D., Digiammarino, E. L., . . .Bromberg, K. D. (2017). Discovery of a selective catalytic p300/CBP inhibitorthat targets lineage-specific tumours.Nature, 550(7674), 128-132. doi:10.1038/nature24028)。尽管这部分结构抑制剂具有较强的结合能力,但是其低选择性和较差的细胞通透性限制其临床应用(He, Z. X., Wei, B. F., Zhang, X., Gong, Y. P.,Ma, L. Y.,&Zhao, W. (2021). Current development of CBP/p300 inhibitors in thelast decade.Eur J Med Chem, 209, 112861. doi:10.1016/j.ejmech.2020.112861)。因此开发更为有效的p300特异性抑制剂,探索其在肥胖中的治疗价值是非常重要的。
发明内容
因此,本发明要解决的技术问题在于提供一种p300蛋白表达的抑制剂和制备方法及其用途。
为此,本发明提供了如下的技术方案:
一种p300蛋白表达的抑制剂,包括shRNA;所述shRNA是依据如SEQ ID NO.1所示的靶序列设计的。
可选的,所述shRNA的核苷酸序列如SEQ ID NO.2所示。
一种重组载体,所述重组载体包含所述的shRNA。
一种慢病毒颗粒,包含所述的重组载体。
可选的,所述慢病毒颗粒由包括PG-P1-VSVG、PG-P2-VSVG和PG-P3-VSVG的慢病毒辅助包装质粒辅助包装而成。
一种所述的慢病毒颗粒的制备方法,包括如下步骤:
将所述的重组载体和慢病毒辅助包装质粒共转染至包装细胞中,培养,收集细胞上清液。
可选的,所述慢病毒辅助包装质粒包括3个辅助质粒,分别为PG-P1-VSVG、PG-P2-VSVG和PG-P3-VSVG。
所述的p300蛋白表达的抑制剂、所述的重组载体、所述的慢病毒颗粒或所述的慢病毒颗粒的制备方法制备得到的慢病毒颗粒具有制备缓解或治疗由P300蛋白引起的相关疾病的产品中的用途。
可选的,所述由P300蛋白引起的相关疾病包括代谢性疾病或肿瘤。
可选的,所述的用途包括如下任意一个:
(1)在制备抑制脂肪组织的脂肪累积的产品中的用途;
(2)在制备缓解脂质代谢紊乱的产品中的用途;
(3)在制备缓解肥胖的产品中的用途。
可选的,所述的产品为药物或生物制剂。
本发明技术方案,具有如下优点:
1.本发明提供的一种p300蛋白表达的抑制剂,通过选择如SEQ ID NO.1所示核苷酸序列作为靶序列,依据该靶序列设计的shRNA特异性高,能够显著抑制p300蛋白的表达,从而可以缓解或治疗由p300蛋白引起的疾病,在代谢性疾病方面,可以调节脂质代谢紊乱,抑制脂肪组织脂肪累积,从而可以起到缓解肥胖的作用。
2. 本发明提供的一种p300蛋白表达的抑制剂,依据如SEQ ID NO.1所示核苷酸序列设计的shRNA如SEQ ID NO.2所示,能够高特异性干扰p300蛋白的表达,能够显著的抑制p300蛋白的表达。
3.本发明提供的一种慢病毒颗粒,较传统的小分子制剂具有更好的细胞穿透性和更直接的对靶细胞基因表达进行干扰的作用。
4.本发明提供的一种慢病毒颗粒,所述慢病毒辅助包装质粒包括3个辅助质粒,分别为PG-P1-VSVG、PG-P2-VSVG和PG-P3-VSVG;本发明制备慢病毒颗粒的制备方法中采用四质粒体系,其中三个辅助质粒不仅提供病毒包装需要的反式作用因子,同时采用“自我灭活”修饰,阻断子代病毒的自我复制和转移,从而保证其安全性。同时慢病毒载体未发现致肿瘤活性。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是本发明实施例1的shRNA慢病毒穿梭质粒图谱;
图2是本发明实验例1干预后的P300的免疫荧光检测图;
图3是本发明实验例1干预后的P300的mRNA表达检测图;
图4是本发明实验例2应用于高脂饮食的C57/6J小鼠四周后的外观及解剖后脂肪组织对比;
图5是本发明实验例2应用于高脂饮食的C57/6J小鼠四周后的体重变化对比;
图6是本发明实验例2应用于高脂饮食的C57/6J小鼠四周后血清胆固醇和低密度脂蛋白的变化;
图7是本发明实验例2应用于高脂饮食的C57/6J小鼠四周后的脂肪组织和肝脏组织的HE染色对比;
图8是本发明实验例2应用于高脂饮食的C57/6J小鼠四周后的脂肪组织的中脂质代谢相关基因的mRNA水平的变化。
具体实施方式
提供下述实施例是为了更好地进一步理解本发明,并不局限于所述最佳实施方式,不对本发明的内容和保护范围构成限制,任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品,均落在本发明的保护范围之内。
实施例中未注明具体实验步骤或条件者,按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规试剂产品。
实施例1
本实施例提供了一种慢病毒颗粒的制备方法,包括如下步骤:
(1)穿梭质粒的构建
根据如SEQ ID NO.1所示的靶序列片段,构建的shRNA的核苷酸序列如SEQ IDNO.2所示(编号LV4174):然后根据shRNA设计引物序列如下表:
表1、靶序列和对应的shRNA序列
表2、编号LV4174的shRNA设计引物序列
采用吉玛公司(GenePharma)慢病毒干扰载体LV-3 (pGLVH1/GFP+Puro)构建慢病毒穿梭质粒,质粒图谱见图1,构建过程由吉玛公司完成。具体步骤如下:
1)、shRNA DNA oligo采用Designer 3.0(Genepharma)设计(如上表),合成引物由吉玛基因提供,DNA oligo浓度为100μM,取相应的正义链和反义链oligo溶液配置退火反应体系(50μL):包括正义链(100μM)、反义链(100μM)、10×shDNA 退火缓冲液(吉玛基因提供)各5μL,其余用ddH2O补齐。退火条件为95℃保持5min,85℃保持5min,75℃保持5min,获得10μM的shRNA的DNA模板,稀释至200nM以用于连接反应。
2)、构建重组载体:酶切体系(100μL):10μL 2×Buffer Tango、5μL BamHI(#ER0051,MBI Fermentas)、5μL EcoRI(#ER0271,MBI Fermentas)及10μg LV-3载体,其余用ddH2O补齐。酶切1h后电泳,使用Agarose Gel DNA Purification Kit 2.0(DP209-03,Tangen)回收并稀释至50ng/μL使用;连接反应体系(20μL):2μL 10×Ligation Buffer、1μLLV-3(BamHI + EcoRI双酶切产物,50ng/μL)、1μL shRNA的DNA模板(100nM)、1μL T4 DNA 连接酶、其余用ddH2O补齐。混匀后至于22℃反应1h,后转移至感受态细胞进行筛选。
3)、连接产物转化:将10μL连接产物加入感受态细胞(大肠杆菌,采用氯化钙法制备,100μL/管冻存),冰中放置30min,42℃90s,冰中3min后加入培养基LB培养基(不含抗生素),37℃培养45min复苏,然后采用含50μg/mL的抗生素平板上进行培养筛选,对阳性克隆菌落进行质粒的抽提和测序。
4)、对载体质粒进行测序。对经过测序验证正确的质粒进行提取和纯化,获取高质量的不含内毒素的 shRNA慢病毒穿梭质粒,构建的穿梭质粒图谱如图1所示。
(2)慢病毒包装
慢病毒包装过程由吉玛公司完成,采用的包装细胞为293T细胞,按照比例将慢病毒穿梭质粒和辅助质粒共转染293T细胞,培养并收集病毒,并进行浓缩和纯化。包装过程如下:
1)将293T细胞接种至15cm培养皿培养(培养基为含10% FBS 的DMEM 培养基)过夜;
2)将上述构建的shRNA慢病毒穿梭质粒与包装质粒(pGag/Pol、pRev、pVSV-G)加入装有1.5mL DMEM培养基的反应管中;同时将300μL RNAi-Mate加至装有1.5mL DMEM的新反应管中,室温放置5min后将上述两管液体混匀,室温放置20~25min;
3)除去培养皿中的培养基,加入8ml无血清培养基,后将2)中混合物滴入混匀,培养箱中培养4-6h;
4)去转染液,后加入带血清培养基,继续培养72h;
5)收集细胞上清液,用0.45μm的过滤器过滤,后超速离心(条件为2000rpm,2h,4℃)获取慢病毒浓缩液。
对比例1
本对比例和实施例1的区别在于,shRNA和对应的靶序列如下表中所示,涉及的引物序列如下。
表3 、对比例的靶序列和对应的shRNA序列
表4、LV2406 的shRNA设计引物序列
对比例2
本对比例和实施例1的区别在于,shRNA的核苷酸序列和对应的靶序列如下表所示,涉及的引物序列如下表。
表5、对比例的靶序列和对应的shRNA序列
表6、LV4536 shRNA设计引物序列
实验例1
将实施例1、对比例1、对比例2获得的抑制P300蛋白表达的慢病毒颗粒的滴度调整为1x109TU/ml,同时设置空慢病毒颗粒NC(按照实施例1实施,仅是将慢病毒穿梭质粒替换为慢病毒干扰载体LV-3)作为对照。然后在293T细胞中验证上述慢病毒颗粒下调P300表达水平的能力,方法如下:
(1)293T细胞培养,常规培养使用含10%FBS的MEM培养基中,37℃、5%CO2饱和湿度培养箱中进行培养。
(2)将状态良好的293T细胞消化后重悬,取适量细胞铺板至24孔板中,加入0.5ml完全培养基/孔,培养箱中过夜,细胞生长至50-60%。
(3)稀释病毒,稀释液(完全培养基)400μl含终浓度5μg/ml聚凝胺(Polybrene),将20μl慢病毒原液加入稀释液中。移去步骤(2)中的细胞培养液,加入稀释后的慢病毒液,同时建立其他毒株干预及对照组,在前述培养箱中过夜。
(4)24h后移去病毒液,加入0.5ml完全培养基,然后在前述培养箱中继续培养。
(5)根据细胞状态采用试剂盒(RN easy minikit,74101,QIAGEN)提取细胞中的RNA并反转录为cDNA(G592,Abm),设计P300 mRNA引物进行qPCR验证mRNA的表达水平;或分出部分继续培养进行P300蛋白免疫荧光实验验证。P300 mRNA引物进行qPCR的引物F、R的序列分别如SEQ ID NO.13~14所示,PCR扩增体系为(10μl):0.3μl的引物F(浓度为10μM),0.3μl的引物R(浓度为10μM)、5μl的qPCR masterMix(G891,abm)、4.4μl的cDNA;PCR扩增程序为:50℃保持2min,95℃保持10min;95℃保持15s,60℃保持30s(40个循环);95℃保持15s,60℃保持30s,95℃保持15s。
结果如图2所示,由图2中可以看到LV4174干预后的293T细胞中表达的P300蛋白水平明显下降,免疫荧光显示本发明对细胞P300表达的抑制效果好于对比例1、2。由图3中可以看到本发明的抑制P300蛋白表达的慢病毒颗粒能够显著下调P300的mRNA表达水平,具有统计学差异(p<0.05),而对比例1、2的慢病毒颗粒与空白组无显著差异。
实验例2 在高脂饮食诱导的肥胖小鼠模型中验证慢病毒颗粒的减肥和调节脂代谢的作用
1.实验动物
实验对象为12只为C57BL/6J雄性小鼠(8周龄),购自维通利华公司。
2.实验方法
(1)、 高脂饮食干预:对小鼠进行8周的高脂饮食(高脂饲料:D12942, ResearchDiets)干预,自由饮食。
(2)、慢病毒干预:将12只小鼠随机分为两组,其中6只为实验组(P300-),其余为对照组(HFD)。实验组于腹股沟脂肪垫注射病毒液,取实施例1中的慢病毒原液100μl/只,加入生理盐水中进行稀释。稀释方案为400μl生理盐水+100μl病毒原液(1x109TU/ml)+终浓度为5μg/ml的聚凝胺。每侧脂肪垫注射250μl稀释病毒液一次。两组小鼠继续4周高脂饮食。
(3)、取材:于慢病毒干预4周后处死小鼠,留取血清、脂肪、肝脏以备后续实验。
(4)、血清总胆固醇和低密度脂蛋白的测量:采用ELISA试剂盒(南京建成,A11-1-1,A113-1-1)对两组小鼠血清的总胆固醇和低密度脂蛋白水平进行比较。
(5)、 HE染色:对留取的肝脏、脂肪组织采用多聚甲醛固定并包埋,蜡块切片后进行苏木精伊红染色。
(6)、脂肪RNA-seq:每个样本提供脂肪组织200mg(n=3)提取RNA并进行测序,对两组差异基因进行富集分析。
3.实验结果
(1)小鼠体重变化
结果如图4所示和图5所示,实验组较对照组的小鼠体重明显下降,在P300-慢病毒干预4周后,图4中实验组小鼠外观体型(图中第一行)及解剖后的脂肪组织(为棕色脂肪、睾周脂肪及肾周脂肪)(图中第二行)大小较对照组具有明显差异。表示p<0.05,/> 表示p<0.01。
(2)血清总胆固醇及低密度脂蛋白
结果如图6所示,实验组较对照组的血清总胆固醇及低密度脂蛋白显著下降(表示p<0.05)。
(3)HE染色
结果如图7所示,实验组较对照组脂肪组织HE染色可观察到明显的脂肪细胞大小的下降(图中第一行),肝脏组织中可以观察到脂肪浸润的减少(图中第二行)。
(4)脂质代谢相关基因
进一步对实验组和对照组脂肪中脂质代谢相关基因的mRNA水平进行比较,结果如图8所示,实验组较对照组,脂肪酸合成基因Acaca、脂质储存基因Plin1、脂质转运基因Gpihbp1、脂肪酸代谢基因cyp2e1以及脂肪细胞肥大基因Mt1的相对表达水平高,其中脂质转运基因Gpihbp1以及脂肪细胞肥大基因Mt1具有显著性差异(表示p<0.05)。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (7)
1. 一种p300蛋白表达的抑制剂,其特征在于,包括shRNA;所述shRNA的核苷酸序列如SEQ ID NO.2所示。
2.一种重组载体,其特征在于,所述重组载体包含如权利要求1中所述的shRNA。
3.一种慢病毒颗粒,其特征在于,包含权利要求2所述的重组载体。
4.根据权利要求3所述的慢病毒颗粒,其特征在于,所述慢病毒颗粒由包括PG-P1-VSVG、PG-P2-VSVG和PG-P3-VSVG的慢病毒辅助包装质粒辅助包装而成。
5.一种如权利要求3或4所述的慢病毒颗粒的制备方法,其特征在于,包括如下步骤:
将权利要求2所述的重组载体和慢病毒辅助包装质粒共转染至包装细胞中,培养,收集细胞上清液。
6.根据权利要求5所述的慢病毒颗粒的制备方法,其特征在于,所述慢病毒辅助包装质粒包括3个辅助质粒,分别为PG-P1-VSVG、PG-P2-VSVG和PG-P3-VSVG。
7.权利要求1所述的p300蛋白表达的抑制剂、权利要求2所述的重组载体、权利要求3或4所述的慢病毒颗粒或权利要求5或6所述的慢病毒颗粒的制备方法制备得到的慢病毒颗粒在制备缓解或治疗肥胖的药物中的用途。
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