CN118109577A - mRNA DCAF5的医药用途 - Google Patents
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Abstract
mRNA DCAF5的医药用途,其可用作冠状动脉粥样硬化的生物标志物,用于冠状动脉粥样硬化性心脏病生物学鉴定及临床检测,应用该基因表达水平进行诊断,可以早期发现冠心病患者,降低冠心病死亡率,改善患者预后。目前冠状动脉粥样硬化性心脏病的主要诊断方法存在手术危险系数高、辐射大等风险,通过收集患者外周血进行检测,具有创伤小、可重复性好等优点,更易于被接受。
Description
技术领域
本发明属于分子标志物技术领域,具体涉及一种mRNA DCAF5的医药用途。
背景技术
泛素化是指泛素(一类低分子量的蛋白质)分子在一系列特殊的酶作用下,将细胞内的蛋白质分类,从中选出靶蛋白分子,并对靶蛋白进行特异性修饰的过程。这些特殊的酶包括泛素激活酶、结合酶、连结酶和降解酶等。泛素化在蛋白质的定位、代谢、功能、调节和降解中都起着十分重要的作用。同时,它也参与了细胞周期、增殖、凋亡、分化、转移、基因表达、转录调节、信号传递、损伤修复、炎症免疫等几乎一切生命活动的调控。
DCAF蛋白家族是一类在DNA损伤修复和细胞周期调控中发挥重要作用的蛋白质。它们可以与DDB1和CUL4等蛋白质构成E3泛素连接酶复合物,将泛素分子连接到靶蛋白上,从而对底物进行泛素化修饰。DCAF家族蛋白的主要作用是参与调控细胞生命周期中的各个环节,如细胞增殖、分化、凋亡等,同时也与肿瘤发病存在紧密的关联性。
DCAF5(DDB1-and CUL4-associated factor 5)蛋白是DCAF蛋白家族的一个成员,是mRNA DCAF5在蛋白水平的表达产物,与DDB1(DNA damage-binding protein 1)和CUL4(Cullin 4)等蛋白质相互作用,形成CRL4(Cullin-RING ligase 4)E3泛素连接酶复合物的一部分。CRL4复合物在细胞内发挥着重要的调控作用,涉及DNA损伤修复、细胞周期控制、基因转录调节等多个生物学过程。DCAF5作为CRL4复合物的底物受体,通过识别并结合特定的靶蛋白,介导这些蛋白的泛素化修饰。泛素化是一种将泛素分子连接到靶蛋白上的过程,通过改变靶蛋白的稳定性、定位或功能来调控细胞内的各种生理活动。DCAF5蛋白的功能和调控机制目前仍在深入研究中。然而,已有研究表明DCAF5在某些生物过程中起着重要的作用。例如,DCAF5可以参与DNA复制和修复的调控,维护基因组的稳定性。此外,DCAF5还可能参与细胞周期的调控,影响细胞的增殖和分化。DCAF5表达水平异常可能与冠心病的发生和发展之间关系尚不明确。
发明内容
解决的技术问题:本发明提供一种mRNA DCAF5作为分子标志物在制备检测冠状动脉粥样硬化产品中的应用。
技术方案:核酸序列如SEQ ID NO.1-5顺序拼接所示的mRNA DCAF5作为分子标志物在制备检测冠状动脉粥样硬化产品中的应用。
上述产品为诊断试剂盒。
上述试剂盒中含有检测mRNA DCAF5表达量的实时定量PCR扩增引物。
上述引物如SEQ ID NO.6和SEQ ID NO.7所示。
检测冠状动脉粥样硬化的试剂盒,含有核酸序列如SEQ ID NO.1-5顺序拼接所示的mRNA DCAF5。
上述试剂盒还含有如SEQ ID NO.6和SEQ ID NO.7所示的引物。
检测制剂通过检测被试者PBMC细胞中DCAF5的表达,与健康对照组相比较表达显著上调,以此来判断被试者有冠状动脉粥样硬化。
诊断冠状动脉粥样硬化的标志物,所述标志物为mRNA基因DCAF5,核苷酸序列如SEQ ID NO.1-5所示。
一种冠状动脉粥样硬化的诊断试剂,包括:检测mRNA基因DCAF5表达量的制剂,所述RNA基因DCAF5的核苷酸序列如SEQ ID NO.1-5所示。
一种冠状动脉粥样硬化的诊断试剂盒,所述诊断试剂盒中包括所述的诊断试剂。
进一步的,诊断试剂盒采用以下步骤进行检测:
(1)从待测样品中分离PBMCs,从PBMCs中提取纯化RNA;
(2)去除步骤2中的获得产物中的gDNA,加入逆转录试剂,进行逆转录反应;
(3)以步骤2获得的cDNA为模板,加入扩增引物,进行实时荧光定量聚合酶链式反应;
(4)对步骤3扩增得到的DNA产物进行凝胶电泳分析或进行测序;
(5)根据步骤4的结果与正常对照组基因表达水平进行对比,根据基因表达水平差异,分析待测样品中是否存在该基因差异表达。
进一步的,扩增引物中正向引物核苷酸序列如SEQ ID NO.6所示,反向引物核苷酸序列如SEQ ID NO.7所示。
有益效果:本发明发现DCAF5基因通过在冠状动脉粥样硬化过程中表达量具有显著差异,该基因在冠状动脉粥样硬化患者及正常对照中表达差异具有显著统计学意义。该基因位于人类第14号染色体,其表达量在冠状动脉粥样硬化患者中显著下降(P<0.001)。通过比较基因组学研究,对比该基因在不同人群中的表达,可区分冠心病患者及非冠心病的对照组,作为诊断的生物标志物。本发明提供了一种适用于冠心病患者分子鉴定的标准基因及分子鉴定方法,该基因能有效的区分非冠心病患者和冠心病,应用该基因表达水平进行诊断,可以早期发现冠心病患者,降低冠心病死亡率,改善患者预后。目前冠状动脉粥样硬化性心脏病的主要诊断方法存在手术危险系数高、辐射大等风险,通过收集患者外周血进行检测,具有创伤小、可重复性好等优点,更易于被接受。
附图说明
图1mRNA DCAF5在临床标本中表达特征。
图2是实施例2中mRNA DCAF5在临床标本中受试者工作特征曲线分析结果。
具体实施方式
为了使本技术领域的人员更好地理解本申请方案,下面将结合本申请实施例中的附图,对本申请实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本申请一部分的实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本申请保护的范围。
需要说明的是,本申请的说明书和权利要求书及上述附图中的术语“包括”和“具有”以及他们的任何变形,意图在于覆盖不排他的包含,例如,包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元,而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
实施例1:判断lncRNA DCAF5作为诊断标志物的效能
(1)入组患者:入组经冠脉造影证实的连续性冠心病患者114例与正常对照组患者120例。根据造影结果,选择至少一条冠状动脉狭窄>程度50%的受试者为患者组,所有冠状动脉狭窄程度均<50%的受试者为对照组。
(2)分离样品:通过密度梯度离心分离PBMCs,样品与淋巴细胞分离液体积为1:1,2000rpm×20min,分层后使用移液器插入云雾层,吸取单个核细胞,然后将其保存在TRIzol试剂中,总RNA用氯仿12000g离心15min提取,用等体积异丙醇离心12000g×10min沉淀,用75%乙醇离心7500g×5min纯化,最后溶于无RNA酶的水中。
(3)设计引物:基于如SEQ ID NO.1-5所示的序列设计了PCR引物,引物序列如下:F:5’-CCCATGAGACTTCCAGACCTTG-3’,核苷酸序列如SEQ ID NO.6所示R:5’-GCCTCAAGTCATCTATGCTCCTG-3’,核苷酸序列如SEQ ID NO.7所示。
(4)逆转录:使用Strand cDNA Synthesis SuperMix for qPCR试剂进行逆转录,反应在冰上进行。
(5)扩增:以步骤(3)扩增的cDNA作为模板,qPCR SYBR Green Master Mix试剂配置反应体系,两步法进行实时定量PCR,反应程序为:95℃/5min预变性,95℃/10s变性,60℃/30s退火/延伸,40个循环重复,单个样品设置三个复孔,并通过ΔΔCt法计算相对表达量。
(6)验证结果:统计分析结果表明,mRNA DCAF5(核苷酸序列如SEQ ID NO.1所示)在两组间存在显著表达差异(p<0.001),该基因在冠心病组中存在低表达。受试者工作特征曲线分析结果,其曲线下面积大于0.5,表明该基因对于冠状动脉粥样硬化具有一定的诊断能力。
上述结果表明,lncRNA DCAF5参与了冠状动脉粥样硬化过程中,通过检测该基因表达水平诊断及鉴别冠状动脉粥样硬化患者具有可行性。应理解,本发明仅以举例方式加以描述并可同时在本发明的范围和精神内进行修改。以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术无需创造性劳动就可以根据本发明的构思做出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的试验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
Claims (6)
1.核酸序列如SEQ ID NO.1-5顺序拼接所示的mRNA DCAF5作为分子标志物在制备检测冠状动脉粥样硬化产品中的应用。
2.根据权利要求1所述的应用,其特征在于,所述产品为诊断试剂盒。
3.根据权利要求2所述的应用,其特征在于,所述试剂盒中含有检测mRNA DCAF5表达量的实时定量PCR扩增引物。
4.根据权利要求3所述的应用,其特征在于,所述引物如SEQ ID NO.6和SEQ ID NO.7所示。
5.检测冠状动脉粥样硬化的试剂盒,其特征在于,含有核酸序列如SEQ ID NO.1-5顺序拼接所示的mRNA DCAF5。
6.根据权利要求5所述试剂盒,其特征在于,还含有如SEQ ID NO.6和SEQ ID NO.7所示的引物。
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