CN108085383B - 基因检测组合物及其应用 - Google Patents
基因检测组合物及其应用 Download PDFInfo
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Abstract
本发明涉及基因检测组合物及其应用，具体涉及TMED2、CCDC72和ERC1基因组合物在检测绝经后骨质疏松及六味地黄丸临床用药中的应用。发明人通过高通量测序技术，对绝经后骨质疏松患者服用六味地黄丸前、后外周血样本和健康人对照外周血样本进行高通量测序，显示候选基因TMED2、CCDC72和ERC1与绝经后骨质疏松，并且经后骨质疏松患者服用六味地黄丸后上述3个基因发生明显变化，进一步，通过分子生物学实验验证了高通量数据分析结果。本发明为临床精准诊断绝经后骨质疏松及精准用药提供基因组合物，具有很好的市场应用价值。
Description
技术领域
本发明涉及生物医药领域，具体涉及基因检测组合物及其应用，更具体的涉及TMED2、CCDC72和ERC1基因组合物在检测绝经后骨质疏松及六味地黄丸临床用药中的应用。
背景技术
目前对于TMED2功能的研究多集中在其参与蛋白质在内质网与高尔基体之间的囊泡转运过程。近两年中，TMED2被证明为G蛋白偶联受体(GPCR)的特异转运受体，并控制着GPCR的生物合成途径中的转运过程，在星形胶质细胞中调控GPCR信号通路。另外，有文献报道TMED2在人的整个妊娠期的胎盘中及绒毛膜癌细胞系中均表达，在小鼠中TMED2发生突变导致胚胎和胎盘发育缺陷，提示其可能在胎盘发育中发挥作用(Histone deacetylaseinhibitors sensitize glioblastoma cells to TRAIL-induced apoptosis by c-myc-mediated downregulation of cFLIP.Oncogene,2012Jan 23.)。
CCDC72首次是在人类CD34+造血干细胞发现，对维持DPC凝集性生长及DPCS其他特性可能发挥重要作用。王继文等发现CCDC72基因的功能域与T2FA基因同源，即可能在基因转录过程中发挥转录起始因子的作用，对细胞分裂、核内基因复制、修复和重组发挥作用(毛乳头细胞凝聚性生长差异表达基因的筛选及HSPC016全长克隆的生物信息学分析.中华医学杂志.2004,84(18):1513-1517.)；CCDC72基因在人体组织中广泛表达，并且这些组织均具有高代谢、细胞分裂旺盛的特点。
ERC1基因研究的不多，2016年有一篇关于先天性膈疝相关基因的筛选的文章显示ERC1基因可能与先天性膈疝相关，但是相关性未经证实(先天性膈疝相关基因的筛选，山东医药，2016年第56卷第19期)。
我们之前的研究显示，TMED2基因与椎间盘病变次相关(椎间盘退行性病变诊治标志物，CN 2015109830657)，CCDC72基因与骨髓瘤相关(一种诊治多发性骨髓瘤的分子标志物，CN 201610602127X)，ERC1与下咽癌相关(ERC1在制备诊断或治疗下咽癌工具中的应用，CN 2016107521916)。
本发明中，发明人通过高通量测序技术，对绝经后骨质疏松患者服用六味地黄丸前、后外周血样本和健康人对照外周血样本进行高通量测序，显示候选基因TMED2、CCDC72和ERC1与绝经后骨质疏松，并且经后骨质疏松患者服用六味地黄丸后上述3个基因发生明显变化，发明人通过分子生物学实验验证了高通量数据分析结果，验证结果与测序结果一致。本发明为临床精准诊断绝经后骨质疏松及精准用药提供基因组合物，具有很好的市场应用价值。
发明内容
本发明的目的在于提供一种基因组合物，其包括下列基因中的一种或几种：TMED2、CCDC72和ERC1。
进一步，上述基因组合物或其表达产物在制备绝经后骨质疏松临床用药基因检测制剂中的应用。
进一步，适用六味地黄丸的样本组中TMED2基因或TMED2蛋白高表达，或样本组中ERC1基因或ERC1蛋白高表达，或样本组中CCDC72基因或CCDC72蛋白低表达。
为实现上述目的，本发明首先通过高通量测序结合生物信息学方法筛选到候选基因TMED2、CCDC72和ERC1，骨质疏松患者服用六味地黄丸后TMED2和ERC1基因表达量明显下降，CCDC72基因表达量明显上升，显示上述基因既与绝经后骨质疏松相关，还是六味地黄丸的作用靶点，可用于制备绝经后骨质疏松诊断制剂和临床用药基因检测靶点，具有重要的临床应用价值。
进一步，检测制剂采用下列方法中的一种或者几种检测TMED2、CCDC72和/或ERC1基因的表达：荧光定量PCR方法、基因芯片方法、高通量测序方法。
优选的，基因芯片包括与TMED2、CCDC72和/或ERC1基因的核酸序列杂交的探针。
优选的，荧光定量PCR方法采用特异的引物对，所述引物对选自：SEQ ID NO.1和SEQ ID NO.2组成的引物对；或SEQ ID NO.3和SEQ ID NO.4组成的引物对；SEQ ID NO.5和SEQ ID NO.6组成的引物对。
本发明目的是提供一种绝经后骨质疏松检测试剂盒，所述的检测试剂盒检测TMED2、CCDC72和/或ERC1基因及其蛋白的表达。进一步的，所述的试剂盒还包括其他检测试剂。
本发明目的是提供一种检测绝经后骨质疏松的基因芯片，所述的基因芯片包括与TMED2、CCDC72和/或ERC1基因的核酸序列杂交的探针。
本发明目的是提供一种绝经后骨质疏松临床用药基因检测试剂盒，所述试剂盒检测TMED2、CCDC72和/或ERC1基因及其蛋白的表达。TMED2和/或ERC1基因及其蛋白高表达的样本组推荐使用六味地黄丸，CCDC72基因和/或CCDC72蛋白低表达的样本组推荐使用六味地黄丸。
本发明目的是提供一种绝经后骨质疏松临床用药基因检测基因芯片，所述基因芯片包括与TMED2、CCDC72和/或ERC1基因的核酸序列杂交的探针。TMED2和/或ERC1基因及其蛋白高表达的样本组推荐使用六味地黄丸，CCDC72基因和/或CCDC72蛋白低表达的样本组推荐使用六味地黄丸。
本发明的目的在于提供TMED2抑制剂、ERC1抑制剂和/或CCDC72促进剂在制备治疗绝经后骨质疏松药物中的应用。
进一步，所述治疗绝经后骨质疏松药物抑制TMED2和/或ERC1基因的表达和/或促进CCDC72基因的表达。本领域人员熟知抑制基因的表达通常可以采用下述方法中的一种和/或几种：通过激活TMED2和/或ERC1基因的抑制基因、激活TMED2和/或ERC1基因的抑制基因表达的蛋白、采用RNA干扰技术抑制TMED2和/或ERC1基因表达、激活促进TMED2和/或ERC1基因mRNA降解的microRNA、导入促进TMED2和/或ERC1基因编码蛋白降解的分子、抑制促进TMED2和/或ERC1基因表达的因子及蛋白的表达。本领域人员熟知促进基因的表达通常可以采用下述方法中的一种和/或几种促进基因表达：通过DNA水平调控CCDC72基因：包括但不限于增加CCDC72基因的拷贝数、转染含CCDC72基因的过表达载体；通过转录水平调控CCDC72基因：包括但不限于激活CCDC72基因的表达、激活调控CCDC72基因表达的启动子、抑制负调控CCDC72基因表达的转录因子、采用RNA干扰技术对抑制CCDC72基因表达的抑制子进行干扰；通过转录后水平调控CCDC72基因：包括但不限于抑制促进CCDC72基因mRNA降解的microRNA转录表达、导入促进CCDC72基因表达的microRNA；通过翻译后水平调控CCDC72基因：包括但不限于导入促进CCDC72基因编码蛋白的分子、抑制负调控CCDC72基因表达的蛋白、促进CCDC72基因表达的因子及蛋白的表达。
RNA干扰(RNAi)是指外源性和内源性双链RNA在生物体内诱导同源靶基因的mRNA特异性降解，导致转录后基因沉默的现象，是一种使用小的双链RNA高效、特异地阻断体内某种特定基因的表达，促使mRNA降解，使细胞表现出特定基因缺失表型的技术。siRNA设计完成后可以采用直接合成法或者构建siRNA表达载体，制备好的siRNA可以通过磷酸钙共沉淀法、电穿孔法、DEAE-葡聚糖和polybrene法、显微注射或基因枪等机械法、阳离子脂质体试剂法等途径转染细胞。
本发明的目的在于提供一种治疗绝经后骨质疏松药物，所述治疗绝经后骨质疏松药物抑制TMED2和/或ERC1基因及其蛋白的表达，促进CCDC72基因及其蛋白的表达。
进一步，治疗绝经后骨质疏松药物还包含药剂学上能接受的载体。
包含在本发明的药剂学上能接受的载体为在制剂时通常利用的载体，该载体包含乳糖(lactose)、右旋糖(dextrose)、蔗糖(sucrose)、山梨醇(sorbitol)、甘露醇(mannitol)、淀粉、阿拉伯橡胶、磷酸钙、藻酸盐(alginate)、凝胶(gelatin)、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮(polyvinylpyrrolidone)、纤维素(cellulose)、水、糖浆、甲基纤维素(methyl cellulose)、羟基苯甲酸甲酯(methyl hydroxybenzoate)、丙基羟基苯甲酸丙酯(propyl hydroxybenzoate)、滑石、硬脂酸镁(stearic acid magnesium)及矿物油(mineral oil)等，但并非局限于此。
本发明的组合物除了上述成分以外还可以包含润滑剂、湿润剂、甜味剂、香味剂、乳化剂、悬浮剂、防腐剂等。药剂学上能接受的载体和制剂详细记载于雷明登氏药学全书。
本发明的组合物能通过口服或非口服进行给药，作为非口服给药时，能通过静脉内注射，鼻腔内注射，局部注射，脑室内注射，脊髓腔注射，皮下注射，腹腔注射，经皮给药等方式进行给药。
本发明的组合物的适合的给药剂量根据制剂化方法、给药方式、患者的年龄、体重、性别、病态、食物、给药时间、给药途径、排泄速度及反应灵敏性之类的因素而可以进行多种处方，通常，熟练的医生能够容易地决定及处方对所希望的治疗或预防有效的给药剂量。
本发明的目的在于提供检测TMED2、CCDC72和/或ERC1基因及其蛋白的试剂在制备绝经后骨质疏松诊断制剂中的应用。
进一步，TMED2和/或ERC1基因及其蛋白在绝经后骨质疏松样本中高表达，CCDC72基因或其蛋白在绝经后骨质疏松样本中低表达。
进一步，绝经后骨质疏松诊断制剂检测外周血中TMED2、CCDC72和/或ERC1基因及其蛋白的表达情况。
进一步，绝经后骨质疏松的诊断制剂采用下列方法中的一种或者几种检测TMED2、CCDC72和/或ERC1基因的表达：荧光定量PCR方法、基因芯片方法、高通量测序方法。
荧光定量PCR法是通过荧光染料或荧光标记的特异性的探针，对PCR产物进行标记跟踪，实时在线监控反应过程，结合相应的软件可以对产物进行分析，计算待测样品模板的初始浓度。荧光定量PCR的出现，极大地简化了定量检测的过程，而且真正实现了绝对定量。多种检测系统的出现，使实验的选择性更强。自动化操作提高了工作效率，反应快速、重复性好、灵敏度高、特异性强、结果清晰。
基因芯片又称为DNA微阵列(DNA microarray)，可分为三种主要类型：1)固定在聚合物基片(尼龙膜，硝酸纤维膜等)表面上的核酸探针或cDNA片段，通常用同位素标记的靶基因与其杂交，通过放射显影技术进行检测。2)用点样法固定在玻璃板上的DNA探针阵列，通过与荧光标记的靶基因杂交进行检测。3)在玻璃等硬质表面上直接合成的寡核苷酸探针阵列，与荧光标记的靶基因杂交进行检测。基因芯片作为一种先进的、大规模、高通量检测技术，应用于疾病的诊断，其优点有以下几个方面：一是高度的灵敏性和准确性；二是快速简便；三是可同时检测多种疾病。
高通量测序(High-throughput sequencing)又称下一代测序技术(nextgeneration sequencing)是对传统测序一次革命性的改变，一次对几十万到几百万条DNA分子进行序列测定，极大提高了测序效率。这类大规模测序技术极大的提高了多个物种遗传信息的解读速度，为获取所有mRNA的序列信息，解密mRNA图谱提供了保证。同时高通量测序使得对一个物种的转录组和基因组进行细致全貌的分析成为可能，所以又被称为深度测序。高通量测序平台的代表是罗氏公司(Roche)的454测序仪(Roch GSFLX sequencer)，Illumina公司的Solexa基因组分析仪(Illumina Genome Analyzer)和ABI的SOLiD测序仪(ABI SOLiD sequencer)。
所述用于荧光定量PCR方法检测绝经后骨质疏松中TMED2、CCDC72和/或ERC1基因的产品含有特异性扩增TMED2、CCDC72和/或ERC1基因的引物对；所述的基因芯片包括与TMED2、CCDC72和/或ERC1基因的核酸序列杂交的探针。
进一步，绝经后骨质疏松的诊断制剂还包括用免疫方法检测TMED2、CCDC72和/或ERC1蛋白的表达。优选所述免疫方法检测绝经后骨质疏松中TMED2、CCDC72和/或ERC1蛋白表达为western blot和/或ELISA和/胶体金法。
酶联免疫吸附实验(ELISA)即将已知的抗原或抗体吸附在固相载体表面，使酶标记的抗原抗体反应在固相表面进行的技术。该技术可用于检测大分子抗原和特异性抗体等，具有快速、灵敏、简便、载体易于标准化等优点。ELISA检测试剂盒根据检测目的和操作步骤可分为间接法、双抗夹心法、竞争法、双位点一步法、捕获法测IgM抗体、应用亲和素和生物素的ELISA。ELISA检测试剂盒中生色底物可选择辣根过氧化物酶(HRP)或者碱性磷酸酶(AP)。
常用的免疫胶体金检测技术：(1)免疫胶体金光镜染色法细胞悬液涂片或组织切片，可用胶体金标记的抗体进行染色，也可在胶体金标记的基础上，以银显影液增强标记，使被还原的银原子沉积于已标记的金颗粒表面，可明显增强胶体金标记的敏感性。(2)免疫胶体金电镜染色法可用胶体金标记的抗体或抗抗体与负染病毒样本或组织超薄切片结合，然后进行负染。可用于病毒形态的观察和病毒检测。(3)斑点免疫金渗滤法应用微孔滤膜作载体，先将抗原或抗体点于膜上，封闭后加待检样本，洗涤后用胶体金标记的抗体检测相应的抗原或抗体。(4)胶体金免疫层析法将特异性的抗原或抗体以条带状固定在膜上，胶体金标记试剂(抗体或单克隆抗体)吸附在结合垫上，当待检样本加到试纸条一端的样本垫上后，通过毛细作用向前移动，溶解结合垫上的胶体金标记试剂后相互反应，当移动至固定的抗原或抗体的区域时，待检物与金标试剂的结合物又与之发生特异性结合而被截留，聚集在检测带上，可通过肉眼观察到显色结果。该法现已发展成为诊断试纸条，使用十分方便。
进一步，所述检测TMED2、CCDC72和/或ERC1蛋白的ELISA法为使用ELISA检测试剂盒。所述试剂盒中的抗体可采用市售的TMED2、CCDC72和ERC1单克隆抗体。进一步，所述的试剂盒包括：包被TMED2、CCDC72和/或ERC1单克隆抗体的固相载体，酶标二抗，酶的底物，蛋白标准品，阴性对照品，稀释液，洗涤液，酶反应终止液等。
进一步，所述检测TMED2、CCDC72和ERC1蛋白的胶体金法为使用胶体金试纸条，抗体采用市售的TMED2、CCDC72和ERC1单克隆抗体。进一步，所述胶体金试纸条采用胶体金免疫层析技术或胶体金渗滤法。进一步，所述胶体金试纸条硝酸纤维素膜上的检测区(T)喷点有抗TMED2、CCDC72和ERC1单克隆抗体、质控区(C)喷点有免疫球蛋白IgG。
本发明的目的在于提供一种检测绝经后骨质疏松的荧光定量PCR试剂盒，其特征在于，所述试剂盒检测基因TMED2、CCDC72和/或ERC1，采用特异的引物对。
进一步，该PCR试剂盒适合于目前存在市场上的所有类型荧光定量基因扩增仪，灵敏度高，定量快速准确、稳定性好，具有良好的应用前景。
进一步，上述荧光定量PCR试剂盒组分包括：特异性引物对、内参引物、荧光定量PCR反应液。
所述的试剂盒还包含RNA抽提试剂。优选
Reagent进行样本RNA提取。
本发明的组合物根据本发明所属技术领域的普通技术人员可以容易实施的方法，利用药剂学上能接受的载体和/或赋形剂来进行制剂化，从而能够以单位用量形态制备或者内装在多容量容器内来制备。此时，剂型是油性或者水性介质中的溶液、悬浮液或乳化液形态，或者也可以是浸膏剂、粉末剂、颗粒剂、片剂或者胶囊剂形态，还可以包括分散剂或者稳定剂。
除非另有定义，本发明中所有的技术和科学术语具有与本发明所属技术领域的普通技术人员所通常理解的含义。
附图说明
图1是TMED2、CCDC72和ERC1基因在绝经后骨质疏松患者服用六味地黄丸前后外周血中相对表达量图
图2是TMED2、CCDC72和ERC1基因在绝经后骨质疏松外周血及健康人外周血中相对表达量图
具体实施方式
下面结合具体实施例，进一步阐述本发明，仅用于解释本发明，而不能理解为对本发明的限制。本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件实施检测。
实施例1高通量测序及分析
分别收集3例绝经后骨质疏松患者服用六味地黄丸前、后外周血样本和3例健康人对照外周血样本，进行RNA提取，RNA提取后琼脂糖凝胶电泳，从电泳结果可以初步判定提取的RNA样品质量合格与否，是否可以用于进一步的转录组分析。进而通过NanoDrop1000分光光度计检测RNA样品的提取情况，RNA-seq测序的样品要求：OD260/OD280为1.8-2.2。
测序平台为Illumina公司的HiSeq 2500高通量测序平台，进行高通量转录组深度测序，测序后我们运用Fast-QC软件对测序数据的质量进行整体评估，包括碱基的质量值分布，质量值的位置分布，GC含量，PCR duplication含量，kmer的frequency等。在差异基因表达分析时，根据得到的FPKM值，采用国际公认算法EBSeq进行差异筛选。其中，筛选时，LOG2FC>1或<-1,FDR<0.05。为了更好的理解差异表达基因的功能，我们对差异表达基因进行了Gene Onlogy和信号通路分析，并对差异表达基因进行功能注释和蛋白质互相作用网络分析，鉴于以上数据分析的结果，结合文献我们筛选了差异表达基因TMED2、CCDC72和ERC1。TMED2和ERC1在骨质疏松组高表达，并且在服用六味地黄丸后低表达，CCDC72在骨质疏松组低表达，并且在服用六味地黄丸后高表达。
实施例2绝经后骨质疏松患者服用六味地黄丸前后外周血TMED2、CCDC72和ERC1基因表达情况
一、材料和方法
1、材料
征集22例绝经后骨质疏松患者的同意，在其检测初期抽取外周血，服用六味地黄丸半年后再次收取其外周血，对其进行分组及编号。
2、方法
2.1绝经后骨质疏松外周血及健康人外周血总RNA的提取
采用
Reagent进行样本RNA提取，实验操作按产品说明书进行。
RNA质量判定标准：RNA样本的OD260/OD280值为1.8-2.2之间；总RNA电泳图谱有清晰的28S、18S条带；70℃水浴保温1小时后的电泳图谱与水浴保温前的图谱无明显差异。
2.2逆转录合成cDNA
采用
III Reverse Transcriptase(invitrogen，货号18080-044)进行cDNA反转录，实验操作按产品说明书进行，具体操作如下：
使用逆转录试剂盒，用逆转录缓冲液对lμg总RNA进行逆反录合成cDNA。采用25μl反应体系，每个样品取1μg总RNA作为模板RNA，在PCR管中分别加入以下组分：
5×逆转录缓冲液5μl，10mmol/l dNTP 1.25μl，0.1mmol/l DTT 2.5μl，30μmmol/lOligodT 2μl,200U/μl MMLV 1.25μl，模板RNA 1μg，加入灭菌水至总体系25μl。42℃孵育1小时，72℃10分钟，短暂离心。cDNA保存放-20℃冰箱备用。
2.3Real-Time PCR
2.3.1仪器及分析方法
用ABI 7500型荧光定量PCR仪，采用2-△△CT法进行数据的相对定量分析。2.3.2引物设计
采用在线引物设计软件，模板序列为NM_001321445.1(TMED2)、NM_001301248.1(ERC1)和NM_001329417.1(CCDC72)，引物设计后由invitrogen公司合成。具体引物序列如下：
TMED2引物：
5’-CCATCAGTGTCAGCATTAG-3’(SEQ ID NO.1)
5’-CCATCATCAGAGAACAAGAG-3’(SEQ ID NO.2)
ERC1引物：
5’-GGCTATCTTCACCTACTCTTCAG-3’(SEQ ID NO.3)
5’-AATCAGGAGACAGTTGGTTACAT-3’(SEQ ID NO.4)
CCDC72引物：
5’-CTTACAGTGGCTCATCATC-3’(SEQ ID NO.5)
5’-AGACTCTGGCAAGATTCT-3’(SEQ ID NO.6)
操作过程如下：
反应体系：2×mix 10μl；上游引物(10uM)和上下游引物(10uM)各0.5μl；模板2μl；加入灭菌蒸馏水补平至25μl。
用Power
Green PCR Master Mix(invitrogen，货号4367659)进行扩增，实验操作按产品说明书进行。
扩增程序为：95°10min，(95℃15sec，58℃60sec)×35个循环。
样品RealTimePCR检测：将各样品cDNA 10倍稀释后取2μl作模板，分别用目的基因引物和内参基因引物进行扩增。同时在60-95℃进行溶解曲线分析。二、实验结果
实时定量PCR扩增曲线拐点清楚，扩增曲线整体平行性好，表明各反应管的扩增效率相近，极限平而无上扬现在，曲线指数期斜率较大，说明扩增效率较高；样本扩增产物溶解曲线都是单峰，说明扩增产物只有一条，为特异性扩增；根据qRT-PCR的相对定量公式，比较TMED2、CCDC72和ERC1基因在绝经后骨质疏松患者服用六味地黄丸前后外周血中TMED2、CCDC72和ERC1表达情况，服用六味地黄丸后TMED2、CCDC72和ERC1基因表达量比服用前均有差异，分别为服用前的0.61、1.31和0.58(见图1)，与高通量测序结果趋势一致。
实施例3绝经后骨质疏松患者外周血及健康人外周血中TMED2、CCDC72和ERC1基因表达情况
1、材料
收集32例绝经后骨质疏松患者外周血及29例健康人外周血，对其进行分组及编号。
2、方法
具体步骤参照实施例3。
3、结果
实时定量PCR结果显示(具体见图2)：qRT-PCR扩增结果稳定，其中TMED2、CCDC72和ERC1在绝经后骨质疏松外周血中的表达水平与健康人外周血中的表达水平差异显著，3个基因前者约为后者的1.22倍、0.83倍和1.39倍，以上结果验证了高通量测序的结果。
本发明采用高通量测序筛选出绝经后骨质疏松致病相关基因TMED2、CCDC72和ERC1，进一步分析显示，该基因与六味地黄丸用药效果相关，在临床指导用药方面方面具有重要意义，发明人结合分子细胞生物学实验验证，证实了TMED2、CCDC72和ERC1与绝经后骨质疏松病具有很好的相关性。本发明为绝经后骨质疏松临床诊疗提供了新的诊断靶点和用药靶点，具有很好的临床应用前景。
序列表
<110> 北京泱深生物信息技术有限公司
<120> 基因检测组合物及其应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccatcagtgt cagcattag 19
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ccatcatcag agaacaagag 20
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggctatcttc acctactctt cag 23
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aatcaggaga cagttggtta cat 23
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cttacagtgg ctcatcatc 19
<210> 6
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agactctggc aagattct 18
Claims (4)
1.基因组合物或其表达产物在制备绝经后骨质疏松六味地黄丸用药基因检测制剂中的应用，其特征在于，基因组合物包括下列基因中的一种或几种：TMED2、CCDC72和ERC1。
2.根据权利要求1所述的应用，其特征在于，适用六味地黄丸的样本组中TMED2基因或TMED2蛋白高表达，或样本组中ERC1基因或ERC1蛋白高表达，或样本组中CCDC72基因或CCDC72蛋白低表达。
3.根据权利要求1所述的应用，其特征在于，检测制剂采用下列方法中的一种或者几种检测TMED2、CCDC72和ERC1基因的表达：荧光定量PCR方法、基因芯片方法、高通量测序方法。
4.根据权利要求1所述的应用，其特征在于，基因芯片包括与TMED2、CCDC72和/或ERC1基因的核酸序列杂交的探针；荧光定量PCR方法采用特异的引物对，所述引物对选自：SEQID NO.1和SEQ ID NO.2组成的引物对；或SEQ ID NO.3和SEQ ID NO.4组成的引物对；SEQID NO.5和SEQ ID NO.6组成的引物对。
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Non-Patent Citations (5)
| Title |
|---|
| Bioinformatics Analysis Reveals the Altered Gene Expression of Patients with Postmenopausal Osteoporosis Using Liuweidihuang Pills Treatment.;Rui Gong等;《Biomed Res Int.》;20190127;1-11 * |
| GenBank NM_001301248.1;Held RG等;《GenBank》;20171113;1-6 * |
| GenBank NM_001321445.1;Nagae M等;《GenBank》;20171003;1-3 * |
| GenBank NM_001329417.1;Song Z等;《GenBank》;20171004;1-2 * |
| 六味地黄丸对绝经后骨质疏松症肾阴虚证JAK/STAT信号通路基因的影响;谢丽华等;《中国骨质疏松杂志》;20140720;741-746 * |
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