CN1896235A - 一种抑制骨桥蛋白表达的小分子rna药物及其表达系统 - Google Patents
一种抑制骨桥蛋白表达的小分子rna药物及其表达系统 Download PDFInfo
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Abstract
本发明公开了一种能抑制骨桥蛋白OPN表达的小分子RNA药物及其表达系统。本发明提供的抑制OPN表达的方法是RNAi技术，具体提供了两条能特异抑制OPN表达的shRNA序列，并提供了包含上述两条shRNA的质粒pcDNA3.0+EGFP+U6/OPN－RNAi－1和OPN－RNAi－2及其构建方法。本发明可用于治疗一些与OPN表达密切相关的疾病，如动脉粥样硬化、骨质疏松症、风湿性关节炎、糖尿病及多种高转移性的肿瘤等。本发明改造的RNAi载体也可用于制备研究其它基因功能的试剂或是其它疾病的治疗药物。
Description
技术领域
本发明涉及基因工程领域中一种抑制骨桥蛋白(OPN)表达的方法。
背景技术
OPN是一分子量为44KD的分泌型糖基化磷蛋白，主要通过与其受体整合素和CD44相互作用，参与多器官多组织的生理病理过程，具有多种功能。研究表明OPN的表达和动脉粥样硬化、骨质疏松症、风湿性关节炎、糖尿病及多种肿瘤的发生发展密切相关。
恶性肿瘤已成为引起人类死亡的主要原因，其死亡率仅次于心血管疾病，而肿瘤的转移是恶性肿瘤死亡率高、预后差的主要原因。很多研究表明细胞中OPN的高表达与恶性肿瘤细胞的转移潜能密切相关，OPN在肿瘤细胞的黏附、移行、浸润、血管新生以及肿瘤的微环境中起着关键作用。临床上OPN高表达的肿瘤患者一般复发和转移率高，预后较差。因此抑制肿瘤细胞中OPN的表达，有可能降低肿瘤细胞的转移潜能，从而提高肿瘤患者的生存率。
RNAi(RNA interference，RNA干扰)是近几年发展起来的一种转录后基因沉默技术，与反义技术相比其抑制基因表达更有效、特异且持久。RNAi现象由Fire等在线虫中首次发现并报道，随后在果蝇、植物和动物细胞中都证实了这种现象的存在。随后Tuchl等发现体外合成的21bp的siRNA可以在哺乳动物细胞中特异有效的抑制基因表达，不久Brummelkamp等将含有19-29bp的shDNA的表达载体转染到哺乳动物细胞中，成功转录出shRNA并能特异抑制目标基因的表达，之后RNAi技术在抗肿瘤、抗病毒和基因功能研究的领域中都得到了广泛的应用。
目前在实验室一般使用合成的dsRNA或shRNA表达载体来实现目标基因的沉默，合成RNA成本高，容易降解，不能长期有效的抑制目标基因的表达；而一些早期开发的RNAi载体如pSilencer和pSUPER等都缺少指示标记和抗性筛选基因，给其使用带来很多不便。因此我们构建的RNAi载体可以有效的解决这些问题，另外虽也有文献报道使用RNAi技术在小鼠结肠癌细胞中抑制OPN表达，但我们设计的是针对人的OPN基因的不同抑制位点，并且抑制效果很好。
发明内容
本发明的目的是提供一种抑制骨桥蛋白(OPN)表达的方法。
本发明提供的抑制骨桥蛋白(OPN)表达的方法是使用RNAi技术。
本发明提供了两条shRNA序列：
shRNA1
CGACTCTGATGATGTAGATGACACTTTCAAGAGAAGTGTCATCTACATCATCAGAGTCG
ShRNA2
GCGAGGAGTTGAATGGTGCATACAATTCAAGAGATTGTATGCACCATTCAACTCCTCGC
包含上述两条shRNA的质粒及其细胞系均属于本发明的保护范围。具体的质粒为：pcDNA3.0+GFP+U6/OPN-RNAi-1和pcDNA3.0+GFP+U6/OPN-RNAi-2。
质粒的通用结构如图1所示。
质粒的构建方法为：设计三条引物：U6-F、U6-R+shRNA1和U6-R+shRNA2，U6-F引物含有BamH I的酶切位点及保护碱基，U6-R+shRNA1和2分别含有shDNA1和shDNA2序列，并且含有Xba I酶切位点和保护碱基。用人的基因组DNA为模板，分别用引物U6-F和U6-R+shRNA1及U6-R+shRNA2进行扩增，得到U6+shRNA的PCR产物，用BamH I和Xba I酶切之后连接到pcDNA3.0的BamH I和Xba I酶切位点之间。EGFP(增强绿色荧光蛋白)插入到pcDNA3.0的Kpn I和BamH I酶切位点之间。
本发明提供了两种shRNA序列，并在pcDNA3.0的基础上进行了改造，使之可表达EGFP，可作为更好的转染标记，不共转染更方便和经济。
本发明筛选的含有OPN-shRNA的稳定细胞系为研究OPN在肿瘤转移过程中的作用机制提供了极好的研究材料，也属于本发明的保护范围，并将在恶性肿瘤的治疗研究中起到重要作用。
附图说明
图1：质粒pcDNA3.0+EGFP+U6/OPN-RNAi-1和OPN-RNAi-2的通用图谱。
图2：稳定细胞系中OPN表达水平检测图。
具体实施方式
实施例1：针对OPN基因RNAi载体的构建
一、RNA干扰片段的选择
首先从GeneBankTM得到OPN的mRNA序列，根据文献提供的经验利用Ambion网站设计siRNA软件选取了两条siRNA片段，核苷酸序列如下：
siRNA1：CGACTCTGATGATGTAGATGACACT
siRNA2：GCGAGGAGTTGAATGGTGCATACAA
根据这两条siRNA片段和U6启动子序列设计以下三条引物：
U6-F’：CAT
GGATCCTCGGGCAGGAAGAGGGCCTA
U6-R+shRNA1：5’-CAT
TCTAGAAAAAACGACTCTGATGATGTAGATGACACT
AGTGTCATCTACATCATCAGAGTCGCGGAATTCCGCGTCCTTTCCACAAG3’
U6-R+shRNA2：5’-CAT
TCTAGAAAAAAGCGAGGAGTTGAATGGTGCATACAA
TTGTATGCACCATTCAACTCCTCGCCGGAATTCCGCGTCCTTTCCACAAG-3’
带有下划线的为酶切位点，下划线之前的为保护碱基，方框中为siRNA之间的Loop结构，前后反向互补序列形成shRNA结构。
二、U6+shRNA片段和EGFP的扩增
用人的基因组DNA为模板，分别用U6和U6-R+shRNA1或shRNA2进行扩增，得到的产物即为U6启动子+shRNA片段；用PWEN100为模板，根据EGFP的序列设计引物进行PCR，得到的PCR产物即为EGFP。
三、RNAi载体的构建
将载体pcDNA3.0和EGFP分别用Kpn I和BamH I进行双酶切，酶切后切胶回收目的片段，将载体和EGFP的酶切片段连接。按照常规方法制备大肠杆菌感受态，将连接好的产物进行转化，涂板后37℃培养过夜，挑出单菌落摇菌培养，提取质粒并用Kpn I和BamH I双酶切鉴定，阳性克隆进一步用T7引物测序鉴定，扩增并纯化阳性克隆，即得到pcDNA3.0+EGFP质粒。
将载体pcDNA3.0+EGFP和U6+shRNA分别用BamH I和Xba I进行双酶切，回收、连接、转化及鉴定同上，阳性克隆进一步用U6-F引物测序，扩增并纯化阳性克隆，即得到pcDNA3.0+EGFP+U6+shRNA的RNAi载体。
实施例2：RNAi载体的转染和稳定细胞系的筛选
以5×104/孔的密度在24孔板上铺SK-Hep-1细胞，24小时后进行转染操作。将带有OPN基因序列的RNAi载体和带有无关序列的负对照载体各500ng转染细胞，转染试剂使用Lipofectamine(Invitrogen)，具体操作步骤见Invitrogen公司脂质体转染试剂使用说明。转染48小时后以1∶20的比例传代转染细胞，加500ug/ml的G418进行稳定细胞系的筛选，10-14天后在荧光显微镜下标记表达绿色荧光蛋白的细胞集落，转移至孔板继续扩大培养，冻存能稳定表达绿色荧光蛋白的稳定细胞系，以备继续研究之用。
实施例3：目标基因OPN在稳定细胞系中表达的比较分析
提取稳定细胞系及SK-Hep-1的RNA，提取RNA使用试剂RNA提取试剂Trizol(Invitrogen)，具体操作步骤见Invitrogen公司Trizol试剂使用说明。用2ug的RNA进行RT-PCR；RT使用M-MLV逆转录酶(Promega)，用随机引物进行RT，具体见Promega公司的M-MLV逆转录酶说明书。PCR时所用OPN引物为根据OPN的Exon序列设计而得，用HPBDG作为内源对照，引物序列如下：
OPN-F：AGCCTTCTCAGCCAAACG OPN-R：GACTTACTTGGAAGGGTCTGTG
HPBDG-F：TCTGGTAACGGCAATGCGG HPBDG-R：GCAGATGGCTCCGATGGTG
用55℃退火，扩增27个循环，PCR完成之后用1.5％的琼脂糖凝胶进行电泳，拍照分析，结果见图2，由图可知，在含pcDNA3.0+EGFP+U6/OPN-RNAi-1和2的稳定细胞系中，OPN的表达水平都有明显的下调，而在含负对照载体的稳定细胞系中，OPN的表达水平无变化。
由此可见，本发明中的OPN-RNAi序列和根据pcDNA3.0所改造使用的载体，在体外细胞实验中能够有效的抑制目标基因的表达，并可以用于转染后目标基因下调的稳定细胞系的筛选。因此该RNAi序列可以用来有效的抑制OPN的表达，可以作为一种抑制OPN表达的小分子RNA药物，故可用于治疗与OPN表达密切相关的一些疾病，如动脉粥样硬化、骨质疏松症、风湿性关节炎、糖尿病及多种肿瘤等；本发明建立的抑制OPN表达的稳定细胞系及其负对照稳定细胞系也可用来做为研究OPN在肝癌转移过程中作用机制的模型；本发明改造的RNAi载体也可用于制备研究其它基因功能的试剂或是其它疾病的治疗药物。
Claims (6)
1、一种抑制骨桥蛋白OPN表达的核苷酸序列，其特征在于：其包含有siRNA1或siRNA2序列。
2、按权利要求1所述的一种抑制骨桥蛋白OPN表达的核苷酸序列，其特征在于：所述的核苷酸序列是siRNA1或siRNA2。
3、一种抑制骨桥蛋白OPN表达的shRNA，其特征在于：其序列如shRNA1或shRNA2所示。
4、一种包含权利要求3所述shRNA的载体。
5、按权利要求4所述的载体，其特征在于：所述载体为pcDNA3.0+GFP+U6/OPN-RNAi-1和pcDNA3.0+GFP+U6/OPN-RNAi-2。
6、一种遗传工程化的宿主细胞，其特征在于，其含有权利要求4或5所述的载体。
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| EP2140268A4 (en) * | 2007-04-17 | 2011-05-04 | Pfizer | METHOD FOR CONTROLLING GLUCOSE TAKING AND INSULIN SENSITIVITY |
| CN102757499A (zh) * | 2011-04-26 | 2012-10-31 | 中国人民解放军第二军医大学 | 抗骨桥蛋白嵌合抗体、其制备方法及其在制备治疗骨质疏松药物中的用途 |
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| EP2140268A4 (en) * | 2007-04-17 | 2011-05-04 | Pfizer | METHOD FOR CONTROLLING GLUCOSE TAKING AND INSULIN SENSITIVITY |
| CN102757499A (zh) * | 2011-04-26 | 2012-10-31 | 中国人民解放军第二军医大学 | 抗骨桥蛋白嵌合抗体、其制备方法及其在制备治疗骨质疏松药物中的用途 |
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