CN117603973B - 一种靶向抑制Agrin基因的shRNA及其应用 - Google Patents
一种靶向抑制Agrin基因的shRNA及其应用 Download PDFInfo
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Abstract
本发明涉及生物医学领域,特别是涉及一种靶向抑制Agrin基因的shRNA及其应用。本发明提供了一种靶向抑制Agrin基因的shRNA,所述shRNA的正义链的核苷酸序列如SEQ ID NO.1所示,反义链的核苷酸序列如SEQ ID NO.2所示。本发明所述shRNA能够有效沉默Agrin基因,进而促进体内低密度脂蛋白受体的表达,最终达到治疗高胆固醇血症及其诱导的动脉粥样硬化的效果。
Description
技术领域
本发明涉及生物医学领域,特别是涉及一种靶向抑制Agrin基因的shRNA及其应用。
背景技术
心血管疾病是目前全球范围内死亡的主要原因,其中低密度脂蛋白胆固醇(LDL-C)为血脂的重要管理指标。众多循证医学证据表明,将LDL-C的水平控制在理想范围,有助于延缓血管动脉粥样硬化斑块形成,降低心梗、脑梗等严重心脑血管事件的发生风险。因此,研发更有效的降胆固醇药物,提高低密度脂蛋白胆固醇达标率,对降低心血管疾病的发生率和死亡率至关重要。
集聚蛋白(Agrin)是一种硫酸肝素蛋白多糖,分子量约为400kDa。作为一种细胞外基质蛋白,Agrin在神经肌肉接头、心肌、肾脏、血管等组织中均有表达。研究报道,小鼠心脏中的Agrin与心肌细胞受体肌营养不良蛋白聚糖(α-dystroglycan,DAG)结合,激活Hippo-Yap通路促进心肌细胞增殖。研究发现Agrin在刚出生小鼠心脏ECM内含量较高,而在7天时表达明显下调,从而心肌细胞再生修复能力减弱,在成年小鼠心梗模型中外源性注射Agrin能够促进心肌细胞增殖和血管新生形成。此外,Agrin在调控肿瘤微环境中的血管生成中扮演着关键的角色。Agrin通过与其受体复合物结合,稳定了内皮细胞血管内皮生长因子受体2(VEGFR2),同时激活了细胞粘附激酶(FAK)。VEGFR2的稳定进一步激活了内皮细胞一氧化氮合酶(eNOS)-Akt-ERK1/2(细胞外信号调节激酶1/2)信号通路,从而在肿瘤组织中持续促进血管发芽和血管生成。总之,这些发现表明Agrin在心血管系统中具有重要的功能。然而,目前Agrin在高胆固醇血症及高胆固醇血症引起的动脉粥样硬化中的作用机制尚未有报道。
发明内容
本发明的目的是提供一种靶向抑制Agrin基因的shRNA及其应用,以解决上述现有技术存在的问题。本发明所述shRNA能够有效沉默Agrin基因,进而促进体内低密度脂蛋白受体的表达,最终达到治疗高胆固醇血症及其诱导的动脉粥样硬化的效果。
为实现上述目的,本发明提供了如下方案:
本发明提供了一种靶向抑制Agrin基因的shRNA,所述shRNA的正义链的核苷酸序列如SEQ ID NO.1所示,反义链的核苷酸序列如SEQ ID NO.2所述。
本发明提供了一种载体,所述载体包括上述的shRNA。
优选的,所述载体包括腺病毒载体。
本发明提供了一种重组菌,所述重组菌包括上述的载体。
优选的,所述重组菌的基础菌包括大肠杆菌。
本发明提供了上述的shRNA、上述的载体或上述的重组菌在制备和/或筛选抗高胆固醇血症的药物中的应用。
优选的,所述抗高胆固醇血症是通过所述shRNA抑制Agrin基因表达,提高体内低密度脂蛋白受体的表达量。
优选的,所述药物包括核酸分子、脂类、小分子化合物、抗体、多肽、蛋白质或腺相关病毒。
本发明提供了上述的shRNA、上述的载体或上述的重组菌在制备和/或筛选抗高胆固醇血症引起的动脉粥样硬化的药物中的应用。
优选的,所述药物包括核酸分子、脂类、小分子化合物、抗体、多肽、蛋白质或腺相关病毒。
本发明公开了以下技术效果:
本发明针对Agrin基因设计得到了靶向抑制Agrin基因的shRNA,该shRNA能够有效沉默Agrin基因,促进低密度脂蛋白受体(LDLR)的表达,最终达到治疗高胆固醇血症及其诱导的动脉粥样硬化的效果。在本发明具体实施例中,通过连续12周高脂喂养ApoE-/-小鼠来构建高脂小鼠模型,对小鼠模型注射腺相关病毒包装得到的腺相关病毒(AAV-Agrin-shRNA),达到了降低血清胆固醇以及缓解主动脉根部粥样硬化的作用,从而为制备或筛选抑制Agrin基因表达的药物或制剂作为抗高胆固醇血症及其诱导的动脉粥样硬化的备选药物或制剂提供了理论依据。
药物筛选主要是针对未知药物,将药物作用于靶标基因,根据该药物是否可以抑制靶标基因的表达筛选得到抗高胆固醇血症及其诱导的动脉粥样硬化的药物;药物制备主要是以靶标基因为基础,有针对性地制备或构建抗高胆固醇血症及其诱导的动脉粥样硬化的药物以抑制靶标基因的表达。本发明以Agrin基因沉默为作用靶标,制备或筛选抗高胆固醇血症及其诱导的动脉粥样硬化的药物,对筛选或制备的药物在高胆固醇血症及其诱导的动脉粥样硬化的治疗具有重要意义。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,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为Agrin-shRNA腺相关病毒载体信息;
图2为AAV-Agrin-shRNA注射小鼠肝脏组织中Agrin、低密度脂蛋白受体LDLR蛋白的表达(A)以及血脂水平(B);
图3为小鼠尾静脉注射AAV-Agrin-shRNA后主动脉根部粥样硬化斑块负荷情况;
图中,COH代表胆固醇,LDL代表低密度脂蛋白,HDL代表高密度脂蛋白,TG代表甘油三酯,AAV-shNC或AAV-sh-NC代表对照组,AAV-shAgrin或AAV-sh-Agrin代表实验组;β-actin广泛分布于各种组织中的细胞,为内参蛋白。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1:小鼠腺相关病毒(腺相关病毒AAV-Agrin-shRNA)包装
一):腺相关病毒重组载体构建
1.合成小鼠Agrin基因(NM_020569.3)的shRNA区序列,添加ccgg与aatt接头序列,选择EcoRI/AgeI酶切位点,通过T4 ligase(T4连接酶)将shRNA与骨架连接在一起。
shRNA序列如下:
正义链的核苷酸序列如SEQ ID NO.1所示,具体为:GATCCGCACTGTTTGTGTGTTATTAATTCAAGAGATTAATAACACACAAACAGTGCTTTT TTA;
茎环结构的核苷酸序列如TTCAAGAGA所示;
反义链的核苷酸序列如SEQ ID NO.2所示,具体为:AGCTTAAAAAAGCACTGTTTGTGTGTTATTAATCTCTTGAATTAATAACACACAAACAGT GCG;
shRNA识别的靶点序列如SEQ ID NO.3所示,具体为:5’-GCACTGTTTGTGTGTTATTAA-3’。
2.酶切连接
将合成shRNA的引物进行退火,退火体系如表1所示。
表1退火体系
反应液成分 | 体积 |
shRNA-F(100μM) | 10μL |
shRNA-R(100μM) | 10μL |
Total | 20μL |
注:shRNA-F的序列如SEQ ID NO.1所示,shRNA-R的序列如SEQ ID NO.2所示。
加样混匀后置于95℃5min,缓慢降温至37℃。将合成产物进行凝胶电泳,回收目的基因片段。
腺相关病毒载体pAV-U6-shRNA-CMV-GFP质粒(图1,购自山东维真生物科技有限公司)用表2中的酶切体系酶切,然后胶回收质粒片段。
表2酶切体系
反应液成分 | 体积 |
质粒(0.5μg/μL) | 4μL |
10×Buffer | 5μL |
EcoRI | 1.5μL |
AgeI | 1.5μL |
ddH2O | 38μL |
Total | 50μL |
3.连接
将回收的目的基因片段与经过同样双酶切的pAV-U6-shRNA-CMV-GFP质粒片段连接,连接体系如表3所示。
表3连接体系
成分 | 体积 |
目的基因片段 | 5μL |
质粒片段 | 3μL |
10×T4Buffer | 1μL |
T4DNA连接酶(10U/μL) | 1μL |
Total | 10μL |
混匀后微离心,22℃连接1h,得到连接产物。
4.转化
连接产物分别转化大肠杆菌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:
从-80℃取出提前制备好的大肠杆菌DH5a感受态细胞置于冰浴中。
待大肠杆菌DH5a感受态细胞融化后,将5μL连接产物和20μL大肠杆菌DH5a感受态细胞充分混匀,冰浴中静置15min。
将离心管放入42℃水浴锅中40s(期间不要摇动离心管),然后快速移至冰浴中,静置2min。
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℃,220rpm,振摇1h。目的是使质粒上相关的抗性标记基因表达,使菌体复苏。
涂布到相应抗性的固体培养基平皿中。
37℃培养箱中培养过夜。
5.测序
挑取单菌落培养后提取质粒进行酶切鉴定阳性克隆,测序验证,验证正确的质粒大提后用于后续实验。
二):腺相关病毒包装:
1.细胞复苏的步骤为:
1)在直径为10cm的培养皿中加10mL新鲜DMEM培养基,放培养箱预热至37℃。
2)从液氮中取出冷冻管,迅速投入37℃-38℃水浴中,使其融化(1-2min左右)。
3)冻存管中溶液融化后,200×g离心5min,弃掉液体并吸取1mL预热的培养基将细胞沉淀轻轻吹起,转移到直径为10cm的培养皿中。
4)摇晃均匀,置于培养箱培养。
5)培养过夜,更换新鲜培养基(除去冻存液中DMSO对细胞的毒害作用)。
2.细胞传代(以直径为10cm的培养皿为例)的步骤为:
1)生物安全柜紫外灭菌0.5h。
2)灭菌期间,将含有FBS和青链霉素混合液的DMEM培养基(该培养基中FBS的体积百分含量为10%,青链霉素混合液的体积百分含量为1%)和PBS置于37℃水浴锅中预热;0.25%胰酶放置室温,不可水浴加热。
3)取汇合度接近100%且活性较好的HEK293T细胞,吸弃培养盘中培养基,加入约5mL1×PBS,摇晃几下,吸弃PBS。
4)加1mL 0.25%胰酶在生物安全柜内消化1min,室温低时可放置于培养箱中消化。消化时间不宜过长,否则影响细胞再次贴壁效率与活性。
5)加入约5mL预热的培养基,终止消化。
6)用移液管吹打均匀(吹打过程中不可用力过大,否则会吹跑),按照1:3的比例(1mL胰酶+5mL预热的培养基终止消化=6mL,取2mL至3个新的培养皿)传代。各取2mL培养基至新的直径为10cm的培养皿中,再加入8mL预热的DMEM培养基。
注意:传代盘数较多时,要先将预热的DMEM培养基加入到直径为10cm的培养皿中,再加入含细胞的培养基,以避免细胞分布不均匀。置于培养箱前轻轻混匀培养皿中的培养基,使细胞均匀分散于培养基中。
3.AAV病毒包装(以直径为10cm的培养皿为例)的步骤为:
第一天:汇合度90%以上的HEK293T细胞按1:3比例传盘(每盘大约2.5×106个),培养基为Hydone高糖DMEM培养基(含10%FBS,厂家为Hydone公司)。
第二天:转染前1-2h左右,换成无血清培养基。
按照表4比例配制转染试剂:
表4转染试剂配制体系
Mix1和Mix 2分别混合后,室温5-10min,后将Mix 1和Mix 2混合,室温20min,逐滴加入至直径为10cm的培养皿中(此时细胞汇合度在80%-90%比较适宜,细胞过少会影响转染效率)。
第三天:质粒转染24h后,换新的无血清培养基。
第五天:转染72h收毒,将产毒的细胞连同培养基一起收集至50mL离心管中,离心,分别收获培养基上清与细胞沉淀(PEG8000沉淀培养基上清中的病毒);裂解细胞沉淀收毒;合并从细胞沉淀和上清中得到的AAV。
4.AAV病毒纯化与浓缩
4.1纯化-碘克沙醇密度梯度离心
①配制不同浓度的碘克沙醇;
②取一个超离管,用电动移液器逐层、缓慢加入不同浓度的碘克沙醇;③将处理好的病毒液加入到最上层;④配平后超速离心,超速离心的条件参数为:18℃、48000rpm超速离心2.5h。
4.2浓缩
①离心完毕后,将超滤管底用针头刺破,收集腺相关病毒所在层至15mL管中;
②将收集的病毒液注入浓缩柱中,加PBS+0.001%PF68至满,混匀;
③4000rpm、10℃离心1h;
④将超滤管中剩下的液体反复吹打后吸至病毒储存管中,最后加入病毒储存液,得到腺相关病毒AAV-Agrin-shRNA,标明名称和日期;
⑤将收集起来的病毒涡旋震荡混匀后离心,吸10μL病毒液(腺相关病毒AAV-Agrin-shRNA)进行滴度检测。
5.病毒滴度检测方法
实时定量PCR法是一种简单的、高通量的测定纯化病毒样本中腺相关病毒颗粒数量的方法。每个模板的Ct值与该模板起始拷贝数的对数存在线性关系,利用已知起始拷贝数的标准品可作出标准曲线,最后通过标准曲线对未知模板进行定量分析。
5.1去除游离DNA分子
先将病毒液稀释10倍,以保证样品中游离的DNA充分降解:取5μL病毒液至45μLPBS缓冲液中,充分混匀。按表5的比例配制混合体系。
表5混合体系
37℃孵育30min,95℃加热5min,使DNA酶失活。
5.2去除病毒蛋白外壳
向上述体系中再加入1μL蛋白酶K(5μg/μL)。37℃孵育30min;再加30μL超纯水稀释至40μL(至此病毒原液稀释100倍),95℃加热5min使蛋白酶K失活,然后12000rpm,离心2min,取上清进行qPCR检测。
37℃孵育30min,95℃加热5min,使DNA酶失活。
5.3qPCR
将步骤5.2得到的上清,取5μL进行10倍梯度稀释,即病毒原液稀释了1000倍。分别取2μL待测样品及标准品作为模板进行qPCR检测。
qPCR反应体系为:2×SYBR Green mix 10μL,Primers-F 0.8μL,Primers-R 7.2μL,DNA 2μL,Total 20μL;Primers-F的核苷酸序列如SEQ ID NO.1所示;Primers-R的核苷酸序列如SEQ ID NO.2所示。
qPCR反应程序:95℃预变性3min,95℃5s,60℃15s,72℃15s,39个循环。
实施例2:Agrin基因的腺相关病毒对高胆固醇血症及其诱导的动脉粥样硬化的作用
将ApoE-/-小鼠分为两组,每组6只,在10周龄时进行处理,分别尾静脉注射实施例1制备得到的腺相关病毒AAV-Agrin-shRNA及对照AAV空载体(AAV-sh-NC,购自山东维真生物科技有限公司)。
两组的小鼠及处理情况如下:
实验组:ApoE-/-小鼠,注射AAV-Agrin-shRNA;
对照组:ApoE-/-小鼠,注射AAV-sh-NC;
尾静脉注射后,两组小鼠给予连续12周高脂喂养,后安乐死小鼠,取上述小鼠肝脏组织,加蛋白裂解液研磨样品,各取40μg蛋白上样进行Western blot验证,分别用Agrin和LDLR抗体检测Agrin和LDLR蛋白的表达情况,结果如图2所示,发现小鼠尾静脉注射腺相关病毒AAV-Agrin-shRNA可以抑制Agrin蛋白表达,促进LDLR蛋白的表达,从而降低血清胆固醇,特别是LDL水平。
取小鼠主动脉根部切片,进行油红O染色,小鼠主动脉根部粥样斑块负荷情况如图3所示,小鼠尾静脉注射腺相关病毒AAV-Agrin-shRNA后能够显著缓解主动脉根部粥样硬化。
以上结果证实了Agrin基因在调控肝细胞中LDLR表达及血清胆固醇水平中的关键作用,通过给ApoE-/-小鼠尾静脉注射腺相关病毒AAV-Agrin-shRNA的方法加以治疗,能够降低小鼠血清胆固醇水平,缓解小鼠主动脉根部粥样硬化程度。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (5)
1.靶向抑制Agrin基因的shRNA或含靶向抑制Agrin基因的shRNA的载体在制备抗高胆固醇血症的药物中的应用,其特征在于,所述shRNA的正义链的核苷酸序列如SEQ ID NO.1所示,反义链的核苷酸序列如SEQ ID NO.2。
2.根据权利要求1所述的应用,其特征在于,所述抗高胆固醇血症是通过所述shRNA抑制Agrin基因表达,提高体内低密度脂蛋白受体的表达量。
3.根据权利要求1所述的应用,其特征在于,所述药物包括核酸分子、脂类、小分子化合物、抗体或腺相关病毒。
4.靶向抑制Agrin基因的shRNA或含靶向抑制Agrin基因的shRNA的载体在制备抗高胆固醇血症引起的动脉粥样硬化的药物中的应用,其特征在于,所述shRNA的正义链的核苷酸序列如SEQ ID NO.1所示,反义链的核苷酸序列如SEQ ID NO.2。
5.根据权利要求4所述的应用,其特征在于,所述药物包括核酸分子、脂类、小分子化合物、抗体或腺相关病毒。
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