CN115181756B - 一种重组慢病毒载体、重组慢病毒质粒、细胞模型及相关应用 - Google Patents
一种重组慢病毒载体、重组慢病毒质粒、细胞模型及相关应用 Download PDFInfo
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Abstract
本发明公开了一种重组慢病毒载体、重组慢病毒质粒、细胞模型及相关应用，涉及生物医药技术领域，本发明提供的重组慢病毒载体含有双荧光素酶报告基因和VGLL4基因，双荧光素酶报告基因可以作为检测指标，准确有效地反应VGLL4基因的表达或VGLL4基因受药物调控的情况。采用该载体构建的细胞模型可用于筛选VGLL4调控剂或以VGLL4为靶点的抗肿瘤产品，为VGLL4基因的功能研究、其调控剂和以VGLL4为靶点的抗肿瘤药物的开发提供了新的途径。此外，本发明利用上述载体构建的细胞模型筛选出了新的VGLL4小分子调控剂。
Description
技术领域
本发明涉及生物医药技术领域，具体而言，涉及一种重组慢病毒载体、重组慢病毒质粒、细胞模型及相关应用。
背景技术
Hippo通路是维持组织稳定的关键信号通路，其失调将引起细胞增殖过度而凋亡不足，组织器官过度增生，从而大大增加人体罹患癌症的风险。YAP是Hippo信号通路下游的效应分子，其表达量上调与多种类型肿瘤的发生、耐药和肿瘤干细胞自更新密切相关，是潜在的肿瘤治疗靶点。YAP可通过与TEADs相互作用来调控靶基因的表达，从而促进细胞增殖，抑制细胞凋亡。与YAP的作用方式相似，哺乳动物退变样蛋白(Vestigial-like proteins，VGLL4/VGL4)也可以通过与TEADs的结合来发挥其转录调控功能。由此可见，YAP与VGLL4是互相竞争的关系，过表达或上调VGLL4可以阻碍YAP与TEADs的结合，从而抑制其下游靶基因的表达和肿瘤生长，还可以避免直接抑制YAP功能可能导致的毒副作用，如图1。从文献报道来看，采用VGLL4的多肽类似物对胃癌模型进行治疗，可以有效抑制癌蛋白YAP的活性，得到较好的阳性治疗效果；采用转染VGLL4过表达载体的方法在肺癌细胞中上调VGLL4表达水平，可以显著抑制肿瘤的生长。上述研究结果均显示，VGLL4可以通过与YAP竞争性结合TEADs来抑制YAP的活性，进而抑制Hippo信号通路异常导致的肿瘤发生和进展。
然而，目前上调VGLL4的治疗方法尚停留在使用VGLL4多肽类似物和基因过表达阶段，针对VGLL4靶点的抗肿瘤试剂主要包括；重组溶瘤基因-腺病毒Ad-sp-E1A-ΔE1B-VGLL4、miR-130a反义核酸及其衍生物、VGLL4蛋白类似物等，存在成药性低，不易储存的缺点。
鉴于此，特提出本发明。
发明内容
本发明的目的在于提供一种重组慢病毒载体、重组慢病毒质粒、细胞模型及相关应用。
本发明是这样实现的：
第一方面，本发明实施例提供了一种重组慢病毒载体，其包括：双荧光素酶报告基因慢病毒载体和位于双荧光素酶报告基因载体中的VGLL4基因。
第二方面，本发明实施例提供了一种慢病毒载体系统，其包括：包装质粒和如前述实施例所述的重组慢病毒载体。
第三方面，本发明实施例提供了一种重组慢病毒质粒，其由如前述实施例所述的慢病毒载体系统经慢病毒包装后获得。
第四方面，本发明实施例提供了如前述实施例所述的重组慢病毒质粒的构建方法，其包括：采用如前述实施例所述的慢病毒载体系统进行慢病毒包装。
第五方面，本发明实施例提供了一种细胞模型，其含有前述实施例所述的重组慢病毒质粒。
第六方面，本发明实施例提供了如前述实施例所述的细胞模型的构建方法，其采用如前述实施例所述的重组慢病毒质粒转染细胞后，筛选获得高表达VGLL4基因的细胞。
第七方面，本发明实施例提供了如前述实施例所述的重组慢病毒载体或如前述实施例所述的慢病毒载体系统或如前述实施例所述的重组慢病毒质粒或如前述实施例所述的细胞模型在筛选VGLL4调控剂或以VGLL4为靶点的抗肿瘤产品中的应用。
第八方面，本发明实施例提供了目标化合物在制备VGLL4调控剂或以VGLL4为靶点的抗肿瘤产品中的应用，所述目标化合物包括PI3K/mTOR抑制剂、mTOR抑制剂、VEGFR抑制剂、甘草酸铵(Ammonium Glycyrrhizinate)和GSK-3β抑制剂中的至少一种。
第九方面，本发明实施例提供了一种以VGLL4为靶点的抗肿瘤产品，其活性成分包括前述实施例所述的目标化合物和其他VGLL4调控剂。
本发明具有以下有益效果：
本发明提供的重组慢病毒载体，含有双荧光素酶报告基因和VGLL4基因，双荧光素酶报告基因可以作为检测指标，准确有效地反应VGLL4基因的表达情况或VGLL4基因受药物调控的情况。采用该载体系统构建的细胞模型可用于筛选VGLL4调控剂或以VGLL4为靶点的抗肿瘤产品，为VGLL4基因的功能研究、其调控剂和以VGLL4为靶点的抗肿瘤药物的开发提供了新的途径。
此外，本发明利用上述载体构建的细胞模型筛选出了目标化合物可作为新的VGLL4小分子调控剂，小分子调控剂可以在细胞水平探究VGLL4表达调控机制及对细胞生物学功能的影响，也可以进行动物实验探究药物作用和治疗效果。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，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相关的附图。
图1为Hippo通路的核心分子及其调控方式；
图2为在细胞模型中进行的双荧光素酶报告基因检测的部分筛选结果；
图3为Dactolisib在Huh7细胞中对VGLL4，以及YAP-TEAD下游靶点表达的影响；
图4为三种PI3K/mTOR抑制剂的双荧光素酶报告基因检测结果；A.采用不同浓度的Dactolisib分别处理A549-VGLL4细胞24h、48h和72h后对VGLL4表达的影响；B.采用不同浓度的Dactolisib、GSK1059615和Wortmannin分别处理A549-VGLL4细胞24h后对VGLL4表达的影响；C.采用不同浓度的Dactolisib、GSK1059615和Wortmannin分别处理A549-VGLL4细胞48h后对VGLL4表达的影响；D.采用不同浓度的Dactolisib、GSK1059615和Wortmannin分别处理A549-VGLL4细胞72h后对VGLL4表达的影响。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
首先，本发明实施例提供了一种重组慢病毒载体，其包括：双荧光素酶报告基因慢病毒载体和位于双荧光素酶报告基因载体中的VGLL4基因。
在一些实施例中，所述VGLL4基因位于双荧光素酶报告基因载体的酶切位点之间。
在一些实施例中，所述双荧光素酶报告基因载体为CV060载体，CV060载体带有目的基因依赖的萤火虫荧光素酶(Firefly Luciferase)报告基因和胸苷激酶启动子驱动的海肾荧光素酶(Renilla Luciferase)报告基因。
在一些实施例中，所述VGLL4基因位于CV060载体的PacI酶切位点和NheI酶之间。
在一些实施例中，所述VGLL4基因的核苷酸序列如SEQ ID No.1所示。
本发明实施例还提供了一种慢病毒载体系统，其包括：包装质粒和如前述任意实施例所述的重组慢病毒载体。
在一些实施例中，所述包装质粒包括pHelper 1.0载体和pHelper 2.0载体。pHelper 1.0载体中含有HIV病毒的gag基因和pol基因，分别用于编码病毒主要的结构蛋白和特异性的酶，还含有rev基因，用于编码调节gag和pol基因表达的调节因子；pHelper 2.0载体中含有单纯疱疹病毒来源的VSV-G基因，用于提供病毒包装所需要的包膜蛋白。在其他实施例中，包装质粒还可以选用现有其他的一代包装质粒、二代包装质粒和三代包装质粒中的任意一种。
本发明实施例还提供了一种重组慢病毒质粒，其由如前述任意实施例所述的慢病毒载体系统经慢病毒包装后获得。
本发明实施例还提供了如前述任意实施例所述的重组慢病毒质粒的构建方法，其包括：采用如前述任意实施例所述的慢病毒载体系统进行慢病毒包装。
在一些实施例中，所述所述慢病毒包装包括：将所述慢病毒载体系统转染入宿主细胞。优选地，所述宿主细胞选自293T细胞。
可选地，慢病毒包装采用三质粒系统进行，除了含有目的基因的载体质粒外，另外两种包装质粒可以为pHelper 1.0载体和pHelper 2.0载体。在进行慢病毒包装前，分别对三种载体质粒进行高纯度无内毒素抽提。包装时，收集对数生长期生长状态良好的包装细胞，并以适宜浓度接种于细胞培养皿中，待细胞覆盖面达到70％-80％时开始转染。收集转染46～50h后的293T细胞培养液，离心纯化，浓缩并保存。
本发明实施例还提供了一种细胞模型，其含有前述任意实施例所述的重组慢病毒质粒。
该细胞模型不仅可以从小分子数据库(如ChemDiv化合物库)中筛选出VGLL4小分子调控剂，同时也适用于其他化合物库的筛选。
利用本发明所述的细胞模型筛选出的VGLL4小分子调控剂，相较于重组溶瘤基因-腺病毒Ad-sp-E1A-ΔE1B-VGLL4、miR-130a反义核酸及其衍生物、VGLL4蛋白类似物等目前针对VGLL4靶点的抗肿瘤试剂而言，VGLL4小分子调控剂具有成药性更高、稳定、更易于批量生产、易于储存的优越性。
本发明实施例还提供了如前述任意实施例所述的细胞模型的构建方法，其采用如前述任意实施例所述的重组慢病毒质粒转染细胞后，筛选获得高表达VGLL4基因的细胞。
优选地，所述细胞包括A549细胞和HEK293细胞中的任意一种。
可选地，所述转染包括：接种A549细胞于12孔板，待细胞融合度达到约15％～25％时，加入重组慢病毒质粒感染，2～4天后进行单克隆挑选，扩增后检测，选择报告基因表达水平较高的单克隆作为构建成功的细胞模型(A549-VGLL4细胞)，用于后续应用。
本发明实施例还提供了如前述任意实施例所述的重组慢病毒载体或如前述任意实施例所述的慢病毒载体系统或如前述任意实施例所述的重组慢病毒质粒或如前述任意实施例所述的细胞模型在筛选VGLL4调控剂或以VGLL4为靶点的抗肿瘤产品中的应用。
本发明实施例还提供了目标化合物在制备VGLL4调控剂或以VGLL4为靶点的抗肿瘤产品中的应用，所述目标化合物包括PI3K/mTOR抑制剂、mTOR抑制剂、VEGFR抑制剂、甘草酸铵和GSK-3β抑制剂中的至少一种。
在一些实施例中，所述PI3K/mTOR抑制剂包括Dactolisib。经过体外细胞水平活性的验证，利用本发明所述的细胞模型对小分子候选试剂进行筛选，首次发现PI3K/mTOR抑制剂Dactolisib具有上调VGLL4的功效，在Huh7中可浓度依赖性地上调VGLL4的表达水平，且伴随着VGLL4表达量的升高，YAP-TEAD下游靶蛋白CTGF和Survivin表达显著降低，从侧面反映了癌蛋白YAP的活性受到了抑制。
在一些实施例中，所述mTOR抑制剂包括WYE-354和KU-0063794中的至少一种，所述VEGFR抑制剂包括Linifanib(ABT-869)，所述GSK-3β抑制剂包括TWS119。
在一些实施例中，所述VGLL4调控剂包括VGLL4小分子调控剂。
在一些实施例中，所述VGLL4调控剂包括上调VGLL4表达量的试剂和下调VGLL4表达量的试剂。
在一些实施例中，筛选VGLL4调控剂或以VGLL4为靶点的抗肿瘤产品可以选用ChemDiv化合物库为候选药物和临床试验药物库。
筛选的方法可基于本领域常规的采用细胞模型筛选药物的步骤。具体可以参考以下步骤：将培养基稀释的待测药物加入对数生长期的A549-VGLL4细胞(细胞模型)中，进行双荧光素酶检测，萤火虫荧光素酶含量与VGLL4的表达有很好的相关性，海肾荧光素酶含量可反映细胞数量，以萤火虫荧光素酶活性与海肾荧光素酶活性的比值来反映单位细胞中VGLL4的表达水平。
所述筛选VGLL4调控剂还包括对筛选出的VGLL4调控剂进行功能性验证，具体可步骤包括：收集对数生长期的肿瘤细胞并接种于6孔板中，待细胞贴壁后，加入浓度梯度的VGLL4调控剂(Dactolisib)处理细胞48h，提取总RNA和总蛋白分别进行qRT-PCR和WesternBlot实验，从基因和蛋白水平对Dactolisib上调VGLL4，并抑制YAP-TEAD下游靶点(CTGF，Survivin和CYR61)的功效进行验证。
此外，本发明实施例还提供了一种以VGLL4为靶点的抗肿瘤产品，其活性成分包括前述任意实施例所述的目标化合物和其他VGLL4调控剂。
在一些实施例中，所述产品可以为试剂、试剂盒、药物、保健品中的任意一种类型。
在一些实施例中所述其他VGLL4调控剂可以为现有已知的或市售的VGLL4调控剂。
在一些实施例中，所述VGLL4调控剂包括VGLL4小分子调控剂。
在一些实施例中，所述VGLL4调控剂包括上调VGLL4表达量的试剂和下调VGLL4表达量的试剂。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
一、材料和试剂
试剂：DMED培养基(购于GIBCO)，胰蛋白酶(购于GIBCO)，链霉素/青霉素双抗(购于GIBCO)，胎牛血清(购于GIBCO)，FBS(购于GIBCO)，RNA提取试剂盒(购于TIANGEN)，cDNA合成试剂盒(购于BIO-RAD)，双荧光素酶试剂盒(购于promega)，SsoAdvancedTM 
Greensupermix(172-5260)试剂盒(购于BIO-RAD)；
细胞株：A549细胞株(购于ATCC)，HCC细胞株Huh7。
二、实验方法步骤
1、VGLL4过表达慢病毒载体的构建
VGLL4过表达慢病毒载体的构建和鉴定实验流程概述如下：首先，设计并合成引物，从含有目的基因的质粒中采用PCR法扩增目的基因片段并纯化；采用限制性内切酶PacI/NheI酶切纯化的目的基因质粒以及表达载体CV060(该载体带有目的基因依赖的萤火虫荧光素酶(Firefly Luciferase)报告基因和胸苷激酶启动子驱动的海肾荧光素酶(Renilla Luciferase)报告基因)，并将酶切产物进行定向克隆连接；将连接产物于感受态细胞中转化，挑选单克隆扩增并抽提质粒，采用PCR法鉴定阳性转化子，并对阳性克隆测序，与目的基因序列进行比对，以最终确定含目的基因的慢病毒载体是否构建成功。
2、VGLL4重组慢病毒质粒的包装
VGLL4重组慢病毒质粒的包装采用三质粒系统进行，除了含有目的基因的载体质粒外，另外两种包装质粒分别为pHelper 1.0载体和pHelper 2.0载体。其中，pHelper 1.0载体中含有HIV病毒的gag基因和pol基因，分别用于编码病毒主要的结构蛋白和特异性的酶，还含有rev基因，用于编码调节gag和pol基因表达的调节因子；pHelper 2.0载体中含有单纯疱疹病毒来源的VSV-G基因，用于提供病毒包装所需要的包膜蛋白。在进行慢病毒包装前，首先按Promega中抽试剂盒的操作说明，分别对三种载体质粒进行高纯度无内毒素抽提。包装时，收集对数生长期生长状态良好的293T包装细胞，并以适宜浓度接种于细胞培养皿中，待细胞覆盖面达到70-80％时开始转染。转染前2h将细胞培养基更换为无血清培养基，转染时，将VGLL4重组慢病毒质粒、pHelper 1.0载体质粒和pHelper 2.0载体质粒按20μg：15μg：10μg的量加入到Opti-MEM培养基中，调整总体积到2.5mL作为A液，室温孵育5min；另取100μL Lipofectamine 2000试剂加入到2.4mL Opti-MEM培养基中作为B液，室温孵育5min；将A、B液混合并轻轻颠倒混匀，在室温下孵育20min已形成DNA与Lipofectamine 2000的转染复合物；将转染复合物加入到293T细胞培养液中混匀，放入细胞培养箱中培养；8h后，移除含有转染混合液的培养基，并将细胞轻轻漂洗两次，再加入完全培养基，继续放入孵箱培养；收集转染48h后的293T细胞培养液，其内含有包装病毒，将含病毒的培养基离心纯化和浓缩，保存于-80℃用于后续滴度测定和细胞感染。
3、慢病毒感染A549细胞
收集对数生长期生长状态良好的A549细胞接种于12孔板中，待细胞融合度达到约20％时，加入适宜量的病毒进行感染。16h后更换细胞培养基继续培养，并于感染3天后将各组细胞分别接种到96孔板中进行单克隆挑选。将挑选出的单克隆扩增后检测各自报告基因表达情况，选择报告基因表达水平较高的单克隆作为构建成功的A549-VGLL4细胞，用于后续的药物筛选。
4、药物筛选
收集对数生长期的A549-VGLL4稳转细胞并以3000个细胞/100μL的浓度接种于96孔板中，次日，向板中加入培养基稀释的待测药物，放入细胞培养箱中继续培养48h。在进行双荧光素酶报告基因检测前，按照试剂盒操作说明，将Luciferase Assay Buffer和Stop&
Buffer分别加入到Luciferase Assay Substrate和Stop&/>
Substrate中，完全溶解底物，配制好的试剂分装保存于-80℃并尽快使用。检测时，取出药物处理48h后的细胞，移除培养基，向每孔加入20μL 1×裂解缓冲液，在室温下于摇床上反应10-20min，以使细胞完全裂解。从每孔吸取5μL细胞裂解液加入到白板中，将白板置于Promega化学发光仪中，并通过仪器的自动加样装置依次向每孔加入30μL的萤火虫荧光素酶分析试剂和海肾荧光素酶分析试剂，然后分别读数以测定细胞中萤火虫荧光素酶含量和海肾荧光素酶含量。其中，萤火虫荧光素酶含量可反映目标蛋白VGLL4的表达水平，海肾荧光素酶作为内参可反映各孔中细胞数量，以检测出的萤火虫荧光素酶活性与海肾荧光素酶活性的比值(F/R)来反映单位细胞中VGLL4的表达水平，并制定如下筛选标准：a.以药物处理组中F/R值对溶剂对照组的比值，即(F/R)药物处理组/(F/R)DMSO，来反映每批筛选的药物对VGLL4靶点的调控水平；b.规定该比值>2的药物为VGLL4的潜在激动剂，该比值<0.8为VGLL4的潜在抑制剂。筛选到的潜在激动剂有Dactolisib、Linifanib、Vandetanib、Cabozantinib、KU-0063794、WYE-354、AEE788、TWS119、Duloxetine HCl、Ammonium Glycyrrhizinate潜在抑制剂有Masitinib、Pazopanib HCl、Imatinib Mesylate、KU-55933、Brivanib、Ki8751、DAPT、NPS-2143、Mirabegron、SANT-1。结果见表1和图2。
表1.筛选数据
图2显示的即筛选的部分数据，五角星代表具有上调VGLL4表达功效的小分子药物，结果显示，在10μM药物浓度下处理48h后，Dactolisib处理组上调VGLL4表达效果最显著，是潜在的VGLL4调控剂，选择其进入后续的验证。
5、目标化合物在细胞水平上的活性验证
分别采用RT-PCR、Western Blot实验验证目标化合物在HCC细胞株Huh7中对VGLL4基因水平和蛋白水平表达的影响，以及YAP-TEAD下游靶点CTGF和Survivin表达的影响。
在进行RT-PCR和Western Blot实验前，收集对数生长期的Huh7细胞并接种于6孔板中，待细胞贴壁后，加入药物处理细胞48h。后续对Huh7细胞RNA的提取按照TIANGEN RNA提取试剂盒(RNAsimple Total RNA Kit，DP419)操作说明进行。
RNA提取完后需尽快将其反转录成cDNA，cDNA合成实验采用BIO-RAD的cDNA合成试剂盒(iScript cDNA Synthesis Kit，170-8891)进行，反应体系及反应条件如下。
表1 cDNA合成反应体系与反应条件
将得到的cDNA放入-20℃保存备用。
RT-PCR实验采用BIO-RAD SsoAdvancedTM
Green supermix(172-5260)试剂盒进行，反应体系及反应条件如下：
表2 RT-PCR反应体系与反应条件
表3 RT-PCP实验所用引物列表
结果显示，Dactolisib在Huh7中可浓度依赖性地上调VGLL4的表达水平，且伴随着VGLL4表达量的升高，YAP-TEAD下游靶蛋白CTGF和Survivin表达显著降低，从侧面反映了癌蛋白YAP的活性受到了抑制。这一结果与在细胞筛选模型中进行的双荧光素酶报告基因检测结果一致，进一步确证了Dactolisib具有上调VGLL4表达的功效，如图3。
Dactolisib是PI3K/mTOR抑制剂，为验证Dactolisib是否通过抑制PI3K/mTOR来调控VGLL4的表达，选取了另外两种PI3K/mTOR抑制剂GSK1059615、Wortmannin，采用双荧光素酶报告基因模型检测，与Dactolisib进行了对比研究。结果如图4所示，A549-VGLL4经不同浓度的药物处理不同时间后，仅Dactolisib处理组中VGLL4表达水平显著升高，其它两种PI3K/mTOR抑制剂并无显著上调VGLL4表达的功效，表明Dactolisib并非通过PI3K/mTOR通路来介导VGLL4表达的调控。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (3)
1.潜在激动剂或潜在抑制剂在制备VGLL4调控剂或以VGLL4为靶点的抗肿瘤产品中的应用，其特征在于，
所述潜在激动剂选自：Dactolisib、Linifanib、Vandetanib、Cabozantinib、KU-0063794、WYE-354、AEE788、TWS119、Duloxetine HCl或Ammonium Glycyrrhizinate；
所述潜在抑制剂选自：Masitinib、Pazopanib HCl、Imatinib Mesylate、KU-55933、Brivanib、Ki8751、DAPT、NPS-2143、Mirabegron或SANT-1。
2.根据权利要求1所述的应用，其特征在于，所述VGLL4调控剂包括VGLL4小分子调控剂。
3.一种以VGLL4为靶点的抗肿瘤产品，其特征在于，其活性成分包括如权利要求1或2中所述的潜在激动剂或潜在抑制剂。
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