CN116144705B - 一种高效制备电压依赖性钙离子通道细胞模型的方法 - Google Patents
一种高效制备电压依赖性钙离子通道细胞模型的方法 Download PDFInfo
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Abstract
本发明属于细胞生物学领域,具体公开了一种高效制备电压依赖性钙离子通道细胞模型的方法。公开了一组共表达人Cav1.2钙离子通道蛋白的载体,以及以此为基础高效制备电压依赖性钙离子通道细胞模型的方法;通过多西环霉素诱导,本发明之细胞系具有稳定表达、且具有功能性的人类Cav1.2钙离子通道的特性。本发明提供人类心肌细胞之外的替代细胞系—Cav1.2钙离子通道表达HEK293T细胞系,作为有效且经济的药物体外心安全性评价。
Description
技术领域
本发明属于细胞生物学领域,具体公开了一种高效制备电压依赖性钙离子通道细胞模型的方法。
背景技术
大约20种不同的离子电流协同作用了心脏动作电位,这些电流可以分为去极化和再极化电流。细胞膜去极化期间,激活电压门控钙离子通道扮演极重要之角色。特别是L型Cav通道的成员Cav1.2,心脏动作电位的平台期主要通过电压门控L型Cav1.2通道向细胞内运输钙离子。
电压活化的Cav1.2钙离子通道特异性高表达于神经与心肌细胞膜上,由三个亚单元组成的复合物,包含α1、α2/δ1和β亚单元,比例为1:1:1。Cav1.2钙离子通道是由α1亚单元形成主孔,α1亚单元的C端非共价结合细胞质内的β辅助亚单元和α2/δ1亚单元组成的异质聚合物。
Cav1.2α1单元由CACNA1C基因编码,CACNA1C基因突变可导致Long-QT综合征与Timothy综合征。α2/δ1亚单元由CACNA2D1基因编码,CACNA2D1基因突变可导致心脏缺陷和Short-QT综合征。β亚单元由CACNB2基因编码,CACNB2基因突变可导Brμgada综合征与Long-QT综合征。
Tetracycline(Tet,四环霉素)调控基因表达系统是以大肠杆菌Tn10转座子上Tet抗性操纵子为基础而建立的。Tet阻遏蛋白(Tetrepressorprotein,TetR)与Tet操纵子(Tetoperator,TetO)DNA序列能够特异性结合。当细胞内无四环霉素存在时,TetR会与TetO结合,从而阻断下游抗葯性基因表达。反之,当四环霉素存在时,四环霉素与TetR结合使构象发生改变,导致TetR与TetO分离,使下游抗葯性基因得以表达,细菌从而获得耐药性。利用TetR和TetO特异性结合的特点,多种类型的Tet调控系统逐渐发展起来,如应用较为广泛的Tet-On激活型系统。Tet-On系统由调节和反应表达两个载体组成。调节表达载体包含一个由CMV启动子激活的反义四环霉素转录活化因子(rtTA),反应表达载体则包含四环霉素激活启动子(TetONpromoter)及目的基因。由于四环霉素激活启动子缺少CMV增强子,因此rtTA未与四环霉素激活启动子上的TRE结合时,目的基因不表达;当rtTA与TRE结合时,rtTA上的VP16会使最小CMV启动子活化从而使下游基因表达。在四环霉素或其类似物(如:多西环霉素,doxycycline)存在时,rtTA与多西环霉素形成复合物进而与TRE结合,使得目的基因表达。反之,多西环霉素不存在时,rtTA不能与TRE结合,导致基因表达被抑制。
现有技术中,表达Cav1.2钙离子通道蛋白,通常利用传统三载体设计,分别表达三个Cav1.2亚单元。此一设计缺点在于三载体共转染细胞比例过低,且须经三抗药性长时间筛选,成功难度较高;或可利用单一多顺反子载体表达三个钙离子通道亚单元,三个亚单元间以2A肽连接。如此,理论上单一载体能表达三个亚单元,于筛选更具优势。但实际上以2A肽连接连接多个(>2)基因表达,最下游末端的基因表达量相对低或不表达,如此一来容易使得钙离子通道表达不稳定。
发明内容
为了解决上述问题,本发明公开了一种高效制备电压依赖性钙离子通道细胞模型的方法。
本发明的技术方案如下:
一组共表达人Cav1.2钙离子通道蛋白的载体,包括载体1和载体2,所述人Cav1.2钙离子通道蛋白包括α1、α2/δ1和β三个亚基;
所述载体1含有TetON启动子,α2/δ1和β亚基的表达序列位于TetON启动子下游;同时载体1具备rtTA表达元件,由CMV启动子驱动表达,载体1还含有抗性基因1;
所述载体2含有TetON启动子,EGFP和α1亚基的表达序列位于TetON启动子下游;载体2还含有抗性基因2。
电压活化的Cav1.2钙离子通道特异性高表达于神经与心肌细胞膜上,由三个亚单元组成的复合物,包含α1、α2/δ1和β亚单元,比例为1:1:1。Cav1.2钙离子通道是由α1亚单元形成主孔,α1亚单元的C端非共价结合细胞质内的β辅助亚单元和α2/δ1亚单元组成的异质聚合物。为增加细胞转染效率,本发明将三载体表达三个亚单元的传统上手段近一步优化成双载体系统(表达三亚单元),并且携带报告基因。
进一步的,上述一组共表达人Cav1.2钙离子通道蛋白的载体,α2/δ1和β亚基的表达序列之间通过T2A肽的表达序列连接;EGFP和α1亚基的表达序列之间通过P2A肽的表达序列连接。
发明人发现,可利用IRES序列替代P2A肽序列,连接两个钙离子通道亚单元或报告基因与钙离子通道亚单元。以相同的策略“双多顺反子载体介导钙离子通道Cav1.2表达”加上报告基因分选,理论上能得到相似成果。但发明人实际操作却发现,以IRES序列连接两个钙离子通道亚单元造成EGFP报告基因持续表达,不受多西环霉素诱导调控(图6、A与B)。并且只有10%的细胞具有电压门控钙离子通道特性(图6、C),结果显示本发明使用2A肽连结Cav1.2钙离子通道亚单元,为优选设计。
进一步的,上述一组共表达人Cav1.2钙离子通道蛋白的载体,所述抗性基因1为抗嘌呤霉素基因;所述抗性基因2为抗潮霉素基因。
进一步的,上述一组共表达人Cav1.2钙离子通道蛋白的载体,所述α1亚基的表达序列如NM_199460.4所示;所述α2/δ1的表达序列如NM_000722.4所示;所述β亚基的序列如NM_201596.3所示。
进一步的,一种表达细胞,上述任一项所述的一组载体。
进一步的,上述表达细胞属于HEK293T细胞系。在一些实施例中,透过脂质体介导转染,HEK293T细胞共转染表达CACNB2与CACNA2D1以及EGFP与CACNA1C的两个载体DNA。利用分别位于两个载体上的抗药性基因puromycineR与hygromycineR筛选共转染细胞,具备功能之Cav1.2至少须由CACNA1C、CACNB2与CACNA2D1表达之三个亚单元组成。
进一步的,一种高效制备电压依赖性钙离子通道细胞模型的方法,包括以下步骤:
1)使用上述任一项所述的一组载体转染表达细胞;
2)使用抗性基因1和抗性基因2所针对的双重抗药性进行转染细胞的筛选;
3)使用多西环霉素诱导转染细胞表达人Cav1.2钙离子通道蛋白;
4)使用流式细胞仪分选EGFP高、中度表达细胞亚群。
进一步的,上述一种高效制备电压依赖性钙离子通道细胞模型的方法,所述步骤3)中多西环霉素诱导条件为多西环素2μg/mL处理20小时。
在一些实施例中,本发明利用EGFP作为報告基因,筛选三亚单元共表达之细胞(只有共转两个载体之细胞能成功表达EGFP与三个Cav1.2亚单元)。具备puromycine与hygromycine抗性的共转染细胞,以多西环霉素诱导EGFP与钙离子通道Cav1.2表达。通过荧光显微镜分析EGFP表达,并以流式细胞仪分选EGFP高、中度表达细胞亚群。
进一步的,上述一种高效制备电压依赖性钙离子通道细胞模型的方法,还包括以下步骤:
5)对步骤4)筛选获得的EGFP高、中度表达细胞亚群分别进行二次多西环霉素诱导。
在一些实施例中,EGFP高与中度表达HEK293T细胞亚群分选后,培养于无多西环霉素培养基。扩增后再次铺板于6孔细胞培养板,再次以多西环霉素(2μg/mL)处理20小时诱导EGFP与钙离子通道Cav1.2表达。除以流式细胞仪再次分析EGFP表达之外,透过膜片钳技术操控细胞膜电位,验证Cav1.2通道的钙离子通道功能特性。
一种电压依赖性钙离子通道细胞模型,由上述方法制备获得。
相比现有技术,本发明具有如下有益效果:
本发明根据NCBI提供的CACNA1C、CACNA2D1与CACNB2人类基因序列[NM_199460.4(577..7239)、NM_000722.4 (319..3591)与NM_201596.3 (314..2293) ]合成编码优化全基因序列。为增加细胞转染效率,本发明将三载体表达三个亚单元的传统上手段近一步优化成双载体系统(表达三亚单元),并且携带报告基因。本发明将CACNB2与CACNA2D1构建于同一多顺反子载体,实现同一个启动子控制下的两蛋白共翻译。两亚单元间由T2A肽序列连接,位于TetON启动子下游由其驱动表达。此载体同时具备rtTA表达元件,由CMV启动子驱动(如附图1所示)。为增加细胞分选选便利性,本发明将EGFP与CACNA1C构建于同一多顺反子载体,两者间由P2A肽序列连接,位于TetON启动子下游由其驱动表达(如附图2所示)。由于CMV病毒启动子驱动的rtTA元件只构建于一载体上,实现只有双载体共转细胞始能表达EGFP与组成Cav1.2钙离子通道所必需的亚单元(如附图3所示)。其中α1亚单元為24次穿膜蛋白表达于细胞膜上形成钙离子通道主孔,细胞质内的β辅助亚单元被吸引至α1亚单元的C端与之非共价结合,并防止α1亚单元被泛素/蛋白酶体系统降解,借此促进Cav1.2通道的细胞表面密度。反之,α2/δ1亚单元位于细胞膜表面通过糖基磷脂酰肌醇锚点附着在α1亚单元上。在α2/δ1亚单元共表达的条件下,Cav1.2通道产生之电流密度峰值显著增加, Cav1.2通道激活阀值更低(使Cav1.2通道在更强烈负电压环境下也能激活)。虽然机制尚未明确,但α2/δ1亚单元在细胞表面的密度确实决定了钙离子通过L型Cav1.2通道向细胞内的净流入。
具备功能之Cav1.2至少须由CACNA1C、CACNB2与CACNA2D1表达之三个亚单元组成,本发明利用EGFP作为报告基因,筛选三亚单元共表达之细胞(只有共转两个载体之细胞能成功表达EGFP与三个Cav1.2亚单元)。具备puromycine与hygromycine抗性的共转染细胞,以多西环霉素诱导EGFP与钙离子通道Cav1.2表达。通过荧光显微镜分析EGFP表达,并以流式细胞仪分选EGFP高、中度表达细胞亚群。
通过多西环霉素诱导,本发明之细胞系具有稳定表达、且具有功能性的人类Cav1.2钙离子通道的特性。本发明提供人类心肌细胞之外的替代细胞系—Cav1.2钙离子通道表达HEK293T细胞系,作为有效且经济的药物体外心安全性评价。
附图说明
图1为Cav1.2钙离子通道β与α2/δ1亚单元表达载体图谱;
图2为EGFP与Cav1.2钙离子通道α1亚单元表达载体图谱;
图3为P2A肽介导双多顺反子载体造成钙离子通道Cav1.2诱导性表达;(A)HEK293T细胞单独转染表达EGFP与CACNA1C的载体(左,不含rtTA),或共转染表达CACNB2与CACNA2D1(含rtTA)以及EGFP与CACNA1C的两个载体(右),多西环霉素(w/ Dox,2μg/mL)诱导20小時,荧光显微镜观察EGFP表达细胞;(B)荧光显微镜观察多西环霉素(w/ Dox)诱导共转染HEK293T细胞EGFP表达;(C)流式细胞仪分析多西环霉素(w/ Dox)诱导共转染HEK293T细胞EGFP表达,高与中度表达细胞亚群占比活细胞12.3与26.2%;
图4为Cav1.2表达HEK293T细胞分选;EK293T细胞共转染表达CACNB2与CACNA2D1以及EGFP与CACNA1C的两个载体,多西环霉素(w/ Dox)诱导下流式细胞仪分析显示EGFP表达细胞,分选富集EGFP高与中度表达细胞亚群;培养后的细胞不给予多西环霉素(w/o Dox),EGFP高与中度细胞亚群含有少量EGFP阳性细胞,给予多西环霉素(w/ Dox)再次诱导EGFP高与中度分选细胞分别含有75.2与66.2%EGFP阳性细胞;
图5为人源Cav1.2钙离子通道功能性分析;HEK293T细胞共转染表达CACNB2与CACNA2D1以及EGFP与CACNA1C的两个载体,多西环霉素(2μg/mL)诱导20小時;膜片钳技术纪录Cav1.2通道开启时的细胞内电流变化;(A)、(B)、(C)三个代表性细胞钙离子通道诱导电流;
图6为IRES介导多顺反子载体造成钙离子通道Cav1.2非诱导性表达;HEK293T细胞共转染以IRES连接CACNB2与CACNA2D1以及EGFP与CACNA1C的两个表达载体,(A)有、无多西环霉素(w/, w/o Dox)诱导下,荧光显微镜皆观察到EGFP表达细胞(B)流式细胞仪分析转染双载体HEK293T高度表达EGFP,且不受多西环霉素(w/ 或w/o)诱导;(C)膜片钳技术诱导Cav1.2通道的钙离子通道开启,仅10%细胞(n=10)具有电流纪录。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,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它实施例,都属于本发明保护的范围。
本发明实施例中使用的试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规试剂产品。
本发明中缩略语和关键术语定义见下表1
表1缩略语和关键术语定义
实施例1
人源Cav1.2钙离子表达载体构建
根据NCBI提供的人类基因CACNA1C、CACNA2D1与CACNB2编码序列[NM_199460.4(577..7239)、NM_000722.4 (319..3591)与NM_201596.3 (314..2293) ]合成转译优化编码序列。编码优化之CACNA1C、CACNA2D1与CACNB2序列分别连接至载体pcDNA3.1-Hygro、pcDNA3.1-Puro与pcDNA3.1-Zeo,而获得pcDNA3.1-Hygro-CACNA1C、pcDNA3.1-Puro-CACNA2D1与pcDNA3.1-Zeo-CACNB2,并于合成序列的5’端预留NheI,AflII,HindIII及3’端预留BamHI,EcoRI,XbaI,NotI,XhoI,XbaI等酶切位点。首先构建 pcDNA3.1_TetOn_CACNB2-T2A-CACNA2D1载体,透过T2A串联CACNB2和CACNA2D1同时表达,并在编码序列的5’端引入四环霉素响应元件(TRE),以及上游反义四环霉素转录活化因子(rtTA)。分别用以下四对引物:
V5-CACNB2_F =SEQ ID NO:1=(5’-GTACCACTTCCTACCCTCGTAAACTTAAGGCCACCATGGGCAAGCCCATCCCCAACCCCCTGCTGGGCCTGGATAGCACAGTGCAGAGAGACATGAGCAAGTCC-3’)
和T2A-CACNB2_R=SEQ ID NO:2=(5’-CTTGTAATCCATGGCGGCTGGGCCAGGATTCTCCTCGACGTCACCGCATGTTAGCAGACTTCCTCTGCCCTCGGAGGACTGTCTGATGTACACATCTCTGTTCCAC-3’);
FLAG-CACNA2D1_F = SEQ ID NO:3=(5’-CCAGCCGCCATGGATTACAAGGATGACGATGATAAGGATTACAAGGACGACGATGACAAGGACTACAAGGACGATGACGACAAGGCCGCTGGCTGTCTGCTGG-3’)
和STOP-CACNA2D1_R=SEQ ID NO:4= (5’-CACTGTGCTGGATATCTGCAGAATTCTCACAGCAGTCTGTGTGTGCTGCC-3’);
rtTA_F =SEQ ID NO:
5=(5’-CCAAGCTGGCTAGCGTTTAAACGCCACCATGTCTAGGCTGGACAAGAGCAAAG-3’)
和BGH-Ter_R =SEQ ID NO:
6=(5’-CAGATGTAATGAAAATAAAGATATTTTATTCCATAGAGCCCACCGCATCC-3’);
Blocker_F = SEQ ID NO:7=(5’-AATAAAATATCTTTATTTTCATTACATCTGTGTG-3’)
和TRE_R = SEQ ID NO:8=(5’-GCCATGGTGGCAAGCTTAAGTTTACGAGGGTAGGAAGTGGTA-3’)。
分别以pcDNA3.1-Zeo-CACNB2、pcDNA3.1-Puro-CACNA2D1、TetOn-hVEGF载体为模板,利用聚合酶Q5® High-Fidelity 2X Master Mix (NEB) 进行PCR扩增4 DNA片段(V5-tagged CACNB2-T2A、Flag-tagged CACNA2D1-STOP、rtTA-BGH terminator与 TRE-Transcription blocker terminator)。以限制酶AflII和EcoRI剪切pcDNA3.1-Puro-CACNA2D1,经胶体回收cDNA3.1-Puro载体骨架(移除CACNA2D1序列)。将上述四个DNA片段混合pcDNA3.1-Puro骨架,利用NEBuilder® HiFi DNA Assembly Master Mix (NEB) 进行重组,获得pcDNA3.1-TetOn_CACNB2-T2A-CACNA2D1载体(图1)。
其次,构建pcDNA3.1-X-TRE_EGFP-P2A-CACNA1C载体,以pcDNA3.1-Hygro-CACNA1C为骨架,在CACNA1C编码序列5’端先置入四环霉素响应元件(TRE)。以TetOn-hVEGF载体为模板、引物TRE-NheI fus_F = SEQ ID NO:9=(5’-CTATAGGGAGACCCAAGCTGGCTAGCAATAAAATATCTTTATTTTCATTACATCTGTGTG-3’)和TRE-KpnI fus_R = SEQ ID NO:10=(5’-TTCTCATTCACCATGGTGGCGGTACCTTTACGAGGGTAGGAAGTGGTA-3’),利用聚合酶Q5® High-Fidelity 2XMaster Mix (NEB) 进行PCR扩增DNA (NheI-TRE-KpnI)片段。以限制酶NheI和HindIII剪切pcDNA3.1-Hygro-CACNA1C,并胶体回收pcDNA3.1-Hygro-CACNA1C骨架。将PCR扩增的NheI-TRE-KpnI片段混合pcDNA3.1-Hygro-CACNA1C骨架,利用NEBuilder® HiFi DNA AssemblyMaster Mix (NEB) 进行重组,获得pcDNA3.1-TRE-CACNA1C载体。接着以P2A串联EGFP和CACNA1C,EGFP用於验证四环素诱导CACNA1C表达。以TetOn-hVEGF载体为模板、引物EGFP-P2A-CACNA1C_F = SEQ ID NO:11=(5’-CCACTTCCTACCCTCGTAAAGGTACCGCCACCATGGTGAGCAAGG-3’)和EGFP-P2A-CACNA1C_R = SEQ ID NO:12=(5’-CTGGTGTTCTCATTCACCATAGGGCCAGGGTTTTCCTCC-3’),利用聚合酶Q5® High-Fidelity 2X Master Mix (NEB) 进行PCR扩增EGFP-P2A DNA片段。将经KpnI 酶切的pcDNA3.1-TRE-CACNA1C骨架混合EGFP-P2A片段,利用NEBuilder® HiFi DNA Assembly Master Mix (NEB) 进行组装,获得pcDNA3.1-TRE-EGFP-P2A-CACNA1C。为了免除上游CMV启动子导致的调控干扰,以NheI和MLuI酶切去除CMV启动子片段,利用DNA Polymerase I, Large (Klenow) Fragment (NEB) 抹平酶切端点,再利用T4 DNA黏合酶连接,从而获得pcDNA3.1-X-TRE_EGFP-P2A-CACNA1C_Hygro 载体(图2)。
实施例2
双质粒DNA介导人源Cav1.2钙离子表达载体細胞转染与筛选
将多顺式表达CACNB2与CACNA2D1以及EGFP与CACNA1C的两个载体DNA以3:1比例混合,加入脂质体(Lipofectamine3000,Invitrogen)形成脂质体/DNA复合物,置于10厘米培养皿。將汇合率达到90%的HEK293T細胞以胰酶消化、计算细胞数,加入含脂质体/DNA复合物的OptiMEM培养基。培养于10厘米培养皿4小時,將培养基置换成含5%胎牛血清DMEM,24小时后加入puromycine(2μg/mL)与hygromycine(200μg/mL),通过抗药性筛选1周。双载体共转染细胞经多西环霉素(2μg/mL)处理20小时,高度表达EGFP。反之,转染表达EGFP与CACNA1C之单载体的细胞,多西环霉素无法诱导EGFP表达(图3A)。共转染细胞(表达EGFP与Cav1.2通道HEK293T细胞)以多西环霉素处理,大量EGFP阳性细胞;无多西环霉素处理下,极少量细胞表达EGFP(图3A、B)。符合预期地,多西环霉素处理后EGFP高与中度表达细胞亚群占比活细胞12.3与26.2%(图3C)。
实施例3
多西环霉素诱导人源Cav1.2钙离子通道表达细胞分选
由于EGFP与CACNA1C的两基因以P2A肽连接,位于同一多顺反子载体,两者的表达量关系为1:1,本发明利用EGFP表达量分析Cav1.2通道在HEK293T细胞的表达量。透过流式细胞分选,本发明将EGFP高与中度表达细胞亚群分别分选,并给予第二次的给予多西环霉素诱导。给予多西环霉素再次诱导令EGFP高与中度分选细胞分别含有75.2与66.2% EGFP阳性细胞,显示分选EGFP阳性细胞,细胞可表达更高量的Cav1.2(图4)。
实施例4
人源Cav1.2钙离子通道功能性分析
EGFP高与中度表达细胞亚群,给予第二次多西环霉素诱导Cav1.2离子通道表达。膜片钳技术操控细胞膜电位,使得Cav1.2离子通道开启,并纪录细胞内电流变化。结果显示EGFP高度表达细胞亚群75% (3/4)具备功能性Cav1.2钙离子通道,比起EGFP中度表达细胞亚群(17%,1/6),有更高比例的细胞具备功能性Cav1.2钙离子通道(图5)。
以上所述实施例仅表达了本发明的有限几种优选实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (8)
1.一组共表达人Cav1.2钙离子通道蛋白的载体,其特征在于,包括载体1和载体2,所述人Cav1.2钙离子通道蛋白包括α1、α2/δ1和β三个亚基;
所述载体1含有TetON启动子,α2/δ1和β亚基的表达序列位于TetON启动子下游;同时载体1具备rtTA表达元件,由CMV启动子驱动表达,载体1还含有抗性基因1;
所述载体2含有TetON启动子,EGFP和α1亚基的表达序列位于TetON启动子下游;载体2还含有抗性基因2;
所述α2/δ1和β亚基的表达序列之间通过T2A肽的表达序列连接;
所述EGFP和α1亚基的表达序列之间通过P2A肽的表达序列连接。
2.根据权利要求1所述的一组共表达人Cav1.2钙离子通道蛋白的载体,其特征在于,所述抗性基因1为抗嘌呤霉素基因;所述抗性基因2为抗潮霉素基因。
3.根据权利要求1所述的一组共表达人Cav1.2钙离子通道蛋白的载体,其特征在于,所述α1亚基的表达序列如NM_199460.4所示;所述α2/δ1的表达序列如NM_000722.4所示;所述β亚基的表达序列如NM_201596.3所示。
4.一种表达细胞,其特征在于,含有如权利要求1-3任一项所述的一组载体。
5.根据权利要求4所述的表达细胞,其特征在于,所述表达细胞属于HEK293T细胞系。
6.一种高效制备电压依赖性钙离子通道细胞模型的方法,其特征在于,包括以下步骤:
1)使用如权利要求1-3任一项所述的一组载体转染表达细胞;
2) 使用抗性基因1和抗性基因2所针对的双重抗药性进行转染细胞的筛选;
3)使用多西环霉素诱导转染细胞表达人Cav1.2钙离子通道蛋白;
4)使用流式细胞仪分选EGFP高、中度表达细胞亚群。
7.根据权利要求6所述的一种高效制备电压依赖性钙离子通道细胞模型的方法,其特征在于,所述步骤3)中多西环霉素诱导条件为多西环素2μg/mL处理20小时。
8.根据权利要求7所述的一种高效制备电压依赖性钙离子通道细胞模型的方法,其特征在于,还包括以下步骤:
5)对步骤4)筛选获得的EGFP高、中度表达细胞亚群分别进行二次多西环霉素诱导。
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