CN113684186A - 一种可支持乙肝病毒复制和感染的细胞模型及建立方法 - Google Patents
一种可支持乙肝病毒复制和感染的细胞模型及建立方法 Download PDFInfo
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Abstract
本发明提供了一种可支持乙肝病毒复制和感染的细胞模型及建立方法。本发明的发明人在实验中发现BEL‑7404细胞具有较高的转染效率,在过表达人牛磺胆酸钠共转运体多肽(hNTCP)和肝细胞核因子(HNF4α)基因后可以较好的支持乙肝病毒的复制和感染,以此构建了可稳定表达hNTCP和HNF4α的7404NT‑HNF4α细胞模型,该培养系统可通过在培养基中添加强力霉素(Doxycycline,DOX)来调控HNF4α的表达进而调控HBV的复制和感染,为乙肝病毒的研究提供一种新的细胞模型。
Description
技术领域
本发明涉及细胞改造技术领域,具体涉及在BEL-7404细胞上构建的高表达hNTCP和HNF4α基因的7404NT-HNF4α细胞模型在研究乙肝病毒复制和感染中的应用。
背景技术
乙型肝炎病毒(Hepatitis B virus,HBV)属于嗜肝性的含不完整双链DNA的包膜病毒。据报道全球慢性HBV感染者至少有2.57亿人,HBV慢性感染可以造成慢性肝炎、肝硬化和肝细胞癌等临床疾病,极大地危害着人类生命健康。现有的抗病毒治疗(干扰素和核苷(酸)类似物)只能在一定程度上抑制病毒的复制,但并不能有效清除病毒,尚不能达到治愈慢性乙肝的目的。
HBV通过结合人细胞膜表面的钠离子-牛磺胆酸共转运蛋白(human Na+-taurocholate cotransporting polypeptide,hNTCP)进入肝细胞后,其包裹在核衣壳中的3.2kb环状部分双链DNA(rcDNA)基因组被导入细胞核,并转换为共价闭合环状DNA(cccDNA),随后以cccDNA为模板,进行下游基因组mRNA的转录。由四个启动子(Cp、Sp1、Sp2和Xp)与增强子(Enhancer I,EnI)和增强子II(Enhancer II,EnII)共同作用调控病毒的转录。核心启动子(Cp)在病毒生命周期中起着关键作用,启动3.5kb preC/pregenomic mRNA的合成,编码e抗原、核心蛋白和聚合酶。实验证实启动子区域存在大量肝特异性和泛性转录因子的结合位点,转录因子HNF4α(hepatocyte nuclear factor 4α)是肝富集转录因子家族的重要成员,可参与调控Cp的转录,从而调控HBV的3.5kb preC/pregenomic RNA的产生,进而对HBV复制发挥调控作用。
HBV在体内的感染和复制具有很强的嗜肝性和明确的种属特异性。虽然现在已经建立了一些支持HBV复制和感染的细胞模型,但均存在一定的缺陷。人原代肝细胞(PHH)在体外可以被HBV感染,但价格昂贵,很难获取且很快就容易失去易感性;HepaRG细胞需要经过长期的分化过程才能支持HBV的感染且培养条件比较复杂;hNTCP被证明是乙肝受体后,基于肝癌细胞系HepG2、Huh7构建了HBV感染细胞模型,但这两种细胞转染效率较低,感染效果不理想。对于HBV的研究,仍然需要寻找更多新的细胞模型。
BEL-7404是一株肝癌细胞株,来源于一名69岁中国男性,常被用于肝癌研究,尚未见到其在HBV复制和感染研究中的应用。
发明内容
鉴于相关技术的上述问题和/或其他问题,本发明主要通过体外实验评价了基于BEL-7404构建的7404NT-HNF4α细胞模型在HBV研究中的应用。体外实验主要包括细胞转染效率的测定,HBV复制的检测,构建能够稳定表达hNTCP和HNF4α的7404NT-HNF4α细胞模型,HBV的感染实验等。本发明旨在解决目前亟需能够支持HBV复制和感染的细胞模型的问题,提供了7404NT-HNF4α在HBV复制和感染研究中的新用途。
本发明的技术方案为:通过构建hNTCP慢病毒表达系统,转导BEL-7404细胞,经实验验证和抗生素筛选出BEL-7404-hNTCP细胞。之后使用Tet-on系统构建了可受强力霉素(Doxycycline,DOX)诱导调控的HNF4α慢病毒表达系统,感染上一步构建的BEL-7404-hNTCP细胞,经抗生素筛选和单克隆纯化,得到BEL-7404-hNTCP-Tet-on-HNF4α(命名为7404NT-HNF4α)细胞。之后进行支持HBV复制和感染的实验验证。
具体的,本发明的技术方案如下:
本发明第一个方面公开了一种细胞模型,所述细胞模型为BEL-7404-hNTCP细胞。
优选的,所述的BEL-7404-hNTCP细胞包括表达hNTCP的BEL-7404细胞。
优选的,在所述的BEL-7404细胞中,hNTCP高表达。
优选的,所述的表达包括mRNA和/或蛋白表达。
优选的,hNTCP的Gene ID:6554;RefSeq NM_003049。
在本发明的一些具体实施例中,基于BEL-7404细胞株构建的可支持HBV复制和感染的7404NT-HNF4α细胞模型。
优选的,在BEL-7404细胞中增强了hNTCP和HNF4α基因的表达。优选的,hNTCP的Gene ID:6554;RefSeq NM_003049。
优选的,HNF4α的Gene ID:3172;RefSeq NM_001287182。
本发明第二个方面公开了一种构建上述的细胞模型的方法,所述构建方法包括:通过构建hNTCP慢病毒表达系统,转导BEL-7404细胞,经抗生素筛选,得到BEL-7404-hNTCP细胞。
本发明第三个方面公开了一种基于上述的细胞模型制备得到的细胞,所述细胞为BEL-7404-hNTCP-Tet-on-HNF4α细胞。
本发明第四个方面公开了一种制备上述细胞的方法,包括:使用HNF4α慢病毒表达系统感染BEL-7404-hNTCP细胞,经过抗生素筛选和单克隆纯化得到BEL-7404-hNTCP-Tet-on-HNF4α细胞。
优选的,使用Tet-on表达系统构建可受强力霉素诱导调控的HNF4α慢病毒系统。
优选的,包括:
S1:构建hNTCP和HNF4α重组慢病毒质粒;
S2:用hNTCP重组慢病毒感染BEL-7404细胞;
S3:使用Tet-on系统构建含HNF4α的慢病毒感染系统;
S4:使用S3得到的含HNF4α的慢病毒感染系统感染BEL-7404-hNTCP细胞,经过筛选得到纯化的BEL-7404-hNTCP-Tet-on-HNF4α细胞。
本发明第五个方面公开了一种基于上述的细胞模型制备得到的细胞,所述细胞为7404NT-HNF4α细胞。
优选的,所述的7404NT-HNF4α细胞包括表达hNTCP和HNF4α的BEL-7404细胞。
优选的,在所述的7404NT-HNF4α细胞中,hNTCP高表达。
优选的,在所述的7404NT-HNF4α细胞中,HNF4α高表达。
优选的,所述的表达包括mRNA和/或蛋白表达。
优选的,hNTCP的Gene ID:6554;RefSeq NM_003049。
优选的,HNF4α的Gene ID:3172;RefSeq NM_001287182。
本发明第六个方面公开了一种根据上述的细胞模型、上述的细胞在乙肝病毒研究中的应用。
优选的,所述的研究为体外研究。
优选的,所述的研究为非诊断性研究。
优选的,所述的研究为非治疗性研究。
优选的,所述应用为研究乙肝病毒复制和感染的应用。
优选的,所述细胞模型或细胞用于筛选对HBV有抑制作用的药物。
在符合本领域常识的基础上,上述各优选条件,可任意组合,而不超出本发明的构思与保护范围。
本发明针对7404NT-HNF4α细胞株提供一种作为HBV复制和感染研究的细胞模型的新用途。BEL-7404具有较高的转染效率且培养条件比较简单等优点,在过表达hNTCP和HNF4α基因后,可以支持产生完整的HBV复制并被HBV感染。HNF4α的表达可通过在培养基中添加DOX进行调控,因此,HBV在7404NT-HNF4α细胞模型中的转录、复制、cccDNA形成及HBV感染也可通过DOX进行调控。
本发明相对于现有技术具有如下的显著优点及效果:
本发明基于BEL-7404较高的转染效率且培养条件比较简单等优点,基于hNTCP和HNF4α在HBV感染复制过程中的关键作用,建立了可以稳定表达hNTCP和HNF4α的7404NT-HNF4α细胞模型及方法。另外本发明提供了7404NT-HNF4α在HBV复制和感染研究中的新用途。
附图说明
图1为通过荧光显微镜和流式细胞实验观察在BEL-7404中转染pCMV-EGFP质粒后的荧光强度及转染效率的实验结果示意图。
图2为通过Southern blot实验检测在BEL-7404中过表达HNF4α基因后可支持完整的HBV复制的实验结果示意图。
图3为构建7404NT-HNF4α细胞系相关示意图,实时定量PCR(qPCR)和Western blot检测在细胞中hNTCP和HNF4α的表达水平。
图4为通过酶联免疫吸附实验(ELISA)检测细胞上清HBsAg和HBeAg表达水平示意图(Southern blot实验检测HBV在7404NT-HNF4α细胞中的复制,ELISA检测HBV在7404NT-HNF4α细胞中的感染情况,验证该细胞模型支持HBV复制和感染的作用)。
图5为构建hNTCP重组慢病毒质粒构建示意图。
图6为使用Tet-on系统构建的HNF4α重组慢病毒质粒构建示意图。
术语
在本发明中,术语“hNTCP”是指牛磺胆酸钠共转运体多肽。
在本发明中,术语“HNF4α”是指肝细胞核因子4α。
在本发明中,术语“BEL-7404”、“BEL-7404CELL LINE”与“人肝癌细胞系”可互换使用。
具体实施方式
下面结合附图1-6和实施例对本发明的技术方案进行详细描述,但并不因此将本发明限制在所述的实施例范围之中。
本发明的发明人在可支持HBV复制和感染的细胞系筛选过程中,发现BEL-7404细胞系在过表达hNTCP和HNF4α基因后可以较好的支持HBV的复制和感染,具有较高的转染效率,可以应用于对HBV复制和感染机制的研究以及筛选对HBV有抑制作用的药物。
本发明的一个具体实施方案中提供了BEL-7404在HBV研究中的新用途。
在本发明的一个优选实施方案中,提供了一种基于BEL-7404构建的可支持HBV复制和感染的7404NT-HNF4α细胞模型,可以应用于对HBV复制和感染机制的研究以及筛选对HBV有抑制作用的药物。
另外,在本发明揭示了在7404NT-HNF4α细胞模型在HBV研究中的新用途,本领域技术人员在获知该启示的基础上,可以开发出以7404NT-HNF4α细胞模型为前体的其他细胞模型,以及7404NT-HNF4α细胞模型在生物学研究中可接受的其他应用等。
下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。本发明所用试剂和原料均市售可得。
实施例
hNTCP的信息(Gene ID:6554;RefSeq NM_003049)
HNF4α的信息(Gene ID:3172;RefSeq NM_001287182)
实施例1
通过荧光显微镜和细胞流式实验观察在BEL-7404转染pCMV-EGFP质粒后的转染效率:
1.在6孔板中接种BEL-7404细胞(该BEL-7404细胞由中国科学院细胞库/干细胞库提供,目录号为TCHu64)悬液(8×105个/孔),将培养板放在培养箱中预培养12小时,细胞贴壁生长。
2.每孔使用Turbofect transfection reagent(Thermo Fisher Scientific,Waltham,USA)转染试剂4μl与2μg pCMV-EGFP质粒进行转染实验,37℃培养48小时,在荧光显微镜下观察荧光表达效果,实验结果如图1A所示。本步骤中的pCMV-EGFP质粒由申请人自己构建,具体方法为:使用PCR结合限制性酶切与连接的方法将EGFP cDNA序列克隆到pCDN3.1(V79520,Invitrogen)载体中。
3.将细胞在孔板中用胰酶消化下来,用移液枪吹打混匀制成单细胞悬液,吸取到1.5ml的EP管中,在EP管管底肉眼可见细胞沉淀,弃去上清。
4.在细胞沉淀中加入1ml的Cell Staining Buffer,重悬细胞后,350g离心5min,弃去上清,重复两次。
5.用Cell Staining Buffer重悬细胞,计数活细胞数,按照细胞个数为5×106cells/ml将这些细胞悬液以100μl/管分装至新的1.5ml EP管中,将样品转移到上机检测的玻璃管中,做好标记,接下来用流式细胞仪进行分选测定,之后用FlowJo软件进行数据分析。实验结果如图1B所示。
实验结果显示,BEL-7404细胞转染pCMV-EGFP质粒后比Huh7细胞系有更高的转染效率且荧光表达强度较高。
实施例2
Southern blot实验检测在BEL-7404中过表达HNF4α基因后可支持完整HBV的复制,具体实验包括:
1.在60mm培养皿中接种BEL-7404细胞悬液(1×106个/皿),将培养皿放在培养箱中预培养12小时,细胞贴壁生长。
使用16μl Turbofect转染试剂将4μg pHNF4α与4μg p1.3×HBV复制子质粒共同转染进BEL-7404细胞中,对照组转染4μg空载质粒和4μg p1.3×HBV复制子质粒。96h后提取细胞内HBV DNA进行Southern blot实验。其中,pHNF4α的构建方法为使用PCR结合限制性酶切与连接的方法将HNF4αcDNA序列克隆到pCDN3.1(V79520,Invitrogen)载体中。
p1.3×HBV复制子质粒的构建方法为以HBV毒株序列为模板,用PCR结合限制性酶切的方法分步将1.3拷贝HBV基因组克隆到pUC18(Cat.3218,Takara)载体。序列已上传至NCBI官网,序列编号为KR232337(参考文献:Shen,Z.;Yang,H.;Yang,S.;Wang,W.;Cui,X.;Zhou,X.;Liu,W.;Pan,S.;Liu,Y.;Zhang,J.;Zhang,J.;Xie,Y.;Liu,J.,Hepatitis Bvirus persistence in mice reveals IL-21and IL-33as regulators of viralclearance.Nat Commun 2017,8,(1),2119.)
2.提取细胞内HBV核心颗粒内的DNA
1)细胞裂解:将转染后细胞弃上清,用预冷的PBS洗2遍,每100mm培养皿用400μl裂解缓冲液(0.5%V/V NP40,1mM EDTA,50mM NaCl,10mM Tris-HCl,pH 7.9)裂解细胞15min。
2)去除残余质粒和细胞基因组DNA:收集细胞裂解液,14000g离心5min,收集上清,弃细胞残骸。加入4μl 1M氯化镁,8μl 10mg/ml DNase I,37℃水浴中消化30min。
3)PEG沉淀病毒颗粒:消化产物经14000g离心,保留上清。加入12μl 0.5M EDTA和100μl 35%PEG8000/1.75M氯化钠,混匀后,4℃沉淀过夜。14000g离心10min弃上清留沉淀。
4)第二次去除残余质粒和细胞基因组DNA:将沉淀用100μl DNase I溶液重悬(1μl1M Tris-HCl,pH7.9,1μl 10mg/ml DNase I,1M氯化镁,剩余用水补平)。37℃消化30min。
5)蛋白酶K消化,去除病毒衣壳:在上述溶液中,加入300μl SDS/蛋白酶K溶液,37℃消化过夜。
6)苯酚/氯仿抽提,沉淀病毒DNA:用等体积的苯酚/氯仿抽提两次,再加入2μl20mg/ml糖原、1/10体积的3M醋酸钠(pH 5.2)溶液和等体积的异丙醇,混匀后置于-20℃沉淀过夜。15000g离心15min,弃上清,沉淀用75%乙醇洗两遍并弃尽乙醇,静置待残留乙醇挥发后,用20μl灭菌蒸馏水溶解。
3.Southern blot检测HBV复制
琼脂糖电泳:将提取的细胞内HBV DNA进行1%琼脂糖凝胶电泳(100V,1.5h)。
2)变性:将电泳后的凝胶置于新鲜配置的变性液(0.5M NaOH和1.5M NaCl)中,室温震荡变性1h。
3)中和:弃变性液,倒入中和液(1.5M NaCl和1M Tris-HCl,pH 7.4)中和,室温震荡中和两次,每次30min。
4)转膜:使用向下毛细管转移法,转移系统由下至上分别是:吸水纸、Parafilm膜、2层3mm滤纸、尼龙膜、含DNA的琼脂糖凝胶、2层3mm滤纸、两端浸在20×SSC缓冲液(3M NaCl和0.3M柠檬酸钠)里的盐桥。常温转膜8h以上。
5)DNA交联:转膜后,取出尼龙膜,尼龙膜用2×SSC浸泡5min,沥干多余液体,将尼龙膜置于两张滤纸之间,紫外交联90s。
预杂交:将膜放入杂交管中,加入杂交液5ml,42℃预杂交30min。
7)探针变性和杂交:取适量探针,于100℃金属浴变性5min,然后迅速将变性的探针放置于冰上5min。回收预杂交液,换入新鲜的杂交液5ml,加入变性好的探针,42℃杂交6~8h。
8)洗去除未结合的探针:先用2×SSC室温洗2遍,每遍5min。再用0.5×SSC,68℃洗2遍,每遍15min;
9)封闭:用Maleic acid buffer稀释10×Blocking solution至1×封闭工作液,加入适量封闭液,37℃封闭30min。
10)孵育抗体:用上述1×封闭液稀释anti-DIG AP抗体(抗体按1:10000稀释),弃封闭液并换入适量抗体孵育液,37℃孵育30min。
11)洗去未结合抗体:用washing buffer在37℃洗膜2次,每次15min。
12)显色:先用detection buffer平衡膜5min,同时用detection buffer稀释、配制1×CSPD显色液。将平衡好的尼龙膜置于Parafilm膜上,正面朝上,均匀滴加显色液,再盖一层Parafilm膜,去除尼龙膜正面的气泡和多余的显色液,避光室温放置5min,用化学发光检测仪器检测累积信号,保存结果。
实验结果如图2所示,BEL-7404经pHNF4α和HBV复制子质粒共同转染后可检测到有较强的HBV复制,单独转染p1.3×HBV复制子未检测到复制产生。
实施例3
构建7404NT-HNF4α细胞系,通过实时定量PCR(qPCR)和Western blot检测在细胞中hNTCP和HNF4α的表达水平
1.构建7404NT-HNF4α细胞系:用慢病毒感染的方式分步进行稳转细胞系的构建。
hNTCP和HNF4α重组慢病毒质粒构建示意图分别如图5和图6所示。其中,图5是基于Tet-on表达调控系统构建的HNF4α质粒图谱。此系统表达需要Tet-on和TRE-HNF4α两种基因表达。原理如下:TRE为Tet-on的应答启动子,在四环素(DOX)存在时,可以活化Tet-on表达,从而激活TRE启动子启动HNF4α的转录,因此,在细胞系构建过程中进行了两步慢病毒感染过程,图6中左图为第一步使用的Tet-on质粒表达图谱,右图为TRE-HNF4α质粒表达图谱(FLAG为方便检测的标签基因)。二者是使用的是相同的慢病毒表达载体,但插入的表达基因不同。
1)制备hNTCP,HNF4α重组慢病毒浓缩液,利用阳离子聚合物聚乙烯亚胺(Polyethylenimine,PEI)基因转染技术,将慢病毒包装质粒psPAX2(Addgene,Watertown,USA),pMD2.G(Addgene,Watertown,USA)和hNTCP过表达质粒pCDH-hNTCP-Blast或pLVX-Tet-on 3G或pLVX-TRE-Flag-HNF4α-Puro按质量比3:1:4同时转染到HEK293T细胞中,转染后72h收集含慢病毒的培养上清,用0.45μm滤膜过滤后浓缩,-80℃保存。
2)用制备的hNTCP慢病毒感染BEL-7404细胞,经抗生素筛选鉴定。
3)使用Tet-On系统(Clontech,Mountain View,USA)构建含HNF4α的慢病毒感染系统,使用该系统可以通过四环素(doxycline,DOX)诱导HNF4α的表达,以此来达到调控目的。在上一步筛选出的BEL-7404-hNTCP细胞上进行感染,经抗生素筛选后得到纯化的BEL-7404-NTCP-Tet-on-HNF4α(7404NT-HNF4α)细胞系。
2.使用实时定量PCR(qPCR)方法检测细胞中hNTCP和HNF4α的转录水平:
1)使用传统的Trizol提取法进行RNA抽提。
2)RNA逆转录采用天根FastKing一步法合成cDNA(Tiangen,KR118)。
3)实时定量PCR采用天根SYBR Green I嵌合荧光法进行(Tiangen,FP205)。扩增引物采用针对hNTCP和HNF4α基因的特异性引物:hNTCP上游引物:5’-AAGGACAAGGTGCCCTATAAAGG-3’(SEQ ID NO:1);下游引物:5’-TTGAGGACGATCCCTATGGTG-3’(SEQ ID NO:2);HNF4α上游引物:5’-CGAAGGTCAAGCTATGAGGACA-3’(SEQ ID NO:3);下游引物:5’-ATCTGCGATGCTGGCAATCT-3’(SEQ ID NO:4)。结果如图3A所示。
3.使用Western blot方法检测细胞中hNTCP和HNF4α的蛋白表达水平
1)使用SDS裂解液将细胞裂解,收集细胞裂解液,离心后取上清,加入蛋白上样缓冲液,100℃变性5min。
2)进行SDS-聚丙烯酰胺凝胶电泳,转膜,封闭,抗体孵育[anti-β-actin(Sigma-Aldrich),anti-HNF4α(C11F12,Cell Signaling Technology,Beverly,USA)and anti-SLC10A1(Sigma-Aldrich)],进行相应的二抗孵育后用ECL Blotting Substrate(Millpore)进行显色。结果如图3B-3C所示。
结果显示,构建的7404NT-HNF4α细胞具有较高的hNTCP和HNF4α表达水平,且HNF4α的表达可通过在培养基中加入DOX进行调控。
实施例4
通过酶联免疫吸附实验(ELISA)与Southern blot检测HBV在7404NT-HNF4α细胞中的HBsAg与HBeAg表达及复制水平,ELISA检测HBV在7404NT-HNF4α细胞中的感染水平:
1.在7404NT-HNF4α细胞中转染p1.3×HBV复制子后,使用上海科华的ELISA检测试剂盒检测上清中HBsAg与HBeAg表达水平,结果如图4A所示。使用实施例2中的方法检测HBV的复制,结果如图4B所示。
2.使用浓缩的HBV(MOI=1000)感染7404NT-HNF4α细胞,感染体系中加入2.5%DMSO和4%PEG8000。12h后换液,PBS洗五次,加入含2.5%DMSO的完全培养基,之后48h进行换液并收集上清,ELISA检测上清中HBeAg表达水平。结果如图4C所示。
结果显示,构建的7404NT-HNF4α细胞在DOX的诱导下,HBsAg与HBeAg表达水平显著提升,可以支持HBV的复制,还可以被HBV感染。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (9)
1.一种细胞模型,其特征在于,所述细胞模型为BEL-7404-hNTCP细胞。
2.一种构建权利要求1所述的细胞模型的方法,其特征在于,所述构建方法包括:通过构建hNTCP慢病毒表达系统,转导BEL-7404细胞,经抗生素筛选,得到BEL-7404-hNTCP细胞。
3.一种基于权利要求1所述的细胞模型制备得到的细胞,其特征在于,所述细胞为BEL-7404-hNTCP-Tet-on-HNF4α细胞。
4.一种制备权利要求3所述的细胞的方法,其特征在于,包括:使用HNF4α慢病毒表达系统感染BEL-7404-hNTCP细胞,经过抗生素筛选和单克隆纯化得到BEL-7404-hNTCP-Tet-on-HNF4α细胞。
5.根据权利要求4所述的方法,其特征在于,使用Tet-on表达系统构建可受强力霉素诱导调控的HNF4α慢病毒系统。
6.根据权利要求4所述的方法,其特征在于,包括:
S1:构建hNTCP和HNF4α重组慢病毒质粒;
S2:用hNTCP重组慢病毒感染BEL-7404细胞;
S3:使用Tet-on系统构建含HNF4α的慢病毒感染系统;
S4:使用S3得到的含HNF4α的慢病毒感染系统感染BEL-7404-hNTCP细胞,经过筛选得到纯化的BEL-7404-hNTCP-Tet-on-HNF4α细胞。
7.根据权利要求1所述的细胞模型、权利要求3所述的细胞在乙肝病毒研究中的应用。
8.根据权利要求7所述的应用,其特征在于,所述应用为研究乙肝病毒复制和感染的应用。
9.根据权利要求7所述的应用,其特征在于,所述细胞模型或细胞用于筛选对HBV有抑制作用的药物。
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