稳定表达人ACE2的Huh-7细胞系的建立及应用  

Establishment and Application of Huh-7 Cell Line with Stable Expression of Human ACE2

作　　者：郎巧利 黄楠 李莉萍 杨希 刘春麟[3] LANG Qiaoli;HUANG Nan;LI Liping;YANG Xi;LIU Chunlin(Chongqing Academy of Animal Sciences,Chongqing 402460,China;Chongqing Research Center for the Development and Utilization of Medical Animal Resources,Chongqing 402460,China;Department of Ophthalmology,the Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China)

机构地区：[1]重庆市畜牧科学院,中国重庆402460 [2]重庆市医用动物资源的开发与利用工程技术研究中心,中国重庆402460 [3]重庆医科大学附属第二医院眼科,中国重庆400010

出　　处：《生命科学研究》2023年第3期189-195,共7页Life Science Research

基　　金：重庆市自然科学基金面上项目(cstc2021jcyj-msxmX0881);畜禽养殖关键技术研究与示范推广项目(22509C,22503C,22513C,21504);重庆市科研机构绩效激励引导专项项目(cstc2021jxjl0017,cstc2021jxjl80009);国家自然科学基金资助项目(32000130)。

摘　　要：为了构建稳定表达人血管紧张素转换酶2(human angiotensin converting enzyme 2,hACE2)的Huh-7细胞系,本研究构建了不带荧光的pWPXL-neo-hACE2慢病毒载体,并利用psPAX2和pMD2.G-VSVG共同转染HEK293T细胞获得慢病毒;将包装好的慢病毒感染Huh-7细胞后,利用潮霉素B筛选获得可表达hACE2的Huh-7细胞;通过间接免疫荧光法和蛋白质印迹法检测Huh-7细胞中hACE2蛋白的表达;采用流式细胞术分析Huh-7-hACE2细胞与严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARSCoV-2)刺突(spike,S)蛋白受体结合结构域(receptor-binding domain,RBD)的结合情况,并用SARS-CoV-2假病毒进一步测定Huh-7-hACE2细胞对SARS-CoV-2的易感性,最后利用鉴定过的Huh-7-hACE2细胞测定SARS-CoV-2中和抗体REGN10987对假病毒的中和效价。结果显示,hACE2蛋白在Huh-7细胞中成功表达,且表达的hACE2蛋白能与SARS-CoV-2 S蛋白RBD结合;相较于Huh-7细胞,Huh-7-hACE2细胞对假病毒的易感性显著提高,且假病毒中和效价测定结果与文献报道的真病毒中和效价相似。总之,本研究成功构建了稳定表达hACE2的Huh-7细胞系,并且其能应用于新型冠状病毒感染(corona virus disease 2019,COVID-19)治疗性单抗的活性评价,是研究SARS-CoV-2的致病机制、开发抗病毒药物及疫苗的有利工具。To construct Huh-7 cell line stably expressing human angiotensin converting enzyme 2(hACE2),the pWPXL-neo-hACE2 lentiviral vector without a fluorescent protein gene was constructed and co-transfected into HEK293T cells with psPAX2 and pMD2.G-VSVG to generate lentivirus.The obtained lentivirus particles were used to infect Huh-7 cells,and the cells that could stably express hACE2 protein were selected with hygromycin B.The expression of hACE2 in Huh-7 cells was detected by indirect immunofluorescence assay and Western-blot.Furthermore,the binding activity of Huh-7-hACE2 cells to the receptorbinding domain(RBD)of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike(S)protein was analyzed by flow cytometry,and the susceptibility of Huh-7-hACE2 cells to SARS-CoV-2 was further determined with SARS-CoV-2 pseudovirus.The identified Huh-7-hACE2 cells were finally used in evaluating the neutralizing capacity of SARS-CoV-2 neutralizing antibody REGN10987 against pseudovirus.The results showed that the hACE2 protein was successfully expressed in Huh-7 cells,and the expressed hACE2 protein could bind to SARS-CoV-2 S protein RBD.Compared with Huh-7 cells,Huh-7-hACE2 cells were more susceptible to SARS-CoV-2 pseudovirus infection,and the neutralizing activity evaluated with pseudovirus was similar to that with SARS-CoV-2 reported in the literature.In conclusion,the study demonstrated that the Huh-7 cell line stably expressing hACE2 was successfully established,and could be used to evaluate the activity of corona virus disease 2019(COVID-19)therapeutic monoclonal antibody.The established cell line is a useful tool to study the pathogenesis of SARS-CoV-2 and develop antiviral drugs and vaccines.

关 键 词：严重急性呼吸综合征冠状病毒2(SARS-CoV-2) 人血管紧张素转换酶2(hACE2) HUH-7细胞 慢病毒感染 稳定转染细胞系 治疗性抗体 

分 类 号：Q78[生物学—分子生物学]