稳定表达人ACE2的Huh-7细胞系的建立及应用

郎巧利; 黄楠; 李莉萍; 杨希; 刘春麟

生命科学研究 ›› 2023, Vol. 27 ›› Issue (3) : 189 -195.

新冠肺炎专栏

稳定表达人ACE2的Huh-7细胞系的建立及应用

郎巧利, 黄楠, 李莉萍, 杨希, 刘春麟

作者信息 +

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

Author information +

文章历史 +

PDF (2873K)

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, SARS- CoV-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。

Abstract

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-trans-fected 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 se-lected with hygromycin B. The expression of hACE2 in Huh-7 cells was detected by indirect immunofluo-rescence assay and Western-blot. Furthermore, the binding activity of Huh-7-hACE2 cells to the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) pro-tein 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.