Establishment and Application of Huh-7 Cell Line with Stable Expression of Human ACE2
1. Chongqing Academy of Animal Sciences, Chongqing 402460, China; 2. Chongqing Research Center for the Development and Utilization of Medical Animal Resources, Chongqing 402460, China; 3. Department of Ophthalmology, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China
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.
郎巧利, 黄 楠, 李莉萍, 杨 希, 刘春麟. 稳定表达人ACE2的Huh-7细胞系的建立及应用[J]. 生命科学研究, 2023, 27(3): 189-195. LANG Qiaoli, HUANG Nan, LI Liping, YANG Xi, LIU Chunlin. Establishment and Application of Huh-7 Cell Line with Stable Expression of Human ACE2. Life Science Research, 2023, 27(3): 189-195.
