稳定表达hACE2的293T细胞系的建立及功能分析  

Establishment and Functional Analysis of 293T Cell Lines Stably Expressing Human ACE2

作　　者：黄楠 郎巧利 李莉萍 杨希 刘春麟[2] HUANG Nan;LANG Qiaoli;LI Liping;YANG Xi;LIU Chunlin(Chongqing Academy of Animal Sciences,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,重庆402460 [2]重庆医科大学附属第二医院眼科,重庆400010

出　　处：《中国细胞生物学学报》2022年第2期306-313,共8页Chinese Journal of Cell Biology

基　　金：重庆市自然科学基金面上项目(批准号:cstc2021jcyj-msxmX0876、cstc2020jcyj-msxm X0776);重庆市科研机构绩效激励引导专项(批准号:cstc2021jxjl0017);国家自然科学基金(批准号:32000130)资助的课题。

摘　　要：该研究旨在利用hACE2真核表达载体,通过电转法获得具有新型冠状病毒易感性的稳定表达hACE2的293T细胞株。将线性表达质粒pCMV-hACE2电转至293T细胞,经潮霉素B筛选后,利用间接免疫荧光(IFA)、RT-PCR、Western blot、流式分析法鉴定hACE2的表达,S-RBD蛋白和SARS-CoV-2假病毒分析细胞功能活性。利用250 μg/mL潮霉素B筛选获得了稳定高表达hACE2的细胞系293T-hACE2-D4。在该细胞中,RT-PCR检测到了大量hACE2基因的mRNA,Western blot、间接免疫荧光和流式分析法检测细胞表面hACE2蛋白表达情况(阳性细胞率为86.86%)。功能分析中,293T-hACE2-27-D4细胞不仅能够与SARS-Cov-2 S-RBD蛋白相结合(结合率达84.26%),并且能够成功感染新型冠状病毒假病毒。该研究构建的稳定表达hACE2的293T细胞不仅能与S-RBD蛋白结合,还对新冠假病毒具有易感性,是探究病毒感染机制的有利工具。The aim of this study was to generate a 293T cell line stably expressing human ACE2 and analyze its function.Stably transfected cells were selected by Hygromycin B resistance,and expression of hACE2 in them was determined by IFA (indirect immunofluorescence),RT-PCR,Western blot and FACS (flow cytometry).SARS-CoV-2-S-RBD protein and SARS-CoV-2 S pseudovirus were used to analyze stably transfected function of cell lines.A 293T cell line stably expressing human ACE2 (named as 293T-hACE2-D4) was generated in this study.The mRNA of hACE2 was expressed in 293T-hACE2-27-D4 cells.Human ACE2 protein was highly expressed on surface of 293T-hACE2-27-D4 cells by flow cytometry (the rate of ACE2-positive cells was 86.86%).Furthermore,most 293T-hACE2-27-D4 cells (the binding rate was 84.26%) showed high binding activity with SARS-CoV-2 S-RBD protein by FACS analysis.Results of pseudovirus infection showed that SARS-CoV-2 S pseudovirus could successfully infect 293T-hACE2-27-D4 cells,but not infect 293T cells.This study provides a useful tool for exploring the mechamism of SARS-CoV-2 virus infection.

关 键 词：hACE2 稳定转染 293T细胞 新型冠状病毒 

分 类 号：R563.1[医药卫生—呼吸系统]