中国免疫学杂志2024，Vol.40Issue(4)：811-817,7.DOI:10.3969/j.issn.1000-484X.2024.04.024
用于中和抗体评价和进入抑制剂筛选的多变异株SARS-CoV-2假病毒体系构建与评估
Construction and evaluation of a multi-variant pseudovirus system of SARS-CoV-2 for antibody evaluation and virus entry inhibitors screening
摘要
Abstract
Objective:A SARS-CoV-2 pseudovirus(PsV)system was established for neutralizing antibody evaluation and virus entry inhibitor screening.Methods:Lentiviral vector plasmids psPAX2,pCDH-Luc and SARS-CoV-2 Spike(S)protein expres-sion plasmids were co-transfected,and harvested pseudoviral supernatant was used to infect ACE2-293T cells.Protein content of p24 was determined to reflect titer of PsV,and expression of S protein in PsV was detected by Western blot.Neutralization capacity of an S protein monoclonal antibody was evaluated using original strain,D614G,Gamma,Delta,Omicron PsV.Two reported virus entry inhibitors,chloroquine and carrageenin,were used to detect effect on entry of Omicron PsV.Results:Lentiviral vector successfully incorporated S protein.Western blot results showed that S protein mutated at 665Y showed a different cleavage form(90 kD)than wild-type full-length S protein(180 kD).Titer of PsV packaged by three plasmids system was higher.Ratio of S protein expression plasmid,transfer plasmid and packaging plasmid at 1∶3∶3 was optimum condition for viral packaging.Titer of PsV packaged under this condi-tion was over 20 ng/ml.PsV could effectively infect ACE2-293T cells,and double reporter gene GFP and firefly luciferase were expressed obviously,whose chemiluminescence values reached 106.Monoclonal antibodies of S protein effectively neutralized four types of PsVs,but neutralization of original strain was 10～30 times greater than that of variant PsV.Virus entry inhibitors,chloroquine and ι-carrageenan significantly inhibited entry of Omicron PsV.Conclusion:SARS-CoV-2 PsV infection system we conducted can simu-late entry of SARS-CoV-2 successfully.Effective pharmacodynamic evaluation of neutralizing antibodies and virus entry inhibitors can be performed efficiently by the system,which can provide a technical platform for evaluation of neutralizing antibody of SARS-CoV-2 and screening of virus entry inhibitors,and would benefit R&D of anti-SARS-CoV-2 drugs.关键词
SARS-CoV-2/假病毒/S蛋白/中和抗体/病毒进入抑制剂Key words
SARS-CoV-2/Pseudovirus/S protein/Neutralizing antibody/Virus entry inhibitor分类
基础医学引用本文复制引用
李静璇,刘峰,王莹,程军平,肖智勇,周文霞..用于中和抗体评价和进入抑制剂筛选的多变异株SARS-CoV-2假病毒体系构建与评估[J].中国免疫学杂志,2024,40(4):811-817,7.基金项目
天津市科技计划项目(22ZYJDSS00080). （22ZYJDSS00080）
