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  • 彭浩然,江亮亮,何燕华,肖爱军,唐海琳,戚中田,赵平.严重急性呼吸综合征冠状病毒2假病毒的制备及验证[J].第二军医大学学报,2020,41(4):359-364    [点击复制]
  • PENG Hao-ran,JIANG Liang-liang,HE Yan-hua,XIAO Ai-jun,TANG Hai-lin,QI Zhong-tian,ZHAO Ping.Preparation and verification of severe acute respiratory syndrome coronavirus 2 pseudoparticles[J].Acad J Sec Mil Med Univ,2020,41(4):359-364   [点击复制]
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严重急性呼吸综合征冠状病毒2假病毒的制备及验证
彭浩然,江亮亮,何燕华,肖爱军,唐海琳,戚中田,赵平
0
(1. 海军军医大学(第二军医大学)海军医学系生物医学防护教研室, 上海 200433;
2. 上海捷瑞生物工程有限公司, 上海 201615
*通信作者)
摘要:
目的 建立严重急性呼吸综合征冠状病毒2(SARS-CoV-2)假病毒(SARS-CoV-2 pps)的制备方法。方法 设计、合成序列优化的SARS-CoV-2刺突蛋白(S)基因,构建表达质粒,转染293T细胞,用免疫荧光检测S蛋白的表达;将该质粒与基于慢病毒基因骨架的含增强绿色荧光蛋白(EGFP)报告基因的假病毒包装质粒共转染293T细胞,收集细胞培养上清,感染Vero E6和Huh7细胞,观察细胞内EGFP的表达;将制备的SARS-CoV-2 pps感染Vero E6细胞,检测膜融合抑制剂氯喹和盐酸阿比朵尔、SARS-CoV-2 S1蛋白单克隆抗体及新型冠状病毒肺炎患者恢复期血清对假病毒感染性的影响。结果 构建的SARS-CoV-2 S质粒转染的293T细胞可与S1蛋白单克隆抗体及新型冠状病毒肺炎患者恢复期血清反应。SARS-CoV-2 S质粒与HIV假病毒包装质粒共转染293T细胞的上清加入Vero E6细胞和Huh7细胞36 h和72 h后可分别观察到细胞内表达的EGFP,EGFP阳性的Huh7细胞数量明显多于Vero E6细胞。2种膜融合抑制剂、1株人源S1单克隆抗体及2份新型冠状病毒肺炎患者恢复期血清均可有效抑制SARS-CoV-2 pps对Vero E6细胞的感染(P均<0.01)。结论 成功制备了SARS-CoV-2 pps,其可用于后续抗SARS-CoV-2药物筛选与疫苗评价。
关键词:  严重急性呼吸综合征冠状病毒2  假病毒  膜融合抑制剂  中和抗体
DOI:10.16781/j.0258-879x.2020.04.0359
投稿时间:2020-03-18修订日期:2020-03-24
基金项目:国家重点研发计划(2016YFC1200401),国家科技重大专项(2017ZX10304403-003).
Preparation and verification of severe acute respiratory syndrome coronavirus 2 pseudoparticles
PENG Hao-ran,JIANG Liang-liang,HE Yan-hua,XIAO Ai-jun,TANG Hai-lin,QI Zhong-tian,ZHAO Ping
(1. Department of Biomedical Defense, Faculty of Naval Medicine, Naval Medical University (Second Military Medical University), Shanghai 200433, China;
2. Shanghai Generay Biotech Co., Ltd, Shanghai 201615, China
*Corresponding author)
Abstract:
Objective To establish a method for preparing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudoparticles (SARS-CoV-2 pps). Methods The optimized sequence of spike (S) gene of SARS-CoV-2 was designed, synthesized and used to construct the mammalian cell expression plasmid. The resultant plasmid was used to transfect 293T cells, and the expressed S protein was detected using immunofluorescence. The S expression plasmid was further used to transfect 293T cells together with lentiviral genome backbone based pseudoparticles package plasmids containing enhanced green fluorescent protein (EGFP) reporter gene. The supernatant of 293T cells was collected and used to infect Vero E6 cells and Huh7 cells. The intracellular expression of EGFP was observed. The confirmed SARS-CoV-2 pps was used to infect Vero E6 cells, and then we observed the effects of membrane fusion inhibitors chloroquine and arbidol hydrochloride, monoclonal antibodies to S1 protein of SARS-CoV-2 and convalescent sera of patients with coronavirus disease 2019 on the pseudoparticles infection. Results The 293T cells transfected with SARS-CoV-2 S plasmid could react with monoclonal antibodies to S1 protein of SARS-CoV-2 and convalescent sera. SARS-CoV-2 S plasmid and human immunodeficiency virus (HIV) pseudoparticles package plasmid were used to transfect 293T cells together, adding the supernatant to Vero E6 and Huh 7 cells, the intracellular expression of EGFP was observed at 36 h and 72 h, respectively. Compared with Vero E6 cells, there were more EGFP-positive cells for Huh7 cells. Two membrane fusion inhibitors, one human monoclonal antibody against SARS-CoV-2 S1 protein and two convalescence sera could effectively inhibit the infection of SARS-CoV-2 pps of Vero E6 cells. Conclusion We have established a method for preparing SARS-CoV-2 pps, which can be used for the anti-SARS-CoV-2 drug screening and vaccine evaluation.
Key words:  severe acute respiratory syndrome coronavirus 2  pseudoparticles  membrane fusion inhibitors  neutralizing antibody