新型冠状病毒614D和614G假病毒生物学特性研究  

Biological specificity of 2019 novel coronavirus 614D and 614G pseudovirus

作　　者：麻粉莲[1] 雒晓艺 王超[1] 宋敬东[1] 谢志萍[1] 丛姗姗 黄益曼 郑丽舒[1] Ma Fenlian;Luo Xiaoyi;Wang Chao;Song Jingdong;Xie Zhiping;Cong Shanshan;Huang Yiman;Zheng Lishu(NHC Key Laboratory of Medical Virology and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 100052,China;Department of Health Toxicology,School of Public Health,China Medical University,Shenyang 110122,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所国家卫生健康委员会医学病毒与病毒病重点实验室,北京100052 [2]中国医科大学公共卫生学院卫生毒理学教研室,沈阳110122

出　　处：《中华实验和临床病毒学杂志》2022年第4期397-401,共5页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的利用HIV慢病毒包装系统构建新型冠状病毒(2019 novel coronavirus,2019-nCoV)原始株614D和突变株614G假病毒,并初步研究其生物学特性。方法将重组表达质粒pCDNA3.1-614D和pCDNA3.1-614G分别与慢病毒质粒psPAX2和pLenti CMV Puro LUC瞬时共转染293T细胞,72 h后收集上清,进行20%蔗糖垫层超速离心,检测假病毒的滴度、形态、S蛋白表达和中和活性。结果间接免疫荧光检测可见S蛋白特异性荧光,Western blot分析可见2019-nCoV 614D和614G假病毒S蛋白表达,透射电镜下可见假病毒颗粒具有明显刺突。614D和614G假病毒的滴度分别为1.12×10^(4)和2.52×10^(4) TCID_(50)/ml,均能够中和S蛋白兔多克隆抗体,表明假病毒具有特异性。结论本研究成功构建了2019-nCoV 614D和614G假病毒,为建立基于假病毒的体外中和抗体检测平台奠定基础。Objective To construct 2019 novel coronavirus(2019-nCoV)614D and 614G pseudovirus by HIV lentivirus packaging system and explore their biological specificity.Methods The recombinant expression plasmids pCDNA3.1-614D and pCDNA3.1-614G were transiently cotransfected with psPAX2 and pLenti CMV Puro LUC into 293T cells respectively.After 72 hours,the supernatant was collected and ultracentrifuged with 20%sucrose cushion.The titer,morphology,protein expression and neutralizing activity of pseudovirus were determined.Results S protein specific fluorescence was detected by indirect immunofluorescence test,Western blot analysis showed S protein was expressed,and the spike of pseudovirus was observed under transmission electron microscope.The titers of pseudovirus 614D and 614G were 1.12×10^(4) and 2.52×10^(4) TCID_(50)/ml,respectively.The pseudovirus 614D and 614G could be neutralized by S rabbit polyclonal antibody,indicating that the pseudovirus has high specificity.Conclusions In this study,2019-nCoV 614D and 614G pseudovirus was successfully constructed,which laid the foundation for the establishment of in vitro neutralizing antibody detection platform based on pseudovirus.

关 键 词：新型冠状病毒 假病毒 中和活性 

分 类 号：R373[医药卫生—病原生物学]