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

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.