新型冠状病毒假病毒制备方法的优化及应用

Optimization and Application of SARS-CoV-2 Pseudovirus Production System

该文旨在建立一种稳定高效的新型冠状病毒(SARS-CoV-2)假病毒构建方法,并将所制备的假病毒用于评估抗体中和水平。通过比较不同穿梭质粒、质粒配比以及转染试剂对假病毒滴度的影响,优化制备高滴度假病毒的条件;通过制备不同的SARS-CoV-2突变株假病毒,测试该方法的稳定性;利用所制备的假病毒对SARS-CoV-2抗体的中和能力进行评价,测试该方法的可靠性。优化结果表明,当使用LipofectamineTM3000作为转染试剂时,pCDH-CMV-MCS-EF1-copGFP?psPAX2?pcDNA3.1-S以0.300μg?0.225μg?0.240μg的质量比转染获得的假病毒滴度最高。进一步,发现利用该方法包装的三种突变株的假病毒均可达到较高滴度。利用该方法制备的假病毒可以用于SARS-Co V-2抗体的中和活性检测,测得IC50值为0.126μg/m L。该研究成功建立了一种简单高效的制备新型冠状病毒(SARS-CoV-2)假病毒的方法,其可用于体外评估SARS-CoV-2抗体中和活性,为研发SARS-CoV-2相关疫苗和药物的体外评价提供了技术基础。

The aim of this study was to establish an efficient and stable SARS-CoV-2 pseudovirus production system to evaluate the neutralizing ability of antibodies to SARS-CoV-2 in vitro.To produce high-titer pseudovirus,transfer plasmid types,the proportion of transfected plasmid and transfection methods were investigated in this study.The optimization results showed that the highest pseudovirus titer was obtained when 293T cells were transfected with the plasmids pCDH-CMV-MCS-EF1-copGFP,psPAX2 and pcDNA3.1-S at a mass radio of 0.300μg׃0.225μg׃0.240μg using Lipofectamine™3000.Moreover,this optimized pseudovirus production system was also applicable to other SARS-CoV-2 variants,and three kinds of high-titer SARS-CoV-2 variants pseudoviruses were generated.Furthermore,pseudovirus neutralization assay was performed using the pseudovirus obtained in this study to test neutralizing activity of a reported antibody,and the IC50 value was 0.126μg/mL.In summary,this study successfully established a simple and efficient method to generate high-titer SARS-CoV-2 pseudovirus,which could be used to evaluate the neutralizing activity of SARS-CoV-2 antibodies in vitro.This study provided a good foundation for the research of SARS-CoV-2 vaccines and drugs.