新型冠状病毒假病毒的构建及其在血清中和抗体测定中的应用  

Construction of SARS-CoV-2 pseudovirus and its application in measuring the neutralization activity of serum antibodies

作　　者：梁婉欣 刘淑燕 段炼 周雪峰 邬林枫 刘文齐 张更伟 朱华晨[1,4] 张国良[2] LIANG Wan-xin;LIU Shu-yan;DUAN Lian;ZHOU Xue-feng;WU Lin-feng;LIU Wen-qi;ZHANG Geng-wei;ZHU Hua-chen;ZHANG Guo-liang(Guangdong-Hong Kong Joint Laboratory of Emerging Infectious Diseases/Joint Laboratory for International Collaboration in Virology and Emerging Infectious Diseases(Ministry of Education),Shantou University Medical College,Shantou,Guangdong 515041,China;Institute for Hepatology,Shenzhen Third People's Hospital,Shenzhen,Guangdong 518116,China;Guangdong Medical University,Dongguan,Guangdong 523808,China;State Key Laboratory of Emerging Infectious Diseases,The University of Hong Kong,Hong Kong 999077,China)

机构地区：[1]汕头大学医学院粤港新发传染病联合实验室,教育部病毒学与新发传染病国际合作联合实验室,广东汕头515041 [2]深圳市第三人民医院肝病研究所,广东深圳518116 [3]广东医科大学,广东东莞523808 [4]香港大学新发传染性疾病国家重点实验室,中国香港999077

出　　处：《中国热带医学》2022年第3期240-245,共6页China Tropical Medicine

基　　金：广东省科技计划项目(No.2020B1111340076);深圳市科技计划项目(No.JSGG20200225150702770);深圳湾实验室开放课题(No.SZBL202002271003);广东省基础与应用基础研究基金(No.2020A1515010977)。

摘　　要：目的构建新型冠状病毒(SARS-CoV-2)假病毒,优化假病毒制备条件并应用于中和抗体活性检测。方法优化合成SARS-CoV-2刺突蛋白(S)基因,进行假病毒包装;通过Western blot检测S基因的表达;应用定量ELISA检测接种新冠灭活疫苗后血清中新型冠状病毒IgG抗体的滴度,同时应用假病毒中和实验对接种新冠疫苗后血清中和抗体活性进行评价。结果成功构建了整合SARS-CoV-2 S基因的表达载体pcDNA3.1-S,确定pNL4-3.Luc.R-E-∶pcDNA3.1-S最佳转染比例为2∶1,可成功包装出高滴度的假病毒粒子。Western blot结果表明S蛋白已成功地在假病毒中进行表达。SARS-CoV-2假病毒可感染Vero、Huh7.5、A549-hACE2和293T-hACE2等4种靶细胞,表明所构建的假病毒具有感染活力,且具有广泛的宿主范围,其中293T-hACE2细胞感染假病毒后相对其他细胞株可检测出更高的萤火虫荧光素酶活性。ELISA检测接种新冠疫苗后收集的血清,结果表明接种第二针灭活疫苗一周后可检测出较高血清IgG滴度(S/CO=10.27±3.33),半年后血清IgG滴度降低(S/CO=2.36±2.25);假病毒中和实验结果表明1例IgG阳性(S/CO=10.32)免疫后血清可有效抑制SARS-CoV-2假病毒的感染活力,中和效价达到1/1066。结论成功构建SARSCoV-2假病毒,该假病毒可用于后续有关SARS-CoV-2中和抗体活性检测和疫苗接种后体液免疫效果的评价研究。Objective To construct SARS-CoV-2 pseudovirus, optimize its preparation protocol, and apply it to theevaluation of antibody neutralization activity.Methods The optimized sequence of spike(S) gene of SARS-CoV-2 wassynthesized, the pseudovirus titers were measured, and the expressed Sprotein was then detected by Western blot. Finally,quantitative ELISA was used to measure the serum IgG antibody titers in recipients who had received either one or two doses ofCOVID-19 inactivated vaccine. Meanwhile, the sera were tested for their reactivity with the pseudovirus using neutralizationtests.ResultsS gene was confirmed to have been successfully cloned into the vector, generating the pcDNA3.1-S plasmid.Co-transfection of pNL4-3.Luc.R-E-and pcDNA3.1-S at a ratio of 2∶1 could lead to higher packing efficacy and pseudovirustiters. Expression of the S protein was verified by Western blot. Moreover, this SARS-CoV-2 pseudovirus showed a broad hostinfectivity in Vero, Huh7.5, A549-hACE2 and 293T-hACE2 cells, with the highest relative luciferase unit(RLU) in 293T-hACE2. Comparing the IgG levels measured by ELISA in sera collected from COVID-19 vaccine recipients, we observed ahigher titer in those who received two doses of inactivated vaccine(S/CO=10.27±3.33), measured one week after the secondshot. However, the IgG level significantly dropped( S/CO=2.36±2.25)half year post-vaccination. Amongst the serum samplestested, one with an S/CO of 10.32 could successfully inhibit the infection of SARS-CoV-2 pseudovirus in 293T-hACE2 cells at a dilution of 1/1 066.Conclusion We have established a method for preparing the SARS-CoV-2 pseudovirus, which can beused for detection of the neutralizing antibodies and the evaluation of humoral immune response post-vaccination.

关 键 词：新型冠状病毒 假病毒 刺突蛋白 中和抗体 疫苗接种 

分 类 号：R563.1[医药卫生—呼吸系统]