中东呼吸综合征冠状病毒假病毒系统的建立及其在中和抗体检测中的应用

目的:为了避免中东呼吸综合征冠状病毒(MERS-CoV)感染与中和试验中操作活病毒带来的生物安全隐患,构建只具有一次感染能力而无复制能力的MERS假病毒,建立MERS假病毒系统,并应用于中和抗体检测。方法:构建含有MERS-CoV S基因的重组质粒pcDNA3.1-MERS-S,与缺失Env基因、含有萤光素酶报告基因的HIV-1骨架质粒pNL4-3.Luc.RE共转染293T细胞,收获含有假病毒的上清;通过Western印迹、细胞感染实验和血清中和试验,确定是否包装出MERS假病毒,及是否能有效应用于细胞感染与中和试验。结果:MERS假病毒pMERS-S培养上清经Western印迹鉴定出相对分子质量为25×103的HIV-1 P24蛋白和相对分子质量为180×103的MERS-CoV S蛋白;与阴性对照假病毒pEnv-相比,pMERS-S能有效感染MERS-CoV敏感细胞系Huh-7,在感染细胞中产生荧光信号,感染细胞的假病毒量与产生的荧光信号呈明显的量效关系;在MERS假病毒中和试验中,pMERS-S能被MERS-CoV中和抗体中和而失去感染力,反映抗体对MERS-CoV的中和活性。结论:建立了不依赖于BSL-3高等级生物安全条件的MERS假病毒系统,并有效应用于中和抗体检测,为MERS-CoV疫苗、药物评价及病毒致病机制研究提供了良好的技术支撑手段。

Construction of Middle East Pseudovirus System Applied in Respiratory Syndrome Coronavirus Detecting Neutralizing Antibodies

Objective： In order to bypass biosecurity risk in operation of infection and neutralization assay with alive Middle East respiratory syndrome coronavirus（MERS-CoV）, we produced pseudo-type MERS-CoV which lost infectivity following one infection, and constructed pesudovirus system applied in detecting neutrafizing antibodies. Methods： 293T cells were co-transfected with Env-defective and luciferase-expressing HIV-1 backbone plasmid pNL4-3.Luc.RE and recombinant plasmid pcDNA3.1-MERS-S containing MERS-CoV S gene. Supernatant contain ing pseudovirus was harvested, and was subjected to Western blotting, viral infectivity and serum neutralization as say to confirm the package of MERS-CoV pseudovirus and effective application to infection and neutralization as say. Results： The S protein of relative molecular weight 180x10^3 on pMERS-S was identified by Western blotting, indicating pseudo-type MERS-CoV（pMERS-S） was packaged. The pMERS-S, in contrast to negative control pseu dovirus pEnv-, could infect MERS-CoV-sensitive cell line Huh-7 and made enhanced light signals from the infect ed cells, which showed significant dose-effect relationship. Pseudovirus-based neutralization assay showed that pMERS-S lost the infectivity by MERS-CoV-neutralizing antibody, suggesting pMERS-S could be used to reflect neutralizing activity of antibody to MERS-CoV. Conclusion： This study established a MERS-CoV pseudovirus sys tem which can be effectively applied in detecting neutralizing antibodies out of BSL-3 high-level biosafety conditions, providing good technical support for evaluations of vaccines and drugs against MERS-CoV but also viral pathogenic mechanism study.