SARS和MERS中和抗体假病毒检测方法的建立

Development of SARS and MERS Neutralization Methods Based on Pseudoviruses

严重急性呼吸综合征冠状病毒(SARS-CoV)和中东呼吸综合征冠状病毒(MERS-CoV)均属冠状病毒科(Coronaviridae),能够引起人类严重疾病,特别是SARS-CoV可以导致严重急性呼吸综合征,而且容易造成暴发式流行,引起社会恐慌。本研究利用含有SARS-CoV和MERS-CoV棘突蛋白(Spike protein,S)基因的表达质粒pcD-NA3.1-SS、pcDNA3.1-MS,成功构建了高滴度假病毒,在此基础上通过对病毒接种量进行优化,建立了稳定可靠的假病毒中和抗体检测方法,并通过检测SARS恢复期病人血清以及免疫后小鼠血清对方法的特异性、稳定性以及重复性进行分析,充分证明了该方法是有效的血清中和抗体的检测手段。本研究成功构建了不依赖于BSL-3级生物安全条件的SARS和MERS假病毒,不仅可应用于血清中和抗体检测,还为后续单克隆抗体及抗病毒药物筛选和评价、疫苗研发及病毒感染机制研究等奠定了基础。

The severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) belong to Coronaviridae, a family of enveloped, positive-sense, single-stranded RNA viruses that can cause serious human diseases.In particular, an outbreak of SARS caused by SARS-CoV can lead to social panic.We constructed stable pseudoviruses using pcDNA3.1-SS and pcDNA3.1-MS containing the S genes of SARS-CoV and MERS-CoV.On this basis, a method for detecting neutralizing antibodies was established by optimizing the inoculation amount of pseudoviruses.The specificity, stability and repeatability of this method were evaluated using the sera of patients convalescing from SARS and sera from im-munized mice.Results demonstrated that this method could be used to detect neutralizing antibodies in sera.Construction of SARS and MERS pseudoviruses independent from a Biological Safety Level-3 laboratory could be applied for detection of neutralizing antibodies from sera.Such construction could also lay a foundation for subsequent screening and evaluation of monoclonal antibodies and antiviral drugs, vaccine development, and research on the mechanism of viral infection.