基于VSV载体的新冠假病毒文库构建及免疫逃逸株筛选研究

曹高杰; 许晨晖; 王琳茜; 柴柯柯; 吴蓓蓓

微生物与感染 ›› 2025, Vol. 20 ›› Issue (3) : 139-149.

论著

基于VSV载体的新冠假病毒文库构建及免疫逃逸株筛选研究

曹高杰¹,许晨晖¹,王琳茜²,柴柯柯¹,吴蓓蓓³

作者信息 +

Research on the Construction of a SARS-CoV-2 Pseudovirus Library Based on the VSV Vector and the Screening of Immune Escape Strains

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文章历史 +

摘要

本研究旨在构建基于水疱性口炎病毒（VSV）载体的新型冠状病毒S蛋白假病毒突变文库，以筛选潜在的免疫逃逸株并解析其关键突变位点，为疫苗研发和疫情防控提供科学依据。采用随机突变试剂盒对新冠病毒Wuhan株S蛋白的受体结合域（RBD）进行随机突变，构建假病毒质粒文库，并利用VSV载体实现病毒拯救。通过血清加压和有限稀释法筛选免疫逃逸株，并对其突变位点进行测序验证。结果显示成功构建新冠假病毒质粒文库，阳性率高达98%。筛选出携带E484K、F486I、G339S、S477R等单突变，以及E484K+K444Q、E484K+K462R等双突变的免疫逃逸株。部分逃逸株对中和血清表现出显著的抵抗能力，其中E484K单突变及E484K相关双突变株的中和效价显著下降。本研究证实通过构建新冠假病毒突变文库成功筛选出具有免疫逃逸能力的变异株的可行性，并揭示其潜在的逃逸机制和流行特征。这些发现为理解新冠病毒的变异趋势提供了重要参考，并为疫苗研发和疫情防控策略的优化提供了科学支持。

Abstract

This study aims to construct a pseudovirus mutation library of the SARS-CoV-2 spike (S) protein based on the vesicular stomatitis virus (VSV) vector system, in order to screen potential immune escape variants and identify critical mutation sites, thereby providing scientific evidence for vaccine development and epidemic prevention and control. A random mutagenesis kit was employed to introduce random mutations into the receptor-binding domain (RBD) of the Wuhan strain S protein, followed by the construction of a pseudovirus plasmid library and subsequent virus rescue using the VSV vector system. Immune escape variants were screened through serum pressure and limited dilution methods, with their mutation sites validated by sequencing. Results demonstrated the successful construction of the SARS-CoV-2 pseudovirus plasmid library, achieving a positive rate of 98%. Immune escape variants carrying single mutations such as E484K, F486I, G339S, and S477R, as well as double mutations including E484K+K444Q and E484K+K462R, were identified. Some escape variants exhibited significant resistance to neutralizing sera, with a marked reduction in neutralizing titers observed for the E484K single mutant and E484K-related double mutants. This study confirms the feasibility of screening immune escape variants through the construction of a SARS-CoV-2 pseudovirus mutation library and elucidates their potential escape mechanisms and epidemic characteristics. These findings provide important insights into understanding the evolutionary trends of SARS-CoV-2 and offer scientific support for optimizing vaccine development and epidemic prevention strategies.

导出引用

曹高杰许晨晖王琳茜柴柯柯吴蓓蓓. 基于VSV载体的新冠假病毒文库构建及免疫逃逸株筛选研究[J]. 微生物与感染. 2025, 20(3): 139-149

Research on the Construction of a SARS-CoV-2 Pseudovirus Library Based on the VSV Vector and the Screening of Immune Escape Strains[J]. Journal of Microbes and Infections. 2025, 20(3): 139-149

中图分类号： R373

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