基于LLR载体表达SARS-CoV-2 RBD蛋白rLLR/NSP3-CoV-2/RBD重组病毒的拯救及鉴定  

Rescue and Identification of the Recombinant rLLR/NSP3-CoV-2/RBD Expressing SARS-CoV-2 RBD Based on LLR Backbone

作　　者：刘夏飞 任伟宏[3] 李杉 余俊杰 朱武洋[2] 段招军[2] LIU Xiafei;REN Weihong;LI Shan;YU Junjie;ZHU Wuyang;DUAN Zhaojun(The First Clinical Medical Institute,Henan University of Chinese Medicine,Zhengzhou 450000,China;National Institute for Viral Disease Control and Prevention,Chinese Center for Diseases Control and Prevention,Beijing 102206,China;The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000,China;College of Public Health,Gansu University of Traditional Chinese Medicine,Lanzhou730000,China)

机构地区：[1]河南中医药大学第一临床医学院,郑州450000 [2]中国疾病预防控制中心病毒病预防控制所,北京102206 [3]河南中医药大学第一附属医院,郑州450000 [4]甘肃中医药大学公共卫生学院,兰州730000

出　　处：《病毒学报》2024年第5期952-960,共9页Chinese Journal of Virology

基　　金：国家资助博士后研究人员计划B档资助(项目号:GZB:20240207),题目:G9P[8]型轮状病毒感染性克隆的建立及细胞适应性的分子机制。

摘　　要：2019年12月以来,严重急性呼吸综合征冠状病毒2(Severe acute respiratory syndrome coronavirus 2,SARSCoV-2)席卷全球,但在控制SARS-CoV-2大流行的综合策略中一直空缺3岁以下儿童的免疫接种。兰州羔羊株轮状病毒(Lanzhou lamb rotavirus,LLR)是我国获批上市用于预防婴幼儿轮状病毒胃肠炎的常规减毒活疫苗,本研究利用反向遗传学技术将SARS-CoV-2的受体结合结构域(Receptor-binding domain,RBD)蛋白插入LLR/NSP3基因的ORF后进行重组病毒拯救,通过结合致细胞病变效应(cytopathic effect,CPE)、dsRNA PAGE硝酸银染色和NSP3基因RT-PCR扩增证明我们成功拯救出rLLR/NSP3-CoV-2/RBD重组病毒,且重组病毒的NSP3基因在P5代以内出现了与预期相符的条带迁移。本研究进一步通过Western blot和间接免疫荧光证明rLLR/NSP3-CoV-2/RBD重组病毒可以在P5代以内稳定表达SARS-CoV-2 RBD蛋白。间接免疫荧光和qRT-PCR扩增的结果显示虽然rLLR/NSP3-CoV-2/RBD的滴度和在不同时间点的基因组拷贝数略低于亲本株rLLR,但不影响病毒的感染和复制。本研究通过反向遗传学技术成功拯救出能在P5代以内稳定表达SARS-CoV-2 RBD蛋白的rLLR/NSP3-CoV-2/RBD重组病毒,为进一步探索开发适合3岁以下儿童使用的口服轮状病毒和SARS-CoV-2联合疫苗提供了思路。The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has swept the world since December 2019.However,the immunization of children under 3 years old was absent from comprehensive strategies.Lanzhou lamb rotavirus(LLR)has been approved as a routine vaccine for the prevention of infantile Rotavirus enteritis in China.In this study,a reverse genetic technique was used to insert the receptor-binding domain(RBD)protein of SARS-CoV-2 into the ORF of LLR/NSP3 gene for recombinant virus rescue.The cytopathic effect(CPE),silver nitrate staining of dsRNA PAGE and RT-PCR amplification of NSP3 genes all verified our successful rescue of rLLR/NSP3-CoV-2/RBD recombinant virus.The NSP3 genes of the recombinant virus rLLR/NSP3-CoV-2/RBD showed obvious migration within P5 generations as expected.Furthermore,Western blot and indirect immunofluorescence had demonstrated the ability of recombinant rLLR/NSP3-CoV-2/RBD to stably express RBD of SARS-CoV-2 within P5 generations.The results of indirect immunofluorescence and qRT-PCR amplification showed that the slightly lower titer and GCEs of rLLR/NSP3-CoV-2/RBD than those of the parental rLLR at different time points did not affect the infection and replication.In this study,a reverse genetic technique was used to rescue the recombinant rLLR/NSP3-CoV-2/RBD,which could stably express the RBD protein of SARS-CoV-2 within P5 generations.This provides a new reference for further development of oral rotavirus and SARS-CoV-2 combined vaccines suitable for children under 3 years old.

关 键 词：新冠病毒 受体结合结构域 兰州羔羊株轮状病毒 反向遗传学 重组病毒 

分 类 号：R373.2[医药卫生—病原生物学]