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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。
Abstract：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.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004584
中图分类号:R392-33
引用信息:
[1]刘夏飞,任伟宏,李杉等.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[J].病毒学报,2024,40(05):952-960.DOI:10.13242/j.cnki.bingduxuebao.004584.
基金信息:
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~~