| 144 | 1 | 377 |
| 下载次数 | 被引频次 | 阅读次数 |
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.
[1]潘锋.疫苗接种是预防传染病的最有效措施——访中国疾病预防控制中心曾光教授[J].中国当代医药，2019, 26(5):1-3. DOI:10. 3969/j. issn. 1674-4721. 2019. 05. 002.
[2]葛新斌，渠淇淦，王泽光，等.严重急性呼吸综合征冠状病毒2(SARS-CoV-2)疫苗研究进展[J].细胞与分子免疫学杂志，2023, 39(10):946-951.
[3] Cates JE, Tate JE, Parashar U. Rotavirus vaccines:progress and new developments[J]. Expert Opin Biol Ther, 2022, 22(3):423-432. DOI:10. 1080/14712598. 2021. 1977279.
[4] Desselberger U. Rotaviruses[J]. Virus Res, 2014,190:75-96. DOI:10. 1016/j. virusres. 2014. 06. 016.
[5] Komoto S, Sasaki J, Taniguchi K. Reverse genetics system for introduction of site-specific mutations into the double-stranded RNA genome of infectious rotavirus[J]. Proc Natl Acad Sci USA, 2006, 103(12):4646-4651. DOI:10. 1073/pnas. 0509385103.
[6] Kanai Y, Komoto S, Kawagishi T, et al. Entirely plasmid-based reverse genetics system for rotaviruses[J]. Proc Natl Acad Sci USA, 2017, 114(9):2349-2354. DOI:10. 1073/pnas. 1618424114.
[7] Kawagishi T, Nurdin JA, Onishi M, et al. Reverse genetics system for a human group A rotavirus[J]. J Virol, 2020, 94(2):e00963-e00919. DOI:10. 1128/jvi. 00963-19.
[8] Komoto S, Fukuda S, Kugita M, et al. Generation of infectious recombinant human rotaviruses from just 11cloned cDNAs encoding the rotavirus genome[J]. J Virol, 2019, 93(8):e02207-e02218. DOI:10. 1128/jvi. 02207-18.
[9] Sánchez-Tacuba L, Feng N, Meade NJ, et al. An optimized reverse genetics system suitable for efficient recovery of Simian, human, and murine-like rotaviruses[J]. J Virol, 2020, 94(18):e01294-e01220. DOI:10. 1128/jvi. 01294-20.
[10]Kanda M, Fukuda S, Hamada N, et al. Establishment of a reverse genetics system for avian rotavirus A strain PO-13[J]. J Gen Virol, 2022, 103(6). DOI:10. 1099/jgv. 0. 001760.
[11]Diebold O, Gonzalez V, Venditti L, et al. Using species a rotavirus reverse genetics to engineer chimeric viruses expressing SARS-CoV-2 spike epitopes[J]. J Virol, 2022, 96(14):e0048822. DOI:10. 1128/jvi. 00488-22.
[12]刘夏飞，李慧莹，杜文静，等. SA11轮状病毒反向遗传学方法的初步应用[J].病毒学报，2022, 38(4):850-857. DOI:10. 13242/j. cnki. bingduxuebao. 004161.
[13]Shen S, Burke B, Desselberger U. Rearrangement of the VP6 gene of a group A rotavirus in combination with a point mutation affecting trimer stability[J]. J Virol,1994, 68(3):1682-1688. DOI:10. 1128/jvi. 68. 3. 1682-1688. 1994.
[14]Kojima K, Taniguchi K, Kawagishi-Kobayashi M, et al. Rearrangement generated in double genes, NSP1and NSP3, of viable progenies from a human rotavirus strain[J]. Virus Res, 2000, 67(2):163-171. DOI:10. 1016/S0168-1702(00)00139-8.
[15]Settembre EC, Chen JZ, Dormitzer PR, et al. Atomic model of an infectious rotavirus particle[J]. EMBO J,2011, 30(2):408-416. DOI:10. 1038/emboj. 2010. 322.
[16]Arnold MM, Brownback CS, Taraporewala ZF, et al.Rotavirus variant replicates efficiently although encoding an aberrant NSP3 that fails to induce nuclear localization of poly(A)-binding protein[J]. J Gen Virol, 2012, 93(pt 7):1483-1494. DOI:10. 1099/vir. 0. 041830-0.
[17]Ballard A, McCrae MA, Desselberger U. Nucleotide sequences of normal and rearranged RNA segments 10of human rotaviruses[J]. J Gen Virol, 1992, 73(Pt3):633-638. DOI:10. 1099/0022-1317-73-3-633.
[18]Kojima K, Taniguchi K, Urasawa T, et al. Sequence analysis of normal and rearranged NSP5 genes from human rotavirus strains isolated in nature:implications for the occurrence of the rearrangement at the step of plus strand synthesis[J]. Virology, 1996, 224(2):446-452. DOI:10. 1006/viro. 1996. 0551.
[19]Philip AA, Perry JL, Eaton HE, et al. Generation of recombinant rotavirus expressing NSP3-UnaG fusion protein by a simplified reverse genetics system[J]. J Virol, 2019, 93(24):e01616-e01619. DOI:10. 1128/jvi. 01616-19.
[20]Philip AA, Patton JT. Expression of separate heterologous proteins from the rotavirus NSP3 genome segment using a translational 2A stop-restart element[J]. J Virol, 2020, 94(18):e00959-e00920. DOI:10. 1128/jvi. 00959-20.
[21]Philip AA, Patton JT. Rotavirus as an expression platform of the SARS-CoV-2 spike protein[J/OL].bioRxiv, 2021. DOI:10. 1128/JVI. 00959-20
[22]Kawagishi T, Sanchez-Tacuba L, Feng N, et al.Mucosal and systemic neutralizing antibodies to norovirus and rotavirus by oral immunization with recombinant rotavirus in infant mice[J]. bioRxiv,2022:2022. 09. 01. 505917. DOI:10. 1073/pnas. 2214421120
[23]白植生，陈冬梅，申硕.口服轮状病毒活疫苗LLR-85株的选育及鉴定[J].中国生物制品学杂志，1994(2):49-52.
[24]唐海军. SARS-CoV-2变异体的感染特征及抗病毒药物的筛选和机制研究[D].北京：北京协和医学院，2022.
[25]李启明，梁宇，苏计国，等.一种新型冠状病毒S-RBD三聚体蛋白疫苗，其制备方法和应用.CN202110348881. 6[P].[2024-05-20].
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~~