2025 01 v.41 79-88
轮状病毒NSP1和NSP3区插入并表达荧光素酶基因的研究
基金项目(Foundation):
国家自然科学基金（项目号：21934005）,题目：中国母乳糖组分离分析及与轮状病毒蛋白相互作用研究~~
邮箱(Email):
zhaojund@126.com;
DOI:
10.13242/j.cnki.bingduxuebao.004677
中文作者单位:
甘肃中医药大学公共卫生学院;中国疾病预防控制中心病毒病预防控制所;伊诺维康(北京)生物科技有限公司;河南中医药大学第一临床医学院;
摘要(Abstract):
轮状病毒（Rotavirus,RV）是引起全球婴幼儿急性胃肠炎的重要病因。目前我国使用的轮状病毒疫苗为兰州生物研究所研制的口服活疫苗LLR。LLR是由羊源G10P[15]轮状病毒制备的减毒毒株。疫苗株的深入研究对新型疫苗开发具有重要意义。本研究将荧光素酶（NanoLuc Luciferase, NLuc）报告基因插入LLR/NSP1基因的ORF C端，并利用反向遗传学技术进行重组病毒拯救。通过结合dsRNA-PAGE硝酸银染色和NSP1基因RT-PCR扩增，证明成功拯救出rLLR/NSP1-NLuc重组病毒，并进一步通过间接免疫荧光、qRT-PCR、荧光素酶活性检测及病毒传代后的dsRNA-PAGE硝酸银染色实验与本实验室前期获得的重组轮状病毒rLLR/NSP3-NLuc进行病毒滴度、生长动力学、NLuc表达活性及遗传稳定性进行比较。结果显示rLLR/NSP1-NLuc的基因组拷贝数及荧光素酶活性低于rLLR/NSP3-NLuc，且两株重组病毒的P1～P5代均可检测到插入的NLuc基因片段。表明LLR的NSP1和NSP3基因节段均可稳定插入和表达NLuc，但NSP3插入NLuc后获得的重组病毒活力较NSP1更好，其表达NLuc的能力更强。本研究为进一步开展以LLR轮状病毒为疫苗载体的研究奠定了基础。
关键词(KeyWords):
轮状病毒;载体;反向遗传学
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参考文献
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[25]王明雯. A组轮状病毒相关荧光定量RT-PCR检测方法的建立及应用[D].兰州：甘肃中医药大学，2022.
[26]Barro M, Patton JT. Rotavirus nonstructural protein 1subverts innate immune response by inducing degradation of IFN regulatory factor 3[J]. Proc Natl Acad Sci USA, 2005, 102(11):4114-4119. DOI:10. 1073/pnas. 0408376102.
[27]Wei J, Radcliffe S, Pirrone A, et al. A novel rotavirus reverse genetics platform supports flexible insertion of exogenous genes and enables rapid development of a high-throughput neutralization assay[J]. Viruses, 2023, 15(10):2034. DOI:10. 3390/v15102034.
[28]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.
[29]余俊杰，刘夏飞，李杉，等.完全基于质粒的轮状病毒反向遗传技术进展[J].病毒学报，2024, 40(4):897-906. DOI:10. 13242/j. cnki. bingduxuebao. 004538.
[2] Patton JT. Rotavirus diversity and evolution in the postvaccine world[J]. Discov Med, 2012, 13(68):85-97.
[3]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.
[4]ávila-Pérez G, Nogales A, Martín V, et al. Reverse genetic approaches for the generation of recombinant zika virus[J]. Viruses, 2018, 10(11):597. DOI:10. 3390/v10110597.
[5] Neumann G, Kawaoka Y. Reverse genetics systems for the generation of segmented negative-sense RNA viruses entirely from cloned cDNA[J]. Curr Top Microbiol Immunol, 2004, 283:43-60. DOI:10. 1007/978-3-662-06099-5_2.
[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] Shimomura O, Johnson FH, Saiga Y. Extraction,purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea[J]. J Cell Comp Physiol, 1962, 59:223-239. DOI:10. 1002/jcp. 1030590302.
[8] Chalfie M, Tu Y, Euskirchen G, et al. Green fluorescent protein as a marker for gene expression[J].Science, 1994, 263(5148):802-805. DOI:10. 1126/science. 8303295.
[9] Doyle TC, Burns SM, Contag CH. In vivo bioluminescence imaging for integrated studies of infection[J]. Cell Microbiol, 2004, 6(4):303-317.DOI:10. 1111/j. 1462-5822. 2004. 00378. x.
[10]Agapov EV, Frolov I, Lindenbach BD, et al.Noncytopathic Sindbis virus RNA vectors for heterologous gene expression[J]. Proc Natl Acad Sci U S A, 1998, 95(22):12989-12994. DOI:10. 1073/pnas. 95. 22. 12989.
[11]Tsetsarkin K, Higgs S, McGee CE, et al. Infectious clones of Chikungunya virus(La Réunion isolate)for vector competence studies[J]. Vector Borne Zoonotic Dis, 2006, 6(4):325-337. DOI:10. 1089/vbz. 2006. 6. 325.
[12]McGee CE, Shustov AV, Tsetsarkin K, et al.Infection, dissemination, and transmission of a West Nile virus green fluorescent protein infectious clone by Culex pipiens quinquefasciatus mosquitoes[J]. Vector Borne Zoonotic Dis, 2010, 10(3):267-274. DOI:10. 1089/vbz. 2009. 0067.
[13]Hall MP, Unch J, Binkowski BF, et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate[J]. ACS Chem Biol,2012, 7(11):1848-1857. DOI:10. 1021/cb3002478.
[14]Sun C, Gardner CL, Watson AM, et al. Stable, highlevel expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease[J]. J Virol, 2014, 88(4):2035-2046. DOI:10. 1128/JVI. 02990-13.
[15]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.
[16]Gault E, Schnepf N, Poncet D, et al. A human rotavirus with rearranged genes 7 and 11 encodes a modified NSP3 protein and suggests an additional mechanism for gene rearrangement[J]. J Virol, 2001,75(16):7305-7314. DOI:10. 1128/JVI. 75. 16. 7305-7314. 2001.
[17]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):e00959-20. DOI:10. 1128/JVI. 00959-20.
[18]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(5):952-960.DOI:10. 13242/j. cnki. bingduxuebao. 004584.
[19]刘夏飞.轮状病毒感染性克隆构建及应用的研究[D].中国疾病预防控制中心，2023.
[20]Sen A, Rothenberg ME, Mukherjee G, et al. Innate immune response to homologous rotavirus infection in the small intestinal villous epithelium at single-cell resolution[J]. Proc Natl Acad Sci U S A, 2012, 109(50):20667-20672. DOI:10. 1073/pnas. 1212188109.
[21]Eaton HE, Kobayashi T, Dermody TS, et al. African swine fever virus NP868R capping enzyme promotes reovirus rescue during reverse genetics by promoting reovirus protein expression, virion assembly, and RNA incorporation into infectious virions[J]. J Virol, 2017,91(11):e02416-16. DOI:10. 1128/JVI. 02416-16.
[22]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):e01294-20. DOI:10. 1128/JVI. 01294-20.
[23]Ryan MD, Drew J. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein[J]. EMBO J, 1994, 13(4):928-933. DOI:10. 1002/j. 1460-2075. 1994. tb06337. x.
[24]Chng J, Wang T, Nian R, et al. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells[J]. MAbs, 2015, 7(2):403-412. DOI:10. 1080/19420862. 2015. 1008351.
[25]王明雯. A组轮状病毒相关荧光定量RT-PCR检测方法的建立及应用[D].兰州：甘肃中医药大学，2022.
[26]Barro M, Patton JT. Rotavirus nonstructural protein 1subverts innate immune response by inducing degradation of IFN regulatory factor 3[J]. Proc Natl Acad Sci USA, 2005, 102(11):4114-4119. DOI:10. 1073/pnas. 0408376102.
[27]Wei J, Radcliffe S, Pirrone A, et al. A novel rotavirus reverse genetics platform supports flexible insertion of exogenous genes and enables rapid development of a high-throughput neutralization assay[J]. Viruses, 2023, 15(10):2034. DOI:10. 3390/v15102034.
[28]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.
[29]余俊杰，刘夏飞，李杉，等.完全基于质粒的轮状病毒反向遗传技术进展[J].病毒学报，2024, 40(4):897-906. DOI:10. 13242/j. cnki. bingduxuebao. 004538.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004677
中图分类号:R373.2
引用信息:
[1]余俊杰,李杉,刘夏飞等.轮状病毒NSP1和NSP3区插入并表达荧光素酶基因的研究[J].病毒学报,2025,41(01):79-88.DOI:10.13242/j.cnki.bingduxuebao.004677.
基金信息:
国家自然科学基金（项目号：21934005）,题目：中国母乳糖组分离分析及与轮状病毒蛋白相互作用研究~~
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