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目的 为开发新型猪轮状病毒候选疫苗，本研究以猴轮状病毒rSA11株为骨架，利用反向遗传技术构建表达猪轮状病毒NJ2012株（G9P[7]型）VP4、VP7及VP4/6/7基因的三种重组病毒（rSA11-NJ2012-VP4、rSA11-NJ2012-VP7、rSA11-NJ2012-VP4/6/7），并开展体外生物学特性测定及小鼠体内免疫原性分析。方法 以NJ2012株为模板构建其VP4、VP6和VP7基因的感染性克隆质粒，分别替换SA11株的相应基因，利用完全基于质粒的反向遗传操作系统构建重组病毒。利用western blot、间接免疫荧光、dsRNA-PAGE、一步生长曲线、蚀斑实验及透射电镜等对拯救的重组病毒进行体外生物学特性鉴定，经免疫小鼠后结合ELISA和中和试验评价其免疫原性。结果细胞病变观察、基因测序、dsRNA-PAGE、间接免疫荧光等实验证实三种重组病毒拯救成功。一步生长曲线、噬斑实验及透射电镜验证了重组病毒与亲本毒株之间生物学特性存在一定差异。三种重组病毒可有效刺激小鼠持续产生高水平特异性IgG，诱导的中和抗体对同型毒株具有良好中和活性。结论 本研究成功构建并拯救出表达猪源VP4/VP7基因的重组轮状病毒，其在小鼠体内具有优良免疫原性，为新型猪轮状病毒候选疫苗的快速研发奠定了实验基础。
Abstract：Objective To develop novel candidate vaccines against porcine rotavirus, this study used the simian rotavirus strain rSA11 as the backbone, and constructed three recombinant viruses(rSA11-NJ2012-VP4, rSA11-NJ2012-VP7, rSA11-NJ2012-VP4/6/7) expressing the VP4, VP7, and VP4/6/7 genes of porcine rotavirus strain NJ2012(G9P[7]) via reverse genetics technology. Furthermore, the in vitro biological characteristics and in vivo immunogenicity in mice of these recombinant viruses were determined and analyzed. Methods Infectious clone plasmids carrying the VP4, VP6, and VP7 genes of strain NJ2012 were constructed using NJ2012 as the template. These plasmids were used to replace the corresponding genes of strain SA11, and the recombinant viruses were rescued using a fully plasmid-based reverse genetics system. The in vitro biological characteristics of the rescued recombinant viruses were identified by western blot, indirect immunofluorescence assay(IFA), double-stranded RNA polyacrylamide gel electrophoresis(dsRNAPAGE), one-step growth curve analysis, plaque assay, and transmission electron microscopy(TEM). After immunizing mice with the recombinant viruses, their immunogenicity was evaluated by enzyme-linked immunosorbent assay(ELISA) and neutralization test. Results Observations of cytopathic effect(CPE), gene sequencing, dsRNA-PAGE, and indirect immunofluorescence assay confirmed the successful rescue of the three recombinant viruses. One-step growth curve analysis, plaque assay, and transmission electron microscopy revealed certain differences in biological characteristics between the recombinant viruses and the parental strain. All three recombinant viruses effectively stimulated mice to produce sustained high levels of specific IgG, and the induced neutralizing antibodies exhibited potent neutralizing activity against the homologous viral strain. Conclusion In this study, recombinant rotaviruses expressing porcine-derived VP4/VP7 genes were successfully constructed and rescued. These recombinant viruses showed excellent immunogenicity in mice, which lays a solid experimental foundation for the rapid development of novel candidate vaccines against porcine rotavirus.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250187
中图分类号:S852.651
引用信息:
[1]程曦,陶然,邓慧,等.以猴轮状病毒SA11株为骨架表达猪轮状病毒VP4/VP7基因重组病毒的构建及其免疫原性评价[J].病毒学报,2026,42(01):228-241.DOI:10.13242/j.cnki.bingduxuebao.250187.
基金信息:
国家自然科学基金（项目号：32302892）,题目：PAXIP1通过抑制干扰素信号通路促进轮状病毒增殖的分子机制; 江苏省农业科技自主创新资金（项目号：CX(23)1029）,题目：猪腹泻病新型高效疫苗创制; 江苏省重点研发计划（现代农业）（项目号：BE2023317）,题目：猪轮状病毒三价（G9、G5和G4）mRNA疫苗关键技术创新及疫苗创制; 江苏省卓越博士后计划（项目号：2022ZB766）,题目：猪轮状病毒反向遗传操作平台的建立及应用~~
2025-10-27
2025-10-27
2025-10-27