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带荧光素酶基因的乙脑病毒SA14-14-2感染性克隆的构建 |
Construction of JEV SA14-14-2 infectious clone with luciferase gene |
投稿时间:2019-10-30 修订日期:2019-10-30 |
DOI: |
中文关键词: 荧光素酶 乙脑病毒感染性克隆 重组乙脑病毒 |
英文关键词: Luciferase Infective clone of JEV Recombinant JEV |
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中文摘要: |
目的: 构建乙脑病毒感染性克隆,并拯救获得带有荧光素酶基因的乙脑病毒,用于 乙脑病毒致病机理、SA14-14-2病毒株在小鼠体内动态分布以及乙脑疫苗免疫效果评价的高通量方法建立。 方法:应用反向遗传学技术和病毒感染性克隆技术,在乙脑病毒SA14-14-2 基因组中插入荧光素酶报告基因F-LUC,构建含有F-LUC的SA14-14-2乙脑病毒全长感染性克隆,线性化后体外转录为RNA并转染BHK21细胞。 结果:成功构建带有F-LUC报告基因的SA14-14-2乙脑病毒全长感染性克隆,拯救获得重组病毒。基因序列分析及细胞与动物水平证明拯救的病毒为带有荧光素酶基因的乙脑病毒。 结论:本研究构建出带有荧光素酶报告基因F-LUC的SA14-14-2感染性克隆,并拯救获得带有荧光素酶的重组乙脑病毒,为乙脑血清中和抗体的高通量筛选,以及SA14-14-2在小鼠体内的动态分布和致病机理奠定了基础。 |
英文摘要: |
Objective:To construct the Japanese encephalitis virus (JEV) SA14-14-2 infectious clone and rescue the JEV with luciferase gene, which is used for establish a high-throughput method for evaluating the immune effectiveness of JE vaccine and study the dynamic distribution of JEV SA14-14-2 virus in mice. Methods:Using reverse genetics and virus infectious cloning technology, the luciferase reporter gene F-LUC was inserted into the genome of JEV SA14-14-2 infectious clone to construct SA14-14-2 full-length infectious clones containing F-LUC,which was linearized and transcribed into RNA in vitro and transfected into BHK21 cells. Results:The full-length infectious clone of SA14-14-2 with F-LUC reporter gene was successfully constructed. The rescue of recombinant JEV was successfully identified at the cellular and animal levels besides gene sequence analysis. Conclusion:In this study, JEV SA14-14-2 infectious clone with luciferase reporter gene F-LUC was constructed, and the recombinant JEV with luciferase was rescued, which laid the foundation for the high-throughput test of neutralizing antibody against JE vaccine, the dynamic distribution in mice and the pathogenic mechanism of JEV. |
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