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为建立新城疫病毒（NDV）强毒株NA-1微型基因组（minigenome，MG）双质粒高效拯救系统，本研究将NDV NA-1株基因组3'-Leader、5'-Tailer序列以及增强型绿色荧光蛋白（Enhanced Green Fluorescent Protein ，EGFP）基因序列，反向克隆至T7启动子调控的表达质粒pcDNA3.1，同时在微型基因组表达质粒3'端引入丁肝核酶和T7终止子序列，构建成NDV NA-1株微型基因组转录质粒pcDNA3.1-NDV-MG。将分别表达NP、P和L的三个辅助质粒优化为能够同时表达NP、P和L蛋白的多启动子单质粒pcDNA3.1-NPL，并与pcDNA3.1-NDV-MG组成双质粒拯救系统共转染表达T7 RNA聚合酶的BSR细胞，通过荧光显微镜观察EGFP报告基因的表达鉴定拯救是否成功。结果显示，共转染48 h后，BSR细胞内可观察到特异绿色荧光信号，表明NDV强毒株微型基因组双质粒拯救系统构建成功，并且与传统四质粒系统相比，所构建的双质粒系统表现出更高的拯救效率。本研究成功构建了高效的NDV微型基因组双质粒拯救系统，不仅为NDV高效反向遗传操作平台的建立奠定了基础，也为后续致病机制研究、新型疫苗研制及病毒活载体开发提供了强有效的工具。
Abstract：To establish an efficient two-plasmid rescue system for the minigenome of the virulent Newcastle disease virus (NDV) strain NA-1， the 3'-leader and 5'-tailer sequences， flanked by the hepatitis delta virus ribozyme sequence (HdvRz) and T7 terminator sequences at the 3' end were cloned， along with enhanced green fluorescent protein (Enhanced Green Fluorescent Protein ， EGFP) gene. These components were assembled into a single construct， Tailer-EGFP-Leader-HDV Rz-T7Ter， and inserted into pcDNA3.1 vector under the control of the T7 promoter， resulting in pcDNA3.1-NDV-MG. The three helper plasmids expressing NP， P， and L proteins were optimized into a single multi-promoter plasmid， pcDNA3.1-NPL， capable of simultaneously expressing NP， P， and L proteins. The two-plasmid system， comprising pcDNA3.1-NDV-MG and pcDNA3.1-NPL， was co-transfected into BSR cells expressing T7 RNA polymerase. The expression of the EGFP reporter gene was used to evaluate the success of the rescue system via fluorescence microscopy. Results showed that specific green fluorescence signals were observed in BSR cells 48 hours post-transfection， indicating the successful construction of the two-plasmid rescue system for the NDV virulent strain minigenome. Compared to the traditional four-plasmid system， the two-plasmid system demonstrated higher rescue efficiency. This study successfully developed an efficient two-plasmid rescue system for the NDV minigenome， providing a powerful tool for establishing a highly efficient NDV reverse genetics platform and facilitating future research on pathogenic mechanisms， novel vaccine development， and viral vector applications.
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基本信息:
中图分类号:S852.65
引用信息:
[1]朱广美,宗宪春,徐哲,等.新城疫病毒强毒株微型基因组双质粒拯救系统的构建[J].经济动物学报().
基金信息:
吉林省科技发展计划项目(20220101314JC)
2025-10-29
2025-10-29
2025-10-29