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口蹄疫病毒3D基因的真核表达及其
表达产物的功能分析
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鲁彬
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(武汉大学 生命科学学院 病毒学国家重点实验室，湖北 武汉430072)
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摘要：
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利用RT-PCR技术扩增口蹄疫病毒(FMDV)编码RNA依赖的RNA聚合酶的3D基因，经测序确认后将其克隆到真核表达质粒载体pcDNA3.1(+)中，重组质粒pcDNA3.1(+)-3D经Hind Ⅲ、Xba Ⅰ双酶切后转染至BHK-21细胞，用纯化的目的蛋白进行Western-blotting鉴定，并预测该3D基因表达产物的三级结构。结果显示，获得分子质量约55ku的单一3D基因表达产物，其三级结构较为保守。利用RNA细胞内复制体系和荧光定量RT-PCR技术，证明3D基因表达产物在细胞内可促进口蹄疫病毒基因组的复制。 |
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关键词：
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口蹄疫病毒；RNA依赖的RNA聚合酶；真核表达；链特异性荧光定量RT-PCR |
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中图分类号：S 852.659.6：Q 786 文献标识码：
A 文章编号：1673-4696(2006)10-0800-05 |
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Eukaryotic expression of foot-and-mouth disease virus 3D gene
and functional analysis of the expressed product |
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LU Bin |
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(State Key Laboratory of Virology/College of Life Sciences, Wuhan University, Wuhan 430072, China) |
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Abstract: The 3D gene encoding RNA-dependent RNA polymerase(RdRp) of foot-and-mouth disease virus was amplified by RT-PCR using pfu ultraTM High-Fidelity DNA polymerase, and the amplicon was cloned into the eukaryotic expression vector pcDNA3.1(+) to construct recombinant expression plasmid. Then the constructed recombinant plasmid was sequenced and transfected into BHK-21 cells. Western-blotting analysis confirmed that the purified recombinant protein RdRp was expressed correctly in soluble form. Tertiary structure of the expressed RdRp was predicted to be conservative. Activity of the protein was determined by the genomic RNA replication assay in BHK-21 cells. The RNA product of the replication system in cells was quantified by strand-specific real-time RT-PCR. The results suggested that the RdRp could dramatically improve the synthesis of RNA. |
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Key words: foot-and-mouth disease virus(FMDV); RNA-dependent RNA polymerase; eukaryotic expression; strand-specific real-time RT-PCR |
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