| 摘要: |
| 【目的】研究苜蓿银纹夜蛾核型多角体病毒(AcMNPV)晚期表达因子lef-10在宿主细胞Sf9中的时空表达及定位，为进一步研究lef-10的功能提供理论基础。【方法】利用不同的特异引物，通过PCR方法扩增lef-10及innateP-lef-10目的片段，将目的片段分别克隆到载体pTriEx-SP-GFP上，得到重组质粒pTriEx-lef10-GFP和pTriEx-innateP-lef10-GFP，经过双酶切检测构建载体。将重组质粒分别与Bacmid共转染Sf9细胞，用得到的重组病毒分别于4，8，16，24，36 h再次感染宿主细胞；用激光共聚焦显微镜观察lef-10的表达及亚细胞定位情况。【结果】在感染早期，lef-10就已开始表达；随感染时间的推移，lef-10逐渐移动至细胞核，且在晚期和极晚期时相，lef-10主要集中于细胞核中。【结论】在天然条件下，lef-10是作为晩期表达因子发挥功能的，并且对其他基因的转录可能起一定的调控作用。 |
| 关键词: AcMNPV lef-10 绿色荧光蛋白 亚细胞定位 |
| DOI： |
| 分类号: |
| 基金项目:陕西省自然科学基础研究计划项目(K332021108) |
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| Expression and cellular localization of Ac-MNPV lef-10 in Sf9 cells |
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LIU Tian-tian,JIANG Xiang,XU Xiao-dong
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| Abstract: |
| 【Objective】The research studied temporal and spatial characteristics and cellular localization of AcMNPV lef-10 expression in Sf9 cells.【Method】Specific primers were designed for amplification of lef-10 sequence and innateP-lef-10 sequence using PCR.Then the target sequences were cloned to vector pTriEx-SP-GFP to obtain recombinant plasmids pTriEx-lef10-GFP and pTriEx-innateP-lef10-GFP,respectively.Recombinant plasmids and Bacmid were used to cotransfect Sf9 cells to generate recombinant baculovirus,which was used to infect Sf9 cells at 4,8,16,24,and 36 h,respectively.Then the expression of lef-10 and subcellular localization were observed using laser scanning confocal fluorescence microscope.【Result】lef-10 expression was observed in the early phase after infection.Then,lef-10 moved to nucleus gradually,and mainly localized in nucleus in the late and very late phases.【Conclusion】Under natural condition,lef-10 works as a late express factor,and may play a regulatory role on other genes transcription. |
| Key words: AcMNPV lef-10 green fluorescent protein subcellular localization |
