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【目的】构建携带有受杆状病毒多角体启动子控制的疱疹性口腔炎病毒糖蛋白(vesicular stomatitis virus glycoprotein,VSVG)和受白斑综合症病毒极早期基因(immediately-early gene1,ie1)启动子控制的绿色荧光蛋白(enhanced green fluorescent protein,EGFP)两个表达阅读框的新型重组病毒vAc-G-EGFP,分析其在无脊椎动物和脊椎动物细胞系中表达报道基因的能力。【方法】利用Bac-To-Bac系统构建重组杆状病毒,利用病毒感染或转导实验介导报道基因在待测细胞系中的表达,用荧光显微镜和免疫印迹技术分析报道基因在待测细胞系中的实时表达情况。【结果】成功构建了分别含VSVG和ie1启动子两个阅读框的重组杆状病毒vAc-G-EGFP,发现vAc-G-EGFP可以在无脊椎和脊椎动物细胞系中有效表达报道基因EGFP,免疫印迹实验显示,在不同时间点EGFP于这两类细胞中的表达存在差异。【结论】基于白斑综合症病毒ie1启动子并携带有VSVG表达框的单一杆状病毒载体可以实现同时在不同种类细胞系中有效表达外源基因。本文构建的新型杆状病毒表达载体有希望普遍应用于基础和应用生物学研究。
【Objective】 To construct a recombinant plasmid carrying vesicular stomatitis virus glycoprotein (VSVG) and the immediate-early gene1 (ie1) promoter controlled by baculovirus polyhedrin promoter Control recombinant green fluorescent protein (EGFP), a new recombinant virus vAc-G-EGFP expressing the reading frame, was used to analyze its ability to express the reporter gene in invertebrate and vertebrate cell lines. 【Method】 Bac-To-Bac system was used to construct recombinant baculovirus. The viral infection or transduction experiments were used to mediate the expression of the reporter gene in the cell line under test. Fluorescent microscopy and Western blotting were used to analyze the expression of the reporter gene in the cells to be tested Department of real-time expression. 【Results】 The recombinant baculovirus vAc-G-EGFP containing the two reading frames of VSVG and ie1 promoters was successfully constructed and found that vAc-G-EGFP could effectively express EGFP in invertebrate and vertebrate cell lines, Western blotting showed that there was difference in the expression of EGFP between these two types of cells at different time points. 【Conclusion】 A single baculovirus vector based on ie1 promoter of white spot syndrome virus carrying VSVG expression cassette can efficiently express foreign genes in different cell lines at the same time. The new baculovirus expression vector constructed in this paper is promising for general and applied biology research.