基于对虾白斑综合症病毒极早期启动子的新型杆状病毒表达载体的构建与分析

Construction and characterization of a novel baculovirus vector based on the White spot syndrome virus immediately-early promoter

【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 universal bacttlovirus vector for efficient gene expression in both invertebrate and vertebrate cell lines. [Methods] Using the Bac-To-Bac system, we genetically engineered the immediately-early 1 gene promoter （iel promoter） from White spot syndrome virus into a baeulovirus vector that was pseudotyped with Vesicular stomatitis virus glycoprotein （VSV G）. We placed the enhanced green fluorescent protein （EGFP） gene under the control of iel promoter in the baculovirus vector to get the reporter recombinant baculovirus, vAe-G-EGFP. We tested the reporter EGFP gene expression in tested cell lines through virus infection or transduction experiments using direct fluorescence microscopy and Western blot analysis. [ Results] Under the control of iel promoter, vAc-G-EGFP could efficiently express the EGFP reporter gene in both invertebrate and vertebrate cells. The steady-state expression level of EGFP in vertebrate cell lines were different from that in invertebrate Sf9 cells as reflected by Western blot assays. [ Conclusion] The iel promoter-based and VSV G-pseudotyped baculovirus vector presents a unique and effective tool to express target genes simultaneously in various cell systems; the novel baculovirus-mediated gene expression system developed in this study has the potential to be widely used in both basic and applied research.