多角体包埋异源蛋白杆状病毒表达系统的构建

目的]探索多角体病毒的包装特性,构建能形成多角体包裹外源蛋白的polyh+Bac-to-Bac表达系统。方法]通过酶切连接的方式,将4个元件片段:作为标记的绿色荧光基因EGFP,Bm CPV自身的多角体蛋白基因,用于基因表达的H1信号肽柔性连接肽,插入到质粒Fast Bac Dual中,利用Ac MNPV Bac-to-Bac系统,得到整合表达重组Bacmid:Ac-polyh/EGFP。将其感染Sf9细胞后,检测蛋白表达情况。结果]利用该系统构建了一种能大量表达绿色荧光蛋白(EGFP)和能在胞质形成多角体的重组病毒v Bm Bac(polyh+)-EGFP。感染的Sf9细胞在荧光显微镜下观察发现EGFP与多角体可以在同一细胞中同时表达;从感染的Sf9细胞中收集纯化多角体,观察到多角体能被激发出绿色荧光,进一步利用Western blot证实多角体中含有EGFP。结论]该表达系统为解决活性蛋白长期保存提供了新方法,同时拓宽了杆状病毒在开发含有毒素蛋白的新型生物杀虫剂和新型疫苗的转运载体等领域的应用前景。

Construction of Polyhedrin-plus Bac-to-Bac System for Embedded Exogenous Protein

Objective] To make clear the packing mechanism of the BmNPV polyhedra,a polyhedrin-plus Bac-to-Bac expression systemfor application in silkworm was constructed.Method] On the basis of plasmid FastBacDual,four components were integrated,which were EGFP gene as resistant marker,H1 signal peptide,polyhedrin gene from BmCPV and a flexible linker peptide.After enzyme digestion and ligation,high copy recombinant expression vector pFastBac Dual/polyh-EGFP was constructed.pFastBac Dual/polyh-EGFP was linearized and Ac-Polyh/EGFP transferred into Sf9 cells.Result] Using this new system,vBmBac (polyh +)-EGFP was constructed that the foreign gene and polyhedrin were both expressed at higher level and large amount of polyhedra were formed.Fluorescent microscopic observation demonstrated that EGFP and polyhedrin were expressed simultaneously,and the EGFP expression and polyhedra formation occurred in most of the jointly infected cells.Analysis of the purified polyhedra from jointly infected Sf9 cells showed that the foreign proteins were present in the polyhedra.Conclusion] The result provide a new inspiration for efficient preservation of active proteins and development of new biopesticide with toxin protein and new delivery vector system for vaccines.