绿色荧光蛋白基因在昆虫细胞中的克隆与表达

EXPRESSION OF GREEN FLUORESCENT PROTEIN WITH BACULOVIRUS VECTOR IN INSECT CELLS

将绿色荧光蛋白(GFP)基因亚克隆到转移载体pVLneo的多角体蛋白基因(ocu)启动子下游,与杆状病素AcNPV DNA共转染昆虫细胞,通过同源重组和G418筛选,构建了整合有GFP基因的重组病毒。在昆虫细胞中表达的GFP,MW为30kDa,在荧光显微镜下呈现美丽的绿色,荧光光谱表明其激发波长395nm,发射波长509nm。Southern blot杂交证明,重组病毒的1kb EcoRI片段与GFP cDNA探针有很强的杂交信号,这是GFP基因在杆状病毒基因组中整合的直接证据。

The green fluorescent protein (GFP) gene was subcloned into the transfer vector pVLneo downstream of the polyhedrin gene (ocu) promoter. Insect cells were cotransfected with recombinant plasmid and Autographa califomica Nuclear Polyhedrosis Virus (AcNPV) DNA. In the presence of G418, the recombinant virus containing GFP gene was purified. The GFP expressed in insect cells with a Mw of SOkDa is observable by strong green light under a fluorescent microscope. Excitation and emission spectra of the GFP were 395nm and 509nm respectively. Integration of GFP gene on AcNPV genome was identified directly by Southern blot which gave strong hybridization signal between GFP cDNA probe and 1kb EcoRI fragment of recombinant virus.