融合结构域MM蛋白在杆状病毒中的表达和细胞定位

目的将C-Myb的DNA结合域与MEF的AML1结合区组合成的MM融合基因在杆状病毒中进行表达和细胞定位。方法将氨基酸融合有绿色荧光蛋白的MM基因插入到杆状病毒转移载体中,通过重组质粒和亲本病毒AcPak6共转染昆虫Tn5细胞,获得重组病毒AcNPV-GFP-MM,并对该重组病毒在Tn5细胞中进行表达和定位观察。结果通过荧光显微镜观察,在病毒感染48h后的Tn5细胞中,可见表达产物大量集聚在细胞核内,Westernblot证明表达的融合蛋白在相对分子质量约60000处出现特异条带,与预计的蛋白理论值相符。结论由杆状病毒表达系统表达的MM蛋白比较稳定,能正确地趋向于核内。可以通过这种表达方式获得大量产物。

Expression and Subcellular Location of Fusion Domain MM Protein Using Baculovirus Vector

Objective To express and locate a fusion gene(MM)consisting of c-Myb DNA-binding domain and Myeloid elf-like factor(MEF)AML1-binding region by using baculovirus vector.Methods Insert MM gene with green fluorescence protein(GFP)fused at N-terminus into baculovirus transfer vector BacPak8.Co-transfect insect cell Tn5 with the obtained recombinant plasmid and its parental virus AcPak6.Observe the subcellular location of GFP-MM fusion protein by fluorescent microscopy and determine the expressed product by Western blot.Results A large quantity of congregate expressed proteins were observed in Tn5 cells 48 h after transfection.Western blot showed specific reaction band with a relative molecular mass of about 60 000,which was consistent with that expected.Conclusion The MM protein expressed by baculovirus expression system was stable and located in nuclei correctly.In this way,a large quantity of expressed product might be obtained.