家蚕核型多角体病毒成纤维细胞生长因子基因的表达和定位

为了解杆状病毒基因组中成纤维细胞生长因子基因(fgf)的功能,分离纯化了BmNPV DNA,采用PCR方法获得了BmNPV的fgf基因,并克隆至原核表达载体pET28 a,构建了高效原核表达质粒pET28 af-gf。通过IPTG诱导表达后,经SDS-PAGE检测证实了在27 kD左右有一特异条带,与预测的蛋白质分子量大小一致,并通过N i2+-NTA离子交换树脂亲和纯化了目的蛋白。同时构建了融合GFP的真核表达载体pCDNA3.1-gfp-fgf,通过脂质体转染进入COS-7细胞进行表达和定位观察。fgf基因产物定位于细胞核。

Expression and Subcellular Location of the Fibroblast Growth Factor Gene of Bombyx mori Nucleopolyhedrosis Virus

A gene,whose product is similar to that of the human Fibroblast Growth Factor(FGF),was found in baculovirus.To know more information about this gene,polyhedron particles of Bombyx mori Nuclear Polyhedrosis Virus(BmNPV) were purified from the infected cells of Bombyx mori.The DNA fragment of BmNPV fgf gene was amplified by Polymerase Chain Reaction(PCR) with the specific primers and were cloned into prokaryotic expression plasmid pET28a and eukaryotic expression vector pcDNA3.1,respectively.The expression products emerged using IPTG as the inducer in E.coli strain BL21.By SDS-PAGE there was a specific band whose molecular weight was 27 kD.The fusion FGF protein was purified through Ni~(2 )-NTA affinity chromatography.In eukaryotic expression system,the fgf gene was fused with the green fluorescence protein(GFP).The expression products were located in the nuclei of COS-7 cells.