新型EGFP标记的杆状病毒转移载体的构建及EGFP的制备

目的构建可表达增强型绿色荧光蛋白(EGFP)融合蛋白的昆虫杆状病毒转移载体,利用Sf-9细胞表达、制备EGFP。方法用PCR法从pEGFP质粒中扩增EGFP基因,NcoⅠ/SalⅠ双酶切,克隆入杆状病毒转移载体NcoⅠ/SalⅠ酶切位点之间,获得pFB-EGFP质粒;转化E.coliDH10Bac,通过转座子Tn7的介导,获得重组杆状病毒Ac-EGFP;感染Sf-9昆虫细胞;利用激光共聚焦显微镜和SDS-PAGE观察并检测EGFP的表达。非变性法纯化EGFP。结果荧光显微镜和激光共聚焦显微镜证实感染的Sf-9细胞可表达EGFP;通过SDS-PAGE获得的EGFP,大小为27ku。结论重组pFB-EGFP载体具备表达EGFP的能力,为分子生物学研究提供了一种新型可表达EGFP融合蛋白的昆虫杆状病毒表达载体。

Construction of a novel enhanced green fluorescent (EGFP) tagged insect-baculovirus transference vector and preparation of EGFP

Objective To construct a novel enhanced green fluorescent protein (EGFP) tagged insect-baculovirus transference system and prepare EGFP by Sf-9 cells. Methods After EGFP gene had been amplified from pEGFP plasmid by PCR and digested by NcoⅠ/SalⅠ, it was recombined into baculovirus transference vector pFastbac-Hb, between the NcoⅠ/SalⅠrestriction enzyme sites to create a novel pFB-EGFP vector. After E.coli DH10Bac was transformed with the pFB-EGFP vector, a recombinant baculovirus Ac-EGFP, was obtained with the action of Tn7 transposon. The insect Sf-9 cells were infected with the recombinant baculovirus Ac-EGFP, and then the expression of EGFP was analyzed by Western blot. Meanwhile, the Sf-9 cells infected by the recombinant baculovirus were observed under laser confocal microscope and native-PAGE. EGFP was purified by native-PAGE. Results EGFP expression in infected Sf-9 cells was proved by SDS-PAGE. The bright green fluorescence was demonstrated in plasma and nuclei of infected Sf-9 cells. EGFP could be purified using Ni-resin. Conclusion The novel pFB-EGFP baculovirus expression system can express EGFP efficiently and can become a useful EGFP tagged expression system for detection of the interaction of proteins.