可溶性人干细胞因子与EGFP融合基因的构建及真核表达

Construction and Expression of Recombinant Gene for Fusion of Human Stem Cell Factor and EGFP

目的 可溶性人干细胞因子 (SCF)膜外活性区与增强型绿色荧光蛋白 (EGFP)融合基因表达载体的构建及其表达。方法 应用基因工程技术构建重组载体 ,电穿孔转染CHO K1细胞株 ,荧光分光光度计检测上清液中融合蛋白的表达 ,流式细胞仪检测其活性。结果 融合基因在瞬时转染的真核细胞中获得表达 ,并分泌至上清 ,且SCF EGFP融合蛋白中分子标签EGFP未影响可溶性人SCF膜外活性区的结构及其生物学活性。结论 可溶性人干细胞因子与EGFP融合基因载体构建成功 ,表达的融合蛋白具有生物活性 ,为进一步研究SCF对造血干细胞的生物学作用奠定了基础。

Objective To construct the fusion gene of soluble human stem cell factor (SCF) and enhanced green fluorescent protein (EGFP), and study the expression of the fused SCF-EGFP in CHO-K1 cells. Methods The standard cloning technology was employed to construct the SCF-EGFP fusion gene. The fusion gene was transfected into CHO-K1 cells by electroporation. The expression of the fusion gene was determined by flow cytometry (FCM) and fluorescence spectrophotometer. Results The expression of the fusion gene was observed in transiently transfected CHO-K1 cells. The fusion protein was secreted into supernatant. These data suggested that the EGFP tag did not interfere with the natural assembly and the biological activity of SCF molecule. Conclusion The SCF-EGFP fusion gene was constructed successfully and the fusion protein has activity.