HGF非融合蛋白真核表达载体的构建及其在骨骼肌细胞中的表达

目的构建以HGF为目的基因、以EGFP为报告基因的非融合蛋白真核表达载体pCMV-HGF-IRES-EGFP,并观察HGF基因在原代培养的大鼠骨骼肌细胞中的表达。方法利用BamHI单酶切质粒pcDNA3-HGF,得到HGF基因片段,将其正向插入到真核表达载体pCMV-IRES-EGFP的CMV后方BamHI的单克隆位点。脂质体介导转染至原代培养的大鼠骨骼肌细胞,在荧光显微镜下观察EGFP的表达,并用ELISA法检测HGF的表达情况。结果构建了HGF的非融合蛋白真核表达载体pCMV-HGF-IRES-EGFP,转染原代培养的大鼠骨骼肌细胞24~72h后,见30%的细胞表达EGFP。ELISA检测细胞培养液证实转染后第1天即有HGF的表达,第4天HGF浓度为(5402.0±227.9)ng/L。结论成功构建了非融合蛋白真核表达载体pCMV-HGF-IRES-EGFP,其在原代培养的大鼠骨骼肌细胞中能有效表达。

Construction of HGF Gene Non-fusion Expression Vector and Expression of HGF Protein in Skeletal Muscle Cells

Objective To construct a non-fusion expression vector pCMV-HGF-IRES-EGFP for hepatocyte growth factor (HGF) gene and investigate the expression of HGF protein in the primarily cultured rat skeletal muscle cells. Methods HGF gene was obtained from plasmid pcDNA3-HGF with BamHI digestion and was inserted into the same restriction site of pCMV-IRES-EGFP. The upright insertion was identified by sequencing. Then the recombinant plasmid pCMV-HGF-IRES-EGFP was transfected into the primarily cultured rat skeletal muscle cells. And the expression of HGF protein was observed by fluorescence microscope and determined with ELISA. Results The pCMV-HGF-IRES-EGFP, a non-fusion eukaryotic expression plasmid for HGF gene, was constructed. After it had been transfected into rat skeletal muscle cells for 24-72 hours, the expression of EGFP was observed in 30%of the cells. The expression of HGF protein was detected with ELISA just one days later after transfection, and its concentration went up to 5 402.0±227.9 (ng/L) four days later after transfection. Conclusion With the constructed recombinant plasmid, high levels of HGF protein expression can be obtained in the rat skeletal muscle cells.