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

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

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BamH 1单酶切质粒peDNA3-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BamH 1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。

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 poDNA3-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 tranafected 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 eukaryotie expression plasmid for HGF gane, was consturcted. After it had been transfected into rat skeletal muscle ceils for 24 - 72 hours, the expression of EGFP was observed in 30% of the cells. Thc expression of HGF protein was detected with ELISA just one days later after tmnsfection, 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 ceils.