细胞穿透肽PEP-1介导过氧化氢酶转导大鼠心肌H9C2细胞

目的:探索细胞穿透肽PEP-1介导的人过氧化氢酶转导入H9C2细胞的能力。方法:利用基因工程方法表达和纯化His-tag-PEP-1-CAT及His-tag-CAT融合蛋白后,与体外培养的H9C2细胞株共孵育,利用免疫细胞荧光技术和Westernblot检测PEP-1-CAT融合蛋白是否转导入H9C2细胞内;并检测H9C2细胞内的过氧化氢酶活性。结果:制备的PEP-1-CAT融合蛋白纯度为90%以上,其浓度达0.501mg/mL;PEP-1介导的CAT融合蛋白能高效转导入H9C2细胞内,呈时间及剂量依赖性,6h时达峰值;转导入H9C2细胞内的融合蛋白的过氧化氢酶活性亦呈现出时间及剂量依赖的特点。结论:PEP-1-CAT能高效稳定转导入H9C2细胞内,为PEP-1介导的过氧化氢酶防治心肌缺血/再灌注损伤研究奠定基础。

PEP-1-mediated Transduction of Catalase into Cultured Rat H9C2 Cells

Objective To investigate the ability of cell-penetrating peptide PEP -1 mediating the transduction of catalase into cultured rat H9C2 cells line.Methods His-tag-CAT and His-tag-PEP-1-CAT fusion protein were prepared and purified by genetic engineering.The purified fusion protein and H9C2 cells were cocultured,the transduction of catalase into H9C2 mediated with PEP-1 were analyzed with immunofluorescence technique;the characteristic of PEP-1-CAT fusion protein transduction into H9C2 cells was evaluate with western blot;the activity of catalase after transduction was determined with catalase kit.Results The purity of prepared PEP-1-CAT fusion protein was up to 90% and the concentration was 0.501 mg/mL;PEP-1-mediated catalase fusion protein was effectively transduced into H9C2 cells with a time-and dose-dependent manner,and reached peak at 6h.The activity of successfully transduced catalase was shown a time-and dose-dependent feature.Conclusion PEP-1-CAT fusion protein could be efficiently and stably transduced into H9C2 cells with a native activity.This study would provide a basis for the treatment and prevention of myocardial ischemia-reperfusion injury with PEP-1-CAT fusion protein.