人精子蛋白17增强型绿色荧光蛋白共表达卵巢癌细胞模型的建立

Establishment of an ovarian carcinoma cell model with co-expression of human sperm protein 17 and enhanced green fluorescent protein

目的:建立人精子蛋白17(hSp17)与增强型绿色荧光蛋白(EGFP)共表达的卵巢癌细胞模型。方法:用PCR方法获取hSp17基因片段,HindⅢ/KpnⅠ双酶切、T4 DNA连接酶连接的方法将hSp17基因片段克隆至含报告基因EGFP的真核表达载体pEGFP-N1中,脂质体介导转染卵巢癌细胞HO8910,荧光倒置显微镜观察及Westernblot检测转染细胞中hSp17/EGFP融合蛋白的表达,G418筛选稳定表达的细胞株。结果:酶切和测序结果均证实pEGFP-N1/hSp17重组质粒的构建完全正确。荧光观察与Western blot均证实了hSp17/EGFP蛋白的共表达,且成功筛选出稳定表达的细胞株。结论:成功构建hSp17/EGFP共表达的卵巢癌细胞模型,为研究hSp17异常表达对肿瘤细胞生物学行为的影响,以及hSp17为靶标的肿瘤诊断和生物治疗奠定基础。

Objective： To establish a human ovarian carcinoma （OC） cell model that stably co-expresses the human sperm protein 17 and enhanced green fluorescent protein （hSpl7/EGFP）. Methods： hSpl7 cDNA was obtained by PCR and cloned into the vector pEGFP-N1 that contained a report gene-enhanced green fluorescent protein. The recombinant plasmid was transfected into HO8910 cells with lipofectamine 2000. The expression of fusion hSp17/EGFP was observed under the fluorescent inverted microscope and detected by Western blotting. Cells with stable expression of hSpl7 were selected by G418. Results ： Correct construction of pEGFP-N1/hSpl7 recombinant plasmid was confirmed by restriction enzyme digestion and sequencing. Conclusion： An ovarian carcinoma cell model that highly expressed hSp17/EGFP was successfully established, which may contribute to further studies on the effect of the abnormal hSp17 expression on the biological behavior of tumor cells and its role as a target in the diagnosis and biological treatment of tumors.