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靶向PGRMC1基因sh RNA真核表达载体的构建和鉴定

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摘要 目的构建并鉴定靶向PGRMC1基因的短发夹RNA(shRNA)真核表达载体。方法根据Genbank数据库提供的mRNA序列,选择设计3个能转录短发夹RNA(shRNA)的DNA序列,体外分别合成3对互补并编码mRNA基因的特异性shRNA序列的寡核苷酸,克隆到经MspI和smoi双酶切线性化的质粒载体中。为了确定利用酶切和测序鉴定所构建的重组载体是否正确,采用脂质体转染方法将构建好的3个重组载体导入人卵巢癌细胞株SKOV3中,采用免疫组化法和RT-PCR法检测SKOV3细胞系转染前、后PGRMC1基因表达。结果经限制性酶MsqI酶切位点的酶和Smo I双重消化,PGRMC1成功建立到pcDNA3.1(-)的真核表达载体中。通过RT-PCR和Western印迹分析证实重组质粒已经成功传递到SKOV3细胞系。结论靶向PGRMC1基因shRNA真核表达载体的构建成功,为进一步研究PGRMC1基因在SKOV3细胞中的作用奠定了实验基础。
作者 程艳 余晓红
出处 《河南外科学杂志》 2020年第4期28-30,共3页 Henan Journal of Surgery
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