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盐酸多西环素对重组质粒pBI-EGFP-hHO-1表达的调控

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摘要 目的构建Tet-off调控的血色素氧化酶-1(HO-1)真核表达载体pBI-EGFP-hHO-1,并确定盐酸多西环素对其表达的调控剂量。方法利用分子生物学技术,将hHO-1基因与质粒pBI-EGFP连接构成重组质粒。NheI和MluI双酶切鉴定后采用RT-PCR技术及免疫细胞化学的方法检测其在人肝癌细胞SMMC-7721中的表达情况。利用RT-PCR技术检测Tet-off和pBI-EGFP-hHO-1双转染后不同浓度盐酸多西环素对hHO-1基因mRNA表达量的影响,确定该四环素调控系统的盐酸多西环素诱导量。结果用NheI和MluI双酶切证明hHO-1成功克隆入应答质粒pBI-EGFP;RT-PCR、免疫细胞化学检测结果表明重组质粒在SMMC-7721细胞中表达;经RT-PCR检测显示当盐酸多西环素浓度≥2 000 ng/ml时,HO-1基因mRNA表达降低,与未转染组接近。结论成功构建了由Tet-off调控的真核表达载体pBI-EGFP-hHO-1,并确定盐酸多西环素对其调控剂量为2 000 ng/ml。
作者 杨婧 张海风 王媛 赵勤 张新胜 单杰
机构地区 郑州大学基础医学院生物化学与分子生物学教研室
出处 《中国老年学杂志》 CAS CSCD 北大核心 2011年第21期4193-4196,共4页 Chinese Journal of Gerontology
基金 郑州市科技局(No.083SGYS33263-10)
关键词 血色素氧化酶-1 Tet-off诱导表达系统 盐酸多西环素
分类号 R393 [医药卫生—基础医学]
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参考文献10

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二级参考文献35

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中国老年学杂志
2011年 第21期

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