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Construction and expression of a eukaryotic plasmid containing luciferase gene regulated by partial promoter of osteocalcin
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 22
- DOI:
- 10.3321/j.issn:1000-2790.2002.22.004
- Key Word:
- 骨钙素;启动子;萤光素酶;基因表达;遗传载体
Abstract： 目的构建部分骨钙素启动子控制荧光素酶报告基因的真核表达载体pcDNA3-OCP-Luc,转染细胞后检测报告基因对rhBMP-2的反应,以期用于rhBMP活性的定量测定. 方法用PCR方法扩增骨钙素部分启动子147 bp,克隆入pGEM-3zf(-)中,经酶切鉴定和序列分析正确后,亚克隆入能稳定高效表达荧光素酶的真核表达载体pcDNA3-Luc中,构建真核表达载体pcDNA3-OCP-Luc,然后分别将pcDNA3-Luc和pcDNA3-OCP-Luc转染细胞,并检测其对rhBMP-2的反应. 结果成功构建了部分骨钙素启动子控制荧光素酶报告基因的真核表达载体pcDNA3-OCP-Luc;转染细胞后其报告基因的基础活性较pcDNA3-Luc大为降低,而且报告基因的表达与rhBMP-2剂量在一定范围内成线性正相关. 结论真核表达载体pcDNA3-OCP-Luc的报告基因表达具有特异性,可代替直接测定骨钙素用于rhBMP-2活性的定量测定研究.
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