部分骨钙素启动子控制荧光素酶基因真核载体的构建及表达

Construction and expression of a eukaryotic plasmid containing luciferase gene regulated by partial promoter of osteocalcin

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 pc DNA3- OCP- L uc,转染细胞后检测报告基因对 rh BMP- 2的反应 ,以期用于 rh BMP活性的定量测定 .方法 用 PCR方法扩增骨钙素部分启动子 14 7bp,克隆入p GEM- 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 pc DNA3- L uc中 ,构建真核表达载体 pc DNA3- OCP- L uc,然后分别将 pc DNA3-L uc和 pc DNA3- OCP- L uc转染细胞 ,并检测其对 rh BMP- 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 pc DNA3- OCP- L uc;转染细胞后其报告基因的基础活性较 pc DNA3- L uc大为降低 ,而且报告基因的表达与 rh BMP- 2剂量在一定范围内成线性正相关 .结论 真核表达载体 pc DNA3- OCP- L uc的报告基因表达具有特异性 ,可代替直接测定骨钙素用于 rh BMP- 2活性的定量测定研究 .

AIM To quantitatively assay the biological activities of rhBMP 2 by a eukaryotic expression plasmid, pcDNA3 OCP Luc. METHODS The partial promoter of osteocalcin (147 bp) was amplified from the genomic DNA of C3H10T1/2 by PCR. After the restriction and sequencing analysis, it was subcloned into the plasmid pcDNA3 Luc which had efficiently expressed the luciferase gene in mammalian cells, so the eukaryotic plasmid of pcDNA3 OCP Luc was successfully constructed. The pcDNA3 Luc and pcDNA3 OCP Luc were transfected into different cells respectively and the expression of luciferase gene of pcDNA3 OCP Luc was detected in cells treated by rhBMP 2. RESULTS The basic expression of pcDNA3 OCP Luc was lower than that of pcDNA3 Luc and the expression of luciferase gene of pcDNA3 OCP Luc was correlated with the concentration of rhBMP 2 during certain ranges. CONCLUSION Expression of luciferase gene of pcDNA3 OCP Luc is specific to rhBMP 2 and can be used for the quantitative bioassay of rhBMP 2.