| 摘要: |
| ［摘要］ 目的 构建人MicroRNA-335(has-miR-335)阳性对照质粒表达载体。方法 根据与has-miR-335“种子区”完全匹配的序列设计并合成一对互补的寡聚核苷酸链，经过缓慢降温退火得到目的小片段。将退火产物连接入荧光素酶报告基因载体pMIR-REPORT而得到has-miR-335阳性对照重组质粒。应用Lipofectamine 2000将has-miR-335阳性对照重组质粒、pMIR-REPORT空载体，分别与Pre-miRTMmicroRNA335 Precursor共转染至293T7/17细胞，用双荧光素酶方法检测has-miR-335在真核细胞中表达水平。结果 获得has-miR-335阳性对照重组质粒，经测序结果正确。has-miR-335与其阳性对照结合在真核细胞中的表达下降。结论 成功构建了has-miR-335阳性对照质粒表达载体。该载体可用于确认has-miR-335实验中RNA提取、转染和基因表达检测方法是否可靠。has-miR-335双荧光素酶报告基因检测体系的建立为进一步进行has-miR-335靶基因检测及验证奠定基础。 |
| 关键词: 人MicroRNA-335 阳性对照 质粒 荧光素酶报告基因载体 pMIR-REPORT |
| DOI：10.3969/j.issn.1674-3806.2015.01.01 |
| 分类号:Q 782 |
| 基金项目:广西自然科学基金资助项目（编号:2011GXNSFC018019） |
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| Construction of the eukaryotic expression vector for positive control plasmid of human MicroRNA-335 and it′s significance |
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WEN Zhi-hong, DAI Yan, HE Shuang
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Department of Pediatrics, the People′s Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, China
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| Abstract: |
| ［Abstract］ Objective To construct the eukaryotic expression vector for the positive control plasmid of has-miR-335.Methods One pair of complementary oligomeric chains of exact matching sequence for has-miR-335 seed region were synthesized，and then target fragments were obtained by annealing.The target fragments were inserted into pMIR-REPORT luciferase reporter vector.We cotransfected 293T/17 cell with the recombinant plasmid or pMIR-REPORT plasmid together with Pre-miRTMmicroRNA335 Precursor by Lipofectamine 2000.The expression of has-miR-335 in eukaryotic cells was tested by Luciferase Assay.Results The positive control recombinant plasmid of hsa-miR-335 was comfirmed by sequencing. The expression of has-miR-335 decreases after combined with it′s positive control in eukaryotic cells.Conclusion The positive control plasmid for has-miR-335 can be used to confirm if the methods of experiments in RNA extraction,gene transfection and expression are reliable.The double luciferase reporter gene detection system of has-miR-335 is constructed successfully,which lays the foundation for testing and validation of has-miR-335 target genes. |
| Key words: has-miR-335 Positive control Plasmid Luciferase reporter vector pMIR-REPORT |
