| 微小RNA miR-125b真核表达载体的构建及其对细胞增殖的影响 |
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| 引用本文： | 曾泉,谢小燕,赵爱华,王静雪,陈琳,岳文,裴雪涛.微小RNA miR-125b真核表达载体的构建及其对细胞增殖的影响[J].生物技术通讯,2010,21(4):455-460. |
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| 作者姓名： | 曾泉 谢小燕 赵爱华 王静雪 陈琳 岳文 裴雪涛 |
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| 作者单位： | 军事医学科学院野战输血研究所干细胞与再生医学研究室,北京,100850 |
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| 摘 要： | 目的:构建微小RNA125b(miR-125b)真核表达载体,研究其过表达后对细胞增殖的影响。方法:以pcDNA3.1(-)-myc-his载体为模板,PCR扩增CMV启动子,克隆入pHRS-1cla-EGFP慢病毒载体,构建pHRS-1cla-CMV-EGFP载体;以从人全血中提取的基因组DNA为模板,PCR扩增pri-miR-125b序列,将其克隆到pHRS-1cla-CMV-EGFP载体中,构建pHRS-1cla-miR125b-CMV-EGFP慢病毒表达载体;将pHRS-1cla-miR125b-CMV-EGFP表达载体瞬时转染入293FT细胞,用实时定量PCR技术对miR-125b在转录水平的表达进行检测,用MTT及Brdu法检测miR-125b过表达后对293FT细胞增殖的影响。结果:构建的pHRS-1cla-miR125b-CMV-EGFP真核表达载体经质粒酶切和测序鉴定正确,转染细胞后72h经实时定量PCR检测,成熟miR-125b表达上调约750倍(P0.01),说明其能有效高表达,MTT及Brdu法检测显示细胞增殖受到明显抑制(P0.01)。结论:构建了pHRS-1cla-miR-125b-CMV-EGFP慢病毒真核表达载体,转染293FT细胞后能高效表达成熟miR-125b,同时证明过表达miR-125b能使细胞的增殖受到非常明显的抑制。
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| 关 键 词： | 微小RNA miR-125b 真核表达载体 细胞增殖 |
Construction of microRNA miR-125b Eukaryotic Expression Vector and its Effect on Cell Proliferation |
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| ZENG Quan,XIE Xiao-Yan,ZHAO Ai-Hua,WANG Jing-Xue,CHEN Lin,YUE Wen,PEI Xue-Tao.Construction of microRNA miR-125b Eukaryotic Expression Vector and its Effect on Cell Proliferation[J].Letters in Biotechnology,2010,21(4):455-460. |
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| Authors: | ZENG Quan XIE Xiao-Yan ZHAO Ai-Hua WANG Jing-Xue CHEN Lin YUE Wen PEI Xue-Tao |
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| Institution: | ZENG Quan,XIE Xiao-Yan,ZHAO Ai-Hua,WANG Jing-Xue,CHEN Lin,YUE Wen,PEI Xue-Tao Stem Cell and Regenerative Medicine Lab,Beijing Institute of Transfusion Medicine,Beijing 100850,China |
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| Abstract: | Objective: To construct a eukaryotic expression vector of microRNA 125b(miR-125b), and to study the effect of its overexpression on cell proliferation. Methods: CMV promoter was amplified by PCR from pcDNA3.1(-)-myc-his vector and inserted into pHRS-1cla-EGFP lentiviral vector to obtain pHRS-1cla-CMV-EGFP vector. miR-125b was amplified from human genomic DNA isolated from whole blood, then inserted into pHRS-1claCMV-EGFP vector to generate pHRS-1cla-miR125b-CMV-EGFP lentiviral vector. Subsequently, miR-125b... |
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| Keywords: | miR-125b |
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