大鼠微小RNA miR-16的克隆及其慢病毒表达载体的构建 |
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引用本文: | 虢灿杰,潘勤,李定国.大鼠微小RNA miR-16的克隆及其慢病毒表达载体的构建[J].国外医学:分子生物学分册,2009(5):401-405. |
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作者姓名: | 虢灿杰 潘勤 李定国 |
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作者单位: | 上海交通大学医学院附属新华医院消化科,上海市200092 |
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基金项目: | 上海市科委基础研究重点项目基金(No.075C14044) |
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摘 要: | 目的克隆微小RNArno—miR-一16,构建其慢病毒表达载体pLV—miR-16并包装成慢病毒颗粒,为进一步研究miR一16的功能奠定了实验基础。方法从大鼠细胞基因组中用PCR的方法扩增miR-16的前体,构建了miR-16的重组表达载体pLV—miR-16,脂质体法将重组慢病毒载体和包装质粒混合物(pPACK—GAG、pPACK—REV和pVSV)共转染包装细胞293TN细胞,包装产生慢病毒,以293TN细胞绿色荧光蛋白(green fluorescent protein,GFP)的表达水平测定病毒滴度。结果经PCR扩增检测阳性菌落和测序证实,成功构建携带大鼠miR-16基因重组慢病毒载体。倒置荧光显微镜下观察可见包装细胞293TN呈绿色荧光,并测得10^8〉ifu/ml。结论成功构建大鼠慢病毒载体pLV—miR-16,为深入研究rno—miR-16的生物学功能奠定了基础。
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关 键 词: | rno—miR-16 miRNA 慢病毒 载体构建与表达 |
Cloning of rno-miR-16 and Construction of Its Lentiviral Expression Vector |
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Authors: | GUO Canjie PAN Qin LI Dingguo |
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Institution: | (Digestive Disease Laboratory and Department of Gastroenterology, Xinhua Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200092, China) |
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Abstract: | Objective To construct a mo-miR-161entiviral expression vector. Methods The pre-miR-16 was amplified by PCR and constructed into pLV-miR-16 vector. 293TN cells were cotransfected with the recombinant lentiviral vector together with Lentivirus Package plasmid mix containing pPACK-GAG, pPACK-REV and pVSV. Virus titer was measured according to the expression level of GFP. Results The recombinant pLV-miR-16 vector was confirmed by restriction endonuclease analysis and DNA sequencing. Virus titer reached to 10^8 〉 ifu/ml. Conclusion The established lenti-miR-16 vector may provide a tool for further study on molecular functions of rno-miR-16. |
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Keywords: | rno-miR-16 miRNA lentivirus recombine |
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