大鼠微小RNA miR-16的克隆及其慢病毒表达载体的构建

Cloning of rno-miR-16 and Construction of Its Lentiviral Expression Vector

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。

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.