小鼠微小RNA miR-22真核表达载体的构建及功能初步研究

Construction of eukaryotic expression vector of mouse microRNAs miR-22

目的从基因组DNA扩增miR-22前体对应的基因组片段并克隆到pIRES2-EGFP,构建miR-22的真核表达载体。方法利用PCR技术从基因组DNA扩增miR-22前体对应的基因组片段,克隆到质粒pUCm-T,测序正确后构建到真核表达载体pIRES2-EGFP中,经BamHⅠ和EcoRⅠ双酶切后1.5%琼脂糖凝胶电泳鉴定,并进行序列测定。脂质体Li-pofectamin2000法转染Hela细胞后G418筛选获得稳定转染克隆,提取总RNA,通过RT-PCR方法对neo片段进行鉴定,采用Northern blot技术检测miR-22的表达。结果PCR扩增得到的334bp片段与预期的miR-22前体对应的基因组片段序列一致;成功构建了真核表达载体pIRES2-EGFP/miR-22,测序结果正确。重组质粒转染Hela细胞后,经G418筛选成功获得阳性克隆。Northern blot分析显示miR-22在Hela细胞内高效表达。结论本实验成功克隆了miR-22的前体对应的基因组片段,构建其真核表达载体,并在Hela细胞内获得高表达,为深入研究微小RNA miR-22的功能奠定了基础。

Objective To clone microRNA22 fragment from human genomic DNA and construct it into the eukaryotic expression vector. Methods MicroRNA22 genomic sequence was amplified from mouse genomic DNA by PCR and cloned into pUCm-T plasmid, then sequenced. The sequence was cloned into eukaryotic expression vector pIRES2-EGFP plasmid, then identified with BamH Ⅰ and EcoR Ⅰ in 1.5% agarose gel electrophoresis and sequenced. Hela cells were tansfected with the identified pIRES2-EGFP/miR-22 and pIRES2-EGFP by Lipofectamin 2000. Results The 334-bp genomic fragment amplified by PCR was correct. Eukaryotic expression vector pIRES2-EGFP/miR-22 was successfully constructed and confirmed by sequencing. G418-resistant clones of Hela cells were obtained and confirmed as positive by RT-PCR. Northern blotting showed the expression of miR-22 increased. Conclusion The successful cloning of genomic sequence corresponding to pre-miR-22 and construction of its eukaryotic expression vector has laid the foundation for the further study of miR-22 function.