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小鼠微小RNAmiR-22真核表达载体的构建及功能初步研究

, PP. 803-805

Keywords: 微小RNA,真核表达载体,miR-,基因组片段,Hela,Northern?,blot

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Abstract:

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Lipofectamin2000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Northernblot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。Northernblot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RNAmiR-22的功能奠定了基础。

References

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