南方医科大学学报

南方医科大学学报 ›› 2013, Vol. 33 ›› Issue (02): 202-.

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miRNA-30a/30e腺病毒表达载体的构建

刘强，顾金金，罗敏，施琼   

  • 出版日期:2013-02-20 发布日期:2013-02-20

Construction of adenoviral vectors expressing miR-30a and miR-30e

  • Online:2013-02-20 Published:2013-02-20

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摘要： 目的构建稳定表达成熟miRNA-30a和miRNA-30e腺病毒表达载体。方法小鼠基因组中分别扩增出带有酶切位点的
mmu-miR-30a、mmu-miR-30e 目的基因，目的基因连接到穿梭质粒pSES-HUS，带有目的基因的穿梭质粒重组到骨架质粒
AdEasy1 上，重组质粒转染到Hek-293 细胞中包装成腺病毒载体，反复扩增之后获得高滴度的pAd-mmu-miR-30a、pAd-mmumiR-
30e 腺病毒。用目的腺病毒分3 组（RFP对照组，miR-30a 组，miR-30e 组）感染小鼠Mefs 细胞，用荧光定量PCR方法检测
mmu-miR-30a、mmu-miR-30e 表达情况。结果分别扩增出带有酶切位点的357 bp mmu-miR-30a，324 bp mmu-miR-30e，并成
功连接到pSES-HUS 上，进一步与AdEasy1 重组，包装得到腺病毒表达载体，通过荧光定量PCR 检测，Mefs 细胞中mmumiR-
30a升高26.46±7.46倍，mmu-miR-30e升高2.76±0.25倍。结论成功构建了成熟miRNA的腺病毒表达载体。

Abstract: Objective To construct adenoviral vectors expressing mature miRNA-30a and miRNA-30e. Methods The target
mmu-miR-30a and mmu-miR-30e genes amplified from mouse genome were digested and linked to the shuttle plasmid
pSES-HUS, which was then transformed into competent AdEaseier cells for recombination. The confirmed recombinant
plasmids were transfected into Hek-293 cells for production of the adenoviruses pAd-mmu-miR-30a and pAd-mmu- miR-30e.
The obtained adenoviruses were used to infect Mefs cells, and the cellular expressions of mmu-miR-30a and mmu- miR-30e
were detected using fluorescence quantitative PCR. Results mmu-miR-30a (357 bp) and mmu-miR-30e (324 bp) containing the
restriction sites were amplified and linked to the shuttle plasmid pSES-HUS, which was successfully recombined with
AdEasy1. After packaging in Hek-293 cells, the adenoviral vectors were obtained, which caused an increase of mmu-miR-30a
expression by 26.46±7.46 folds and mmu-miR-30e expression by 2.76±0.25 folds in transfected Mefs cells. Conclusion We have
successfully constructed the adenoviral vectors expressing the mature miRNAs.