[1]刘强,顾金金,罗敏,等.miRNA-30a/30e腺病毒表达载体的构建[J].南方医科大学学报,2013,(02):202.
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miRNA-30a/30e腺病毒表达载体的构建()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2013年02期
页码:
202
栏目:
出版日期:
2013-02-01

文章信息/Info

Title:
Construction of adenoviral vectors expressing miR-30a and miR-30e
作者:
刘强顾金金罗敏施琼
关键词:
miRNA-30amiRNA-30eAdEasy系统荧光定量PCR
Keywords:
miRNA-30a miRNA-30e AdEasy system fluorescence quantitative polymerase chain reaction
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.
更新日期/Last Update: 1900-01-01