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-mmu-miR-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mmu-miR-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。
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 AdEasyl. 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.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2013年第2期202-206,共5页
Journal of Southern Medical University
基金
国家自然科学基金(30872770)~~
