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  • 徐强△,涂丁元△,赵仙先*.荧光素酶-微RNA-126海绵腺病毒的构建及应用[J].第二军医大学学报,2020,41(5):520-526    [点击复制]
  • XU Qiang△,TU Ding-yuan△,ZHAO Xian-xian*.Construction and application of luciferase-microRNA-126 sponge adenovirus[J].Acad J Sec Mil Med Univ,2020,41(5):520-526   [点击复制]
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荧光素酶-微RNA-126海绵腺病毒的构建及应用
徐强△,涂丁元△,赵仙先*
0
(海军军医大学(第二军医大学)长海医院心血管内科, 上海 200433
共同第一作者
*通信作者)
摘要:
目的 建立荧光素酶-miRNA海绵腺病毒的构建方法,并验证该腺病毒的表达产物与miRNA的结合效率。方法 化学合成能与miRNA-126部分互补配对的DNA片段;PCR扩增miRNA-126海绵片段,将其克隆至pMD-18T载体及psiCheck2质粒hRluc的3'端非编码区;将hRluc-miRNA-126海绵克隆至Ad-Track质粒,Pme Ⅰ酶切使Ad-Track-hRluc-miRNA-126 sponge质粒线性化,并转化BJ5183感受态细菌,与Ad-Easy质粒同源重组;重组质粒经Pac Ⅰ酶切鉴定后,转染293细胞,7 d后收获腺病毒;用腺病毒感染人脐静脉内皮细胞(HUVEC),通过细胞划痕实验观察miRNA-126对细胞迁移能力的影响。结果 MiRNA-126海绵与miRNA-126形成碱基互补配对关系;双荧光素酶实验结果表明miRNA-126能够直接作用于hRluc-miRNA-126海绵。将hRluc-miRNA-126海绵克隆至Ad-Track质粒使得同步检测细胞转染效率和miRNA抑制效率成为可能。通过Ad-Track与Ad-Easy同源重组,成功构建Ad-Easy-hRluc-miRNA-126 sponge质粒,酶切后可见4.5 kb片段。将其转染293细胞,7 d后获得荧光素酶-miRNA海绵腺病毒;miRNA-126过表达能够抑制腺病毒表达产物hRluc-miRNA-126海绵(P<0.01),感染miRNA-126海绵腺病毒可以抑制HUVEC的迁移能力(P<0.05)。结论 成功建立了荧光素酶-miRNA海绵腺病毒,miRNA-126与该腺病毒的表达产物有较高的结合效率。
关键词:  荧光素酶  微RNA海绵  腺病毒  微RNA
DOI:10.16781/j.0258-879x.2020.05.0520
投稿时间:2019-09-30修订日期:2020-03-09
基金项目:国家自然科学基金(81570208,81370266).
Construction and application of luciferase-microRNA-126 sponge adenovirus
XU Qiang△,TU Ding-yuan△,ZHAO Xian-xian*
(Department of Cardiovasology, Changhai Hospital, Naval Medical University(Second Military Medical University), Shanghai 200433, China
Co-first authors.
* Corresponding author)
Abstract:
Objective To establish a construction method for luciferase-microRNA (miRNA) sponge adenovirus, and to verify the binding efficiency of the expression product of the adenovirus and miRNA. Methods DNA fragments that can be partly complementary to miRNA-126 were chemically synthesized. The miRNA-126 sponge fragments were amplified by PCR, and cloned into pMD-18T vector and the 3'-end non-coding region of psiCheck2 plasmid; hRluc-miRNA-126 sponge was further cloned into Ad-Track plasmid. After linearization by Pme Ⅰ, Ad-Track-hRluc-miRNA-126 sponge plasmid was transformed into competent Escherichia coli strains BJ5183 to homologous recombinate with Ad-Easy plasmid; recombinant Ad-Easy-hRluc-miRNA-126 sponge plasmid was identified by Pac Ⅰ, and then transfected into 293 cells to produce adenovirus. Adenovirus was used to infect human umbilical vein endothelial cells (HUVECs), and the effect of miRNA-126 on cell migration ability was detected by scratch test. Results The miRNA-126 sponge had a good complementary base-pairing relationship with miRNA-126; double luciferase experiment showed that miRNA-126 could directly act on hRluc-miRNA-126 sponge. Cloning hRluc-miRNA-126 sponge into Ad-Track plasmid made it possible to synchronously monitor the efficiency of cellular transfection and miRNA inhibition. Ad-Easy-hRluc-miRNA-126 sponge plasmid was successfully established by homologous recombination of Ad-Track and Ad-Easy, identified by Pac Ⅰ digestion with 4.5 kb fragments. When transfected into 293 cells, luciferase-miRNA sponge adenovirus was obtained 7 days later. Overexpression of miRNA-126 inhibited the expression of hRluc-miRNA-126 sponge (P<0.01). Infection with miRNA-126 sponge adenovirus inhibited the migration of HUVECs (P<0.05). Conclusion Luciferase-miRNA sponge adenovirus has been successfully established. MiRNA-126 has a high binding efficiency with the expressed product of the adenovirus.
Key words:  luciferase  microRNA sponge  adenovirus  microRNAs