CAJ | 학술논문

目的构建表达miR-10a的重组腺病毒(adenovirus,Ad)载体.方法用红色荧光蛋白mCherry基因替换穿梭质粒pDC316-EGFP-U6的增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因.利用表达siRNA的策略,合成编码miR-10a的长链DNA序列,双链退火后克隆至pDC316-mCherry-U6获得pDC316-mCherry-U6-miR-10a.pDC316-mCherry-U6-miR-10a与骨架质粒pBHGlox△E1,3Cre共转染HEK293细胞,包装成复制缺陷型重组腺病毒Ad-miR-10a,空斑形成实验纯化、扩增、滴定重组腺病毒.Ad-miR-10a感染HeLa细胞后在荧光显微镜下观察mCherry表达,用RT-qPCR检测miR-10a表达.结果构建的pDC316-mCherry-U6-miR-10a序列正确,荧光显微镜下可见mCherry的表达,重组腺病毒Ad-miR-10a滴度为1.8 × 107pfu/mL,感染HeLa细胞后miR-10a表达较mock高40倍.结论成功构建了表达miR-10a的重组腺病毒,siRNA表达策略可用于miRNA的人工表达.
목적 구건표체miR-10a적중조선병독(adenovirus,Ad)재체.방법 용홍색형광단백mCherry기인체환천사질립pDC316-EGFP-U6적증강형록색형광단백(enhanced green fluorescent protein,EGFP)보고기인.이용표체siRNA적책략,합성편마miR-10a적장련DNA서렬,쌍련퇴화후극륭지pDC316-mCherry-U6획득pDC316-mCherry-U6-miR-10a.pDC316-mCherry-U6-miR-10a여골가질립pBHGlox△E1,3Cre공전염HEK293세포,포장성복제결함형중조선병독Ad-miR-10a,공반형성실험순화、확증、적정중조선병독.Ad-miR-10a감염HeLa세포후재형광현미경하관찰mCherry표체,용RT-qPCR검측miR-10a표체.결과 구건적pDC316-mCherry-U6-miR-10a서렬정학,형광현미경하가견mCherry적표체,중조선병독Ad-miR-10a적도위1.8 × 107pfu/mL,감염HeLa세포후miR-10a표체교mock고40배.결론 성공구건료표체miR-10a적중조선병독,siRNA표체책략가용우miRNA적인공표체.
Objective To develop a miR-10a-expressing recombinant adenoviral vector. Methods The EGFP gene in shuttle plasmid pDC316-EGFP-U6 was replaced by red fluorescent protein mCherry gene. Long DNA sequence coding miR-l0a was synthesized. After annealing, the double-stranded miR-10a-coding sequence was inserted into pDC316-mCherry-U6. The resultant pDC316-mCherryU6-miR-10a was co-transfected with adenoviral skeleton plasmid pBHGlox△E1, 3Cre into HEK293 cells. The replication-defective adenovirus Ad-miR-10a was purified, amplified, and tittered by plaque assay. The mCherry expression was detected by fluorescence microscope. The expression of miR-10a by Ad- miR- 10 a in HeLa cells was determined by RT-qPCR. Results The sequence of pDC316-mCherryU6-miR-10 a was confirmed by restriction enzyme digestion and sequencing. mCherry expression could be detected under fluorescence microscope. The titers of Ad-miR-10a was 1.8 × 107 pfu/mL, and the level of miR-10a expression in HeLa cells infected with Ad-miR-10a was 40 times higher than that with Admock. Conclusion A miR-10a-expressing recombinant adenovirs Ad-miR-10a has been successfully constructed. The strategy for artificial expression of siRNA is applicable for expression of miRNA.