CAJ | 학술논문

目的：构建靶向LRP16基因的短发夹RNA（shRNA）慢病毒表达载体，鉴定其在HeLa细胞中对LRP16的抑制效果。方法：构建pWPT-U6-LRP16shRNA-CMV-GFP慢病毒载体，通过病毒感染、细胞筛选、Western印迹等步骤，获得LRP16基因稳定抑制的细胞株。结果：构建了具有LRP16干扰效果的慢病毒载体，感染HeLa细胞后获得了稳定沉默LRP16及对照的细胞株；经克隆筛选，在荧光显微镜下观察到近似100%感染细胞发出绿色荧光；Western印迹证实pWPT-U6-L374-CMV-GFP和pWPT-U6-L668-CMV-GFP均可显著抑制HeLa细胞株中LRP16蛋白的表达，其中pWPT-Gsi-L374-GFP的抑制效果更好。结论：构建了靶向人LRP16基因shRNA慢病毒载体及LRP16稳定抑制的HeLa细胞系。
목적：구건파향LRP16기인적단발협RNA（shRNA）만병독표체재체，감정기재HeLa세포중대LRP16적억제효과。방법：구건pWPT-U6-LRP16shRNA-CMV-GFP만병독재체，통과병독감염、세포사선、Western인적등보취，획득LRP16기인은정억제적세포주。결과：구건료구유LRP16간우효과적만병독재체，감염HeLa세포후획득료은정침묵LRP16급대조적세포주；경극륭사선，재형광현미경하관찰도근사100%감염세포발출록색형광；Western인적증실pWPT-U6-L374-CMV-GFP화pWPT-U6-L668-CMV-GFP균가현저억제HeLa세포주중LRP16단백적표체，기중pWPT-Gsi-L374-GFP적억제효과경호。결론：구건료파향인LRP16기인shRNA만병독재체급LRP16은정억제적HeLa세포계。
Objective: To construct the recombinant lentiviral vector of short hairpin RNA(shRNA) against LRP16 and verified the stable knockdown efficiency of LRP16 in infected HeLa cells. Methods: We constructed lentiviral vector carrying LRP16. After PCR verification, virus infecting, cell screening and Western blotting, we got HeLa cell lines stable expressing LRP16 shRNA. Results: Two lentiviral vectors carrying LRP16 shRNA(L668 and L374) were successfully constructed. LRP16 stable knock-downed HeLa cell lines were establiehed and screened to harvest the pure positive clones which were almost reached 100% infection efficiency. Western blot an-alyze confirmed that both L668 and L374 down-regulated the LRP16 expression in HeLa cells which L374 was more significant. Conclusion: The lentivirus containing shRNA targeting the LRP16 gene has been constructed suc-cessfully. HeLa cell lines stable expressing LRP16 shRNA were successfully established with lentiviral system.