CAJ | 학술논문

目的：构建小鼠IL-35基因靶向shRNA干扰的慢病毒表达载体，抑制小鼠肝癌细胞Hepa1～6中IL-35的表达。方法设计合成IL-35EBI3亚基基因靶向shRNA序列，构建shRNA载体PLKO．1-IL-35 EBI3 shRNA-GFP，测序正确后，三质粒病毒包装系统（质粒载体＋psPAX2＋pMD2．G）包装成表达干扰IL-35 EBI3 shR-NA的慢病毒，慢病毒感染靶细胞Hepa1～6，荧光显微镜下观察感染效率，以实时定量RT-PCR分析对Hepa1～6细胞IL-35 EBI3基因表达的干扰效果。结果测序证实，成功构建了真核表达干扰载体PLKO．1-IL-35 EBI3 shRNA-GFP；并成功包装出表达干扰IL-35 EBI3 shRNA的慢病毒，以MOI值4．6pfu/细胞感染Hepa1～6细胞，镜下显示感染效率约90％；RT-PCR结果表明所构建的3个慢病毒载体PLKO．1-IL-35 EBI3 shRNA-GFP均可以有效干扰IL-35 EBI3的表达，其中E545干扰效率最高，为64％．结论成功构建IL-35EBI3亚基基因的shRNA慢病毒表达载体，该慢病毒表达载体能够在细胞水平有效沉默靶基因。
목적：구건소서IL-35기인파향shRNA간우적만병독표체재체，억제소서간암세포Hepa1～6중IL-35적표체。방법설계합성IL-35EBI3아기기인파향shRNA서렬，구건shRNA재체PLKO．1-IL-35 EBI3 shRNA-GFP，측서정학후，삼질립병독포장계통（질립재체＋psPAX2＋pMD2．G）포장성표체간우IL-35 EBI3 shR-NA적만병독，만병독감염파세포Hepa1～6，형광현미경하관찰감염효솔，이실시정량RT-PCR분석대Hepa1～6세포IL-35 EBI3기인표체적간우효과。결과측서증실，성공구건료진핵표체간우재체PLKO．1-IL-35 EBI3 shRNA-GFP；병성공포장출표체간우IL-35 EBI3 shRNA적만병독，이MOI치4．6pfu/세포감염Hepa1～6세포，경하현시감염효솔약90％；RT-PCR결과표명소구건적3개만병독재체PLKO．1-IL-35 EBI3 shRNA-GFP균가이유효간우IL-35 EBI3적표체，기중E545간우효솔최고，위64％．결론성공구건IL-35EBI3아기기인적shRNA만병독표체재체，해만병독표체재체능구재세포수평유효침묵파기인。
Objective To construct the shRNA lentiviral vectors targeting mice IL-35 gene and detect its effect of gene silence in Hepa1~6 cells.Methods The specific siRNA sequences targeting mice IL-35 gene were designed and cloned into eukaryotic expression vector PLKO.1-IL-35 EBI3 shRNA-GFP.After the correct sequencing identification ,the lentivirus particles targeting mice IL-35 gene were packaged with the three plasmid virus packaging system .The IL-35 gene specific shRNAs were infected into Hepa1~6 cells.Then,infection efficiency were observed by the fluorescence microscope .Real time reverse transcription PCR was performed to determine the expression level of IL-35 EBI3 mRNA.Results Sequencing results revealed that PLKO .1-IL-35 EBI3 shRNA-GFP plasmids were correctly constructed .The lentivirus particles targeting mice IL-35 gene were packaged successfully .The observed infection efficiency by the fluorescence microscope a-bout 90 percent;The results of real time PCR showed that three lentivirus vectors could effectively inhibit IL-35 EBI3 expression,especially E545 interference efficiency was highest ,at 64%.Conclusion The recombinant lentiviral shRNA expression vectors targeting mice IL-35 gene have been constructed successfully .IL-35 EBI3 mRNA can be down-regulated availably in Hepa1~6 cells.