摘要
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Hepal-6中IL-35的表达。方法设计合成IL-35EBl3亚基基因靶向shRNA序列，构建shRNA载体PLKO．1-IL-35EB13shRNA．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-35EB13shR-NA的慢病毒，慢病毒感染靶细胞Hepat-6，镟光显微镜下观察感染效率，以实时定量RT—PCR分析对Hepal-6细胞IL-35EBl3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-35EB13shRNA．GFP；并成功包装出表达干扰IL-35EBl3shRNA的慢病毒，以MOI值4．6pfu／细胞感染Hepal-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-35EBl3shRNA—GFP均可以有效干扰IL-35EBl3的表达，其中E545干扰效率最高，为64％．结论成功构建IL-35EBl3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。
Objective To construct the shRNA lentiviral vectors targeting mice II.-35 gene and detect its effect of gene silence in Hepal - 6 cells. Methods The specific siRNA sequences targeting mice IL-35 gene were designed and cloned into eukaryotic expression vecLor PLKO. 1- IL,-35 EB13 shRNA-（;FP. After the correct sequencing identification,the lentivirus particles targeting mice IL-35 gene were paekaged with the three plasmid virus packaging system. The IL-35 gene specific shRNAs were infected into Hepal -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 ,nice IL-35 gene were packaged successfully. The observed infection efficiency by the fluorescence microscope a- bout 90 percent;The resuhs 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 Hepal -6 cells.
出处
《潍坊医学院学报》
2014年第1期5-8,共4页
Acta Academiae Medicinae Weifang
基金
山东省自然科学基金项目(课题编号:ZR2009CM019)
山东省教育厅资助课题(课题编号:J10LF62)
