中国免疫学杂志Issue(7)：927-932,6.DOI:10.3969/j.issn.1000-484X.2014.07.014
靶向小鼠TNF-α基因RNA干扰慢病毒载体的构建及鉴定
Construction and identification of lentiviral vector harboring interference RNA targetting murine TNF-αgene
摘要
Abstract
Objective:To construct recombinant lentiviral vectors harboring interference RNA ( RNAi ) targetting murine TNF-αgene,so as to lay the foundation on the RNAi gene therapy.Methods: Three small interfering RNA ( siRNA) sequences targeting murine TNF-αgene ( siRNA1,siRNA2,siRNA3) and negative-control siRNA were designed and synthesized.The inhibition effects of siRNAs on TNF-α,IL-1βand IL-6 secretion of LPS-stimulated RAW264.7 macrophages were observed using real-time PCR and ELISA methods.DNA oligo was designed and synthesized according to the most effective siRNA 2 sequence.The recombinant lentiviral shuttle plasmid expressing short hairpin RNA ( shRNA) was constructed and sequenced.The lentiviral shuttle plasmids with packaging plasmids were transfected into 293T cells to produce lentiviral particles.Results: ①The TNF-αmRNA relative expression levels of siRNA1, siRNA2 and siRNA3 were 0.24±0.01,0.16±0.02,0.19±0.01 respectively,significantly lower than that of negative control (0.95± 0.02) (F=531.3,P<0.001).The inhibition rates at mRNA level were 74.26%,83.09%,79.93%,respectively comparing with negative control.No significance was observed in IL-1βor IL-6 mRNA relative expression change after TNF-αsiRNA transfection ( P>0.05).②The TNF-αprotein expression levels of siRNA1,siRNA2 and siRNA3 were (23.95±1.21),(17.27±1.46),(19.07± 1.57)ng/ml respectively,significantly lower than that of negative control (35.37±2.93)ng/ml (F=18.1,P=0.000 6<0.001).The inhibition rates of protein expression were 32.29%, 51.16%, 46.08%, respectively comparing with negative control.③The PCR product electrophoresis showed that recombinant vectors yielded 343 bp fragments,non-constructed vectors yielded 306 bp fragments.DNA sequencing partially showed insertion sequence.④Lentiviral particles were obtained by transfecting 293T cells with recombinant lentiviral shuttle plasmids and lentiviral packaging plasmids.Cells grew well during virus production with strong fluorescence expression.The titer of concentrated virus was 2×106 TU/μl.Conclusion:The lentiviral vector harboring RNAi targeting murine TNF-αgene has been successfully constructed.关键词
RNA干扰/慢病毒载体/巨噬细胞/肿瘤坏死因子-αKey words
RNA interference/Lentiviral vector/Macrophages/Tumor necrosis factor-α分类
医药卫生引用本文复制引用
赵颖杰,王吉波,辛苗苗,梁宏达,刘相萍,杨堃,隋爱华..靶向小鼠TNF-α基因RNA干扰慢病毒载体的构建及鉴定[J].中国免疫学杂志,2014,(7):927-932,6.基金项目
本文为山东省优秀中青年科学家科研奖励基金资助项目（2006BS03005）。 （）
