中国组织工程研究Issue(18)：2923-2927,5.DOI:10.3969/j.issn.2095-4344.2015.18.024
构建大鼠白细胞介素1β基因shRNA腺病毒载体
Construction of a recombinant adenovirus vector expressing shRNA targeting interleukin-1beta gene in rats
摘要
Abstract
BACKGROUND:Specific down-regulation of interleukin 1 beta (IL-1β) may al eviate the pain behaviors effectively after peripheral nervous injury. Compared with smal interference RNA (siRNA), short hairpin RNA (shRNA) could inhibit the expression of target gene more stably and efficiently. However, simple shRNA could not enter target cel s to down-regulate target gene efficiently. Adenovirus vectors have wide host range, high infection efficiency and stable expression in host cel s. <br> OBJECTIVE:To construct recombinant adenovirus vector expressing shRNA targeting IL-1βgene and detect its effect on the expression of target gene. <br> METHODS:Three siRNAs were designed on the basis of the nucleotide sequence of IL-1βobtained from NCBI and then three shRNAs (shRNA1, shRNA2 and shRNA3) were synthesized. The annealed shRNA product and adenovirus vector pHBAd/U6/GFP digested by BamH I and EcoR I were connected to construct the recombinant adenovirus vector shuttle plasmid expressing shRNA targeting IL-1β. After sequencing, HEK 293 cel s were co-transfected by the shuttle plasmid and skeleton vector, and three recombinant adenovirus vector expressing shRNA targeting IL-1β(rAd/shRNA1, rAd/shRNA2 and rAd/shRNA3) were packaged and amplified. Rats H9C2 cel s were infected by recombinant adenovirus vector expressing shRNA targeting IL-1βand fluorescence microscope was used to observe the infection efficiency. The effect of recombinant adenovirus vector expressing shRNA targeting IL-1βon the expression of target gene was detected by western blot assay. <br> RESULTS AND CONCLUSION:The sequencing results showed that the sequences of three shRNAs adenovirus vector shuttle plasmid were consistent with the sequences of three designed shRNAs. rAd/shRNA1, rAd/shRNA2 and rAd/shRNA3 were constructed successful y. rAd/shRNA1, rAd/shRNA2 and rAd/shRNA3 could down-regulate the expression of IL-1βin rat H9C2 cel s and the down-regulation effect of rAd/shRNA2 was the most significant.关键词
实验动物/基因病毒载体及相关因子模型/神经病理性疼痛/白细胞介素1β/基因表达/RNA干扰/小干扰RNA/短发卡RNA/腺病毒载体/载体质粒/HEK293细胞/大鼠H9C2细胞/山西省自然科学基金Key words
Pain/Interleukin-1beta/RNA Interference分类
医药卫生引用本文复制引用
赵晓龙,陈建平,张宇,李航,刘艳芳,高文燕,韩磊,邓亚南..构建大鼠白细胞介素1β基因shRNA腺病毒载体[J].中国组织工程研究,2015,(18):2923-2927,5.基金项目
山西省自然科学基金(2010011049-1)@@@@Funding:the Natural Science Foundation of Shanxi Province, No.2010011049-1 （2010011049-1）
