Construction of a recombinant adenovirus vector expressing shRNA targeting interleukin-1beta gene in rats / 中国组织工程研究

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. OBJECTIVE:To construct recombinant adenovirus vector expressing shRNA targeting IL-1βgene and detect its effect on the expression of target gene. 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. 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.