Construction of the adenovirus expressing NF-κB SiRNA in rats and its effect on Kupffer cells reaction to LPS

Abstract

Objective To construct the incompetent-replication adenovirus expressing NF-κB siRNA in rats and identify its effect on Kupffer cells reaction to lipopolysaccharide (LPS) in vitro. Meth-ods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was cloned into the pShuttleHl vector. Linearized pShuttleH1-siNF-κB of 0. 5 μg was transformed into E. coli BJ5183 cells containing backbone plasmid pAdEasy-1 by electroporation. The recombinant plasmid of 4 μg was transfected into 293 cells to package the adenovirus Ad-siNF-κB. The titers of adenovirus were determined using the specific 50% tissue culture infection dosage (TCID50) method. After virus infected the cultured Kupffer cells with MOI = 10, the effect on LPS-induced NF-κB mRNA and TNF-α, IL-6 ex-pression was observed. Results It was identified that the sequence of gene was correctly inserted into the genome of virus. The titer of recombinant adenovirus was 5.32×109 pfu/ml. NF-κB mRNA and TNF-α, IL-6 expression was greatly reduced after virus infection. Conclusion The recombinant adenovirus ex-pressing NF-κB siRNA in rats were successfully constructed, which probably can be further used in re-search on anti-inflammation effect in vivo. Key words: NF-κB; RNA interference ; Adenovirus ; Tumor necrosis faetor-α; Kupffer cells