Construction of the adenovirus expressing rats expressing TLR4 siRNA and its effect on alveolar macrophage reaction to LPS

Objective To construct the incompetent-replication adenovirus expressing rats TLR4 siRNA and to identify its effect on alveolar macrophage reaction to LPS(Lipopolysaccharide) in vitro. Methods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was cloned into the pShuttleH1 vector.Linearized pShuttleH1-siTLR4 was transformed into E. coli BJ5183 cells containing backbone plasmid pAdEasy-1 by electroporation. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad-siTLR4. The titers of adenovirus were determined using the specific 50% tissue culture infection dosage (TCID50) method. After virus infected the cultured alveolar macrophage, the effect on LPS-induced TLR4 protein and its mRNA expression were observed by western blot and RT-PCR. TNF-α, IL-10 expression were also detected by ELISA. Results It was identified that the sequence of gene was correctly inserted into the genome of virus.The titer of recombinant adenovirus was 5.72×109pfu/mL. TLR4 protein and its mRNA expression were greatly reduced after virus infection. TNF-α, IL-10 expression were also decreased. Conclusion The recombinant adenovirus expressing rats TLR4 siRNA were successfully constructed,which probably can be further used in treatment of acute lung injury in vivo.