Construction of Replication-Deficient Recombinant Adenovirus-Interleukin-10 and its Expression

Objective To construct recombinant replication-deficient adenoviruses carrying rat IL-10 cDNA by using AdEasy system and to investigate its expression in hepatic stellate cells(HSC). Methods Total RNA was extracted from mononuclear cells of SD rat spleen, and cDNA of IL-10 was obtained using reverse transcriptional polymerase chain reaction(RT-PCR) amplification. The shuttle plasmid-pAdTrack-CMV-IL-10 in which IL-10 cDNA was inserted into the downstream of CMV promoter was established by ligation. Then the linearized shuttle plasmid was cotransferred into colibacillus BJ5183 with adenovirus vector AdEasy-1 to generate the recombinant replication-deficient adenovirus plasmid pAdIL-10 through homologous recombination. The linearized pAdIL-10 was transferred into HEK 293 cell by lipofectamin for packing, and the recombinant replication-deficient adenovirus, AdIL-10, was finally obtained. HSCs infected with AdIL-10 were cultured for 3 d. Total RNA was isolated from HSCs and IL-10 mRNA expressions were detected using RT-PCR. Results The pAdIL-10 was confirmed by restriction endonuclease digestion and sequencing. GFP expression could be observed on the third day after packing the linearized pAdIL-10 in 293 cells. GFP expression could be observed on the third day after HSCs infected with AdIL-10, and IL-10 mRNA expression could be detected by RT-PCR. Conclusion The replication-deficient recombinant adenoviruses carring rat IL-10 cDNA, AdIL-10, could be generated by AdEasy system. AdIL-10 could infect HSCs with high efficiency and for a long duration, and IL-10 was expressed successfully.