Construction and identification of a recombinant adenovirus vector with indoleamine 2,3-deoxygenase and chimeric albumin promoter / 中华普通外科杂志

ABSTRACT

Objective To construct a recombinant adenovirus vector encoding for indoleamine 2,3-dioxygenase(IDO)and chimetric albumin promoter,evaluate the mRNA and protein expression levels in Hepa 1-6cell.Methods Full-length mouse derived IDO cDNA was subeloned into pAdTraek-ALB shuttle Plasmid.The product was linearized to homologous recombination with AdEasy-l vector in BJ5183 bacteria.The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing.The recombined adenoviruses DNA were transfected into AD-293 cells for packaging and amplification of Ad-ALB/IDO.The expression of IDO was monitored by RT-PCR and EGFP fluorescence in infected cells.The recombinant viruses with Hepa 1-6 cells were cultured and the mRNA and protein expression levels monitored bv RT-PCR and Western blot, respectively. Results Construction of recombinant andenoviruses containing IDO and albumin promoter was confirmed by restriction endonuclease digestion and sequencing.The expression of IDO was identified by RT-PCR in transfected AD-293 cell.The virus titer was 2.9×10~6 pfu/ml.The IDO mRNA and protein expression levels were detectable after transfected Hepa 1-6 cells by RT-PCR and Western blot. Conclusion A recombinant adenovirus Ad-ALB/IDO was susceessfullyconstructed.