Construction Of Recombinant Adenovirus Vector Of Indoleamine 2,3-dioxygenase And Chimeric Albumin Promoter

Objective To construct a recombinant adenovirus encoding for indoleamine 2, 3-dioxygenase (IDO) and chimetric albumin promoter which drive IDO express in hepatic cell specifically, evaluate the transcriptional and post-transcriptional expression levels in Hepa 1-6 cell.Methods Designed primer of albumin promoter and assembled the albumin promoter by PCR amplified, subcloned into multiple clone site XbaI/HindIII of blank pAdTrack shuttle plasmid, constructed pAdTrack-PmALB vector contain albumin promoter; full-length derived IDO cDNA was subcloned into multiple clone site XhoI/EcoRV fo pAdTrack-PmALB and pAdTrack-CMV respectively. The products were linearized to homologous recombination with AdEasy-1 vector in BJ5183 bacteria. The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing respectively. The recombined adenoviruses DNA were transfected into AD-293cells for packaging and amplification of AD-IDO/PmALB and AD-IDO/CMV. The expression of IDO was monitored by RT-PCR and EGFP fluorescence in infected cells, respectively. In vitro, cultured the recombinant viruses with Hepa 1-6 cells by 2 days, monitored the mRNA and protein expression levels by RT-PCR and Western Blot, respectively.Results Construction of recombinant andenoviruses which contain IDO and albumin promoter were successful confirmed by restriction endonuclease digestion and sequencing. The specific expression of mouse IDO/PmALB was identifie by RT-PCR in AD-293 cell. The virus titer was 2.9×106 pfu/ml. The IDO transcriptional and post-transcriptional expression levels were detectable in transfected Hepa 1-6 cells by RT-PCR and Western Blot respectively, which verified the capability of recombinant adevirus delivery specificity.Conclusion A recombinant adenovirus Ad-IDO/PmALB was susccessfully constructed. The Ad-IDO/PmALB have the capability of infecting the hepatic cells specifically.