Abstract:Objective:To study the expression of the adenoviral vector containing myr-HA-Akt gene in the liver of cirrhotic rats in vivo.
Methods :myr-HA-Akt cDNA obtained from the plasmid pcDNA3.1-myr-HA-Akt was cloned into the plasmid pDC316. Then, pDC316-myr-HA-Akt was cotransferred with adenoviral backbone vector into 293 cells. The recombinant adenovirus was reproduced and purified. The recombinant adenovirus was identified by the cytopathic effect of 293 cells, polymerase chain reaction(PCR),and gene sequencing for myr-HA-Akt cDNA. TCID50 assay was performed to determine the titer of virus. After the adenovirus infected the hepatic cirrhosis rat models via the tail vein, protein and phosphorylation status of Akt were examined by Western blotting.
Results:Infected 293 cells showed significant cytopathic effect. Products of PCR confirmed the presence of recombinant adenovirus. The identification result by DNA sequence analysis showed that myr-HA-Akt cDNA was cloned to pDC316 correctly and homologously recombinated with pBHGloxΔE1, and 3Cre and Ad-myr-HA-Akts were packaged successfully. The titer of virus was 5.5×1011 vp/ml. The expression of Akt in the liver of cirrhotic rats was verified by Western blotting.
Conclusions:The recombinant adenoviral vector containing myr-HA-Akt was constructed and the transgene of hepatic cirrhosis rats expressed Akt gene in vivo successfully. It provides a basis for the further study of treatment for hepatic cirrhosis by Akt gene.