Construction And Identification Of A Recombinant Adenovirus Vector Containing PPARΓ₂ Gene

Objective : To construct a recombinant adenovirus vector containing PPARγ₂ gene for investigation of the functions of PPARγ₂ gene and elucidating the mechanisms that infection of HBV results in hepatocellular carcinoma.Methods: The gene was amplified with polymerase chain reaction (PCR) technique by using pcDNA3 containing PPARγ₂ of mice;then subcloned PPARγ₂ gene into the shuttle vector pAdTrack-CMV, and subsequently cotransformed into 293 cells with an adenoviral backbone plasmid pAdEasy-1, identified the transfection rate, transformed into hepatic cell lines L02 for expressing at last.The first part: The PPARγ₂ gene of mice was amplified with polymerase chain reaction (PCR) technique by using plasmid PPARγ₂-pcDNA3 of mice as the template to obtain the targeted gene, then the fragment of PPARγ₂ gene and pAdTrack-CMV were digested with Sal I and Xho I seperately and linked using T4 DNA ligase,subsequently subcloned the fragment of PPARγ₂ gene into the pAdTrack- PPARγ₂-CMV to construct the shuttle vector.The exact recombinant pAd-track-PPARγ₂ was linearized with Pineâ… , and homologous recombined with pAd Easy-1.Obtaining a 23kb big fragment and a 4.5 kb small characteristic fragment after digestion ,which indicated the successful construction of recombinant adenovirus vector.The second part: the packing, identification and expressing of recombinant adenovirus vector. The pAdTrack-PPARγ₂-CMV and the pAdEasy-1 were cotransformed into HEK293 cells with liposome to obtain pAdTrack-PPARγ₂ adenovirus, and the transfection rate was identified. After detection of adenovirus in supernatant of culture ,the recombinant adenovirus gene was transducted into L02.With RT-PCR and Western blotting ,highly stable expressing of recombinant PPARγ₂ was observed in L02.Results:The result of RT-PCR detection showed that the products of PCR of adenovirus Ad-PPARγ₂ were about 4.5kb in size; Western blotting showed positive result with monoclone antibody against PPARγ₂, so it was proved that there were highly stable expressing of PPARγ₂ of recombinant adenovirus vector in L02.Conclusion:A recombinant adenovirus vector containing PPARγ₂ gene of mice was successfully constructed. It`s the basis to investigate the functions of PPARγ₂ gene and elucidate the mechanisms that PPARγ₂ influences the hepatocellular carcinoma.