Construction, package and identification of replication-deficient recombinant adenovirus expression vector of HCV C

Abstract

AIM:To construct a replication-deficient recombinant adenovirus expression vector of HCV C. METHODS:The HCV core gene was cloned at the downstream of CMV promoter of the adenoviral shuttle plasmid pAd.CMV-link.1, and the resultant recombinant plasmid pAd. HCV-C was cotransfected into 293 cell together with plasmid pJM17 containing adenoviral genome, then the adenovirus expression vector was obtained, and identified by infecting test,electronic microscope observation and PCR co-amplification. The plasmid pAd.HCV-C was identified by endonuclease, PCR and sequencing.The expressive activity of adenovirus vector was identified by immunofluorescence and Western blot. RESULTS:HCV core gene in the inserted DNA of pAd.HCV-C was confirmed by endonuclease, PCR and sequencing. Results of infecting test, electronic microscopic observation and PCR co-amplification showed that the adenovirus vector had been constructed successfully. Expression of HCV core antigen was proved in the HepG2 cells by immunofluorescence and Western blot.