Construction of recombinant adenovirus HBx-shRNA and identification of its inhibition effect

Abstract:

Objective：To construct and identify the recombinant adenovirus vector of a short hairpin RNA（shRNA） targeting HBx. Methods：U6 expression promoter and shRNA of pGenesil-1-HBx-shRNA and pGenesil-1-Scramble sequence，which was construct－ed and identified in our previous experiment，were subcloned to pAdTrack-CMV shuttle plasmid，and further identified by enzyme di－gestion and DNA sequence analysis. Recombinant shuttle plasmids were restrictedly digested with PmeⅠ，and subsequently trans－formed into competent AdEasier E.coli for homologous recombinant to obtain the adenovirus plasmids pAd-U6-HBx-shRNA and pAd-U6-Scramble sequence（control）. Recombinants were digested with PacⅠand finally transfected into AD293 for packaging. Re－combinant adenoviruses Ad-U6-HBx-shRNA and Ad-U6-Scramble sequence were amplified in AD293 and the titers were deter－mined. Expression of HBx gene was identified by RT-PCR and real-time PCR. Results：Recombinant adenovirus plasmid was cor－rectly constructed. Real-time PCR proved that the expression of HBx was reduced by Ad-U6-HBx-shRNA（P=0.01）. Conclusions：Recombinant adenovirus vector Ad-U6-HBx-shRNA is correctly constructed，and it could inhibit HBx expression in HepG2.2.15 cells. This recombinant adenovirus vector provides a useful tool for further study of HBx function.