Construction of a retroviral vector carrying HBX gene and its expression in LO2 human hepatocytes]

To construct a retroviral vector carrying HBX gene and investigate its expression in LO2 human hepatocytes.HBX gene was amplified by PCR and subcloned into the retroviral vector pSEB-Flag to construct a retroviral plasmid (pSEB-Flag-HBX) expressing HBX. The HBX gene insert was confirmed by restriction enzyme digestion, PCR and DNA sequencing. The recombinant retroviruses carrying HBX gene were generated in 293T cells co-transfected with pSEB-Flag-HBX and the packaging plasmids pAmpho, and used to infect LO2 human hepatocyte. After selection with blasticidin, the mRNA and protein expressions of HBx were determined by the reverse transcription-PCR and Western blotting, respectively.The retroviral plasmid (pSEB-Flag-HBX) carrying HBX was constructed successfully. The recombinant retrovirus efficiently delivered HBX gene into LO2 human hepatocyte, resulting in stable expression of HBX mRNA and HBx protein as shown by RT-PCR and Western blotting, respectively.The recombinant retrovirus pSEB-Flag-HBX has been successfully constructed, which is capable of delivering the target gene HBX into LO2 human hepatocytes and results in stable expression of HBx to serve as an ideal model to study the effect of HBx on the development of hepatocellular carcinoma.