Abstract: Objective: To establish a retroviral vector containing the HDV ribozyme and a stable vector production cell line for HBV treatment. Methods: The HDV ribozyme gene was cloned by PCR using ptVHRZ as a template and recombinated with pMSCV/U6, which had Puromycin resistance gene. The recombinant of HDV ribozyme was evaluated by enzyme digestion and sequencing. The pMSCV/U6-HDVRZ was transfected to pack a cell line with lipofectamine 2000. The titer of the recombinant virus in the supernatant was assayed on NIH3T3 cells. The helper virus was tested by both the PCR and a marker rescue assay. The hightiter cell lines were chosen for culture, and then the cleavage activity of the retroviral containing the HDV ribozyme was studied in HepG2215 cells by ELISA. Results: The retroviral vector containing the HDV ribozyme was successfully established. The vector production cell lines were established. The highest titer was 4.0×10⁶ CFU/ml. HDV ribozyme gene integration was detected by PCR. The retrovirus was free of a helper virus. The levels of HBsAg of ribozyme groups were significantly lower than those of the control group, proving the HDV ribozyme had HBV-specific cleavage activity in HepG2215 cells. Conclusion: A retroviral vector containing the HDV ribozyme and a hightiter vector production cell line are successfully constructed. The HBV-specific HDV ribozyme can catalyze the sequence-specific RNA, and suppress the replication of HBV. The retrovirus carrying the ribozyme gene has the potential of being further explored in gene therapy for HBV infection.

Key words: Ribozyme, Packaging viral, HepG2215 cells