Construction Of Eukaryotic Expression Vector With A Site-specific M1GS RNA Ribozyme Targeting On C Region Of HBV Genome

Objective: To construct a eukaryotic expression vector with a site-specific M1GS RNA ribozyme targeting on C region of hepatitis B virus (HBV) genome as a tool for gene theray of HBV.Methods: 2333nt in C region of HBV genome was selected as the site of cleavage. The DNA templates for a site-specific M1GS RNA ribozyme targeting on C region of HBV genome were constructed by the polymerase chain reaction (PCR) with the gene for Ml RNA as found in plasmid pTK117 as a template. Then the DNA templates for M1GS RNA and eukaryotic expression vector pEGFP-C1 were digested with restriction enzyme EcoR I/Sal I. The fragment corresponding to the M1GS RNA was gel purified and ligated to the EcoR I site of the pEGFP-Cl vector. The recombination plasmid was named pEGFP-GS and was amplified by transforming to JM109. Restriction enzyme analysis and DNA sequencing were used to identify the recombinant plasmid.Results: The segment of the recombination plasmid pEGFP-GS digested with restriction enzymes EcoR I and Sal I corresponds to the expection and shows two DNA fragments which is 4700bp and 470bp, respectively. The sequence and the position inserted are accurate according to the DNA sequence coding for E. coli M1 RNA.Conclusion: The eukaryotic expression vector with a site-specific M1GS RNA ribozyme targeting on C region of HBV genome has been successfully conducted.