Anti-hbv Drug Screening Cell Models And Animal Models

Objective: To amplify and clone duck hepatitis B virus (DHBV) genome from Chongqing brown duck and analyze the sequence of it for the further study on evaluation of anti-HBV drugs in DHBV infection model.Methods: DHBV genome DNA, extracted from the serum of Chongqing brown duck naturally infected with DHBV, was amplified by PCR and then inserted into pGEM-T vector through T-A clone method. After being identified by endonucleases digestion, the recombinant plasmid was send to determine the sequence of the inserted fragment, which then was analyzed with such software as DNAtools, ClustalX and Phylip.Results: (1) By PCR with a pair of primers located in S region, we confirmed that the substance extracted from duck serum was DHBV DNA; (2) As expected, a 3.0kb single fragment was obtained by PCR method; (3) Successful insertion of the 3.0kb fragment into pGEM-T vector was identified by endonucleases digestion; (4) Sequence analysis demonstrated that the inserted fragment was DHBV genome DNA exactly. This sequence ,which is 3024bp nucleotides long, at least contains three ORFs encoding P,PreC/Cand PreS/S protein respectively. Most of the conserved regulatory nucleotide and amino acids sequences found in other DHBV were identified in DHBVcq. Theεregion of DHBVcq, which is important for encapsidation of pgRNA and synthesis of minus-strand DNA, differs from that of most of other DHBV strains. Phyletic analysis illustrated reasonably high homology between DHBVcq and seven other DHBV strains extracted from other regions in China. Conclusion: The clone and sequence analysis of DHBV genome from Chongqing brown duck established a solid basis for obtaining DHBV with specific sequence or mutated sequence to screen anti-HBV drugs in DHBV infection model.