Inhibition Of Mimic Peptides On The Replication Of Duck Hepatitis B Virus

Objective:To study the inhibitory effect of mimic peptides, which were specifically bound to the duck hepatitis B virus polymerase (DHBVP) and screened by phage display technology (PDT), on duck hepatitis B virus (DHBV) replication in the model of primary culture of duck hepatocytes. Methods:1. The whole genomic DNA of DHBV of the brown Shaoxing duck was amplified and sequenced.2. Preparation of target molecules for PDT screening: this study used two types of target molecules, recombinant fusion proteins and synthesized peptides. The primers for DHBVP gene amplification were designed according to the genome of DHBV of the brown Shaoxing duck. DHBVP was digested into four fragments and each was cloned into pGEM, a prokaryotic expression vector. The recombinants were transformed into E. coli expression strains. Proteins were expressed with IPTG induction, and then they were harvested and purified. For the synthesized peptide, the sequence including YMDD, which is the functional site of a nucleotide analog, was according to the above mentioned DHBV genome.3. Specific mimic peptides were screened by PDT: DHBVP recombinant proteins and polypeptides with the YMDD nucleotide analog functional site were used as target molecules. They were screened by phage peptide library of M13 for three rounds. The specific mimic peptides detected were sequenced and synthesized.4. Inhibition of replication of DHBV by synthesized mimic peptides. Mimic peptides were added into primary culture of duck hepatocytes infected with DHBV in vitro. DHBV-DNA in the cytoplasm, cell nucleus and medium supernatant was assayed over time.5. Inhibition of replication of DHBV by expressed peptides in eukaryotes. Mimic peptide geneswere cloned into the eukaryote expression vectors, and then transfected into the primary culture ofduck hepatocytes infected with DHBV in vitro. DHBV-DNA in the cytoplasm, cell nucleus andmedium supernatant was assayed over time.6. The data were analysed by Kruskal-Wallis test.Results:1 The whole sequence of DHBV DNA was constructed according to the PCR product sequencesand aligned by DNAssist software. Compared with brown Shanghai duck DHBV published inNCBI (ACCESSION M32990 VERSION M32990.1 GI:325448), the genome sequence ofbrown Shaoxing Duck DHBV had 72 bp differences and the homology was 97.62%, while theDHBVP protein had 24 amino acid differences and the homology was 96.95%.2. The sequences of PCR products of pGEM-DHBVP with correct orientation were the same asthat of brown Shaoxing duck DHBVP. The molecular weights of the proteins expressed from therecombinants were as expected.3 Sixteen mimic peptides were obtained by 3-round screening by PDT and seven of them wereselected by bioinformatics according to the homologies and physicochemical properties.4. There were no significant differences in morphology between groups, whether treated with minic peptides or not, after ten-day culture of duck primary hepatocytes.5. The DHBV-DNA levels of medium supernatant and cytoplasm in the synthesized peptides group (the 3rd and the 6th peptide) and Lamivudine treatment group (positive control) were significantly lower than those of negative group (positive control, 3th peptide, 6th peptide vs negative control, p<0.05). The DHBV-DNA levels in the cytoplasm in the expression group (the 6th peptide) and Lamivudine treatment group were significantly lower than those of negative group (positive control, 6th peptide vs negative control, p<0.05). There were no siginificant differences between the other groups and the negative groups (p>0.05).6. In the synthesized group, the inhibition rates of DHBV-DNA in the group of Lamivudine, 3rd peptide and 6lh peptide were: 97.4%, 93.4% and 97.1% in whole cell and 96.4%, 77.7% and 88.9% in cytoplasm, respectively. In the expression group, the inhibition rates of DHBV-DNA in the group of Lamivudine and 6th peptide were 92.3% and 81.8% in cytoplasm, respectively. Conclusions:1.