Screening And Study Of Peptides Targeting Pol-Tpandhbc Proteins Of Hepatitis B Virus

Objective The coding sequences of HBV POL-TP and HBC genes were separately subcloned into a prokaryotic expression plasmid and transformed into E.Colito induce expression of recombinant HBC and POL-TP proteins.Using the obtained recombinant proteins as substrates,the polypeptides that specifically bind to HBC and POL-TP proteins were screened from the phage polypeptide library.Subsequently,the ability of these polypeptides to regulate HBV replication was verified in a HBV cell model.The polypeptides with anti-viral effects were obtained.Methods The coding sequences of HBC(183aa)and HBV POL-TP(192 aa)were analyzed and subjected to rare codon optimizationby online bioinformatics software.The full-length coding sequences of HBC and POL-TP were synthesized and inserted into aprokaryotic expression vector to obtain pET28a(+)/HBC149 and pET28a(+)/POL-TP recombinant plasmids,respectively.Then the plasmids were transformed into E.Coli,and the recombinant HBC149 protein and POL-TP protein were induced,harvested and purified.These two proteins were used as substrates to screen the phage polypeptide library.The phages with low binding abilityto the target proteins were removed while the phages with high binding ability were retained,and finally enriched.The phage display regions were sequenced and a batch of candidate polypeptides were synthesized according to the sequencing results.The polypeptides were added to HepAD38 stably expressing HBV and the proliferation of the cells was detected and compared.Expression levels of HBV replication intermediate in cell lysate were detected by real-time PCR and Southern blot.Results The recombinant plasmids,pET28a(+)/POL-TP and pET28a(+)/HBC149 wereconstructed by double-restriction enzyme digestion.Recombinant HBC protein and POL-TP protein were expressed in E.coli.And identified by SDS-PAGE and Western blot.By using the phage polypeptide library,a total of 8 polypeptides with the highest binding ability to the target proteins were obtained.The effects of the polypeptides on cell proliferation were detected in the cell line HepAD38 stably expressing HBV.The results showed that at low concentrations,8 peptides had no significant effect on cell proliferation,and at higher concentrations,they had a certain effect on cell proliferation.The expression levels of HBV replication intermediate DNA in HepAD38 after peptide treatment were detected by real-time PCR and Southern blot.The results showed that TP-2,TP-3,TP-4 and TP-5 all inhibited while TP-8 stimulated HBV replication.Conclusion Recombinant HBV-related soluble HBC and POL-TP proteins were successfully expressed,and a batch of polypeptides specifically binding to the recombinant HBC or POL-TP proteins were screened by phage polypeptide library and at the cell level.Three peptides that inhibit HBV replication and one polypeptide that promoted HBV replication were obtained.