TAT-HBV Targeted Ribonuclease: Expression, Purification And Inhibition Of HBV Replication

Introduction of proteins into mammalian cells has been achieved by the transfection of expression vectors, micro injection or infectious virus etc. Although these approaches have been somewhat successful, the classical manipulation methods are not easily regulated and can be laborious. One approach to resolve these problems is the use of PTD(Protein transduction domain)-mediated protein transduction. Linked covalently to proteins, peptides, nucleic acids or as in-frame fusions with full-length proteins, PTD would let them enter any cell type in a receptor and transporter independent fashion. HIV-TAT is a member of PTDS and appears to possess high level of protein-transduction efficiency. TAT fusion proteins were shown to transduce into all cells and tissues present in mice, including those present across the blood-brain barrier. And many, if not most, proteins could be transduced into cells by using this technology. Therefore, TatPTD may let us address new questions in preclinical research work and even help in the treatment of human disease.In this research, HBV targeted ribonuclease (TR) gene, mutant of HBV targeted ribonuclease (TRmut) gene, HBV core protein (HBVc) gene and human eosinophil derived neurotoxin (hEDN) gene were cloned into pTAT prokaryotic expression vector respectively. The recombinants were transformed into E.coli BL21(DE3)LysS and expressed with the induction of IPTG. The expression of the fusion proteins was analyzed by SDS-PAGE and Western blot. Four TAT fusion proteins were purified by Ni-NTA agrose in denatured condition. Urea wasremoved using PD-10 desalting columns. The analysis of purity and concentration of the four TAT fusion proteins indicated they were purified successfully. pET-30a(+)/TR, pET-30a(+)/TRmut, and pET-30a(+)/HBVc were transformed into E.coli BL21(DE3)LysS and the proteins without TAT were purified in the same way as for TAT fusion ones. Transduction of TAT-fusion proteins was detected by immunofluorescence assay in cultured 2.2.15 cells. Strong fluorescence was observed in the cytoplasm of the cells treated with TAT fusion proteins, but not in the control groups. These results showed the high transduction efficiency of TAT fusion proteins. We also investigated anti-HBV activity of TAT-TR by radioimmunoassay. As compared with mock group, concentration of HBeAg in TAT-TR group decreased by 60.3%, which indicated that the purified TAT-TR possessed a potent activity of anti-HBV. There was no significant difference between any other group and mock group, suggesting HBVc, hEDN or TRmut had no inhibitive effect on the replication of HBV. MTT assay was performed to reveal whether the TAT fusion proteins were harmful to 2.2.15 cells, and the result indicated they did not affect the growth of the cells. To conclude, the obtained TAT-TR fusion protein can inhibit the replication of the HBV and has laid the foundation for using TR in the therapy of HBV infection.