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AIM:To prepare and purify TAT-HBV targeted ribonucleasefusion protein,evaluate its transduction activity andinvestigate its effect on HBV replication in 2.2.15 cells.METHODS:The prokaryotic expression vector pTATcontaining TR gene was used in transforming E.coli BL21(DE3) LysS and TR was expressed with the induction ofIPTG.The TAT-TR fusion protein was purified using Ni-NTA-agrose and PD-10 desalting columns,and analyzed by SDS-PAGE.Transduction efficiency of TAT-TR was detected withimmunofluorescence assay and the concentration of HBeAgin the supernatant of the 2.2.15 cells was determined viasolid-phase radioimmunoassay (spRIA).MTT assay was usedto detect the cytotoxicity of TAT-TR.RESULTS:The SDS-PAGE showed that the TAT-TR fusionprotein was purified successfully,and the purity of TAT-TRwas 90%.The visualization of TAT-TR by immunofluorescenceassay indicated its high efficiency in transducing 2.2.15 cells.RIA result suggests that TAT-TR could inhibit the replicationof HBV effectively,it didn’t affect cell growth and had nocytotoxicity.CONCLUSION:TAT-TR possesses a significant anti-HBVactivity and the preparation of TAT-TR fusion protein haslaid the foundation for the use of TR in the therapeutic trialof HBV infection.
AIM: To prepare and purify TAT-HBV targeted ribonucleasefusion protein, evaluate its transduction activity and investigated its effect on HBV replication in 2.2.15 cells. METHODS: The prokaryotic expression vector pTATcontaining TR gene was used in transforming E. coli BL21 (DE3) LysS and TR was expressed with the induction of IPTG. The TAT-TR fusion protein was purified using Ni-NTA-agrose and PD-10 desalting columns, and analyzed by SDS-PAGE. Transduction efficiency of TAT-TR was detected with immunofluorescence assay and the concentration of HBeAgin the supernatant of the 2.2.15 cells was determined viasolid-phase radioimmunoassay (spRIA). MTT assay was used to detect the cytotoxicity of TAT-TR. RESULTS: The SDS-PAGE showed that the TAT-TR fusion protein was purified successfully, and the purity of TAT-TRwas 90%. The visualization of TAT-TR by immunofluorescenceassay indicated its high efficiency in transducing 2.2.15 cells. RIA result suggests that TAT-TR could inhibit the replication of HBV effectively , it did not affect cell growth and had nocytotoxicity. CONCLUSION: TAT-TR possesses a significant anti-HBV activity and the preparation of TAT-TR fusion protein haslaid the foundation for the use of TR in the therapeutic trial of HBV infection.