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Anti-HBV effect of TAT- HBV targeted ribonuclease
Jin Ding1, Jun Liu, Cai-Fang Xue
1Department of Etiology, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
Aim:
To prepare and purify TAT-HBV targeted ribonuclease fusion protein, evaluate its transduction activity and investigate its effect on HBV replication in 2.2.15 cells.
Methods:
The prokaryotic expression vector pTAT containing 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 HBeAg in the supernatant of the 2.2.15 cells was determined via solid-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-TR was 90 %. The visualization of TAT-TR by immunofluorescence assay indicated its high efficiency in transducing 2.2.15 cells. RIA result suggests that TAT-TR could inhibit the replication of HBV effectively, it didn't affect cell growth and had no cytotoxicity.
Conclusion:
TAT-TR possesses a significant anti-HBV activity and the preparation of TAT-TR fusion protein has laid the foundation for the use of TR in the therapeutic trial of HBV infection.