Published online Jul 15, 2003. doi: 10.3748/wjg.v9.i7.1525
Revised: March 28, 2003
Accepted: April 9, 2003
Published online: July 15, 2003
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.