Anti-HBV effect of TAT- HBV targeted ribonuclease.
OBJECTIVE
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.


CONCLUSIONS
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.
Ding
Jin
J
Department of Etiology, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
Liu
Jun
J
Xue
Cai-Fang
CF
Gong
Wei-Dong
WD
Li
Ying-Hui
YH
Zhao
Ya
Y
D002478:Cells, Cultured; D004926:Escherichia coli; D005562:Formazans; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics* / Q000254:growth & development*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D012260:Ribonucleases / Q000235:genetics*; D013778:Tetrazolium Salts; D014161:Transduction, Genetic; D014779:Virus Replication / Q000235:genetics
D005562:Formazans; D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D013778:Tetrazolium Salts; C079782:MTT formazan; D012260:Ribonucleases