Targeted ribonuclease can inhibit replication of hepatitis B virus.
OBJECTIVE
To study the effect of a novel targeted ribonuclease (TN), the fusion protein of HBVc and human eosinophil-derived neurotoxin (hEDN), on the HBV replication in vitro.


METHODS
The gene encoding the targeted ribonuclease was cloned into pcDNA3.1 (-) to form recombinant eukaryotic expression vector p/TN. Control plasmids, including p/hEDN, p/HBVc, and p/TNmut in which a Lys113-Arg mutation was introduced by sequential PCR to eliminate the ribonuclease activity of hEDN, were also constructed. Liposome-mediated transfection of 2.2.15 cells by p/TN, p/TNmut, p/hEDN, p/HBVc, and pcDNA3.1 (-), or mock transfection was performed. After that, RT-PCR was used to verify the transgene expression. Morphology of the transfected cells was observed and MTT assay was performed to detect the cytotoxicity of transgene expression. Concentration of HBsAg in the supernatant of the transfected cells was measured using solid-phase radioimmunoassay.


RESULTS
Transgenes were successfully expressed in 2.2.15 cells. No obvious cytotoxic effect of transgene expression on 2.2.15 cells was found. The HBsAg concentration in the p/TN transfected cells was reduced by 58 % compared with that of mock transfected cells. No such an effect was found in all other controls.


CONCLUSIONS
The targeted ribonuclease can inhibit HBV replication in vitro while it has no cytotoxicity on host cells. The targeted ribonuclease may be used as a novel antiviral agent for human HBV infection.
Liu
Jun
J
Department of Pathogenic Organism, Fourth Military Medical University, Xi'an 710033, Shaanxi Province, China. etiology@fmmu.edu.cn
Li
Ying-Hui
YH
Xue
Cai-Fang
CF
Ding
Jin
J
Gong
Wei-Dong
WD
Zhao
Ya
Y
Huang
Yu-Xiao
YX
D047208:Eosinophil-Derived Neurotoxin; D018390:Gene Targeting; D006512:Hepatitis B Core Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000502:physiology*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D012260:Ribonucleases / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014779:Virus Replication / Q000187:drug effects*
D006512:Hepatitis B Core Antigens; D011993:Recombinant Fusion Proteins; D047208:Eosinophil-Derived Neurotoxin; D012260:Ribonucleases