Inhibition of HBV targeted ribonuclease enhanced by introduction of linker.
OBJECTIVE
To construct human eosinophil-derived neurotoxin(hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly(4)Ser) (3) between them to optimize the molecule folding, which will be used to inhibit HBV replication in vitro.


METHODS
Previously constructed pcDNA3.1(-)/TR was used as a template. Linker sequence was synthesized and annealed to form dslinker, and cloned into pcDNA3.1(-)/TR to produce plasmid pcDNA3.1(-)/HBc-linker. Then the hEDN fragment was PCR amplified and inserted into pcDNA3.1(-)/HBc-linker to form pcDNA3.1(-)/TNL in which the effector molecule and the target molecule were separated by a linker sequence. pcDNA3.1(-)/TNL expression was identified by indirect immunofluorescence staining. Radioimmunoassay was used to analyse anti-HBV activity of pcDNA3.1(-)/TNL. Meanwhile, metabolism of cells was evaluated by MTT colorimetry.


RESULTS
hEDN and HBVc eukaryotic fusion expression vector with a linker (Gly(4)Ser)(3) between them was successfully constructed. pcDNA3.1(-)/TNL was expressed in HepG2.2.15 cells efficiently. A significant decrease of HBsAg concentration from pcDNA3.1(-)/TNL transfectant was observed compared to pcDNA3.1(-)/TR (P=0.036, P<0.05). MTT assay suggested that there were no significant differences between groups (P=0.08, P>0.05).


CONCLUSIONS
Linker introduction enhances the inhibitory effect of HBV targeted ribonuclease significantly.
Gong
Wei-Dong
WD
Department of Pathogenic Organisms, Fourth Military Medical University, Xi'an 710033, Shaanxi Province, China.
Liu
Jun
J
Ding
Jin
J
Zhao
Ya
Y
Li
Ying-Hui
YH
Xue
Cai-Fang
CF
D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor / Q000821:virology; D047208:Eosinophil-Derived Neurotoxin; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D006512:Hepatitis B Core Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms; D016254:Mutagenesis, Insertional / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D012260:Ribonucleases / Q000235:genetics*; D014162:Transfection; D014779:Virus Replication
D006512:Hepatitis B Core Antigens; D011993:Recombinant Fusion Proteins; D047208:Eosinophil-Derived Neurotoxin; D012260:Ribonucleases