Viral Hepatitis
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 15, 2003; 9(7): 1504-1507
Published online Jul 15, 2003. doi: 10.3748/wjg.v9.i7.1504
Inhibition of HBV targeted ribonuclease enhanced by introduction of linker
Wei-Dong Gong, Jun Liu, Jin Ding, Ya Zhao, Ying-Hui Li, Cai-Fang Xue
Wei-Dong Gong, Jun Liu, Jin Ding, Ya Zhao, Ying-Hui Li, Cai-Fang Xue, Department of Pathogenic Organisms, Fourth Military Medical University, Xi’an 710033, Shaanxi Province, China
Author contributions: All authors contributed equally to the work.
Supported by National Natural Scientific Foundation of China, No.30100157 and Innovation Project of FMMU, No.CX99005
Correspondence to: Professor Cai-Fang Xue or Dr. Jun Liu, Department of Pathogenic Organisms, Fourth Military Medical University, Xi’an 710033, Shaanxi Province, China. etiology@fmmu.edu.cn
Received: March 5, 2003
Revised: March 24, 2003
Accepted: April 1, 2003
Published online: July 15, 2003
Abstract

AIM: To construct human eosinophil-derived neurotoxin(hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly4Ser)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 (Gly4Ser)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).

CONCLUSION: Linker introduction enhances the inhibitory effect of HBV targeted ribonuclease significantly.

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