AIM: To construct human eosinophil-derived neurotoxin (hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly4 Ser)3 between them to optimize the molecule folding, which will be used ...AIM: To construct human eosinophil-derived neurotoxin (hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly4 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 NTT 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.展开更多
基金National Natural Scientific Foundation of China,No.30100157Innovation Project of FMMU,No.CX99005
文摘AIM: To construct human eosinophil-derived neurotoxin (hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly4 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 NTT 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.
