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AIM:To construct human eosinophil-derived neurotoxin(hEDN) and HBV core protein (HBVc) eukaryotic fusionexpression vector with a linker (Gly_4Ser)_3 between them tooptimize the molecule folding,which will be used to inhibitHBV replication in vitro.METHODS:Previously constructed pcDNA3.1(-)/TR wasused as a template.Linker sequence was synthesized andannealed to form dslinker,and cloned into pcDNA3.1(-)/TRto produce plasmid pcDNA3.1(-)/HBc-linker.Then the hEDNfragment was PCR amplified and inserted into pcDNA3.1(-)/HBc-linker to form pcDNA3.1(-)/TNL in which the effectormolecule and the target molecule were separated by a linkersequence,pcDNA3.1(-)/TNL expression was identified byindirect immunofluorescence staining.Radioimmunoassaywas used to analyse anti-HBV activity of pcDNA3.1(-)/TNL.Meanwhile,metabolism of cells was evaluated by MTTcolorimetry.RESULTS:hEDN and HBVc eukaryotic fusion expressionvector with a linker (Gly_4Ser)_3 between them wassuccessfully constructed,pcDNA3.1(-)/TNL was expressedin HepG2.2.15 cells efficiently.A significant decrease ofHBsAg concentration from pcDNA3.1(-)/TNL transfectant wasobserved compared to pcDNA3.1(-)/TR (P=0.036,P<0.05).MTT assay suggested that there were no significantdifferences between groups (P=0.08,P>0.05).CONCLUSION:Linker introduction enhances the inhibitoryeffect of HBV targeted ribonuclease significantly.
AIM: To construct human eosinophil-derived neurotoxin (hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly_4Ser) _3 between them tooptimize the molecule folding, which will be used to inhibit HBV replication in vitro. METHODS: Previously constructed The pcDNA3.1 (-) / TR wasused as a template. Linker sequence was synthesized andannealed to form dslinker, and cloned into pcDNA3.1 (-) / TRtoenecine plasmid pcDNA3.1 (-) / HBc-linker.Then the hEDNfragment 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 linkersquence, pcDNA3.1 (-) / TNL expression was identified byindirect immunofluorescence staining. Radioimmunoassaywas used to analyze anti-HBV activity of pcDNA3.1 (-) / TNL.Meanwhile, metabolism of cells was evaluated by MTTcolorimetry .RESULTS: hEDN and HBVc eukaryotic fusion expression vector with a linker (Gly_4Ser) _3 between them wassuccessfully constructed, pcDNA3.1 (-) / TNL was expressed in HepG2.2.15 cells efficiently. A significant decrease of HBsAg concentration from pcDNA3.1 (-) / TNL transfectant wasobserved compared to pcDNA3.1 (-) / TR (P = 0.036, no significant differences between groups (P = 0.08, P> 0.05) .CONCLUSION: Linker introduction enhances the inhibitory effect of HBV targeted ribonuclease significantly.