AIM:Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and T...AIM:Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Ta1 linked by different lengths of (G4S)n(n = 1-3) were constructed and expressed in Pichia pastoris. METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNα2b-(G4S)n-Tα1 (n = 1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex?75 gel filtration and analyzed by SDS-PAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins. RESULTS: DNA sequencing confirmed that the fusion genes of IFNa2b-(G4S)n-Tα1 (n= 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex?75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNa2b monoclonal antibody and Tal polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay. CONCLUSION: The recombinant IFNa2b-(G4S)n-Tα1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNa2b and immunomodulatory activity of Tal in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.展开更多
Background and Aims:A functional cure,or hepatitis B virus(HBV)surface antigen(HBsAg)loss,is difficult to achieve in patients with hepatitis B virus e antigen(HBeAg)-positive chronic hepatitis B.The HBV vaccine and gr...Background and Aims:A functional cure,or hepatitis B virus(HBV)surface antigen(HBsAg)loss,is difficult to achieve in patients with hepatitis B virus e antigen(HBeAg)-positive chronic hepatitis B.The HBV vaccine and granulocyte-macrophage colony-stimulating factor(GM-CSF)have been reported to help reduce HBsAg levels and promote HBsAg loss.In this prospective randomized trial,we evaluated HBsAg loss in patients receiving pegylated interferon α2b(PEGIFN-α2b)and tenofovir disoproxil fumarate(TDF),with and without GM-CSF and HBV vaccination.Methods:A total of 287 patients with HBeAg positive chronic hepati-tis B and seroconversion after nucleot(s)ide analog treat-ment were assigned randomly to three treatment groups for 48 weeks,TDF alone(control),PEGIFN-α2b+TDF,and PEGIFN-α2b+TDF+GM-CSF+HBV vaccine.The prima-ry endpoints were the proportions of patients with HBsAg loss and seroconversion at 48 and 72 weeks.Resu/ts:The cumulative HBsAg loss rates in the control,PEGIFN-α2b+TDF,and PEGIFN-α2b+TDF+GM-CSF+HBV vaccine groups at week 48 were 0.0%,28.3%,and 41.1%,respec-tively.The cumulative HBsAg seroconversion rates in these groups at week 48 were 0.0%,21.7%,and 33.9%,respec-tively.Multivariate regression analysis showed that GM-CSF use plus HBV vaccination was significantly associated with HBsAg loss(p=0.017)and seroconversion(p=0.030).Con-clusions:In patients with HBeAg-positive chronic hepatitis B and seroconversion after nucleot(s)ide analog treatment,immunomodulatory/antiviral treatment regimens effective-ly improved HBsAg loss,and the regimen including GM-CSF and HBV vaccination was most effective.展开更多
文摘AIM:Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Ta1 linked by different lengths of (G4S)n(n = 1-3) were constructed and expressed in Pichia pastoris. METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNα2b-(G4S)n-Tα1 (n = 1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex?75 gel filtration and analyzed by SDS-PAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins. RESULTS: DNA sequencing confirmed that the fusion genes of IFNa2b-(G4S)n-Tα1 (n= 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex?75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNa2b monoclonal antibody and Tal polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay. CONCLUSION: The recombinant IFNa2b-(G4S)n-Tα1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNa2b and immunomodulatory activity of Tal in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.
基金Ministry of science and technology of China(2017ZX10202202)CAMS Innovation Fund for Medical Sciences(2019-I2M-5-045)National Key R&D Program of China(2022YFC2304500).
文摘Background and Aims:A functional cure,or hepatitis B virus(HBV)surface antigen(HBsAg)loss,is difficult to achieve in patients with hepatitis B virus e antigen(HBeAg)-positive chronic hepatitis B.The HBV vaccine and granulocyte-macrophage colony-stimulating factor(GM-CSF)have been reported to help reduce HBsAg levels and promote HBsAg loss.In this prospective randomized trial,we evaluated HBsAg loss in patients receiving pegylated interferon α2b(PEGIFN-α2b)and tenofovir disoproxil fumarate(TDF),with and without GM-CSF and HBV vaccination.Methods:A total of 287 patients with HBeAg positive chronic hepati-tis B and seroconversion after nucleot(s)ide analog treat-ment were assigned randomly to three treatment groups for 48 weeks,TDF alone(control),PEGIFN-α2b+TDF,and PEGIFN-α2b+TDF+GM-CSF+HBV vaccine.The prima-ry endpoints were the proportions of patients with HBsAg loss and seroconversion at 48 and 72 weeks.Resu/ts:The cumulative HBsAg loss rates in the control,PEGIFN-α2b+TDF,and PEGIFN-α2b+TDF+GM-CSF+HBV vaccine groups at week 48 were 0.0%,28.3%,and 41.1%,respec-tively.The cumulative HBsAg seroconversion rates in these groups at week 48 were 0.0%,21.7%,and 33.9%,respec-tively.Multivariate regression analysis showed that GM-CSF use plus HBV vaccination was significantly associated with HBsAg loss(p=0.017)and seroconversion(p=0.030).Con-clusions:In patients with HBeAg-positive chronic hepatitis B and seroconversion after nucleot(s)ide analog treatment,immunomodulatory/antiviral treatment regimens effective-ly improved HBsAg loss,and the regimen including GM-CSF and HBV vaccination was most effective.
