This article reports the design and construction of a multiple-epitope foot and mouth disease virus （FMDV） antigen, designated as OAAT. This recombinant antigen consists of the structural protein VP1 genes from sero...This article reports the design and construction of a multiple-epitope foot and mouth disease virus （FMDV） antigen, designated as OAAT. This recombinant antigen consists of the structural protein VP1 genes from serotypes A and O FMDV, five major VP1 immunodominant epitopes from two genotypes of Asial serotype, and three Th2 epitopes originating from the nonstructural protein, three ABC gene and structural protein VP4 gene. Expressions of target gene from these plasmids in HeLa cells were verified by Western-blot. BALB/c mice were immunized intramuscularly with the DNA vaccines thrice every two weeks. We found that pA could induce simultaneously specific antibodies against serotypes A, Asial, and O FMDV. Compared to those of the controls, the spots of FMDV-specific IFN-7 and cytotoxic activity from mice immunized with pA were significantly increased, pA provided full protection in 2/4 guinea pigs from challenge with FMDV O/NY00 and Asial/YNBS/58, respectively. The results show that although pA did not give full protection in 100% immunized guinea pigs from challenge with type O and Asial FMDV, respectively, OAAT may be potential immunogen against FMDV and pA may be potential DNA vaccines against FMDV.展开更多
The purpose of this study was to construct an eukaryotic DNA vector encoding a multiple-epitope antigen (MFC) of hepatitis C virus (HCV) and a hepatitis B surface antigen (HBsAg) , and explore the effect of HBsAg gene...The purpose of this study was to construct an eukaryotic DNA vector encoding a multiple-epitope antigen (MFC) of hepatitis C virus (HCV) and a hepatitis B surface antigen (HBsAg) , and explore the effect of HBsAg gene on the immunity of HCV multiple-epitope DNA construct in vitro and in vivo in mice. An HCV DNA vector (pVAX1-HBs-MFC) was constructed by fusing HBsAg gene to the N-terminal of an HCV multiple-epitope antigen gene. The pVAX1-HBs-MFC was transfected into HEK 293T cells and its expression was measured by ELISA and Western blotting. BALB/c mice were intramuscularly immunized with the pVAX1-HBs-MFC, and an ELISA approach was applied to determine the specific antibody titers and subtypes in the mouse serum. The cross-reactivity of the antibodies was also checked with two synthesized HCV hypervariable region 1 (HVR1) peptides. The IFN-γ production and cell proliferation of the mouse spleen cells were evaluated by ELISA and MTS (3-[4, 5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assays, respectively. The expression of pVAX1-HBs-MFC was detectable in the transfected HEK 293T cells. The serum antibody response was effectively elicited in BALB/c mice injected with pVAX1-HBs-MFC. The highest titer of antibody against HCV (MFC) was 1∶1280, and the ratio of IgG2a/IgG1 was 1.50±0.12 at the fifth week after first immunization. Moreover, the collected mouse serum antibody had the ability to cross-react with the two synthesized HCV HVR1 peptides. The stimulation index of the mouse splenocytes to MFC was 1.79 ±0.07, and the IFN-γ level was 287±6?pg/ml at week 21 after first immunization. The highest titer of the antibody in control BALB/c mice immunized with pVAX1-MFC was 1∶320, and the ratio of IgG2a/IgG1 was 1.33±0.11 at week 5 post-immunization. Furthermore, the stimulation index of the mouse splenocytes cells to MFC was 1.52±0.06, and the IFN-γ level was 225±9.3?pg/ml at week 21 post-immunization. The HBsAg gene can enhance the effects of an HCV multiple-epitope DNA construct on its humoral and cellular immune responses. This HBsAg enhanced HCV multiple-epitope DNA vector may be of potential use in the development of HCV vaccines.展开更多
基金National High Technology Research and Development Program of China(No.2006AA10A204)Educa-tional Department of Liaoning Province (No.20060527)
文摘This article reports the design and construction of a multiple-epitope foot and mouth disease virus （FMDV） antigen, designated as OAAT. This recombinant antigen consists of the structural protein VP1 genes from serotypes A and O FMDV, five major VP1 immunodominant epitopes from two genotypes of Asial serotype, and three Th2 epitopes originating from the nonstructural protein, three ABC gene and structural protein VP4 gene. Expressions of target gene from these plasmids in HeLa cells were verified by Western-blot. BALB/c mice were immunized intramuscularly with the DNA vaccines thrice every two weeks. We found that pA could induce simultaneously specific antibodies against serotypes A, Asial, and O FMDV. Compared to those of the controls, the spots of FMDV-specific IFN-7 and cytotoxic activity from mice immunized with pA were significantly increased, pA provided full protection in 2/4 guinea pigs from challenge with FMDV O/NY00 and Asial/YNBS/58, respectively. The results show that although pA did not give full protection in 100% immunized guinea pigs from challenge with type O and Asial FMDV, respectively, OAAT may be potential immunogen against FMDV and pA may be potential DNA vaccines against FMDV.
文摘The purpose of this study was to construct an eukaryotic DNA vector encoding a multiple-epitope antigen (MFC) of hepatitis C virus (HCV) and a hepatitis B surface antigen (HBsAg) , and explore the effect of HBsAg gene on the immunity of HCV multiple-epitope DNA construct in vitro and in vivo in mice. An HCV DNA vector (pVAX1-HBs-MFC) was constructed by fusing HBsAg gene to the N-terminal of an HCV multiple-epitope antigen gene. The pVAX1-HBs-MFC was transfected into HEK 293T cells and its expression was measured by ELISA and Western blotting. BALB/c mice were intramuscularly immunized with the pVAX1-HBs-MFC, and an ELISA approach was applied to determine the specific antibody titers and subtypes in the mouse serum. The cross-reactivity of the antibodies was also checked with two synthesized HCV hypervariable region 1 (HVR1) peptides. The IFN-γ production and cell proliferation of the mouse spleen cells were evaluated by ELISA and MTS (3-[4, 5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assays, respectively. The expression of pVAX1-HBs-MFC was detectable in the transfected HEK 293T cells. The serum antibody response was effectively elicited in BALB/c mice injected with pVAX1-HBs-MFC. The highest titer of antibody against HCV (MFC) was 1∶1280, and the ratio of IgG2a/IgG1 was 1.50±0.12 at the fifth week after first immunization. Moreover, the collected mouse serum antibody had the ability to cross-react with the two synthesized HCV HVR1 peptides. The stimulation index of the mouse splenocytes to MFC was 1.79 ±0.07, and the IFN-γ level was 287±6?pg/ml at week 21 after first immunization. The highest titer of the antibody in control BALB/c mice immunized with pVAX1-MFC was 1∶320, and the ratio of IgG2a/IgG1 was 1.33±0.11 at week 5 post-immunization. Furthermore, the stimulation index of the mouse splenocytes cells to MFC was 1.52±0.06, and the IFN-γ level was 225±9.3?pg/ml at week 21 post-immunization. The HBsAg gene can enhance the effects of an HCV multiple-epitope DNA construct on its humoral and cellular immune responses. This HBsAg enhanced HCV multiple-epitope DNA vector may be of potential use in the development of HCV vaccines.
