The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells...The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.展开更多
To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8...To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.展开更多
Background The synthesis of virus-like particles (VLPs) provides an important tool to determine the structural requirements for viral particle assembly and virus-host interactions. Our purpose was to express simultan...Background The synthesis of virus-like particles (VLPs) provides an important tool to determine the structural requirements for viral particle assembly and virus-host interactions. Our purpose was to express simultaneously all three structural proteins of hepatitis C virus (HCV) in insect cells to investigate the proteins assembly into VLPs and the immunogenicity of these particles KH*2/5DMethods HCV gene sequences encoding the structural proteins C, E1, and E2 were amplified with PCR, and recombinant baculoviruses were constructed using recombinant DNA techniques The expression of HCV structural proteins in insect cells was analyzed by immunofluoresceoce and SDS-PAGE The interaction of expressed structural proteins was investigated by immunoprecipitation and immunoblotting The VLPs in the insect cells were visualized by electron microscopy (EM) VLPs were then purified by sucrose gradient centrifugation and used to immunize BALB/c mice Antibodies against HCV were tested for in mouse serum samples by an ELISA assay Results The recombinant baculoviruses reBV/C and reBV/E1-E2 were constructed successfully Insect cells co-infected with reBV/C and reBV/E1-E2 expressed HCV C, E1, and E2 proteins with the expected molecular weights of 20kD, 35kD, and 66kD, respectively The results of immunoprecipitation and immunoblotting assays revealed the coimmunoprecipitation of C, E1, and E2 proteins, indicating association of the three structural proteins Electron microscopy of insect cells co-infected with reBV/C and reBV/E1-E2 demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from serum samples or hepatic tissue samples of HCV infected humans The VLPs were partially purified Antibodies to HCV were detectable in the serum of mice immunized with VLPs Conclusion HCV structural proteins simultaneously expressed in insect cells can interact with each other and assemble into HCV-like particles, which are shown to be immunogenic in mice展开更多
文摘The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.
基金supported by the National Science Foundation of China (30970135)The Key Project of Genetically Modified Organisms Breeding(2009ZX08009-044B)+1 种基金the Natural Science Foundation of Fujian Province of China (No.2006J0065)the Public-interest Scientific Institution Basal Research Fund of Fujian Province (2009R10029-3)
文摘To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.
基金ThisstudywassupportedbyagrantfromtheYunnanCommissionofScienceandTechnology,China (No 2 0 0 2C0 0 74M)
文摘Background The synthesis of virus-like particles (VLPs) provides an important tool to determine the structural requirements for viral particle assembly and virus-host interactions. Our purpose was to express simultaneously all three structural proteins of hepatitis C virus (HCV) in insect cells to investigate the proteins assembly into VLPs and the immunogenicity of these particles KH*2/5DMethods HCV gene sequences encoding the structural proteins C, E1, and E2 were amplified with PCR, and recombinant baculoviruses were constructed using recombinant DNA techniques The expression of HCV structural proteins in insect cells was analyzed by immunofluoresceoce and SDS-PAGE The interaction of expressed structural proteins was investigated by immunoprecipitation and immunoblotting The VLPs in the insect cells were visualized by electron microscopy (EM) VLPs were then purified by sucrose gradient centrifugation and used to immunize BALB/c mice Antibodies against HCV were tested for in mouse serum samples by an ELISA assay Results The recombinant baculoviruses reBV/C and reBV/E1-E2 were constructed successfully Insect cells co-infected with reBV/C and reBV/E1-E2 expressed HCV C, E1, and E2 proteins with the expected molecular weights of 20kD, 35kD, and 66kD, respectively The results of immunoprecipitation and immunoblotting assays revealed the coimmunoprecipitation of C, E1, and E2 proteins, indicating association of the three structural proteins Electron microscopy of insect cells co-infected with reBV/C and reBV/E1-E2 demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from serum samples or hepatic tissue samples of HCV infected humans The VLPs were partially purified Antibodies to HCV were detectable in the serum of mice immunized with VLPs Conclusion HCV structural proteins simultaneously expressed in insect cells can interact with each other and assemble into HCV-like particles, which are shown to be immunogenic in mice
