摘要
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
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
基金
ThisstudywassupportedbyagrantfromtheYunnanCommissionofScienceandTechnology,China (No 2 0 0 2C0 0 74M)
