Background Dengue is currently a significant global health problem but no vaccines are available against the four dengue serotypes virus infections. The development of safe and effective vaccines has been hampered by ...Background Dengue is currently a significant global health problem but no vaccines are available against the four dengue serotypes virus infections. The development of safe and effective vaccines has been hampered by the requirement of conferring complete protection against all four dengue serotypes and the lack of a convenient animal model. Virus-like particles （VLPs） have emerged as a promising subunit vaccine candidate. One strategy of vaccine development is to produce a tetravalent dengue subunit vaccine by mixing recombinant VLPs, corresponding to all four dengue virus serotypes. Towards this end, this study aimed to establish a Pichia pastoris （P. pastoris） expression system for production of dengue virus type 1 （DENV-1） VLPs and evaluate the humoral and cellular immune response of this particle in mice. Methods A recombinant yeast P. pastoris clone containing prM and E genes of DENV-1 was constructed and DENV-1 VLPs expressed by this clone were analyzed by sucrose density gradient centrifugation, Western blotting, and transmission electron microscope. Groups of mice were immunized by these particles plus adjuvant formulations, then mice were tested by ELISA and neutralization assay for humoral immune response, and by lymphocyte proliferation and cytokine production assays for a cellular immune response. Results Our data demonstrated that recombinant DENV-1 VLPs consisting of prM and E protein were successfully expressed in the yeast P. pastoris. Sera of VLPs immunized mice were shown to contain a high-titer of antibodies and the neutralization assay suggested that those antibodies neutralized virus infection in vitro. Data from the T lymphocyte proliferation assay showed proliferation of T cell, and ELISA found elevated secretion levels of interferon IFN-γ and IL-4. Conclusions P. pastoris-expressed DENV-1 VLPs can induce virus neutralizing antibodies and T cell responses in immunized mice. Using P. pastoris to produce VLPs offers a promising and economic strategy for dengue virus vaccine development.展开更多
To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris (P. pastoris).MethodsA codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commerc...To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris (P. pastoris).MethodsA codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commercially and inserted into the P. pastoris pPICZαA expression vector. The recombinant DENV-2 NS1 protein was purified by Ni-NTA affinity chromatography, and its antigenicity was tested.ResultsThe recombinant DENV-2 NS1 protein was secreted as a protein with a molecular weight of ∼45 kDa, and the optimal expression condition was achieved by induction with 2% (v/v) methanol for 72 h. The purified recombinant DENV-2 NS1 protein was able to interact with a monoclonal antibody of NS1 in a commercial rapid test.ConclusionsThe resulting recombinant DENV-2 NS1 protein produced in P. pastoris KM71 is a potential candidate for use in the development of a dengue diagnostic kit and vaccine.展开更多
Heterocapsa circularisquama RNA virus(HcRNAV) is the first single-stranded RNA virus to be characterized that infects dinoflagellates.The ability of HcRNAV coat protein(HcRNAV CP) to self-assemble into virus-like part...Heterocapsa circularisquama RNA virus(HcRNAV) is the first single-stranded RNA virus to be characterized that infects dinoflagellates.The ability of HcRNAV coat protein(HcRNAV CP) to self-assemble into virus-like particles(VLPs) in vitro suggested that heterologous expression was possible,and that the VLPs might be ideal nanocontainers for the targeted delivery of genes and chemicals.In this paper,we report the expression of a codon-optimized HcRNAV 109 CP gene in Pichia pastoris and the production of self-assembled HcRNAV VLPs using large-scale fermentation.The HcRNAV 109 CP gene was synthesized according to the codon preference of P.pastoris and cloned into a pPICZA vector.The recombinant plasmid pPICZA-CPsyns was transformed into P.pastoris by electroporation.The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS polyacrylamide gel electrophoresis.After large-scale fermentation,the yield of HcRNAV CPsyns reached approximately 2.5 g L 1 within 4 d.The HcRNAV VLPs were purified using PEG precipitation followed by cesium chloride density gradient ultracentrifugation,and were subsequently analyzed using UV spectrophotometry and transmission electron microscopy.Fluorescence dye-labeled myoglobin was loaded into the cages of the HcRNAV VLPs and the encapsulation was confirmed by fluorescence spectroscopy.The results point to the possible utilization in pharmacology or nanotechnology of HcRNAV VLPs produced by P.pastoris fermentation.展开更多
基金This work was supported by grants from the National Science Foundation （No. U0632002） and National High Technology Research Development Program of China （No. 2006AA02A223）.
文摘Background Dengue is currently a significant global health problem but no vaccines are available against the four dengue serotypes virus infections. The development of safe and effective vaccines has been hampered by the requirement of conferring complete protection against all four dengue serotypes and the lack of a convenient animal model. Virus-like particles （VLPs） have emerged as a promising subunit vaccine candidate. One strategy of vaccine development is to produce a tetravalent dengue subunit vaccine by mixing recombinant VLPs, corresponding to all four dengue virus serotypes. Towards this end, this study aimed to establish a Pichia pastoris （P. pastoris） expression system for production of dengue virus type 1 （DENV-1） VLPs and evaluate the humoral and cellular immune response of this particle in mice. Methods A recombinant yeast P. pastoris clone containing prM and E genes of DENV-1 was constructed and DENV-1 VLPs expressed by this clone were analyzed by sucrose density gradient centrifugation, Western blotting, and transmission electron microscope. Groups of mice were immunized by these particles plus adjuvant formulations, then mice were tested by ELISA and neutralization assay for humoral immune response, and by lymphocyte proliferation and cytokine production assays for a cellular immune response. Results Our data demonstrated that recombinant DENV-1 VLPs consisting of prM and E protein were successfully expressed in the yeast P. pastoris. Sera of VLPs immunized mice were shown to contain a high-titer of antibodies and the neutralization assay suggested that those antibodies neutralized virus infection in vitro. Data from the T lymphocyte proliferation assay showed proliferation of T cell, and ELISA found elevated secretion levels of interferon IFN-γ and IL-4. Conclusions P. pastoris-expressed DENV-1 VLPs can induce virus neutralizing antibodies and T cell responses in immunized mice. Using P. pastoris to produce VLPs offers a promising and economic strategy for dengue virus vaccine development.
基金"Penelitian Unggulan Strategis Nasional 2013" under the contract number of 0400/I1/B04/SPK-WRRI/VI/2014,Ministry of Research,Technology,and Higher Education of Indonesia,for funding this work
文摘To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris (P. pastoris).MethodsA codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commercially and inserted into the P. pastoris pPICZαA expression vector. The recombinant DENV-2 NS1 protein was purified by Ni-NTA affinity chromatography, and its antigenicity was tested.ResultsThe recombinant DENV-2 NS1 protein was secreted as a protein with a molecular weight of ∼45 kDa, and the optimal expression condition was achieved by induction with 2% (v/v) methanol for 72 h. The purified recombinant DENV-2 NS1 protein was able to interact with a monoclonal antibody of NS1 in a commercial rapid test.ConclusionsThe resulting recombinant DENV-2 NS1 protein produced in P. pastoris KM71 is a potential candidate for use in the development of a dengue diagnostic kit and vaccine.
基金supported by the Pioneer Research Center Program through the National Research Program of Korea funded by the Ministry of Education,Science and Technology,Korea (Grant M1071118001-08M1118-00110)(2010)
文摘Heterocapsa circularisquama RNA virus(HcRNAV) is the first single-stranded RNA virus to be characterized that infects dinoflagellates.The ability of HcRNAV coat protein(HcRNAV CP) to self-assemble into virus-like particles(VLPs) in vitro suggested that heterologous expression was possible,and that the VLPs might be ideal nanocontainers for the targeted delivery of genes and chemicals.In this paper,we report the expression of a codon-optimized HcRNAV 109 CP gene in Pichia pastoris and the production of self-assembled HcRNAV VLPs using large-scale fermentation.The HcRNAV 109 CP gene was synthesized according to the codon preference of P.pastoris and cloned into a pPICZA vector.The recombinant plasmid pPICZA-CPsyns was transformed into P.pastoris by electroporation.The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS polyacrylamide gel electrophoresis.After large-scale fermentation,the yield of HcRNAV CPsyns reached approximately 2.5 g L 1 within 4 d.The HcRNAV VLPs were purified using PEG precipitation followed by cesium chloride density gradient ultracentrifugation,and were subsequently analyzed using UV spectrophotometry and transmission electron microscopy.Fluorescence dye-labeled myoglobin was loaded into the cages of the HcRNAV VLPs and the encapsulation was confirmed by fluorescence spectroscopy.The results point to the possible utilization in pharmacology or nanotechnology of HcRNAV VLPs produced by P.pastoris fermentation.
