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Objective:To express virus-like particles of poliovirus type 2 (PV2-VLP) in insect cells using a recombinant baculovirus expressing P1 and 3CD and to preliminarily evaluate its immunogenicity.Methods:Based on the codon preference of High 5 cells, the sequences of P1 gene and 3CD gene of PV2 were optimized and inserted into pUC57-Amp to construct pUC57-PV2-P1 and pUC57-PV2-3CD. UC57-PV2-P1s mutant that carried P1 gene mutation affecting thermostability was then constructed. Recombinant baculovirus strains of rBac-PV2-P1s-3CD and rBac-PV2-P1-3CD (wild type) were constructed using homologous recombination. The expression of target proteins was detected by Western blot. PV2-VLP was purified by ion exchange chromatography. The structure of VLP was observed under transmission electron microscopy to evaluate the assembly efficiency. The immunogenicity of PV2-VLP was assessed in a rat model.Results:The recombinant baculovirus with stable expression of P1s and 3CD proteins was successfully constructed. Western blot results showed that the yield of VLP was higher after thermostability mutation than that of the wild type. A three-dimensional structure with a diameter of about 30 nm was observed under electron microscopy, indicating that the VLP was successfully assembled. Animal experiment showed that the recombinant PV2-VLP had immunogenicity and could effectively induce the production of neutralizing antibodies.Conclusions:Effective VLP vaccines could be successfully prepared using the insect cell-baculovirus expression system, which provided reference for the development of polio VLP vaccine.
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Objective:In the present study,Bac-to-Bac baculovirus expression system was used to obtain recombinant human Cosmc extracellular domain protein,which can lay the foundation for the research about the structure and function of Cosmc protein in vitro,and simultaneously provide ideas for the research of O-glycosylation and related diseases. Methods: The Cosmc extracellular domain ( Cosmc-ED) gene was cloned into a transfer vector pFastBac1 to form the recombinant donor plasmid pFastBac1-Cosmc ED, which was transformed into competent cells DH10Bac. By using blue-white selection and PCR analysis,we could obtain recombinant shuttle vector rBacmid-Cosmc ED. Then, the recombinant gene DNA of rBacmid-Cosmc ED was used to transfect Sf-9 mediated by cationic lipid formulation,and the recombinant baculovirus bacmid was obtained,which was further used to infect the serum-free cell Sf-9 to express Cosmc-ED in the supernatant. Then the protein of interest was detected by SDS-PAGE and Western blot and purified with Ni-NTA affinity column. Results:SDS-PAGE and Western blot analysis showed a specific band about 33 kD,consistent with the interest protein. Mass spectrometry results further prove that the protein was Cosmc extracellular domain protein. Conclusion: Human Cosmc-ED protein can be successfully expressed in Sf-9 insect cells and laid basis for subsequent studies.
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Objective:To express mycobacterium tuberculosis protein MPB 64 in baculovirus insect cell expression system ,and identify its immunogenicity.Methods:The target gene MPB64 was connected to pFastBac vector ,then the pFastBac-MPB64 plasmid which was harvested would transformed to DH10Bac competent,and the target gene was transposition into Bacmid by Tn7 transposase fragment,therefore Bacmid-MPB64 Shuttle vector was obtained.The shuttle vector was packaged by liposomes and transfected Sf 9 cells to harvest P1-generation virus ,then high titers of P4 generation virus was harvested by repeat transfected Sf 9 cells three times.The target protein MPB64 was purified from the supernatant by Q Sepharose FF and Ni affine chromatography ,which were used to immunize BALB/c mice.Antibody changes in serum would be detected ,and the proliferation of immunized mice spleen cells would be detected by MTT,detected the IFN-γsecretion by MPB64 stimulated spleen cells by ELISA method.Results: MPB64 successfully expressed in insect cells.The purity of target protein was over 90% and yield up to 35 mg/L after purification.Purified protein can effectively stimulate BALB/c mice to produce antibodies , increase the content of IFN-γmedium in mice spleen cells ,and significantly promoting proliferation in spleen cells between 0.2-100 μg/ml.Conclusion: MPB64 which has immunogenicity was successfully expressed in baculovirus insect cell expression system ,that open a new avenue for tuberculosis vaccine production.
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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.
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Objective To express human cytotoxic T lymphocyte associated antigen-4 (CTLA4) extracelluar domain in Bacmid-baculovirus expression system. Methods The cDNA of human CTLA4 extracelluar domain was isolated from plasmid pUC19-CTLA4Ig by PCR.The specific cDNA was subcloned into plasmid pFastBacl. The insertion was confirmed by DNA sequencing, and then transposed into Hz8 Bacmid in DH10B. The recombinant Bacmid was used to transform Hz insect cells. The supernatant and cellular lysate were analyzed by using SDS-PAGE. Results A specific protein in the cellular lysate of Hz insect cells containing human CTLA4 extracelluar domain with MW38 x 103 was found. Conclusion Human CTLA4 extracellular domain is expressed in the Bacmid-baculovirus expression system.
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Objective To produce the recombinant NS3 protease of the hepatitis C virus in insect cells. Methods The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition. After the recombinant bacmid had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells. The bacmid DNA was verified to replicate in insect cells and package into baculovirus particles via PCR and electronic microscope analysis. The insect cells infected by recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-Lauryl Sarcosine Sodium (NLS) and purifed by metal-chelated-affinity chromatography, then the enzymatic activity was measured with the protein substrate NS5ab and the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbent assay. Results The HCV NS3 protease domain was expressed in insect cells at high level and it was partially resolved in NLS;Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5?10 7 cells and both enzymatic activity and antigenicity of the protein were proven to be good. Conclusion The recombinant HCV NS3 protease expressed in insect cells is a membrane-binding protein with good enzymatic activity and antigenicity.
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Transformation efficiency, virus multiplication and foreign gene expression were characterized in the insect cells transformed with Autographa calfornica nuclear polyhedrosis virus (AcNPV) immediate early 1 gene (IE1). Transformation efficiency of insect cells by AcNPV IE1 gene vector horboring foreign gene was approximately 8-fold higher in the Sf9 cells transformed previously with AcNPV IE1 gene than in the normal Sf9 cells. Virus multiplication and foreign gene expression of recombinant baculovirus in the Sf9 cells transformed with AcNPV IE1 gene were similar to those of the normal Sf9 cells. These results suggest that transformed cells displaying foreign gene product by using AcNPV IE1 gene promoter will be useful for the diverse applications of insect cells.
Subject(s)
Baculoviridae , Gene Expression , Insecta , Nucleopolyhedroviruses , Sf9 CellsABSTRACT
Transformation efficiency, virus multiplication and foreign gene expression were characterized in the insect cells transformed with Autographa calfornica nuclear polyhedrosis virus (AcNPV) immediate early 1 gene (IE1). Transformation efficiency of insect cells by AcNPV IE1 gene vector horboring foreign gene was approximately 8-fold higher in the Sf9 cells transformed previously with AcNPV IE1 gene than in the normal Sf9 cells. Virus multiplication and foreign gene expression of recombinant baculovirus in the Sf9 cells transformed with AcNPV IE1 gene were similar to those of the normal Sf9 cells. These results suggest that transformed cells displaying foreign gene product by using AcNPV IE1 gene promoter will be useful for the diverse applications of insect cells.
Subject(s)
Baculoviridae , Gene Expression , Insecta , Nucleopolyhedroviruses , Sf9 CellsABSTRACT
We have now construted a novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) producing polyhedra with Bacillus thuringiensis (Bt) Cry1Ac crystal protein. The recombinant polyhedra produced by the recombinant baculovirus, Btrus, in insect cells was characterized. The recombinant baculovirus has two independent transcription units in opposite orientations with two promoters, p10 or polyhedrin gene promoter each initiating transcription of either native polyhedrin or fusion protein with polyhedrin and Bt Cry1Ac crystal protein. Suprisingly, this recombinant baculovirus stably produced recombinant polyhedra which were nearly similar to those of wild-type AcNPV. The immunogold staining experiment showed that the recombinant polyhedra were assembled with polyhedrin and Bt Cry1Ac crystal protein, and contained virus paticles. Insecticidal toxicity of recombinant polyhedra of Btrus to the fall webworm, Hyphantrea cunea, was strikingly improved in comparison with the wild-type AcNPV.
Subject(s)
Bacillus thuringiensis , Bacillus , Baculoviridae , Insecta , NucleopolyhedrovirusesABSTRACT
We have expressed GFP in Sf9 and Bm5 cells or Bombyx by larvae by using Ac-Bm hybrid virus capable of replicating in both Bm5 and Sf9 cells. Genomic DNA of Ac-Bm hybrid virus expressing P-galactosidase was cotransfected with baculovirus transfer vector containing GFP gene, pBacPAK-GFP in Sf9 cells. The Ac-Bm hybrid virus harboring GFP was named as Ac-Bm hybrid virus-GFP. The Ac-Bm hybrid virus-GFP-infected insect cells were easily selected by detecting the emission of GFP from each well of cell culture dish on the UV illuminator. GFP produced by Ac-Bm hybrid virus-GFP in Sf9 and Bm5 cells or B. mori larvae was confirmed by SDS-PAGE and Western blot analysis using GFP antibody. In addition, B. mori larvae infected with Ac-Bm hybrid virus-GFP was apparently appeared fluorescence from the whole body at 5 days postinoculation. The fluorescence of GFP from the hemolymph and fat body of B. mori larvae infected with Ac-Bm hybrid virus-GFP was also observed by fluorescence microscope. In conclusion, our results demonstrated that in baculovirus expression vector system, use of Ac-Bm hybrid virus have an additional advantage of expanded host range for producing recombinant proteins.
Subject(s)
Animals , Baculoviridae , Blotting, Western , Bombyx , Cell Culture Techniques , DNA , Electrophoresis, Polyacrylamide Gel , Fat Body , Fluorescence , Hemolymph , Host Specificity , Insecta , Larva , Recombinant Proteins , Sf9 Cells , SpodopteraABSTRACT
Preparation of a pure autoantigen by way of recombinant DNA technology has an important value in an accurate diagnosis or prognosis of an autoimmune disease. BCOADC-E2 subunit, a mitochondrial protein, has been known to be the autoantigen of primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, as well as idiopathic dilated cardiomypathy (IDCM), a chronic autoimmune heart disease. Recombinant form of this molecule had been expressed in E. coli but with low yield and severe degradation. Furthermore, sera from IDCM patients failed to recognized BCOADC-E2 molecule produced in prokaryotic expression system. In this study, a recombinant bovine BCOADC-E2 fusion protein has been expressed in insect cells using baculovirus expression system and analyzed anti-BCOADC-E2 reactivity in sera from patients with PBC or with IDCM. Optimal production of the recombinant fusion protein has been achieved at 20 multiplicity of infection (MOI), and the protein was affinity-purified using metal-binding resins. The affinity-purified BCOADC-E2 protein was successfully recognized by sera from PBC patients, but not by sera from IDCM patients suggesting that the different auto-immune response against BCOADC-E2 is needed to be elucidated in terms of epitope recognition.
Subject(s)
Cattle , Humans , Acetyltransferases/metabolism , Acetyltransferases/immunology , Acetyltransferases/genetics , Animals , Baculoviridae/genetics , Cardiomyopathy, Dilated/immunology , Immune Sera , Insecta/cytology , Ketone Oxidoreductases/metabolism , Ketone Oxidoreductases/immunology , Ketone Oxidoreductases/genetics , Liver Cirrhosis, Biliary/immunology , Multienzyme Complexes/metabolism , Multienzyme Complexes/immunology , Multienzyme Complexes/genetics , Protein Engineering/methods , Recombinant Proteins/isolation & purification , Recombinant Proteins/immunology , Recombinant Proteins/geneticsABSTRACT
The baculovirus expression system employing Autagrapha californica nuclear polyhidrosis virus and Spodoptera frugiperda insect cells in culture has proved very popular for high level expression of heterologous genes: In this system, transcription of the foreign gene is usually driven by the hyperactive and temporally regulated polyhedrin gene promoter. Replacement of the polyhedrin gene, which encodes a 29-kDa occlusion protein (non-essential for viral replication), with a gene of interest leads to an occlusion negative phenotype which serves as a visual marker to select for recombinant viruses. Simultaneous expression of multiple genes can also be achieved. The heterologous proteins synthesized in this system are antigenically, immunologically and functionally identical in most respects to their native counterparts. This mini-review will aim at summarizing the potentials and utility of the baculovirus expression vector system and will address some important questions relating to the biology of this system.
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Objective:To express and purify a new fusion protein harboring human epidermal growth factor receptor(EGFR) binding domain and Interleukin-18(IL-18), as well as preliminary assay biological activity of recombinant proteins.Methods:Fusion protein was expressed in insect cells Spodoptera frugiperda cell (Sf9) by using Bac-to-Bac system, and an abbreviation purification procedure was used to purify fusion protein. IFN-?induction assay and EGF Receptor competitive test was used to determine fusion protein's biological activity.Results:SDS-PAGE and western blot assay showed that purified EGF-IL-18 fusion protein had high purity in 20 kD as expected and had the same antigenic specificity as human IL-18. IFN-?induction assay and EGF Receptor competitive test showed that fusion protein induced production of IFN-?in human PBMC and bound with tumor cells.Conclusion:EGF-IL-18 fusion protein has been successfully expressed and purified in insect cells and shows potential to apply in targeting therapy for tumor.