ABSTRACT
The gene encoding the gamma-chain of the mouse Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates prepared from insect cells infected with the produced recombinant virus VL1392-mIL-2R gamma. Kinetic analysis demonstrated that the corresponding protein could be detected as an approximately 50 kDa protein already at 24 h post-infection. Intrinsic labelling with [35S]-methionine/cysteine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma protein could also be determined on the surface of infected insect cells by flow cytometric analysis. Comparison of the molecular weights between baculovirus expressed human and mouse IL-2R gamma chains indicated differences in the glycosylation pattern despite similar numbers of N-linked glycosylation sites.
Subject(s)
Gene Expression Regulation , Receptors, Interleukin-2/genetics , Animals , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Flow Cytometry , Genetic Vectors/genetics , Glycosylation , Humans , Immunoblotting , Lepidoptera , Mice , Nucleopolyhedroviruses/genetics , Occlusion Body Matrix Proteins , Promoter Regions, Genetic , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombination, Genetic , Viral Proteins/genetics , Viral Structural ProteinsABSTRACT
The genes encoding the VHCH1 and VLCL parts of the mouse anti-human IL-2R alpha antibody 7G7B6 were amplified by PCR and the corresponding antibody fragments displayed on the surface of filamentous phages. The expression of Fab fragments was analysed by immunoblotting using HRP-labelled goat anti-mouse Ig antisera. By traditional hybridoma technology, splenocytes from Balb/c mice, immunized with native phage particles, were fused with P3X63-Ag8.653 myeloma cells in order to yield monoclonal antibodies against filamentous phage proteins. The obtained monoclonal antibody IF8 (mu/kappa) recognized the minor coat protein III as a 65-70 kDa protein band by immunoblotting, whereas the monoclonal antibody IVC8 (mu/kappa), in addition to cpIII, recognized a protein with an approximate molecular weight of 38-43 kDa. Both antibodies were employed to determine the binding specificity of the phage-displayed anti-human IL-2R alpha Fab fragments in an ELISA using recombinant baculovirus-expressed human IL-2R alpha proteins as antigens.
Subject(s)
Antibodies, Monoclonal/chemistry , Antigens/metabolism , Capsid/immunology , Coliphages/immunology , Coliphages/metabolism , DNA-Binding Proteins/immunology , Immunoglobulin Fab Fragments/analysis , Receptors, Interleukin-2/immunology , Viral Fusion Proteins/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/biosynthesis , Capsid Proteins , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Protein Binding/immunology , Receptors, Interleukin-2/metabolism , Recombinant Proteins/immunology , Recombinant Proteins/metabolismABSTRACT
The gene encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, ligated to the honeybee melittin signal peptide-encoding sequence, was inserted under transcriptional control of the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant green fluorescent protein was identified by SDS-PAGE gel electrophoresis followed by Coomassie blue staining of lysates from the recombinant baculovirus infected insect cells. Emission and excitation scanning of the recombinant baculovirus infected insect cells gave an emission maximum of 509 nm and excitation maximum of 398 nm. The GFP protein expressed was also detected in infected insect cells by a flow cytometer analysis.
Subject(s)
Gene Expression , Luminescent Proteins/genetics , Recombinant Fusion Proteins/genetics , Animals , Flow Cytometry , Green Fluorescent Proteins , Kinetics , Luminescent Proteins/chemistry , Luminescent Proteins/metabolism , Nucleopolyhedroviruses , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombination, Genetic , Scyphozoa/metabolism , Spectrometry, Fluorescence , Spodoptera/cytologyABSTRACT
The gene encoding the gamma-chain of the human Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates, prepared from insect cells infected with the recombinant virus. At 40 h post infection the corresponding protein was detected as two major bands with apparent molecular weights of 50-60 kDa using a rabbit anti-human IL-2R gamma-receptor specific antiserum. Metabolic labelling with [35S]-methionine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma- protein could also be determined on the surface of infected insect cells by flow cytometer analysis.
