RESUMO
BACKGROUND: The baculovirus expression vector system (BEVS), utilizing the Autographa californica nuclear polyhedrosis virus (AcNPV), has turned out to be an attractive alternative for high-level expression (<600 mg/l) of recombinant proteins. However, there is a shortage of reliable methods for monitoring the infection process in situations where marker proteins cannot be used. METHODS: Three recombinant baculoviruses, FastBac1-wtGFP, VTBac-GFP, and VL1392-hIL-2Ralpha, all having the genes inserted under the transcriptional control of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV), were used to infect Spodoptera frugiperda (Sf9) and Mamestra brassicae (IZD-MB-0503) insect cells. The infection process of the recombinant baculoviruses was monitored by flow cytometric side-scatter and fluorescence intensity analyses over a period of 6-96 h. RESULTS: A clear correlation between the side-scatter (SSC) signal and the relative fluorescence was observed for both of the infected cell lines, compared to noninfected cells. Comparison of SSC histograms from noninfected insect cells with cells infected with the nonfluorescent recombinant baculovirus VL1392-hIL-2Ralpha showed a clear increase of SSC for the infected cells. CONCLUSIONS: The SSC parameter can therefore be utilized for flow cytometric monitoring of a baculovirus infection process in situations where suitable markers are not available.
Assuntos
Baculoviridae/fisiologia , Citometria de Fluxo/métodos , Spodoptera/citologia , Animais , Linhagem Celular , Separação Celular , DNA Viral/análise , Fluorescência , Expressão Gênica , Proteínas de Fluorescência Verde , Indicadores e Reagentes/metabolismo , Proteínas de Insetos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nucleopoliedrovírus/genética , Proteínas Recombinantes de Fusão/metabolismo , Espalhamento de Radiação , Spodoptera/metabolismo , Spodoptera/virologia , TransfecçãoRESUMO
A homogeneous receptor-ligand assay based on fluorescence resonance energy transfer is described. In the assay, recombinant human interleukin 2 (IL-2) and a monoclonal antibody against the human IL-2 receptor alpha chain were labelled with a highly fluorescent europium chelate and Cy5, respectively. As a result of a successful receptor-ligand complex formation, these labels are brought into close proximity, which will thereby allow an energy transfer to occur from the donor (europium) to the acceptor (Cy5), upon excitation of the donor. Utilization of specific non-neutralizing antibodies made it possible to use crude hIL-2R alpha membranes prepared from recombinant baculovirus-infected Sf9 insect cells. The specific energy transfer was measured at the emission wavelength of Cy5 using a time-resolved fluorometer. The data presented, demonstrate that this assay design can be utilized for saturation as well as competitive binding experiments in addition to regular receptor titrations. As a rapid simple homogeneous assay it is particularily suitable for high throughput screening analyses.
Assuntos
Interleucina-2/metabolismo , Receptores de Interleucina-2/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Ligação Competitiva , Carbocianinas/metabolismo , Células Cultivadas , Európio/metabolismo , Técnica Direta de Fluorescência para Anticorpo , Fluorimunoensaio/métodos , Humanos , Ligantes , Espectrometria de Fluorescência , SpodopteraRESUMO
A time-resolved fluorometric, solid phase, receptor ligand interaction assay is described. The assay consists of wells coated with anti-human IL-2 receptor alpha (hIL-2R alpha) monoclonal antibodies (mAb), europium labelled hIL2 (Eu-IL-2) and human recombinant IL-2 receptor alpha subunits expressed in the baculovirus expression vector system (BEVS). In the assay hIL-2R alpha-Eu-IL-2 complexes bind to the solid phase mAb. Receptor bound Eu is dissociated into an enhancer solution where it forms highly fluorescent complexes. The fluorescence is measured in a time-resolved fluorometer. The Kd value calculated from the saturation curve is in good agreement with previously reported values for the low affinity type of IL-2R, making the described assay a simple and nonradioactive alternative for measurement of soluble hIL-2R alpha in biological systems. Furthermore this assay format provides convenient separation of bound ligand from unbound and is therefore suitable for high throughput screenings.
Assuntos
Fluorometria/métodos , Técnicas de Imunoadsorção , Interleucina-2/metabolismo , Receptores de Interleucina-2/metabolismo , Európio , Corantes Fluorescentes , Humanos , Ligantes , Ligação Proteica , Proteínas Recombinantes/metabolismoRESUMO
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.
