Objetivo
Infection with the AlDS-causing human immunodeficiency virus (HIV) still presents one of the current major challenges to mankind. In the context of the wide-spread occurrence of HIV infection, the long asymptomatic carrier state and the lethality of the disease, it is highly desirable to have access to fast, reliable and inexpensive assays to detect anti-HlV antibodies in HlV-infected individuals. The aim of this proposal is to develop new diagnostic probes for simple and fast detection of anti-HlV antibodies in human plasma. The proposed work is based on the previous discovery of Partner 2 that the insertion of antigenic peptides in specihc sites of the bacterial enzyme beta-galactosidase causes a severe reduction of the enzymatic activity, which can be partially recovered upon antibody binding to the inserted antigenic peptide (Benito et al., 1996, J. Biol. Chem. 271, 21251). This phenomenon will be exploited to generate new beta-galactosidase HIV antigenic fusion proteins as enzymatic probes for anti-HlV antibody detection. The recovery of beta-galactosidase activity upon antigen-antibody binding will allow the detection of antibodies by simple colorimetric quantification of beta-galactosidase activity in a homogeneous test system, involving only the antigenic fusion protein, the chromogenic substrate and a serum sample for testing. The beta-galactosidase enzyme (EC 3.2.1.23) encoded by the Eschenchia coli lacZ gene, is a high molecular mass multimeric protein composed of four non-covalently linked subunits. Analysis of beta-galactosidase activity is accomplished by simple colorimetric assays (Miller, 1972, Experiments in Molecular Genetics, Cold Spring Harbour Laboratory, p. 352) and small-scale purification of the enzyme can be carried out in one-step purification procedures (Ullmann, 1984, Gene 29, 27). beta-Galactosidase and recombinant beta-galactosidase fusion proteins are in general stable, non-toxic for the producing cells, and they can be produced in high yields in recombinant bacteria. Moreover, beta-galactosidase does not react with human serum rendering this enzyme ideal for the design of new molecular probes for diagnosis of human infectious diseases. Partner 4, an important European industrial company with a wide and recognized expertise in immunodiagnostics, has suggested specific HIV Env antigenic peptides as a model of commercial relevance for the design, construction and improvement of beta-galactosidase molecular probes. Partner 2, a research laboratory devoted to the design and production of recombinant proteins, will construct fusion proteins with different HIV Env peptides inserted within the activating loop of the enzyme and determine their enzymatic properties. Among those constructs, protein chimeras responsive to antibody-mediated reactivation will be transferred to Partner 3. This research laboratory, mainly working on high cell density production of recombinant proteins and protein characterization, will establish medium- and large-scale production and purification protocols for these proteins, characterize the antibody binding parameters in different constructs and also determine the effects of the peptide insertion on the in vivo and in vitro stability of the chimeric proteins. The most interesting proteins will be transferred to Partner 4 for the evaluation of the diagnostic feasibility of the concept. Partner 2 will also transfer the amino acid sequences of both the responsive and non-responsive proteins to Partner 3. This research group is devoted to the modeling of protein structure. Conformational studies will be done by this partner on selected constructs, focusing on the activating interface and the insertion sites. Three-dimensional data of native beta-galactosidase obtained in Prof. Matthews' laboratory (Jacobson et al. 1994, Nature 369, 761) will be used for this purpose. The aim of this task is to explore the structural basis of reactivation upon antibody binding, to provide a rational for the construction of new modified mutants, especially concerning the appropriate insert length and the insertion sites of the antigenic peptides. From an integrated exchange of information between Partners 1, 2, 3, and 4, sequence modifications on the previous constructs will be suggested to Partner 2, in order to improve critical properties of these proteins, such as increasing structural stability, reducing background activity and improving the enzymatic response. This iterative interaction will result in new sets of proteins to be tested again by Partner 4 who will evaluate these new molecular probes for their applicability in commercial homogeneous assay kits. All the partners in this collaborative research project have agreed to consider this project with high priority within their research activities.
Keywords: HIV infection, diagnostic tests, beta-galacosidase fusion proteins, recombinant Escherichia coli
Ámbito científico (EuroSciVoc)
CORDIS clasifica los proyectos con EuroSciVoc, una taxonomía plurilingüe de ámbitos científicos, mediante un proceso semiautomático basado en técnicas de procesamiento del lenguaje natural. Véas: El vocabulario científico europeo..
CORDIS clasifica los proyectos con EuroSciVoc, una taxonomía plurilingüe de ámbitos científicos, mediante un proceso semiautomático basado en técnicas de procesamiento del lenguaje natural. Véas: El vocabulario científico europeo..
- ciencias naturales ciencias biológicas biología molecular genética molecular
- ciencias naturales ciencias biológicas microbiología virología
- ciencias médicas y de la salud ciencias de la salud enfermedad infecciosa virus de ARN VIH
- ciencias naturales ciencias químicas química orgánica aminas
- ciencias naturales ciencias biológicas bioquímica biomoléculas proteínas enzima
Para utilizar esta función, debe iniciar sesión o registrarse
Programa(s)
Programas de financiación plurianuales que definen las prioridades de la UE en materia de investigación e innovación.
Programas de financiación plurianuales que definen las prioridades de la UE en materia de investigación e innovación.
Tema(s)
Las convocatorias de propuestas se dividen en temas. Un tema define una materia o área específica para la que los solicitantes pueden presentar propuestas. La descripción de un tema comprende su alcance específico y la repercusión prevista del proyecto financiado.
Las convocatorias de propuestas se dividen en temas. Un tema define una materia o área específica para la que los solicitantes pueden presentar propuestas. La descripción de un tema comprende su alcance específico y la repercusión prevista del proyecto financiado.
Convocatoria de propuestas
Procedimiento para invitar a los solicitantes a presentar propuestas de proyectos con el objetivo de obtener financiación de la UE.
Datos no disponibles
Procedimiento para invitar a los solicitantes a presentar propuestas de proyectos con el objetivo de obtener financiación de la UE.
Régimen de financiación
Régimen de financiación (o «Tipo de acción») dentro de un programa con características comunes. Especifica: el alcance de lo que se financia; el porcentaje de reembolso; los criterios específicos de evaluación para optar a la financiación; y el uso de formas simplificadas de costes como los importes a tanto alzado.
Régimen de financiación (o «Tipo de acción») dentro de un programa con características comunes. Especifica: el alcance de lo que se financia; el porcentaje de reembolso; los criterios específicos de evaluación para optar a la financiación; y el uso de formas simplificadas de costes como los importes a tanto alzado.
Coordinador
38124 BRAUNSCHWEIG
Alemania
Los costes totales en que ha incurrido esta organización para participar en el proyecto, incluidos los costes directos e indirectos. Este importe es un subconjunto del presupuesto total del proyecto.