RESUMEN
Peptide signal sequences attached to or embedded into a core protein sequence control its cellular localization and several post-translational modifications. However, misleading or cumbersome results may be generated when expressing recombinant proteins with modified signal peptides or single domains of larger proteins.
Asunto(s)
Procesamiento Proteico-Postraduccional/genética , Señales de Clasificación de Proteína/genética , Transducción de Señal , Células HeLa , Humanos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
The lack of a sufficient number of antibodies represents an obstacle in the research performed using the zebrafish (Danio rerio) as a model organism. On the other hand, high-throughput generation of antibodies, especially those suitable for immunohistochemistry, is not an established methodology. Here we present the results of an immunization experiment with a zebrafish tissue lysate that allowed for the isolation of hundreds of monoclonal antibodies suitable for labeling of a large variety of zebrafish tissue and cell structures. Some of them were further characterized in terms of detailed localization and age-dependent expression. In addition, the antigen recognized by one of them was first immunoprecipitated and then identified by mass spectrometry. Furthermore, immunofluorescence-competent recombinant antibodies were also isolated by panning large repertoire phage display libraries, in both single-chain (scFv) and single-domain (VHH) format. Such selection alternative is simpler to organize and could contribute to limit the costs of antibody screening and production.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Inmunización , Biblioteca de Péptidos , Proteínas de Pez Cebra/inmunología , Factores de Edad , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Western Blotting , Mapeo Epitopo , Hibridomas/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/aislamiento & purificación , Inmunohistoquímica , Inmunoprecipitación , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Proteínas Recombinantes/aislamiento & purificación , Pez Cebra , Proteínas de Pez Cebra/análisisRESUMEN
OBJECTIVES: Growth hormone (GH) plays a role in thymic function, and recombinant GH may stimulate thymopoiesis in HIV-infected individuals. We performed immunologic analyses in 26 antiretroviral-treated children matched for age, pubertal status, clinical parameters, and antiretroviral exposure who did or did not show an impaired response to GH-release stimulation tests with arginine + GH-releasing hormone. RESULTS: The following abnormalities were found in GH-deficient compared with GH-nondeficient children after >4 years of therapy: CD4 count ( P = .02) and percentage ( P = .03), CD4 as percentage of normal cells for age ( P = .003), serum interleukin-7 concentration ( P = .02), and thymic volume ( P = .01). Naive CD4 (4+62+RA+ and 4+CCR7+RA+) and CD8 (8+CCR7+RA+) lymphocytes were lower in GH-deficient children ( P = .003; P = .007; and P = .02, respectively). Postthymic pathways were also impaired in GH-deficient children. Thus, central memory (4+CCR7+RA-) CD4+ cells were reduced ( P = .006), whereas effector memory (4+CCR7-RA-) CD4+ cells ( P = .002) and late effector CD8+ lymphocytes (8+CCR7-RA+ and 8+27-28-) ( P = .009 and P = .002, respectively) were increased in these children. CONCLUSIONS: Growth hormone plays a role in thymic and postthymic pathways, and defective GH production may be associated with incomplete immunoreconstitution. Immunomodulant agents (including GH) could be useful in patients with defective GH production.
