ABSTRACT
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.
Subject(s)
Protein Processing, Post-Translational/genetics , Protein Sorting Signals/genetics , Signal Transduction , HeLa Cells , Humans , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Recombinant Proteins/metabolismABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/isolation & purification , Immunization , Peptide Library , Zebrafish Proteins/immunology , Age Factors , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/genetics , Blotting, Western , Epitope Mapping , Hybridomas/metabolism , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/isolation & purification , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/isolation & purification , Immunohistochemistry , Immunoprecipitation , Mice , Mice, Inbred BALB C , Microscopy, Fluorescence , Recombinant Proteins/isolation & purification , Zebrafish , Zebrafish Proteins/analysisABSTRACT
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.