RESUMO
Because the accurate measurement of a number of cytokine mRNA transcripts provides valuable knowledge about cytokine gene regulation, we have developed the Xplore assay for the quantification of cytokine mRNA. This microplate-based assay is rapid (under four hours), quantitative over three orders of magnitude and carries no risk of false-positive values from contamination with amplified target. Here, we describe the use of Xplore assays to measure the steady-state mRNA levels of TNF-alpha and IL-1 beta produced by mouse WEHI and J774 macrophage-like cell lines.
Assuntos
Interleucina-1/genética , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/genética , Animais , Linhagem Celular , CamundongosRESUMO
Interleukin-12 is a heterodimeric cytokine with potent immunoregulatory properties, making it a potential vaccine adjuvant and an immune response modulator. The study of its function is confounded by its heterodimeric structure. In order to facilitate the study of interleukin-12 in both in vitro and in vivo models, we constructed a single-chain porcine interleukin-12 gene and expressed the recombinant protein in Pichia pastoris. Single-chain porcine interleukin-12 was bioactive in vitro on both human and porcine cells as measured by its ability to induce proliferation of lymphoblasts and interferon-gamma secretion by lymph node cells. In contrast, the p40 subunit of porcine interleukin-12 alone did not induce proliferation or inhibit the activity of the single-chain porcine interkeukin-12. The in vivo bioactivity of single-chain porcine interleukin-12 was demonstrated in an oral immunization model where it increased antigen-specific IgA and IgG in jejunal mucus. These results indicate that binding of interleukin-12 to its receptor and transduction of intracellular signals requires both p40 and p35 subunits. The bioactivity of interleukin-12 expressed as a single polypeptide will facilitate its in vivo delivery and study of its structure and function.
Assuntos
Adjuvantes Imunológicos , Interleucina-12/farmacologia , Conformação Proteica , Administração Oral , Animais , Fenômenos Químicos , Físico-Química , Humanos , Imunização , Imunoglobulina A/biossíntese , Imunoglobulina A/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Interferon gama/metabolismo , Interferon gama/farmacologia , Interleucina-12/química , Interleucina-12/genética , Mucosa Intestinal/imunologia , Jejuno/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Estrutura Terciária de Proteína , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12 , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais , Especificidade da Espécie , Relação Estrutura-Atividade , SuínosRESUMO
A Pichia pastoris expression clone has been developed to produce the human cytokine interleukin-17 (hIL-17). Characterization of purified recombinant hIL-17 made with this clone demonstrated that it shared many characteristics with hIL-17 produced in mammalian cells. The hIL-17 produced in Pichia had the correct N-terminus of natural mature hIL-17 and a glycosylation pattern similar to hIL-17 produced in mammalian cells; both Pichia and human cells add approximately 5 kDa of sugars via N-linked glycosylation and both express a mixture of the glycosylated and nonglycosylated forms. Gel filtration provides evidence that the Pichia produced hIL-17 exists as a dimer in solution. A combination of cation-exchange and gel-filtration chromatography yielded 3.5 mg of highly purified and biologically active hIL-17 from a 10-liter fermentation. These results show that P. pastoris is a useful system to produce recombinant hIL-17 in structure/function studies of this molecule.
