RESUMO
A recombinant soluble bovine tumor necrosis factor receptor type I (sboTNF-RI) was expressed in the methylotrophic yeast Pichia pastoris and evaluated for its ability to inhibit bovine tumor necrosis factor alpha (TNF-alpha) cytotoxicity. A cDNA encoding the extracellular domain of bovine TNF-RI was placed under the control of the powerful and tightly regulated alcohol oxidase1 (AOX1) gene promoter of the pPICZa A vector and the resulting construct integrated into the 5' region of the alcohol oxidase genes of GS115 and KM71 strains of Pichia. Soluble bovine TNF-RI was secreted into the medium following induction of the AOX1 gene promoter with methanol, and purified to greater than 95% purity by ion-exchange chromatography. In in vitro assays, the purified recombinant sboTNF-RI will block the cytolytic activity of bovine TNF-alpha on WEHI 164 cells clone 13 by 50% when used at a concentration of 170 microg/ml, and by nearly 90% when used at a concentration of 310 microg/ml. Results of this study suggest that recombinant sboTNF-RI may have therapeutic value as a TNF inhibitor in cattle with coliform mastitis.
Assuntos
Antígenos CD/biossíntese , Receptores do Fator de Necrose Tumoral/biossíntese , Proteínas Recombinantes/biossíntese , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Antígenos CD/isolamento & purificação , Bovinos , Pichia/genética , Reação em Cadeia da Polimerase , Receptores do Fator de Necrose Tumoral/isolamento & purificação , Receptores Tipo I de Fatores de Necrose Tumoral , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Interleukin-3 (IL-3) is one of the cytokines that act during the early and late stages of blood cell formation. To enable the study of the role of IL-3 in bovine haemopoietic stem cell differentiation, the polymerase chain reaction was used to amplify an IL-3 cDNA from first-strand cDNAs prepared from RNA isolated from 4- and 5-hour concanavalin-A-stimulated peripheral blood lymphocytes (PBL) from N'Dama cattle. An analysis of the cDNA sequence reveals that it contains a 432-nucleotide (nt) open reading frame which codes for 144 amino acids (aa). Cleavage of the putative signal peptide consisting of the first 17 aa yields the mature form of the protein (14.5 kDa). Comparisons of the bovine IL-3 sequence with the sheep, human and mouse IL-3 sequences show that the bovine sequence shares 90.7, 55.8 and 51.9% nt identity, respectively, in the coding region, and 85.4, 35 and 27.7% aa identity, respectively.
Assuntos
Bovinos/genética , Interleucina-3/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Genes , Humanos , Linfócitos , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , OvinosRESUMO
Changes in plasma iron and zinc concentration were studied in rabbits following a needle challenge with Trypanosoma brucei brucei clone ILTat 2.1. The infection resulted in a decrease of the concentration of both trace elements. Plasma iron concentrations decreased gradually and were decreased maximally to 52.3% of pre-infection levels on day 18 post-inoculation. Plasma zinc concentrations, on the other hand, decreased more rapidly and were decreased maximally to 27.4% of pre-infection levels on day 3 post-inoculation. The onset of these decreases coincided with the appearance of parasites in the peripheral blood. Furthermore, the magnitude of their decrease correlated closely with the level and duration of the parasitaemia. Other abnormal findings, namely, anaemia and periods of leucocytosis and leukopenia, were also observed. This study therefore demonstrates that depression in plasma iron and zinc concentrations is part of the acute phase response in rabbits infected with this clone of T. b. brucei.
