RESUMO
IFN-gamma is a cytokine produced primarily by both T lymphocytes and natural killer cells and it is considered to be an attractive therapeutic molecule. In the present study, a DNA sequence encoding the mature murine IFN-gamma (muIFN-gamma) protein was cloned and expressed in the food-grade lactic acid bacterium Lactococcus lactis. The activity of recombinant muIFN-gamma produced by genetically engineered L. lactis was confirmed in an antiviral assay using MoV cells infected with Vesicular Stomatitis Virus. The data provide the first demonstration that a Gram-positive bacterium, L. lactis, is able to produce functional muIFN-gamma. This recombinant strain could lead to the development of a new, well-tolerated vector to deliver active muIFN-gamma at the mucosal level.
Assuntos
Interferon gama/biossíntese , Lactococcus lactis/metabolismo , Animais , Linhagem Celular , Vetores Genéticos/metabolismo , Interferon gama/genética , Interferon gama/farmacologia , Lactococcus lactis/genética , Macrófagos/imunologia , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacosRESUMO
A system for controlled targeting of heterologous protein was developed in the food-grade bacterium Lactococcus lactis. It is composed of the L. lactis strain NZ9000 and of two broad host range expression vectors pCYT:Nuc and pSEC:Nuc for, respectively, cytoplasmic and secreted staphylococcal nuclease (Nuc) nisin-inducible production. The level of intracellular production of Nuc measured with pCYT:Nuc (3 mg x l(-1)) is significantly lower than the one obtained with pSEC:Nuc ( approximately 20 mg x l(-1)). The secretion efficiency (SE) of Nuc is estimated to be approximately 70%, corresponding to approximately 15 mg of secreted Nuc x l(-1). Furthermore, we established that Nuc production continued in L. lactis 10 h after a 1-h nisin-pulse induction. This system was then used for intra- and extracellular production of a protein of therapeutical interest in L. lactis, the ovine interferon-omega (IFN-omega). The SE and the quantity of secreted active IFN-omega were evaluated respectively to be approximately 70% and approximately 1 mg x l(-1) ( approximately two-fold higher than the cytoplasmic form).
Assuntos
Biotecnologia/métodos , Interferons/genética , Lactococcus lactis/genética , Nuclease do Micrococo/genética , Animais , Antibacterianos/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Interferons/metabolismo , Lactococcus lactis/metabolismo , Nuclease do Micrococo/metabolismo , Nisina/farmacologia , Plasmídeos , OvinosRESUMO
Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.