RESUMO
In order to express swine hepcidin gene in Pichia pastoris, a DNA fragment coding hepcidin gene was synthesized with adaptation to yeast codon usage of highly expressed genes. A Kex2 signal cleavage site was fused in the 5' end of the DNA fragment for getting a peptide with the same N-end as native hepcidin. The 96-bp DNA fragment was ligated into the expression plasmid of pGAPZaA to construct pGAPZaA-hepcidin vector, which was transferred into P. pastoris (X33) to express hepcidin gene for extracellular secretion of protein at 86 µg/mL. A band of 2.76 kD molecular mass was detected by Tricine sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) analysis. Through antibacterial assay, the expressed hepcidin displayed obvious antibacterial activity. The minimal inhibitory concentration (MIC) was 5.38 and 2.69 µg/mL for Staphylococcus aureus and Bacillus subtilis prolification inhibitions, respectively.
Assuntos
Clonagem Molecular/métodos , DNA/síntese química , DNA/genética , Hepcidinas/genética , Pichia/genética , Suínos/genética , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Sequência de Bases , Códon/genética , Vetores Genéticos/genética , Hepcidinas/farmacologia , Humanos , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacosRESUMO
In order to increase nutritive values of soybean meal (SBM), 3 species of microbes were used to ferment SBM. Through a 3 × 3 orthogonal design and parameter measurements of soybean peptide and anti-nutritional factor contents in the fermented soybean meal (FSBM), it was estimated that the best microbial proportion of Bacillus subtilis, Hansenula anomala and Lactobacillus casei was 2:1:2 for SBM fermentation (P < 0.05). The further piglet feeding experiment showed that 10% FSBM substitute for SBM had no significant effect on growth performance of suckling piglets (d 7-28) (P > 0.05). However, newly-weaned piglets (d 28-38) fed 10% FSBM and different levels of plasma protein obtained higher average daily gain (ADG) and feed conversion ratio (FCR), compared with those without FSBM but with 6% plasma protein (P < 0.05). Piglets (d 38-68) fed diets supplemented with FSBM and soybean protein concentrate (SBPC) at 3.75% and 7.5% respectively increased nutrient digestibility, fecal enzyme activity and lactic acid bacteria counts, and decreased fecal Escherichia coli counts (P < 0.05), compared with the control. These data indicated that FSBM had positive effects on nutrient digestibility and fecal microflora for piglets.