Small bioactive peptides, with diverse biological functions, have received increasing attention as physiologically beneficial substances in animal production. The main obstacle to wide application of small bioactive peptides is a lack of cost- effective methods for mass production. In this study, we used glycyl-glutamine (Gly-Gln) as a test case to develop a novel mass-production method for small bioactive peptides. The oligonucleotide encoding Gly-Gln pro-peptide (Gpp) was designed and synthesized. Gpp includes 3 Gly-Gln dipeptides and 2 enzymatic sites for pepsin and trypase, allowing direct digestion and absorption of Gly-Gln in the gastrointestinal tract. The Gpp oligonucleotides were linked to generate an oligomeric oligonucleotide segment containing 12 tandem copies of Gpp. This 12Gpp segment was cloned and expressed in Escherichia coli vector pET32a. By optimizing culture conditions [0.1 mmol L-1 isopropyl-β-D- thiogalactopyranoside (IPTG), 50 μg mL-1 ampicillin (Amp), 30°C for 12 h], the thioredoxin fusion peptides reached 40% of total bacterial protein. After purification, the fusion protein was fed to Kunming mice to determine its effect on mouse immune function. The results showed that similar to Gly-Gln dipeptide, Gpp polymer protein could significantly suppress the proliferation of T and B lymphocytes in blood and spleen, and additionally could significantly improve interleukin-2 (IL-2) and interleukin-6 (IL-6) secretion of blood and spleen lymphocytes. These effects were not observed in mice fed a 2 amino acids mix (glycine and glutamine). These evidences indicated that an efficient digestion of Gpp polymer protein could be achieved when ingested into the animal gut. The expression system in this study provides a potential production method for not only Gly-Gln dipeptide but also other short bioactive peptides.
XU Ping-wen FANG Xin-ling SHU Gang ZHU Xiao-tong LUO Zeng-fu JIANG Qing-yan GAO Ping ZHANG Yong-liang
Background: α-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors (such as α-galactoside) in feed. Intestine-specific and substrate inducible expression of α-galactosidase would be highly beneficial for transgenic animal production. Methods: To achieve the intestine-specific and substrate inducible expression of α-galactosidase, we first identified intestine-specific promoters by comparing the transcriptional activity and tissue specificity of four intestine-specific promoters from human intestinal fatty acid binding protein, rat intestinal fatty acid binding protein, human mucin-2 and human lysozyme. We made two chimeric constructs combining the promoter and enhancer of human mucin-2, rat intestinal trefoil factor and human sucrase-isomaltase. Then a modified lac operon system was constructed to investigate the induction of α-galactosidase expression and enzyme activity by isopropyl β-D-1-thiogalactopyranoside (IPTG) and an α-galactosidase substrate, α-lactose. We declared that the research carried out on human (Zhai Yafeng) was in compliance with the Helsinki Declaration, and experimental research on animals also followed internationally recognized guidelines. Results: The activity of the human mucin-2 promoter was about 2 to 3 times higher than that of other intestine-specific promoters. In the lac operon system, the repressor significantly decreased (P < 0.05) luciferase activity by approximately 6.5-fold and reduced the percentage of cells expressing green fluorescent protein (GFP) by approximately 2-fold. In addition, the expression level of α-galactosidase mRNA was decreased by 6-fold and α-galactosidase activity was reduced by 8-fold. In line with our expectations, IPTG and α-lactose supplementation reversed (P < 0.05) the inhibition and produced a 5-fold increase of luciferase activity, an 11-fold enhancement in the percentage of cells with GFP expression and an increase in α-galactosidase mRNA abundance (by about 5-fold) and α-galactosidase activity (by
Both growth hormone-releasing peptide 6 (GHRP-6) and growth hormone-releasing hormone (GHRH) have potent GH-releasing activity in animals. We have previously demonstrated that the administration of a plasmid encoding the GHRH gene to pregnant mice and pig augmented long-term growth in first generation progeny,and that the administration of GHRP-6 results in growth augmentation in mice and rabbits. However,it has not yet been reported if GHRP-6 induces intergenerational growth effects in pigs. Ploy lactic-co-glycolic acid (PLGA) microsphere adsorption of treatment proteins enhances gene expression,genetic immunization and the ability to protect plasmid DNA and peptides from degradation. The cur-rent study was conducted to determine the growth performance of piglets born to gilts treated with GHRP-6 incorporated into thermosensitive PLGA-PEG-PLGA triblock copolymers. Gilts were injected intra-muscularly once at day 85 of gestation with 30 mg of GHRP6-loaded thermosensitive PLGA-PEG-PLGA triblock copolymers. Piglets were weighed periodically between birth and 28 days. Mean body weights of piglets born to GHRP-6-treated gilts were 6. 58% to 18. 89% (P < 0. 05 ) greater than those of piglets born to control gilts. This study confirms that enhanced maternal GHRP-6 mediated by thermosensitive PLGA-PEG-PLGA can augment growth of piglets.
Feed intake control is vital to ensuring optimal nutrition and achieving full potential for growth and development in poultry. The aim of the present study was to investigate the effects of L-leucine, L-glutamate, L-tryptophan and L-arginine on feed intake and the mRNA expression levels of hypothalamic Neuropeptide involved in feed intake regulation in broiler chicks. Leucine, glutamate, tryptophan or arginine was intra-cerebroventricularly (ICV) administrated to 4d-old broiler chicks respectively and the feed intake were recorded at various time points. Quantitative PCR was performed to determine the hypothalamic mRNA expression levels of Neuropeptide Y (NPY), agouti related protein (AgRP), pro-opiomelanocortin (POMC), melanocortin receptor 4 (MC4R) and corticotrophin releasing factor (CRF). Our results showed that ICV administration of L-leucine (0.15 or 1.5 μmol) significantly (P<0.05) increased feed intake up to 2 h post-administration period and elevated both hypothalamic NPY and AgRP mRNA expression levels. In contrast, ICV administration of L-glutamate (1.6μmol) significantly (P<0.05) decreased feed intake 0.25, 0.5 and 2 h post-injection, and increased hypothalamic CRF and MC4R mRNA expression levels. Meanwhile, both L-tryptophan (10 or 100μg) and L-arginine (20 or 200μg) had no significant effect on feed intake. These findings suggested that L-leucine and L-glutamate could act within the hypothalamus to influence food intake, and that both orexigenic and anorexigenic Neuropeptide genes might contribute directly to these effects.
Different amino acids have been shown to affect feed intake when injected directly into the central nervous system of birds. In the present study, we investigated the effects of L-glutamine and L-alanine on feed intake and the mRNA expression levels of hypothalamic neuropeptides involved in feed intake regulation in broiler chicks. L-Glutamine or L- alanine was intra-cerebroventricularly (ICV) administered to 4-d-old broiler chicks and the feed intake were recorded at various time points. Quantitative PCR was performed to determine the hypothalamic mRNA expression levels of neuropeptide Y (NPY), agouti related protein (AgRP), pro-opiomelanocortin (POMC), melanocortin receptor 4 (MC4R) and corticotropin releasing factor (CRF). Our results showed that ICV administration of L-glutamine (0.55 or 5.5 μmol) significantly increased feed intake up to 2 h post-administration period and the hypothalamic NPY mRNA expression levels, while it markedly decreased hypothalamic POMC and CRF mRNA expression levels. In contrast, ICV administration of L-alanine (4 μmol) significantly decreased feed intake for the first 0.5 h post-administration period, and reduced the hypothalamic AgRP mRNA expression levels, while it remarkablely enhanced the mRNA expression levels of MC4R and CRF. These findings suggested that L-glutamine and L-alanine could act within the hypothalamus to influence feed intake in broiler chicks, and that both orexigenic and anorexigenic neuropeptide genes might contribute directly to these effects.
