Rijun Zhang

A safety assessment of a fowlpox-vectored Mycoplasma gallisepticum vaccine in chickens

A recombinant fowlpox virus vaccine expressing key protective Mycoplasma gallisepticum antigens could facilitate in the prevention both of fowlpox virus and M. gallisepticum infections. Vectormune FP-MG vaccine, a recombinant fowlpox virus expressing both M. gallisepticum 40k and mgc genes, was assessed for its safety in 8-wk-old specific-pathogen-free White Leghorn chickens. The vaccine virus was serially passaged 5 times by wing-web inoculation. Based on the postinoculation clinical observation, gross pathological examination of air sacs and peritoneum, genetic stability evaluation, virus shedding and tissue distribution detection, horizontal transmission ability determination, and protection against fowlpox virus challenge, the Vectormune FP-MG vaccine possesses a high level of safety.

High-Level Overproduction of Thermobifida Enzyme in Streptomyces lividans Using a Novel Expression Vector

In this study, we constructed a novel Streptomyces-E.coli shuttle vector pZRJ362 combining the xylose isomerase promoter and amylase terminator. A gene encoding the endoglucanase Cel6A in Thermobifida fusca was amplified by PCR, cloned into Streptomyces lividans host strain using the novel expression vector and Pichia pastoris GS115 host strain using the vector pPICZα-C, respectively. Afterwards, the expression pattern and the maximum expression level were comparatively studied in both expression systems. The maximum enzyme activity of Cel6A-(His)6 secreted in S. lividans supernatant after 84-h of cultivation amounted to 5.56 U/mL, which was dramatically higher than that secreted in P. pastoris about 1.4 U/mL after 96-h of cultivation. The maximum expression level of Cel6A-(His)6 in S. lividans supernatant reached up to 173 mg/L after 84-h of cultivation. The endoglucanase activity staining SDS-PAGE showed that there were some minor proteins in S. lividans supernatant which may be the Cel6A derivant by proteolytic degradation, while there was no proteolytic product detected in supernatant of P. pastoris.

Novel Hybrid Peptide Cecropin A (1-8)-LL37 (17-30) with Potential Antibacterial Activity.

Hybridizing different antimicrobial peptides (AMPs) is a particularly successful approach to obtain novel AMPs with increased antimicrobial activity but minimized cytotoxicity. The hybrid peptide cecropin A (1–8)-LL37 (17–30) (C-L) combining the hydrophobic N-terminal fragment of cecropin A (C) with the core antimicrobial fragment of LL37 (L) was designed and synthesized. C-L showed higher antibacterial activity against all indicator strains than C and L, and no hemolytic activity to sheep erythrocytes was observed. C-L kills bacterial cells and causes disruption of surface structure, as determined by scanning electron microscopy. Synergistic effects were observed in the combination of C-L with several antibiotics (chloramphenicol, thiamphenicol, or neomycin sulfate) against Escherichia coli and Staphylococcus aureus.

A novel antioxidative peptide derived from chicken blood corpuscle hydrolysate

Chicken blood contains copious amounts of edible protein, which is currently underutilized. In this study, chicken blood corpuscle was hydrolyzed using two proteases (papain and flavourzyme) and its hydrolysis conditions were optimized by means of response surface methodology (RSM). The optimal conditions based on obtaining a hydrolysate with maximum antioxidant activity were E/S 2.0%, temperature 50°C, and time 6.0h. Under the above conditions, the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity, superoxide anion scavenging activity and reducing power of papain and flavourzyme hydrolysate (PFH) were 94.99±0.31%, 57.39±2.82%, and 1.83±0.06, respectively. Additionally, PFH showed not only a stable DPPH scavenging activity in the pH ranges of 1 to 7, but also a good tolerance to magnesium (Mg2+), potassium (K+) and calcium (Ca2+). We also found that PFH retained at least 95% of antioxidant capacity under simulated gastrointestinal digestion. Subsequently, PFH was purified sequentially by ultrafiltration, anion exchange chromatography, gel filtration chromatography, and high-performance liquid chromatography (HPLC). Finally, the sequence of the peptide with the highest antioxidant activity was identified to be AEDKKLIQ (943.5Da) using LC-ESI-MS/MS. Further, this peptide showed free radicals scavenging ability and reducing power as well as that of GSH (P>0.05), further evidencing its potential used as an antioxidative ingredient.

Response surface optimization of enzymatic hydrolysis of duck blood corpuscle using commercial proteases

With the rapid development in livestock and poultry husbandry and increasing shortage of protein sources, recycling of wastes from agricultural and food processing such as blood corpuscles has been regarded as an important industrial procedure to obtain protein sources. This study aimed to find an appropriate method for recycling the considerable amounts of blood corpuscle so as to improve its nutritional value and organoleptic quality. An effective production process for enzymatic hydrolysis of duck blood corpuscle was successfully developed and optimized by response surface methodology. Optimal conditions based on achieving a high value of trichloroacetic acid solubility index were substrate concentration of 14 g/100 mL, temperature 51°C, initial pH 7.0, and time 7.5 h. The electrophoretic patterns of the protein hydrolysate were investigated, and a large diffuse band was observed in the vicinity of 5 kDa. The organoleptic quality of spray-dried blood corpuscle hydrolysate was also evaluated, indicating that enzymatic hydrolysis and decoloration methods were feasible and cost-effective to achieve the desirable bright yellow product without bitterness. In vitro protein digestibility of blood corpuscle hydrolysate was 96.32 ± 0.50%, which was better than that of soybean, fish meal, and casein. Based on the amino acid composition and nutritional parameters, we found that the spray-dried blood corpuscle hydrolysate had abundant nutritional value and high potential for application as an ingredient in nonruminant animal feed.

Expression, Purification, and Characterization of a Novel Hybrid Peptide with Potent Antibacterial Activity

The hybrid peptide cecropin A (1–8)–LL37 (17–30) (C–L), derived from the sequence of cecropin A (C) and LL-37 (L), showed significantly increased antibacterial activity and minimized hemolytic activity than C and L alone. To obtain high-level production of C–L, the deoxyribonucleic acid sequence encoding C–L with preferred codons was cloned into pET-SUMO to construct a fusion expression vector, and overexpressed in Escherichia coli (E. coli) BL21 (DE3). The maximum fusion protein (92% purity) was obtained with the yield of 89.14 mg/L fermentation culture after purification with Ni-NTA Sepharose column. The hybrid C–L was cleaved from the fusion protein by SUMO-protease, and 17.54 mg/L pure active C–L was obtained. Furthermore, the purified C–L showed identical antibacterial and hemolytic activity to synthesized C–L. Stability analysis results exhibited that the activity of C–L changed little below 80 °C for 20 min, but when the temperature exceeded 80 °C, a significant decrease was observed. Varying the pH from 5.0 to 10.0 did not appear to influence the activity of C–L, however, pH below 4.0 decreased the antibacterial activity of C–L rapidly. Under the challenge of several proteases (pepsin, trypsin, and proteinase K), the functional activity of C–L was maintained over 50%. In summary, this study not only supplied an effective approach for high-level production of hybrid peptide C–L, but paved the way for its further exploration in controlling infectious diseases of farm animals or even humans.

Gene expression profile changes in the jejunum of weaned piglets after oral administration of Lactobacillus or an antibiotic

The small intestine plays an essential role in the health and well-being of animals. Previous studies have shown that Lactobacillus has a protective effect on intestinal morphology, intestinal epithelium integrity and appropriate maturation of gut-associated tissues. Here, gene expression in jejunum tissue of weaned piglets was investigated by RNA-seq analysis after administration of sterile saline, Lactobacillus reuteri, or an antibiotic (chlortetracycline). In total, 401 and 293 genes were significantly regulated by chlortetracycline and L. reuteri, respectively, compared with control treatment. Notably, the HP, NOX1 and GPX2 genes were significantly up-regulated in the L. reuteri group compared with control, which is related to the antioxidant ability of this strain. In addition, the expression of genes related to arachidonic acid metabolism and linoleic acid metabolism enriched after treatment with L. reuteri. The fatty acid composition in the jejunum and colon was examined by GC-MS analysis and suggested that the MUFA C18:1n9c, and PUFAs C18:2n6c and C20:4n6 were increased in the L. reuteri group, verifying the GO enrichment and KEGG pathway analyses of the RNA-seq results. The results contribute to our understanding of the probiotic activity of this strain and its application in pig production.