Construction of Bacillus subtilis strain engineered for expression of porcine β-defensin-2/cecropin P1 fusion antimicrobial peptides and its growth-promoting effect and antimicrobial activity
0
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
Objective: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine β-defensin-2 (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. Methods: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge. Results: The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and gram-positive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. Conclusion: The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.
Abstract
Objective: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine β-defensin-2 (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. Methods: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge. Results: The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and gram-positive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. Conclusion: The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.
참고문헌 (29)
- Cheng G, "Antibiotic alternatives: the substitution of antibiotics in animal husbandry?", Front Microbiol, 5, pp.217~ (2014)
- Jukes TH, "Antibiotics in meat production", JAMA, 232, pp.292~293 (1975)
- Stanton TB, "A call for antibiotic alternatives research", Trends Microbiol, 21, pp.111~113 (2013)
- Marshall B, "Food animals and antimicrobials: impacts on human health", Clin Microbiol Rev, 24, pp.718~733 (2011)
- Castanon JI, "History of the use of antibiotic as growth promoters in european poultry feeds", Poult Sci, 86, pp.2466~2471 (2007)
- Wang N, "Pollution characteristics and environmental risk assessment of typical veterinary antibiotics in livestock farms in Southeastern China", J Environ Sci Health B, 49, pp.468~479 (2014)
- Collins MD, "Probiotics, prebiotics and symbiotics: approaches for modulating the microbial ecology of the gut", Am J Clin Nutr, 69, pp.1052~1057 (1999)
- Oggioni MR, "Recurrent Septicemia in an Immunocompromised Patient Due to Probiotic Strains of Bacillus subtilis", J Clin Microbiol, 36, pp.325~326 (1998)
- Baker AA, "The effect of a -based direct-fed microbial supplemented to sows on the gastrointestinal microbiota of their neonatal piglets", J Anim Sci, 91, pp.3390~3399 (2013)
- Sang Y, "Antimicrobial peptides and bacteriocins: alternatives to traditional antibiotics", Anim Health Res Rev, 9, pp.227~235 (2008)
- Fellermann K, "Defensinsinnate—immunity at the epithelial frontier", Eur J Gastroenterol Hepatol, 13, pp.771~776 (2001)
- Han F, "Porcine β-defensin 2 attenuates inflammation and mucosal lesions in dextran sodium sulfate-induced colitis", J Immunol, 194, pp.1882~1893 (2015)
- Wei L, "Expression of Ascaris suum cecropin P1 gene in Pichia pastoris and its antimicrobial activity", Chin J Vet Sci (2011)
- Arcidiacono S, "Membrane permeability and antimicrobial kinetics of cecropin P1 against Escherichia coli", J Pept Sci, 15, pp.398~403 (2009)
- Gregory K, "Immobilization of Escherichia coli cells by use of the antimicrobial peptide cecropin P1", Appl Environ Microbiol, 71, pp.1130~1134 (2005)
- Mukherjee S, "Antimicrobial defense of the intestine", Immunity, 42, pp.28~39 (2015)
- Ostaff MJ, "Antimicrobial peptides and gut microbiota in homeostasis and pathology", EMBO Mol Med, 5, pp.1465~1483 (2013)
- Spizizen J, "Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonuclease", Proc Natl Acad Sci USA, 44, pp.1072~1078 (1958)
- Wang X, "Codon optimization enhances secretory expression of Pseudomonas aeruginosa exotoxin A in E. coli", Protein Expr Purif, 72, pp.101~106 (2010)
- Yang W, "Endogenous animal toxin-like human β-defensin 2 inhibits own K+ channels through interaction with channel extracellular pore region", Cell Mol Life Sci, 72, pp.845~853 (2014)
- Harwig SS, "Cryptdins: endogenous antibiotic peptides of small intestinal paneth cells", Adv Exp Med Biol, pp.251~255 (1995)
- Vargues T, "Efficient production of human β-Defensin 2 (HBD2) in Escherichia coli", Protein Pept Lett, 16, pp.668~676 (2009)
- Wu S, "Effects of the antimicrobial peptide cecropin AD on performance and intestinal health in weaned piglets challenged with Escherichia coli", Peptides, 35, pp.225~230 (2012)
- Li C, "Molecular cloning, expression and characterization of the porcine beta defensin 2 in E. coli", Protein Pept Lett, 20, pp.715~723 (2013)
- Bonavita R, "Mucosal immunity induced by gliadin-presenting spores of Bacillus subtilis in HLA-DQ8-transgenic mice", Immunol Lett, 165, pp.84~89 (2015)
- Zokaeifar H, "Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei", Fish Shellfish Immunol, 33, pp.683~689 (2012)
- Sorokulova I, "The safety of two bacillus probiotic strains for human use", Dig Dis Sci, 53, pp.954~963 (2008)
- Saida F, "Expression of highly toxic genes in E. coli: special strategies and genetic tools", Curr Protein Pept Sci, 7, pp.47~56 (2006)
- Tatsuda D, "Expression and purification of cytokine receptor homology domain of human granulocyte-colony-stimulating factor receptor fusion protein in Escherichia coli", Protein Expr Purif, 21, pp.87~91 (2001)