Chief EditorK.M.L. Pathak
Print ISSN 0367-6722
Online ISSN 0976-0555
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Antimicrobial activity of recombinant E. coli expressed chicken AvBD-2 and its mRNA expression in Indian native Aseel and Kadaknath chicken breeds
First Online 26-05-2017|
Avian b defensins (AvBD) are antimicrobial peptides that play a crucial role in the innate immune response in chickens. In the present study, chicken AvBD2 gene was cloned, expressed in E. coli system and the in vitro antimicrobial activity of recombinant peptide was evaluated. The entire mature peptide region of chicken AvBD2 region was amplified and cloned in pUC29 cloning vector. Further, the coding region was sub cloned in pET-28A expression vector. After transformation in E. coli cells, the peptide synthesis was induced and recombinant protein (7.7 kDa) was purified by using Ni-NTA affinity column. The recombinant chicken AvBD2 showed antibacterial activity against S. Pullorum. The minimum bactericidal concentration (MBC) of recombinant chicken AvBD2 evaluated by micro-broth dilution assay was 35 µg/ ml. We also quantified the expression of AvBD2 transcript expression in day-old spleen tissue of Indian native chicken breeds (Aseel and Kadaknath) and White Leghorn. Measurable AvBD2 mRNA expression was found in the spleen of all three breeds. However, no significant difference was found in AvBD2 gene expression between native chickens and White Leghorn.
- Baelmans, R., Parmentier, H.K., Nieuwland, M.G., Dorny, P., Demey, F., Berkvens, D. (2005). Haemolytic complement activity and humoral immune responses to sheep red blood cells in indigenous chickens and in eight German Dahlem Red chicken lines with different combinations of major genes (dwarf, naked neck and frizzled) of tropical interest. Tropical Animal Health Production, 37:173–186.
- Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254.
- Cao, Y., Ma, Q., Shan, A., Dong, N. (2012). Expression in Pichia pastoris and biological activity of avian â-defensin 6 and its mutant peptide without cysteines. Protein and Peptides Letters, 19:1064-1070.
- Evans, E.W., Beac,h F.G., Moore, K.M., Jackwood, M.W., Glisson, J.R., Harmon, B.G., (1995). Antimicrobial activity of chicken and turkey heterophil peptides CHP1, CHP2, THP1, and THP3. Veterinary Microbiology, 47: 295-303.
- Evans, E.W., Beach, G.G., Wunderlich, J., (1994). Harmon, B.G. Isolation of antimicrobial peptides from avian heterophils. Journal of Leukocyte Biology, 56: 661-665.
- Ganz, T., (2003). Defensins: antimicrobial peptides of innate immunity. Nature Review Immunology, 3: 710–720.
- Harmon, B.G., (1998). Avian heterophils in inflammation and disease resistance. Poultry Science, 77: 972-927.
- Harwig, S.S., Swiderek, K.M., Kokryakov, V.N., Tan, L., Lee, T.D., Panyutich, E.A., Aleshina, G.M., Shamova, O.V., Lehrer, R.I., (1994). Gallinacins: cysteine-rich antimicrobial peptides of chicken leukocytes. FEBS Letters, 342: 281-285.
- Haunshi, S., Sharma, D., (2002). Immunocompetence in native and exotic chicken populations and their crosses developed for rural farming. Indian Journal of Poultry Science, 37: 10-15.
- Hellgren, O., Ekblom, R., (2010). Evolution of a cluster of innate immune genes (beta-defensins) along the ancestral lines of chicken and zebra finch. Immunome Research, 6:3.
- Higgs, R., Lynn, D.J., Cahalane, S., Alaña, I., Hewage, C.M., James, T., Lloyd, A.T., O’ Farrelly, C. (2007). Modification of chicken avian beta-defensin-8 at positively selected amino acid sites enhances specific antimicrobial activity. Immunogenetics, 59: 573-580.
- Kannaki, T.R., Verma, P.C., Reddy, M.R., (2012). Differential gene expression of antimicrobial peptides â defensins in the gastrointestinal tract of Salmonella serovar Pullorum infected broiler chickens. Veterinary Research Communications, 36: 57-62.
- Kundu, A., Singh, D.P., Mohapatra, S.C., Dash, B.B., Moudgal, R.P., Bisht, G.S., (1999). Antibody response to sheep erythrocytes in Indian native vis-à-vis imported breeds of chickens. British Poultry Science, 40:40-43.
- Livak, K.J., Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods, 25: 402-428.
- Lynn, D.J., Higgs, R., Gaines, S., Tierney, J., James, T., Lloyd, A.T., (2004). Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken. Immunogenetics, 56:170–177.
- Lynn, D.J., Higgs, R., Lloyd, A.T., O’Farrelly, C., Herve-Grepinet, V., Nys, Y., Brinkman, F.S., Yu, P.L., Soulier, A., Kaiser, P., Zhang, G., Lehrer, R.I., (2007). Avian beta-defensin nomenclature: a community proposed update. Immunology Letters, 110: 86–89.
- Ma, D., Lin, L., Zhang, K., Han, Z., Shao, Y., Wang, R., Liu, S., (2012). Discovery and characterization of Coturnix chinensis avian â- defensin 10, with broad antibacterial activity. Journal of Peptide Science, 18: 224-32.
- Ma, D., Wang, R., Liao, W., Han, Z., Liu, S., (2009). Identification and characterization of a novel antibacterial peptide, avian beta- defensin 2 from ducks. J. Microbiol, 47:610-618.
- Maxwell, A.I., Morrison, G.M., Dorin, J.R., (2003). Rapid sequence divergence in mammalian beta-defensins by adaptive evolution. Molecular Immunology, 40: 413-421.
- Milona, P., Townes, C.L., Bevan, R.M., Hall, J., (2007). The chicken host peptides gallinacins 4, 7 and 9 have antimicrobial activity against Salmonella serovars. Biochemical and Biophysical Research Communication, 356:169–174.
- Peschel, A., (2002). How do bacteria resist human antimicrobial peptides? Trends in Microbiology, 10:179-186.
- Piers, K.L., Brown, M.H., Hancock, R.E., (1993). Recombinant DNA procedures for producing small antimicrobial cationic peptides in bacteria. Gene, 134: 7–13.S
- Rout, P.K., Pani, P.K., Naithani, S., (1992). Genetic susceptibility of indigenous chicks to subgroup A Rous sarcoma virus inoculated via the chorioallantoic membrane. Veterinary Immunology and Immunopathology, 33: 89-102.
- Sadeyen, J.R., Trotereau, J., Protais, J., Beaumont, C., Sellier, N., Salvat, G., Velge, P., Lalmanach, A.C., (2006). Salmonella carrier state in hens: study of host resistance by a gene expression approach. Microbes Infection, 8:1308–1314.
- Sahl, H.G., Pag, U., Bonness, S., Wagner, S., Antcheva, N., Tossi, A., (2005). Mammalian defensins: structures and mechanism of antibiotic activity. Journal of Leukocyte Biology, 77: 466–475.
- Semple, C.A., Taylor, K., Eastwood, H., Barran, P.E., Dorin, J.R., (2006).Beta-defensin evolution: selection complexity and clues for residues of functional importance. Biochemical Society Transactions, 34:257-262.
- Soman, S.S., Arathy, D.S., Sreekumar, E., (2009). Discovery of Anas platyrhynchos avian â-defensin 2 (Apl_AvBD2) with antibacterial and chemotactic functions. Molecular Immunology, 46: 2029-2038.
- Thouzeau, C., Le Maho, Y., Froget, G., Sabatier, L., Le Bohec, C., Hoffmann, J. A., Bulet, P., (2007). Spheniscins, avian beta-defensins in preserved stomach contents of the king penguin, Aptenodytes patagonicus. Journal of Biological Chemistry, 278: 51053–51058.
- Tomita, T., Hitomi, S., Nagase, T., Matsui, H., Matsuse, T., Kimura, S., Ouchi, Y., (2000). Effect of ions on antibacterial activity of human beta defensin 2. Microbiology Immunology, 44:749-754.
- Van Dijk, A., Veldhuizen, E.J., Haagsman, H.P., (2008). Avian defensins. Veterinary Immunology and Immunopathology, 124:1-18.
- Van Dijk, A., Veldhuizen, E.J.A., Kalkhove, S.I.C., Tjeerdsma-van Bokhoven, J.L.M., Romijn, R.A., Haagsman, H.P., (2007). The â defensin gallinacin-6 is expressed in the chicken digestive tract and has antimicrobial activity against food-borne pathogens. Antimicrobial Agents Chemotherapy, 51:912–922.
- Wimmers, K., Ponsuksili, S., Hardge, T., Valle-Zarate, A., Mathur, P.K., Horst, P., (2000). Genetic distinctness of African, Asian and South American local chickens. Animal Genetics, 31:159-165.
- Xiao, Y., Hughes, A.L., Ando, J., Matsuda, Y., Cheng, J.F., Skinner-Noble, D., Zhang, G., (2004). A genome-wide screen identifies a single beta-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins. BMC Genomics, 5:56.
- Yang, D., Chertov, O., Bykovskaia, S.N., Chen, Q., Buffo, M.J., Shogan, J., (1999). Beta defensins: linking innate and adaptive immunity through dendritic and T cell. Science 286: 525–528.
- Yenugu, S., Hamil, K.G., Birse, C.E., Ruben, S.M., French, F.S., Hall, S.H., (2003). Antibacterial properties of the sperm-binding proteins and peptides of human epididymis 2 (HE2) family; salt sensitivity, structural dependence and their interaction with outer and cytoplasmic membranes of Escherichia coli. Biochemistry Journal, 372: 473-483.
- Zekarias, B., Ter Huurne, A.A., Landman, W.J., Rebel, J.M., Pol, J.M., Gruys, E., (2002). Immunological basis of differences in disease resistance in the chicken. Veterinary Research, 33:109-125.
- Zhao, C., Nguyen, T., Liu, L., Sacco, R.E., Brogden, K.A., Lehrer, R.I., (2001). Gallinacin-3, an inducible epithelial beta-defensin in the chicken. Infection Immunity, 69: 2684-2691.
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