| Defensins are cationic cystine-rich cationic antimicrobial peptides that have been identified in animals and plants. The defensins attack a wide rang of microorganisms including Gram-positive and Gram-negative bacteria, fungi, and viruses. Compared with other antibiotic medicines, antimicrobial peptides are more effective and not easy to induce the drug resistance. They may be used as feed additives to provide an alternative to antibiotics in animal feed. In poultry, only the P-defensins (AvBDs) have been reported to exist. AvBDs have been shown to display a broad spectrum of antimicrobial activities and play a pivotal role in the innate immune system of avians.In order to identify potential AvBDs from ducks, mRNAs of five novel AvBDs were isolated from bone morrow and lung tissues of ducks by RT-PCR, respectively. Homologies among the five novel AvBD genes and AvBDs from other avian species were analyzed. The result showed that they were duck AvBD1,3,5,6and16. Sequence analysis showed that duck AvBD1consisted of198bp encoding65amino acids and shared the amino acid homology (78%) with Ostrich AvBD1; duck AvBD3consisted of182bp encoding60amino acids and shared the highest amino acid homology (100%) with its chicken analog; duck AvBD5consisted of201bp encoding66amino acids and shared the highest amino acid homology (97%) with its chicken analog; duck AvBD6consisted of204bp encoding67amino acids and shared the highest amino acid homology (100%) with chicken AvBD6; duck AvBD16consisted of155bp encoding50amino acids and shared the amino acid homology (62%) with chicken AvBD3.The cDNA of duck1,3,5,6and16were cloned into pGEX-6p-1vector to construct recombinant plasmid, which were translated into E. coli BL21and the bacteria were induced with IPTG, respetively. Additionally, putative mature AvBDs were synthesized commercially and purified. Twelve pathogenic bacterial strains were used to investigate the antibacterial activities of GST, rAvBDs and sAvBDs. In addition, effect of ionic strength on the antibacterial activity, and hemolytic activity of the both protein were investigated. It was showed that GST has no antibacterial activities against all of bacteria investigated. Both recombinant and synthetic forms of duck AvBD1,3,5,6and16showed antibacterial activities against most of the bacteria investigated, including Gram-negative and Gram-positive bacteria (P<0.05or P<0.01). In addition, the antibacterial activity of all the AvBDs against M. tetragenus and P. multocida decreased at high salt ions conditions (P<0.05or P<0.01). However, none of the duck AvBDs showed significant hemolytic activity (P>0.05). The dose-dependent survival time of duck embryos inoculated with the rAvBD1,3,5,6,16treated duck hepatitis virus (DHV) was investigated. It was shown that survival time of duck embryos was prolonged significantly by all of the rAvBDs (P<0.05or P<0.01), when compared with the control and GST. The results suggested that the five novel duck AvBDs exhibit significant antiviral activity against DHV in vitro.In an attempt to understand wether the mRNA expressions of AvBD in the tissues of ducks was effected in response to DHV infection, we determined the expression profile of AvBDs in eight tissues, including the liver, bone marrow, kidney, cecal tonsil, spleen, bursa of Fabricius, thymus, and lung of ducklings at24,32,48,72and96h after infection. Additionally, the levels of duck IL-2, IL-18, IFN-γ and TLR-7were measured in six tissues as above except for kidney and lung. The results showed that all of the mRNA expressions were induced or up-regulated in liver in response to DHV infection, except for AvBD1, AvBD3and IFN-y(P<0.05or P<0.01). Furthermore, expression of TLR-7showed high positive relation to the expressions of AvBDs or cytokines in most tissues investigated. The present results suggested that the anti-viral activities of these AvBDs and cytokines against DHV in vivo were partially induced by signaling pathway mediated by TLR-7. |
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