The Preliminary Study On Mechanism Of Antiviral Activity Of Four Novel Pigeon Avian Beta-Defensin Against Newcastle Disease Virus

Avian β-defensins are a class of endogenous cationic antimicrobial peptides that have widely presented in poultry. The main characteristic structure is six cysteine residues that form three pairs of disulfide bridges. They display a wide range of microbicidal or microbiostatic activities against bacteria, viruses, fungi and play an important role in the innate immune system.Four novel avian β-defensins (AvBDs) were isolated from bone marrow, Bursa of fabricius, bone marrow and liver of pigeons by RT-PCR. Homologies among the4pigeon β-defensins (AvBDs) genes and known avian P-defensins (AvBDs) from other avian species or some mammal species were compared. It respectively named pigeon AvBDl, AvBD2, AvBD7, and AvBD10. Sequence analysis resulted that the coding region of pigeon AvBD1contained198bp nucleotides and encoding a polypeptide of65amino acid residues, with the highest amino acid homology of duck AvBDl (81.54%); pigeon AvBD2contained195bp nucleotides and encoding a polypeptide of64amino acid residues, with the highest amino acid homology of turkey AvBD2(85.94%); pigeon AvBD7and AvBD10both contained204bp nucleotides and encoding a polypeptide of67amino acid residues, respectively. Pigeon AvBD7with the highest amino acid homology of chicken and duck AvBD7(64.71%); pigeon AvBD10with the highest amino acid homology of quail AvBD10(82.35%).The cDNA of pigeon AvBD1, AvBD2, and AvBD7were cloned into pProEX-HTa vector, and pigeon AvBD10was cloned into pProEX-HTb vector, respectively. To construct four recombinant expression plasmids which were translated into E. coli Rosseta and the bacteria were expression induced by IPTG. It was demonstrated by Tricine-SDS-PAGE that four recombinant proteins molecular weight was about13kD, and the recombinant proteins were produced as insoluble bodies in the cells. Additionally, the recombinant proteins were purified, Micrococcus tetragenus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella pullorum were used to research the antibacterial activities of AvBDs and His-tagged protein by colony counting method. The results indicated that His-tagged protein has no antibacterial activities against all of bacteria. On the contrary, the recombinant proteins have different degrees of antibacterial activity. Furthermore, the effect of salt concentration on the antibacterial activity and hemolytic activity of the recombinant proteins were investigated. The results indicated that in high salt ions conditions, antibacterial activity of recombinant proteins was significantly decreased. None of the pigeon AvBDs found significant hemolytic activity. In addition, the dose-dependent survival time of chicken embryos inoculated and the rAvBD-treated NDV was investigated in the present study. It was shown that after recombinant protein neutralizing poison disease vaccination chicken embryos, the content of virus was decreased, when compared with the control at different points. The results suggested that the four recombinant proteins have antiviral activity against NDV in vitro.In order to investigate whether the mRNA expression of pigeon AvBDs was constitutive or inducible, in response to NDV infection, and its signal transduction mechanism of antiviral effect. Pigeons were infected orally with NDV. The mRNA expression of AvBDs and TLR2,3,5,7,15of pigeon in the control or at3and7day post-infection (dpi), were examined through the Real-time RT-PCR. The results showed that the expression of AvBD1was significantly up-regulated in Harderian glands, rectum, duodenum, pancreas, glandular stomach, and cerebrum. The mRNA of AvBD2showed strong up-regulated in rectum, large intestine, pancreas, and glandular stomach. The expression of AvBD7was significantly up-regulated in pancreas, lung, and kidney. The mRNA of AvBD10showed strong up-regulated in spleen and liver after infection. The results showed that that the mRNA expression of the pigeon AvBDs were induced, and in most tissues investigated was highly variable in response to NDV infection. In addition, results showed that the body may through TLR7mediated signal transduction pathways which activating transcription factor induced pigeons AvBDs, and play a pivotal role in the initiation of the immune response to protect these tissues from microbial.