Cloning And Initial Characterization Of Two Novel Pigeon Avian β-defensin5

With the more long-term use of in-feed antibiotics, Antimicrobial peptides (AMPs) has come intoour horizon and become a focus for its new type of antibacterial agents in the study of scientists.Defensin is a member of the family of antimicrobial peptides, and it does not only have the efficientlybroad-spectrum antibacterial activity, but also does not induce antibiotic resistance. No matter in theinnate or acquired immune it plays an important role, and has the great potential development as thefeed additive used in animal husbandry and aquaculture.From1980s, it has made breakthrough advances on the Avian β-defensins (AvBDs) of the chicken,ducks, goose, turkeys and the penguins, while there is few reports on the characterization of pigeonβ-defensins. In order to study the new Avian β-defensins further，this test uses the RT-PCR method thatis two novel avian β-defensins (AvBDs) orthologue were isolated from pigeon bone marrow and liver tissues, and make homology analysis among the two novel AvBD genes and AvBDs from other avianspecies or some mammals species and draw a diagram molecular genetic evolutionary tree. Theexperimental result is the complete nucleotide sequence of both cDNAs contained201bp nucleotides,encoding a polypeptide of66amino acids. From the perspective of genetic evolution it was discoveredthat the two pigeon AvBDs shared the highest amino acid homology (87.9%and78.8%)with duckAvBD5, respectively. So it was named as pigeon AvBD5α (bone marrow) and AvBD5β (liver).By the way of addition, these genes have low homology with mammalian β-defensins. The cDNAof pigeon AvBD5α and AvBD5β were cloned into pGEX-6p-1vector to construct recombinant plasmid,which were translated into E. coli BL21and the bacteria were induced with IPTG, respetively. At last,the recombinant protein were expressed and purified. A32-kDa protein was highly expressed. Afterpurification, antibacterial activity of the purified was investigated. Both purified recombinant pigeonAvBD5α and AvBD5β exhibited extensive antimicrobial activities against12bacteria, includingGram-positive and Gram-negative（.p<0.05or p<0.01）. The recombinant pigeon AvBD5α and AvBD5βprotein have high antibacterial activities including P. multocida、P. Aeruginosa、P. Mirabilis and S. Aureus, while low antibacterial activities with S. Pullorum. The antibacterial activities of recombinantpigeon AvBD5α and AvBD5β have no significant difference. Moreover, that GST has no antibacterialactivities against all of bacteria by the investigation.Furthermore, effect of ionic strength on the antibacterial activity, and hemolytic recombinantprotein activity of the purified recombinant protein were investigated. It is found that both AvBD5αand AvBD5β were sensitive to P. multocida in the high salt concentration. The bacterial survival wasdecreased with the salt concentration’s elevation (0～150mM). Both AvBD5α and AvBD5β have nohaemolysis.（p>0.05）, and have no significant difference on the comparison with negative control.