Isolation, characterization, and determination on bioactivity of duck avian beta-defensin 5

Abstract:

To clone, express and characterize the antimicrobial activity of duck AvBD5, reverse transcription polymerase chain reaction (RT-PCR) was used to amplify duck AvBD5 gene from lung of duck in the present study. Furthermore, phylogenetic relationships of the duck AvBD5 with AvBDs from other avian species and some mammalian beta-defensin 5 were analyzed. Sequence analysis showed that the cDNA of duck AvBD5 consisted of 201 bp nucleotides, encoding a polypeptide of 66 amino acids. The β-defensin has six conserved cysteines that form three disulfide bonds in a C1-C5, C2-C4, and C3-C6 conformation. It was demonstrated that duck AvBD5 shared 97% amino acid homology with chicken AvBD5. The cDNA of duck AvBD5 was sub-cloned on pGEX-6p-1 vector to construct recombinant plasmid pGEX-duck AvBD5. The recombinant plasmid was transformed into E. coli BL21 and the bacteria were induced with IPTG. After purification, antibacterial activity of the purified recombinant protein was investigated. In addition, effect of ionic strength on the antibacterial activity, and hemolytic activity of the purified recombinant protein were investigated. It was demonstrated that a 32 kD protein in molecular weight was highly expressed. The purified recombinant duck AvBD5 exhibited extensive antimicrobial activity against 11 strain bacteria, including Gram-positive and Gram-negative investigated. In high salt ions conditions, antibacterial activity of recombinant duck AvBD5 protein was decreased. In addition, the recombinant protein of hemolysis activity was extremely low.