Cloning and bioactivity determination of pigeon avian beta-defensin 1

Abstract:

[Objective] The study was conducted to clone avian beta-defensin1 (AvBD1) gene from pigeon tissues, expression of recombinant AvBD1 protein in Escherichia coli and determine its biological characteristics. [Methods] The AvBD1 gene was amplified from bone marrow of pigeon by RT-PCR, differential mRNA expression of this gene was evaluated by real-time PCR. The cDNA of pigeon AvBD1 was sub-cloned into EcoR I and Xho I sites of pProEX-HTa vector to construct recombinant plasmid pProEX-pigeon AvBD1, the recombinant plasmid was expressed into E. coli, the bacteria induced with IPTG. The recombinant protein was purified, antimicrobial activity, physio-chemical characteristics of the recombinant protein were measured by colony counting assays in vitro. [Results] Sequence analysis showed that the cDNA of pigeon AvBD1 consisted of 198 bp nucleotide, encoding a polypeptide of 65 amino acids. Homology analysis showed that pigeon AvBD1 shared the highest percentage of amino acid homology (81.5%) with duck AvBD1. The mRNA was widely expressed in immune system and digestive system. It was demonstrated by Tricine-SDS-PAGE that a 8.8 kD protein which was equal to pigeon pProEX-HTa AvBD1 protein in molecular weight was expressed. The purified recombinant pigeon AvBD1 exhibit extensive antimicrobial activity. In high salt ions conditions, the antibacterial activity was significantly decreased. In addition, little hemolysis of erythrocytes was observed at any concentration of the peptide. [Conclusion] The pigeon AvBD1 gene was successfully cloned from bone marrow, the mRNA was widely expressed in immune system and digestive system. The recombinant protein exhibit extensive antimicrobial activity, has little hemolytic properties. High salt concentration significantly decreased its antibacterial activity.