mRNA Cloning, Tissue Distribution and Expression of Duck Avian Beta-Defensin 9 in E. coli

【Objective】 The study was conducted to clone avian beta-defensin 9 (AvBD9) gene from duck tissues and determine tissue distribution of the gene in ducks. In addition, expression of recombinant AvBD9 protein in E. coli and determination of its antimicrobial activity were performed. 【Method】 The mRNA of duck AvBD9 was cloned from liver of duck by RT-PCR. In addition, differential mRNA expression of the gene has been demonstrated across a panel of tissues in the duck. The cDNA of duck AvBD9 mRNA was sub-cloned into EcoR I and Sal I sites of pGEX-6p-1 vector to construct recombinant plasmid pGEX-duck AvBD9. The recombinant plasmid was transformed into E. coli BL21 and the bacteria were induced with IPTG. The recombinant fusion protein was purified by affinity chromatograph. Antimicrobial activity, physio-chemical characteristics of the recombinant fusion protein were measured by inhibition zone assay. 【Result】 The sequence analysis showed that the full length cDNA of duck AvBD9 consisted of 204 bp encoding 67 amino acid residues. Duck AvBD9 mRNA was widely expressed in the tissues of duck. It was demonstrated by SDS-PAGE that a 31 kD protein which was equal to duck AvBD9 protein in molecular weight was highly expressed as insoluble bodies in E. coli BL21. The recombinant duck AvBD9 exhibited antimicrobial activity against Escherichiacoli, Lactobacilli acidophilus, Bacillus subtilis, Staphylococcus aureus, and Pasteurella multocida. In addition, the results revealed that the recombinant protein retained its antimicrobial activity under different temperatures (from –70℃ to 100℃) and pH values (from 3 to 10). 【Conclusion】 The full length cDNA of duck AvBD9 consistes of 204 bp encoding 67 amino acid residues. Duck AvBD9 mRNA was widely expressed in the tissues of duck. Duck AvBD9 protein is highly expressed as insoluble bodies in E. coli BL21. The recombinant protein exhibited broad-spectrum antimicrobial activity and retained antimicrobial activity under different temperatures and pH values.