关键词: 鸭, AvBD2, 遗传进化, 重组蛋白, 抗菌活性

Abstract:

【Objective】 The objective of the study was to clone avian β-defensin (AvBD) 2 gene from duck tissues, expression the recombinant AvBD2 protein in E.coli, and determine its antimicrobial activity. 【Method】 The mRNA of duck AvBD2 was cloned from pancreas of duck by RT-PCR. In addition, phylogenetic relationships between amino acid sequence of the duck AvBD2, AvBDs from other avian species, and some mammalian beta-defensin-2 were analyzed. The cDNA of duck AvBD2 was sub-cloned into pGEX-6p-1 vector to construct recombinant plasmid pGEX-duck AvBD2. The recombinant protein was expressed into E. coli. and purified. Antimicrobial activity and physical-chemical stability of the recombinant fusion protein was measured in vitro. 【Result】 The sequence analysis showed that the full length cDNA of duck AvBD2 consisted of 195 bp, encoding 64 amino acid residues. Homology analysis showed that duck AvBD2 shared high amino acid homology with AvBD2 from other avian species. A high expression level of the recombinant duck AvBD2 was noted in E. coli (molecular weight, 32 kD). The recombinant protein exhibited high antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, and Pasteurella multocida, low antimicrobial activity against E. coli., and Salmonella. choleraesuis. The minimal inhibitory concentration (MIC) of the recombinant protein against the above bacteria ranged from 15.25-125 μg?ml-1. The MIC of the recombinant protein against S.choleraesuis was＞400 μg?ml-1. In addition, the results revealed that the recombinant protein retained antimicrobial activity against S. aureus under different temperatures (-20℃ to 100℃) and pH values (3 to 12). 【Conclusion】The AvBD2 gene from ducks was successfully cloned, expressed in E. coli, and purified. The recombinant protein showed antimicrobial activity and retained stability under different temperatures and pH values.

Key words: duck, AvBD2, phylogenetic analysis, recombinant protein, antimicrobial activity