Molecular Cloning, Expression and Characterization of the Porcine β Defensin 2 in E. coli

Chun-li      Li; Yan-cong      Zhao; Xiao-yan      Song; Xian-xian      Huang; Wei-dong      Zhao

doi:10.2174/0929866511320060010

Abstract

Porcine β defensin 2(pBD2)is a cationic 37-amino acid antimicrobial peptide with disulfide bonds. Synthetic pBD2 had broad antimicrobial activity against pathogenic bacteria, and thus pBD2 could be a good candidate as a bactericidal agent for pigs. This study reported the successful recombinant expression of pBD2 in Escherichia coli and analysis of its antimicrobial activity, its hemolytic activity, salt-tolerance and thermal stability as well. The pBD2 gene, obtained by RT-PCR using the tongue total RNA as a template and cloned into pET30a expression vector, was transformed into E. coli BL21 (DE3) plysS. The recombinant pBD2 was expressed after induction by IPTG and purified by His tag affinity column with 95% purity. The recombinant pBD2 exhibited antimicrobial activity against both Gram-positive S. aureus and Gram-negative E. coli including the multi-resistant E. coli. The minimum inhibitory concentration (MIC) of recombinant pBD2 against tested bacteria was 10 μg/mL, and the recombinant pBD2 could kill 50% E. coli at 14.39 μg/mL and S. aureus at 21.1 μg/mL. In addition, pBD2 showed low hemolytic activity, salt-tolerance and thermal stability, the properties would be important for its application in practice.

Keywords: Antimicrobial activity analysis, Porcine β defensin 2, Recombinant expression.

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