title: Molecular cloning, expression and characterization of the porcine β defensin 2 in E. coli.
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.
authors.affiliation: Department of Animal and Veterinary Science, Henan Agricultural University, Zhengzhou, Henan Province, People's Republic of China. hnclli@163.com
authors.forename: Chun-li
authors.initials: CL
authors.lastname: Li
authors.forename: Yan-cong
authors.initials: YC
authors.lastname: Zhao
authors.forename: Xiao-yan
authors.initials: XY
authors.lastname: Song
authors.forename: Xian-xian
authors.initials: XX
authors.lastname: Huang
authors.forename: Wei-dong
authors.initials: WD
authors.lastname: Zhao
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D006461:Hemolysis / Q000187:drug effects; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D012965:Sodium Chloride; D013211:Staphylococcus aureus / Q000187:drug effects; D013552:Swine; D013696:Temperature; D023083:beta-Defensins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D023083:beta-Defensins; D012965:Sodium Chloride