[show abstract][hide abstract] ABSTRACT: Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34°C in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95%). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 μg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5μg/ml and 25μg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86% at the mBD2 concentration of 100 μg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.
Brazilian Journal of Microbiology 07/2011; 42(3):1180-7. · 0.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human influenza A virus (IAV) is a major cause of life-threatening respiratory tract disease worldwide. Defensins are small cationic peptides of about 2-6 kDa that are known for their broad-spectrum antimicrobial activity. Here, we focused on the anti-influenza A activity of mouse beta-defensin 2 (mBD2). The prokaryotic expression plasmid pET32a-mBD2 was constructed and introduced into Escherichia coli Rosseta gami (2) to produce recombinant mBD2 (rmBD2). Purified rmBD2 showed strong antiviral activity against IAV in vitro. The protective rate for Madin-Darby canine kidney cells was 93.86% at an rmBD2 concentration of 100 microg/ml. Further studies demonstrated that rmBD2 prevents IAV infection by inhibiting viral entry. In addition, both pretreatment and postinfection treatment with rmBD2 provided protection against lethal virus challenge with IAV in experimental mice, with protection rates of 70 and 30%, respectively. These results suggest that the mBD2 might have important effects on influenza A virus invasion.
Archives of Virology 03/2010; 155(4):491-8. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mouse beta defensin-1 (mBD-1) is a cationic 37-amino acid antimicrobial peptide with three conserved cysterine disulfied bonds. It exhibits a broad antimicrobial spectrum, but mBD-1 against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) is poorly understood. This study describes the mBD-1 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released mature mBD-1. By constructing the expression plasmid (pET32a-mBD1), high yields of soluble mBD-1 fusion protein (0.67 g/L) could be obtained in E. coli and cleaved by enterokinase. The digested product was further purified and desalted with the final amount of pure mature mBD-1 being 0.14 g/L. Classical fungi growth inhibition assay showed clear antifungal activity against C. albicans and C. neoformans with IC(50) of 5 and 2 microM, respectively. The results show that the mBD-1 control fungal colonization through hyphal induction, direct fungicidal activity, and the activity is suppressed by increasing NaCl concentration. Successful expression of the mBD-1 peptide in E. coli offers a basis for further studying its antifungal mechanisms and may provide significance in developing this peptide to an antifungal drug.
Applied biochemistry and biotechnology 04/2009; 160(1):213-21. · 1.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: Influenza (flu) pandemics have presented a threat to human health in the past century. Because of outbreaks of avian flu in humans in some developing countries in recent years, humans are more eager to find a way to control flu. Mammalian beta-defensins (beta-defensins) are associated primarily with mucosal and skin innate immunity. Previous studies have demonstrated antimicrobial properties of a variety of defensin peptides. We have identified the presence of mouse beta-defensin 1, 2, and 3 genes (Mbd-1, 2, and 3) in trachea and lung tissues by RT-PCR before and after infection with influenza virus. We constructed a eukaryotic expression plasmid containing Mbd-3, pcDNA 3.1(+)/MBD-3, and the plasmid was introduced into Madin-Darby canine kidney (MDCK) cells by transfection. The expression of Mbd-3 in MDCK cells was verified by immunofluorescence test, RT-PCR, and Western blot. The pcDNA 3.1(+)/MBD-3 plasmid was injected into mice to observe its effect against influenza A virus (IAV) in vivo. Mouse beta-defensin genes could be expressed in trachea and lung tissues before IAV infection, but expression of Mbd-2 and Mbd-3 was increased significantly after IAV infection. The survival rate of mice with MBD-3 against IAV challenge was 71.43%, and MDCK cells with MBD-3 could clearly inhibit IAV replication. The results demonstrated that mouse beta-defensins possess anti-influenza virus activity, suggesting that mouse beta-defensins might be used as agents to prevent and treat influenza.
Archives of Virology 02/2009; 154(4):639-47. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mouse beta defensin-1 (mBD-1) is a cationic peptide with broad antimicrobial activity. The mBD-1 gene was cloned and fused with TrxA to construct pET32-mBD1, which was transformed into E. coli BL21 (DE3). The optimal expression conditions of fusion protein TrxA–mBD1 were: cultivation at 32°C in 2×YT medium, induction
with 0.2mM isopropylthio-d-galactoside (IPTG), and post-induction expression for 8h. The fusion protein was highly soluble (90.0%) and accounted for
65% of the total soluble protein; and its volumetric productivity reached 0.67g/l, i.e., 0.14g/l of recombinant mBD-1. At
5μM, purified recombinant mBD-1 killed 50% of Candida albicans.
World Journal of Microbiology and Biotechnology 01/2009; 25(5):917-920. · 1.26 Impact Factor