Antibacterial Activity Of Recombinant Humanβ-defensin3

Human beta-defensin-3(HBD-3) is kind of antimicrobial peptides found in recent studies, the molecular mass is about3-5KDa, it has broadspectrum antimicrobial activity without drug resistance, Various immune adjustment effect was showed to Connect inherent immunity and acquired immune response. Nowdays the antibiotic abuse is so common, the problems such as possible residue in the body, allergic reactions, bacteria resistance, forced humans in urgent need of developing a novel antibacterial substance, therefore the research and development of antimicrobial peptides become a hot spot. Defensin is kind of cationic antimicrobial peptide, containing β-sheet structure, the human defensins can be divided into three categories:α,β, and0defensins. Human β-defensin-3(Human beta-defensin-3, HBD-3) was originally found in psoriatic skin lesions, the tissue extracts of the skin, tonsils, respiratory, reproductive tract epithelial cells, adult skeletal muscle, heart, thymus, esophagus, oral mucosa and placental expression have varying degrees of HBD-3.It has broad spectrum antimicrobial, the small salt concentration impact, low toxicity, and it is effective against the multi-resistant bacteria,so it has high research value.According to E. coli preference codons, compound HBD-3gene was designed, then was cloned into the prokaryotic expression vector pET30a-SPM-ELP, so obtained the recombinant expression vector PET-30a-ELP-SPM-HBD3. Prokaryotic expression system containing elastin like polypeptides is one of the most successful exogenous protein expression system, ELP is a class of synthetic biological macromolecules, containing repeat pentapeptide sequence unit VPGXG (Val-Pro-Gly-Xaa-Gly, Xaa can be any amino acid except Pro), ELP features could be induced at different temperature, higher than a particular phase transition temperature it is precipitation, it is the soluble state when the temperature is lower. ELP fusion protein may have the similar properties, therefore, With temperature control and centrifugation the aim protein can be separated. Self-processing module has a label of the unit within a self-shear characteristics, in the presence of Ca2+executable it presents self-shearing function, so the small protein can be obtained by Cutting fluid include Ca2+.The ELP system has a phase transition temperature, the recombinant bacteria were induced at a low temperature, precipitated by the saline solution repeatedly washing after centrifugation at different temperature, the pure fusion protein detected by SDS-PAGE proved that it expressed the expected molecular weight precursor protein ELP-SPM-HBD3, then it was cut, to obtain the desired protein, It was confirmed that the purified HBD3have antibacterial activity in agar diffusion antibacterial test including Candida albicans and Staphylococcus aureus. MIC of HBD-3againest Staphylococcus aureus was80μg/ml, and it had antibacterial activity in an animal model.It is result that in such a method, separation of the recombinant protein, purification process is greatly simplified and the cost is significantly reduced,so this experiment lay the foundation for further research and development of HBD-3,...