| Antimicrobial peptide is a key element to the Innate immunity.β-defensin contains six conservative cys residues, and was made up by 64 amino acid. It is mainly in epithelial tissue of animals. The mature peptides ofβ-defensin, MW 3.5-6KDa, can inhibit the growth of gram positive bacteria, gram negative bacteria and viruses.Objective: The method that can be obtained through genetic engineering with biological activity, a lot of easy purification of sheep sBD-1 was founded and established.Methods: The psBD-1 that sBD-1 signal sequence contained the original peptide and msBD-1 without signal peptide sBD-1 were expressed in E. coli BL21 (DE3). Then, the production was tested on the Escherichia coli and Staphyloccocus aureus,after metal chelate chromatography, enterokinase cut, desalting and freeze drying. The activity zone of sBD-1 was msBD-1. And the pichia pastoris expression vectors of Ppic9k-msBD-1 and Ppic9k-msBD-1-T whose C terminal was added six His-tag were constructed. The condition of expression time, pH, OD600 and the concentration of methanol was optimized. And these pichia pastoris was fermentation. The fermentation production was purified. And the two peptides were tested to inhibite Escherichia coli, Staphylococcus aureus, Proteusbacillus vulgaris, Pseudomonas aeruginosa, and Shigella flexneri. Meanwhile, the effect on the CCR6-CCL20 in Hek293 cells of msBD-1 and msBD-1-T, and the chemotactic on the Hek293 cells were studied.Results: msBD-1 is the active zone of sBD-1. Then, in the pichia pastoris expression, the high level expression was in the 24h after induced, pH 4.4, methanol concentration 4%. And these pichia pastoris was fermentation. The Ppic9k-msBD-1-T was purified by metal chelate chromatography, Sepdex-G10, freeze drying. Ppic9k-msBD-1 was purified by PBS-cation exchange chromatography, gel-filtration and freeze drying. msBD-1and msBD-1-T can inhibit the growth of E. coli, S. aureus, P. vulgaris, P. aeruginosa and S. flexneri. In the Chemoattractant assay, msBD-1and msBD-1-T can induce the Hek293 cells to move.Conclusion:1 sBD-1 was secreted to the extracellular after proceeded by the enzyme and showed its antimicrobial activity.2 The pichia pastoris expression vector Ppic9k-msBD-1 and Ppic9k-msBD-1-T was Successfully constructed, and transferred into the Pichia pastoris GS115. The Pichia pastoris expression system was amplified from the shake flask to fermentation tan. The level of expansion for further large-scale production provided the foundation3 The purified system of msBD-1and msBD-1-T was built. Especially msBD-1 purification, which obtained for further large-scale sheep matureβ-defensin -1 provided a basis for laboratory.4 msBD-1 and msBD-1-T showed the inhibition role of bacterial growth, indicating that defensin plus six HIS tag in C-terminal is not effect on the role of defensin antimicrobial and since the HIS tag was a positively charged amino acid residues, msBD-1 also enhanced the anti-activity. Meanwhile msBD-1 and msBD-1-T were functions with chemically induced. They can induce the human embryonic kidney cells CCR6 expression level increased. They also can make embryonic kidney cells for directional movement.5 In short, msBD-1 and msBD-1-T showed the same biological function. msBD-1-T was more accessible obtained, which may provide for large-scale industrial production of favorable conditions. |
