| Lysozyme is a small bioactive basic protein, named for its ability to dissolve cell wall of bacteria. Lysozyme is an important effector molecule of nonspecific innate immunity, widely exists in human, animal, plants and microorganisms. It has weak antigenicity, high stability, special antimicrobial mechanism and it is not easy to produce drug resistance and to enter the body without harmful residue. Compared with other lysozyme, human lysozyme (Hlys) has higher security, stronger bactericidal activity. Currently, human lysozyme is mainly got from human milk and placenta. The yield of human lysozyme is low but at great expense. These factors greatly limit the application of human lysozyme. Realize its large-scale and efficient expression is particularly important.This study tried to express human lysozyme efficiently in Pichia pastoris system by genetic engineering method. Viewed the human lysozyme gene sequences (FJ795023.1) in the GenBank database, designed and synthetised human lysozyme gene according to Pichia pastoris codon bias usages, then digested Pichia pastoris expression vector pPICZaA and Hlys gene with EcoRI and Kpnl, recombinant pPICZaA-Hlys plasmid was successfully constructed; linearized pPICZaA-Hlys with SacⅠ, transformed linearized pPICZaA-Hly to Pichia pastoris strain GS115 competent cells, cultivated GS115 cells in YPD plate with Zeocin concentration 100 μ g/mL,500 μ g/mL,1000 μ g/mL. According to the alcohol oxidase of pPICZaA, promoter primers were designed:5’-AOX1 and 3’-AOX1, the positive recombinants were identified by PCR. Six recombined strains containing the target gene were got. Methanol was the carbon source when recombinant strains was induced, the expression of exogenous protein was secreted into the culture medium and detected by SDS-PAGE and Western blot.Recombinant strain number six was selected because it was the best one to express recombinant protein. Optimize the induce conditions in shake flask, the best conditions: pH=7.0, induction time 72h, methanol concentration of 2%. Hlys was induced in fermentor according to the optimum condition in shake flask. The target protein in supernatant can be detected by SDS-PAGE. Electrophoresis gel analysis indicated that the target protein accounted for 30.5% of total protein, the concentration of expressed Hlys was about 669.78 μ g/mL,The fermentation supernatant was concentrated, bacteriostatic test in vitro showed that recombinant Hlys had certain bacteriolysis on Ecoli, Staphylococcus aureus, streptococcus, Haemophilus parasuis, Actinobacillus pleuropneumoniae. The minimum inhibitory concentration (MIC) of recombinant Hlys on multiple strains of Streptococcus was in the range of 96-192 μ g/mL, the minimum bactericidal concentration (MBC) was in the range of 383-766 μ g/mL. MIC of recombinant Hlys on strains of Haemophilus parasuis was 383～766 μg/mL, MBC was above 766 μg/mL.The fermentation supernatant was concentrated by ultrafiltration, then pured by SephadexG-50 gel chromatography so that Hlys protein was pured.The recombinant Hlys activity was 3847.93U/mL, detected by rapid nephelometry measures while Micrococcus lysodeikticus was the substrate. The specific activity was approximately 19233.20U/mg. Freeze-drying the fermentation of Hlys with cryoprotector, dry powder was added to feed and fed mice for one week. Body weight of mice which fed with Hlys increased 3.97% more than group without Hlys, but there was no significant difference between these two groups. Animal test results showed that the recombinant Hlys had prevention effect on Streptococcus infection, but the treatment effect was not obvious. |
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