Expression Of Human Lysozyme-Antimicrobial Peptide Fusion Protein And Its Efficacy Of Treating Mouse Mastitis

To obtain a higher activity of anti-bacterial and anti-virus fusion protein and its efficacy of treating bovine mastitis.The human lysozyme gene was cloned from human placenta and .antibacterial peptide gene was synthetic. Prokaryotic and eukaryotic expression system were used to express human lysozyme-antibacterial peptide fusion gene. Compare biological activity of recombinant fusion protein and then used it to treat experimental mouse mastitis. This results will provide a new strategy for therapy bovine mastitis.1. Fusion gene design and cloneThe human lysozyme gene (hLYZ) was amplified from the clone vector pMD18-T-hLYZ by PCR. The linker and the antibacterial peptide gene were synthetic. The lysozyme gene and antibacterial peptide gene were connected by linker. The research of cloning, sequencing and analyzing this gene suggested that the gene contains 514bp, and encodes 166 amino acid residues. The molecular weight is 18477.02 Da, Isoelectric point is 8.98.2. Expression of the human lysozyme-antibacterial peptide gene in E.coli.The fusion gene was insert into prokaryotic expression vector pGEX-4T-1 and construct fusion gene expression plasmid pGEX-4T-hLYZ-tachyplesins.Transformed correctly positive plasmid into the BL21(DE3), and induced with IPTG. The fusion gene was expressed from the recombinant cell and fusion protein was purified by GST. At the same time, the conditions of induction were optimized. The optimal parameters are 6 hours,25℃,pH 7.2 and the concentration of IPTG 0.8mmol/L.Under such conditions, the expressed recombinant protein existed in form of fusion protein and the expression level of recombinant peptide is 21.704% contrasted the total protein of E.coli. The recombinant fusion protein was purified and its antibacterial activity was tested. The result showed that it could inhibit Staphylococcus aureus (ATCC25923) and E.coli(ATCC25922).3. Expression of human lysozyme-antibacterial peptide gene in Pichia pastorisThe expression system was consisted of the complete secreting expression vector pPICZαA, Pichia pastoris GS115 and the fusion gene. The recombinant pPICZαA-hLYZ-tachyplesins was linearized with Sac I and transformed into competent GS115 for expression. The positive yeasts were screened by the ZeocinTM resistance gene ,nourishment phenotype and PCR. The expression of the recombinant fusion protein with initial purifization and concentration was identified by SDS-PAGE and Western blot . Under induction of 1% methanol, a protein with molecular weight of 18 KDa was successfully expressed by the recombinant yeast, which was specifically recognized by polyclonal antibody against human lysozyme. The assay of the antibacterial activity in vitro. The result was that recombinant fusion protein could inhibit. Staphylococcus aureus, Salmonella, E.coli, Bacillus subtilis, Candida albicans etc. The fusion protein have antibacterial activity like human lysozyme and antibacterial peptide ,its increase Salmonella typhi, K. pneumoniae antibacterial bacteria to recombinant human lysozyme, increase Candida albicans effect to recombinant antimicrobial peptides.4. The biological characteristics of human lysozyme-antibacterial peptide fusion proteinThe fusion protein was purified by molecular sieve and high-performance liquid chromatography. The purified protein was used to detect the optimal reaction temperature, pH, thermal stability properties according to conventional methods of measuring activity.The fusion protein purity was 87% after purification. The heat shock results showed that the fusion protein has cut off the peptidoglycan of the N-acetyl-glucosamine and N-acetylmuramic acid between theβ-1,4 glycosidic bond functions, this is lysozyme activity. Results: The optimum reaction temperature and pH of fusion protein were 40℃and 6.5,respectively.The Km of enzyme for Micrococcus lysodeikticus is 0.04845mg/mL.The thermal stability of the fusion protein is stable. The affects of some ions on this fusion protein were shown. The ions of Cu2+, Fe3+ and others have impact on the enzyme activity, which indicates that the fusion protein has the natural attributes like human lysozyme and antibacterial peptides.5. Therapeutic effects of human lysozyme–antibacterial peptide fusion protein on mouse MastitisIn order to study fusion protein treatment of bovine mastitis, a strain of Staphylococcus aureus and Escherichia coli were isolated from the milk of dairy cows suffering from clinical mastitis to develop mouse mastitis model. The immune response of mammary glands triggered by Staphylococcus aureus and Escherichia coli infection and the effect of fusion protein on mastitis were studied through CFU counts, histological, histochemical, immunohistochemical and ELISA. Results: Fusion protein could inhibit bacterial proliferation in mammary glands of model mouse. It could also alleviate pathological response and inflammatory symptom of mastitis caused by S.aureus and E.coli apparently.It plays a protective role on experimental mouse mastitis caused by S.aureus and E.coli. The S.aureus mammary gland inflammation of the cure rate was 70%, E. coli mastitis was 60%, it is also can make TNF-αreduced significantly (P<0.05).The results will provide a new idea and way to study natural medicine to treat bovine mastitis.