| The treatment of intracellular bacteria is one of the clinical problems. Intracellular bacteria refers to the parasitic bacteria in the body’s phagocytes, Such as Mycobacterium tuberculosis, Salmonella typhi, Legionella pneumophila will cause chronic persistent infection. When these bacteria infect the human body, these bacteria will be engulfed by phagocytes. But phagocytes can not kill the intracellular bacteria like to kill E. coli and other non-intracellular bacteria, such as Mycobacterium tuberculosis and other intracellular bacteria have evolved the mechanism to escape cell lysis, Mycobacterium tuberculosis by blocking where the phagosome acidification process to prevent phagosome-lysosome fusion. So that they can be a long-term parasitic intracellular and infect surrounding cells in the human immune decline. Majority of the antibiotics and other antibacterial drugs can treatment extracellular bacteria very well, but they are hard to kill the intracellular bacteria. The treatment of intracellular bacteria is a long-term therapy with antibiotics and other antibacterial drugs, but easy to produce multidrug resistance.To solve the challenges of intracellular bacteria in the treatment, mannosylated human lysozyme was expressed in Pichia pastoris. Mannose residues of mannosylated human lysozyme are ligands of macrophage cell surface mannose receptors, by receptor-ligand interactions mediated human lysozyme endocytosis to kill intracellular bacteria in macrophages.Macrophages and other phagocytes are the body’s natural immune cells, which constitute the body’s second line of defense. Receptors of Macrophage can recognize invading bacteria, in a large number of surface receptors, the mannose receptor is an important pathogen pattern recognition receptor of macrophage. The macrophage mannose receptor can mediate pathogen endocytosis by receptor-ligand interactions, so mannose residues modified macrophage-targeted drugs have the value to research.Pichia pastoris expression system is one of the successful expression systems of the protein secretion,and Pichia pastoris have functions of a variety of post-translational processing and modification of proteins.Such as glycosylation,specific glycosylation mainly happened in the asparagine of the NXT (X is all the amino acids,but proline outside)sites,and utilization of the characteristic of Pichia pastoris,we can get a mannosylated protein when the protein has N-glycosylation sites.Human lysozyme has the capacity of hydrolysis of β-1.4-glycosidic bond between N-acetyl muramic acid and N-acetyl glucosamine of bacterial peptidoglycan layer, so human lysozyme has potential value of medicine, because it can kill Gram-positive bacteria,and it has ability to inhibit Gram-negative bacteria in organism, however, the natural human lysozyme has no N-glycosylation sites，In theory, using Pichia pastoris expression of the protein does not produce N-glycosylation modification. In order to obtain the mannosylated modified human lysozyme and the related characteristics of it, We will research it as follows:1、 the expression of mannosylated human lysozyme mutants in pichia pastorisIn this research,we obtained the fusion gene of human lysozyme which C-terminal was fused with two N-glycosylation sites,and construction of the pPlCZaA-hLYZ-myc-His plasmid, the plasmid was electroporated into GS115strains, then the recombinant protein was expressed by the recombinant strain GS115. The recombinant protein showed a diffuse band by SDS-PAGE analysis,the relative molecular mass of the recombinant protein is between20-45kDa.The recombinant proteins is the mannosylated human lysozyme which identificated by MALDI-TOF MS,Western Blotting,ConA and PNGase F analysis.2、 The mannosylated human lysozyme can enter macrophages by receptor-mediated endocytosis.The hLYZ and eGFP genes were linked by PCR,in the linker a site recognized by N-Glycosylation was introduced, then cloned into vector pPICZaA. The recombinant plasmid pPICZaA-hLYZ/eGFP was linearized with Sac I, then transformed to P.pastoris strain GS115by electroporation. Multi-copy transformants were screened with Zeocin indentified by PCR,and the positive clones were induced with methanol. Ammonium sulfate precipitation and nickel affinity chromatography purified proteins, and the fusion protein incubating with macrophages(Raw264.7) were observed under a fluorescence microscope,Green fluorescent substance can be seen in the cytoplasm of RAW264.7by confocal microscope image, and Mannan can inhibit the occurrence of this phenomenon. So the hLYZ/eGFP fusion protein can be internalized into macrophages by MMR. 3、Low-mannose type glycosylation engineering yeast strain GJK05expression of human lysozyme and its activity identification.Construction of pPlCZaA-hLYZ vector and the control vector pPlCZaA-hLYZ (N/Q), and these recombinant plasmid was linearized with Sac I, then the linearized plasmid was electroporated into the Pichia pastoris GS115strain and Low-mannose type glycosylation engineering yeast strain GJK05. To construct different recombinant yeast strains to express recombinant protein of the hLYZ and the hLYZ (N/Q) respectively. Using concanavalin A(ConA)detection of the mannosylated modified recombinant proteins; comparison antibacterial activity of the recombinant human lysozyme by Plate antibacterial circle method, and finding that the high-mannose glycosylated recombinant human lysozyme affects its antibacterial activity; the consequence of immunofluorescence assay by interacting of Raw264.7and human lysozyme expressed in Low-mannose type glycosylation engineering yeast strain GJK05shows that Green fluorescent substance can be seen in the cytoplasm of RAW264.7by confocal microscope image.In summary, we obtained the mannosylated recombinant human lysozyme which has antibacterial activity, and in the macrophage mannose receptor mediated endocytosis,the recombinant protein can enter macrophages. This study laid a foundation for further research the mannosylated recombinant human lysozyme intracellular antimicrobial activity. |
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