毕赤酵母表达甘露糖化人溶菌酶研究
发布时间：2019-05-18 14:17
【摘要】：胞内菌的治疗是目前临床医疗的难题之一。如结核分枝杆菌、沙门伤寒菌、嗜肺军团菌等胞内菌,它们通常寄生在人体吞噬细胞内,往往会对人体造成慢性持续性感染。当这些病菌侵染人体后会被吞噬细胞吞噬,但吞噬细胞不能像裂解大肠杆菌等非胞内菌那样将其杀死。例如胞内菌中的结核分枝杆菌已演化出了逃逸吞噬细胞裂解的机制,它通过阻止其所在吞噬泡的酸化进程,来阻止吞噬泡与溶酶体的融合。这样结核分枝杆菌就能长期寄生在胞内,并在人体免疫能力下降时侵染周围细胞。胞内菌的治疗主要是用抗生素等抗菌药长期疗法,大多数的抗生素等抗菌药治疗胞外菌疗效好,但它们很难进入细胞内或入胞浓度低难以杀死胞内寄生菌,并且长期用药也容易产生耐药菌。为探索解决胞内菌的治疗难题,本研究利用毕赤酵母表达甘露糖化人溶菌酶,并利用该酶具有的甘露糖残基是巨噬细胞表面甘露糖受体的配体这一特点,研究利用巨噬细胞受体与配体之间的相互作用介导甘露糖化人溶菌酶内吞,以达到杀死寄生在巨噬细胞内病菌的目的。 巨噬细胞等吞噬细胞是人体的固有免疫细胞,它们构成人体的第二道防线。巨噬细胞能通过细胞表面受体来识别入侵机体的病原体,在众多的表面受体中,甘露糖受体是重要的病原体模式识别受体之一。巨噬细胞能通过甘露糖受体的介导作用,将带有甘露糖糖链修饰的病原体内吞到胞内。因此利用甘露糖残基修饰的药物靶向巨噬细胞是一条值得探索的药物递送途径。巴斯德毕赤酵母表达系统是成功的蛋白分泌表达系统之一,该系统能对蛋白进行多种翻译后加工修饰,如能特异性在NXS/T(X为除脯氨酸以外的所有氨基酸)位点的天冬酰胺上进行外侧链为甘露糖残基的糖基化修饰。利用毕赤酵母的这一特性,可以使具有N-糖基化位点的蛋白进行甘露糖化修饰。人溶菌酶具有水解细菌肽聚糖层中N-乙酰胞壁酸与N-乙酰葡糖胺之间的β-1.4糖苷键能力,它能杀死革兰氏阳性菌,在体内的补体的帮助下对革兰氏阴性菌也有一定的抑杀作用,因此具有潜在的药用价值。但是天然的人溶菌酶没有N-糖基化位点,理论上用毕赤酵母表达该蛋白也不会产生N-糖基化修饰。为获得甘露糖化修饰的人溶菌酶及对其相关特性的研究,本实验从以下几个方面进行： 1、利用毕赤酵母表达甘露糖化修饰的人溶菌酶突变体本研究得到C-端融合2个N-糖基化位点序列的人溶菌酶突变体的融合基因,并以此构建了pPICZaA-hLYZ-myc-His载体,将该载体转化毕赤酵母GS115菌株。得到重组菌分泌表达的蛋白通过SDS-PAGE分析显示为一弥散的条带,弥散蛋白相对分子质量(Mr)位于20-45kDa之间。取其中Mr最小的带做肽指纹图谱分析,结果该重组蛋白的氨基酸序列与人源溶菌酶lysozyme precursor(EC3.2.1.17)的氨基酸重合率达36%；Western印迹表明弥散的重组蛋白均能与鼠抗人溶菌酶单克隆抗体特异性结合；重组蛋白经过PNGase F酶切分析,SDS-PAGE显示弥散条带消失,在理论Mr附近出现一条分子量均一的条带；用伴刀豆蛋白A(ConA)对该重组蛋白进行检测,结果表明该重组蛋白可以与ConA特异性结合。以上结果表明得到的重组蛋白为甘露糖化修饰的人溶菌酶。 2、甘露糖化修饰的人溶菌酶可以被巨噬细胞内化通过构建人溶菌酶与增强型绿色荧光蛋白融合蛋白的融合基因载体pPICZaA-hLYZ/eGFP,电转GS115构建重组菌GS115/pPICZaA-hLYZ/eGFP,该重组菌诱导表达重组融合蛋白,通过硫酸铵沉淀、G25柱脱盐、镍亲和层析纯化获得目的重组融合蛋白,脱去咪唑与盐后冻干浓缩,将该重组融合蛋白与Raw264.7细胞共孵育,用激光共聚焦显微镜观察,发现有绿色荧光物质进入Raw264.7,而在有甘露糖竞争性配体甘露聚糖存在时,观察不到有绿色荧光物质进入Raw264.7细胞。此现象说明甘露糖化人溶菌酶能进入巨噬细胞且这一过程是由甘露糖受体介导的。 3、低甘露糖化糖基酵母工程菌GJK05表达人溶菌酶及其活性鉴定通过构建pPICZaA-hLYZ载体及用作对照的载体pPICZaA-hLYZ(N/Q),电转化低糖基化酵母工程菌及GS115菌株,分别构建重组菌表达重组蛋白hLYZ及hLYZ(N/Q)。用伴刀豆蛋白A(ConA)检测重组蛋白的甘露糖化修饰；平板抑菌圈法比较重组人溶菌酶的活性,发现GS115表达高甘露糖化的重组人溶菌酶影响了其抑菌的活性。低糖基化酵母工程菌GJK05表达的重组人溶菌酶与Raw264.7进行免疫荧光实验,激光共聚焦显微镜可观察到Raw264.7细胞内有荧光物质。 综上述：本研究获得了具有活性的甘露糖化的重组人溶菌酶,该重组蛋白具有体外抑菌活性,并且能够在巨噬细胞表面的甘露糖受体的介导下进入巨噬细胞,为进一步研究甘露糖化人溶菌酶的胞内抑菌活性奠定基础。
[Abstract]:The treatment of intracellular bacteria is one of the current problems in clinical medical treatment. Such as M.tuberculosis, Salmonella, and Legionella pneumophila, which are usually parasitic in the phagocytic cells of the human body and often cause chronic persistent infection to the human body. When these pathogens infect the human body, they can be phagocytosed by phagocytes, but the phagocytes cannot be killed like non-intracellular bacteria such as E. coli. For example, the Mycobacterium tuberculosis in the intracellular bacteria has shown a mechanism to escape the phagocyte lysis, which prevents the fusion of the phagocytic cells from the lysosomes by blocking the acidification process of the phagocytic cells in which it is located. So that the mycobacterium tuberculosis can be parasitically in the cell for a long time, and the surrounding cells can be infected when the immunity of the human body is reduced. The treatment of intracellular bacteria is mainly based on the long-term treatment of antibiotics and the like, and most of the antibacterial drugs such as antibiotics have good curative effect on the external bacteria, but they are difficult to enter the cell or the cell concentration is low to kill the intracellular parasitic bacteria, and the long-term use is also easy to produce the drug-resistant bacteria. In order to solve the problem of the treatment of intracellular bacteria, the present study uses pichia pastoris to express the mannosylated human lysozyme, and uses the mannose residue of the enzyme as the ligand of the mannose receptor on the surface of the macrophage, The interaction between the macrophage receptor and the ligand is used to mediate the endocytosis of the mannosylated human lysozyme, so as to achieve the purpose of killing the bacteria in the macrophage. The phagocytic cells such as macrophages are the natural immune cells of the human body, which form the second defense of the human body Lines. Macrophages can identify the pathogens of the invading organism by the cell surface receptor, and in a large number of surface receptors, the mannose receptor is an important pathogen-recognition receptor. I. The macrophage can be bound by the mannose receptor, and the pathogen with the mannose chain modification can be engulfed into the cell. The drug-targeting macrophages modified with mannose residues are therefore a well-explored drug delivery route The Pichia pastoris expression system is one of the successful expression systems of the protein secretion, which can be used for the post-translational processing of the protein. In the case of a decoration, a glycosylation repair of a mannoose residue can be carried out on the alicyclic amine at the site of NXS/ T (X is all amino acid other than proline). With this characteristic of Pichia pastoris, the protein with the N-glycosylation site can be saccharified and fixed. The human lysozyme is capable of killing gram-positive bacteria and inhibiting Gram-negative bacteria with the help of complement in the body. with, therefore, a potential medicinal price However, the natural human lysozyme has no N-glycosylation site, which is expressed by Pichia pastoris and does not produce N-glycosylation repair. In order to obtain the human lysozyme modified by the mannose and the study on its related properties, the experiment was carried out in the following aspects: Line: 1. The fusion gene of the human lysozyme mutant with the 2 N-glycosylation site sequence of the C-terminal is obtained by using the human lysozyme mutant expressed by the Pichia pastoris to express the mannose-modified human lysozyme mutant, and the pPICZaA-hLYZ-myc-His vector is constructed, and the vector is transformed to the Pichia pastoris GS11. 5. The protein expressed by the recombinant bacteria was analyzed by SDS-PAGE as a dispersed band, and the relative molecular weight (Mr) of the dispersion protein was in the range of 20-45 kD. A. The amino acid sequence of the recombinant protein and the amino acid sequence of the human lysozyme lyszyme precorcursor (EC3. 2.1.17) were 36%. Western blot showed that the recombinant protein could be specific to the mouse anti-human lysozyme monoclonal antibody. The results showed that the recombinant protein could be specific to ConA. The results showed that the recombinant protein could be specific to ConA. The above results show that the recombinant protein obtained is a mannoSaccharide modified person. Lysozyme.2. The human lysozyme modified by the mannose can be internalized by the macrophages to construct the fusion gene vector pPICZaA-hLYZ/ eGFP of the human lysozyme and the enhanced green fluorescent protein fusion protein, and the electrorotation GS115 constructs the recombinant bacteria GS115/ pPICZaA-hLYZ/ eGFP, which induces the expression of the recombinant fusion protein, The recombinant fusion protein was obtained by precipitation, G25 column desalination and nickel affinity chromatography. The recombinant fusion protein was isolated and concentrated by freeze-drying. The recombinant fusion protein was co-incubated with the cells of Raw264.7 cells, and the green fluorescent substance was found to enter the Raw. 264.7. In the presence of a mannose-competitive ligand mannan, no green fluorescent substance was observed to enter the Raw26 4.7 Cells. This phenomenon indicates that the mannosylated human lysozyme can enter the macrophage and this process is caused by mannose Receptor-mediated.3. The expression of human lysozyme and its activity was identified by the construction of pPICZaA-hLYZ vector and vector pPICZaA-hLYZ (N/ Q) used as control. The recombinant protein, hLYZ and hL, was constructed by the transformation of low-glycosylated yeast engineering bacteria and GS115 strain. YZ (N/ Q). Mannose Saccharification of the Recombinant Protein by ConA (ConA); the Activity of Recombinant Human Lysozyme was compared by the method of plate inhibition, and the effect of GS115 on the recombinant human lysozyme was found. The activity of the antibacterial activity of the recombinant human lysozyme and the recombinant human lysozyme expressed by the low-glycosylation yeast engineering strain GJK05 were compared with that of Raw264.7. The laser confocal microscope can observe the fine of Raw264.7. In the present study, recombinant human lysozyme with activity of mannose is obtained, and the recombinant protein has in vitro antibacterial activity and can be used as a mannose receptor on the surface of the macrophage. in ord to further study that cell of the mannosylated human lysozyme,
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378
，
本文编号：2480074
[Abstract]:The treatment of intracellular bacteria is one of the current problems in clinical medical treatment. Such as M.tuberculosis, Salmonella, and Legionella pneumophila, which are usually parasitic in the phagocytic cells of the human body and often cause chronic persistent infection to the human body. When these pathogens infect the human body, they can be phagocytosed by phagocytes, but the phagocytes cannot be killed like non-intracellular bacteria such as E. coli. For example, the Mycobacterium tuberculosis in the intracellular bacteria has shown a mechanism to escape the phagocyte lysis, which prevents the fusion of the phagocytic cells from the lysosomes by blocking the acidification process of the phagocytic cells in which it is located. So that the mycobacterium tuberculosis can be parasitically in the cell for a long time, and the surrounding cells can be infected when the immunity of the human body is reduced. The treatment of intracellular bacteria is mainly based on the long-term treatment of antibiotics and the like, and most of the antibacterial drugs such as antibiotics have good curative effect on the external bacteria, but they are difficult to enter the cell or the cell concentration is low to kill the intracellular parasitic bacteria, and the long-term use is also easy to produce the drug-resistant bacteria. In order to solve the problem of the treatment of intracellular bacteria, the present study uses pichia pastoris to express the mannosylated human lysozyme, and uses the mannose residue of the enzyme as the ligand of the mannose receptor on the surface of the macrophage, The interaction between the macrophage receptor and the ligand is used to mediate the endocytosis of the mannosylated human lysozyme, so as to achieve the purpose of killing the bacteria in the macrophage. The phagocytic cells such as macrophages are the natural immune cells of the human body, which form the second defense of the human body Lines. Macrophages can identify the pathogens of the invading organism by the cell surface receptor, and in a large number of surface receptors, the mannose receptor is an important pathogen-recognition receptor. I. The macrophage can be bound by the mannose receptor, and the pathogen with the mannose chain modification can be engulfed into the cell. The drug-targeting macrophages modified with mannose residues are therefore a well-explored drug delivery route The Pichia pastoris expression system is one of the successful expression systems of the protein secretion, which can be used for the post-translational processing of the protein. In the case of a decoration, a glycosylation repair of a mannoose residue can be carried out on the alicyclic amine at the site of NXS/ T (X is all amino acid other than proline). With this characteristic of Pichia pastoris, the protein with the N-glycosylation site can be saccharified and fixed. The human lysozyme is capable of killing gram-positive bacteria and inhibiting Gram-negative bacteria with the help of complement in the body. with, therefore, a potential medicinal price However, the natural human lysozyme has no N-glycosylation site, which is expressed by Pichia pastoris and does not produce N-glycosylation repair. In order to obtain the human lysozyme modified by the mannose and the study on its related properties, the experiment was carried out in the following aspects: Line: 1. The fusion gene of the human lysozyme mutant with the 2 N-glycosylation site sequence of the C-terminal is obtained by using the human lysozyme mutant expressed by the Pichia pastoris to express the mannose-modified human lysozyme mutant, and the pPICZaA-hLYZ-myc-His vector is constructed, and the vector is transformed to the Pichia pastoris GS11. 5. The protein expressed by the recombinant bacteria was analyzed by SDS-PAGE as a dispersed band, and the relative molecular weight (Mr) of the dispersion protein was in the range of 20-45 kD. A. The amino acid sequence of the recombinant protein and the amino acid sequence of the human lysozyme lyszyme precorcursor (EC3. 2.1.17) were 36%. Western blot showed that the recombinant protein could be specific to the mouse anti-human lysozyme monoclonal antibody. The results showed that the recombinant protein could be specific to ConA. The results showed that the recombinant protein could be specific to ConA. The above results show that the recombinant protein obtained is a mannoSaccharide modified person. Lysozyme.2. The human lysozyme modified by the mannose can be internalized by the macrophages to construct the fusion gene vector pPICZaA-hLYZ/ eGFP of the human lysozyme and the enhanced green fluorescent protein fusion protein, and the electrorotation GS115 constructs the recombinant bacteria GS115/ pPICZaA-hLYZ/ eGFP, which induces the expression of the recombinant fusion protein, The recombinant fusion protein was obtained by precipitation, G25 column desalination and nickel affinity chromatography. The recombinant fusion protein was isolated and concentrated by freeze-drying. The recombinant fusion protein was co-incubated with the cells of Raw264.7 cells, and the green fluorescent substance was found to enter the Raw. 264.7. In the presence of a mannose-competitive ligand mannan, no green fluorescent substance was observed to enter the Raw26 4.7 Cells. This phenomenon indicates that the mannosylated human lysozyme can enter the macrophage and this process is caused by mannose Receptor-mediated.3. The expression of human lysozyme and its activity was identified by the construction of pPICZaA-hLYZ vector and vector pPICZaA-hLYZ (N/ Q) used as control. The recombinant protein, hLYZ and hL, was constructed by the transformation of low-glycosylated yeast engineering bacteria and GS115 strain. YZ (N/ Q). Mannose Saccharification of the Recombinant Protein by ConA (ConA); the Activity of Recombinant Human Lysozyme was compared by the method of plate inhibition, and the effect of GS115 on the recombinant human lysozyme was found. The activity of the antibacterial activity of the recombinant human lysozyme and the recombinant human lysozyme expressed by the low-glycosylation yeast engineering strain GJK05 were compared with that of Raw264.7. The laser confocal microscope can observe the fine of Raw264.7. In the present study, recombinant human lysozyme with activity of mannose is obtained, and the recombinant protein has in vitro antibacterial activity and can be used as a mannose receptor on the surface of the macrophage. in ord to further study that cell of the mannosylated human lysozyme,
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378
，
本文编号：2480074
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