畢赤酵母表達(dá)甘露糖化人溶菌酶研究
發(fā)布時(shí)間：2019-05-18 14:17
【摘要】：胞內(nèi)菌的治療是目前臨床醫(yī)療的難題之一。如結(jié)核分枝桿菌、沙門傷寒菌、嗜肺軍團(tuán)菌等胞內(nèi)菌,它們通常寄生在人體吞噬細(xì)胞內(nèi),往往會(huì)對(duì)人體造成慢性持續(xù)性感染。當(dāng)這些病菌侵染人體后會(huì)被吞噬細(xì)胞吞噬,但吞噬細(xì)胞不能像裂解大腸桿菌等非胞內(nèi)菌那樣將其殺死。例如胞內(nèi)菌中的結(jié)核分枝桿菌已演化出了逃逸吞噬細(xì)胞裂解的機(jī)制,它通過阻止其所在吞噬泡的酸化進(jìn)程,來阻止吞噬泡與溶酶體的融合。這樣結(jié)核分枝桿菌就能長期寄生在胞內(nèi),并在人體免疫能力下降時(shí)侵染周圍細(xì)胞。胞內(nèi)菌的治療主要是用抗生素等抗菌藥長期療法,大多數(shù)的抗生素等抗菌藥治療胞外菌療效好,但它們很難進(jìn)入細(xì)胞內(nèi)或入胞濃度低難以殺死胞內(nèi)寄生菌,并且長期用藥也容易產(chǎn)生耐藥菌。為探索解決胞內(nèi)菌的治療難題,本研究利用畢赤酵母表達(dá)甘露糖化人溶菌酶,并利用該酶具有的甘露糖殘基是巨噬細(xì)胞表面甘露糖受體的配體這一特點(diǎn),研究利用巨噬細(xì)胞受體與配體之間的相互作用介導(dǎo)甘露糖化人溶菌酶內(nèi)吞,以達(dá)到殺死寄生在巨噬細(xì)胞內(nèi)病菌的目的。 巨噬細(xì)胞等吞噬細(xì)胞是人體的固有免疫細(xì)胞,它們構(gòu)成人體的第二道防線。巨噬細(xì)胞能通過細(xì)胞表面受體來識(shí)別入侵機(jī)體的病原體,在眾多的表面受體中,甘露糖受體是重要的病原體模式識(shí)別受體之一。巨噬細(xì)胞能通過甘露糖受體的介導(dǎo)作用,將帶有甘露糖糖鏈修飾的病原體內(nèi)吞到胞內(nèi)。因此利用甘露糖殘基修飾的藥物靶向巨噬細(xì)胞是一條值得探索的藥物遞送途徑。巴斯德畢赤酵母表達(dá)系統(tǒng)是成功的蛋白分泌表達(dá)系統(tǒng)之一,該系統(tǒng)能對(duì)蛋白進(jìn)行多種翻譯后加工修飾,如能特異性在NXS/T(X為除脯氨酸以外的所有氨基酸)位點(diǎn)的天冬酰胺上進(jìn)行外側(cè)鏈為甘露糖殘基的糖基化修飾。利用畢赤酵母的這一特性,可以使具有N-糖基化位點(diǎn)的蛋白進(jìn)行甘露糖化修飾。人溶菌酶具有水解細(xì)菌肽聚糖層中N-乙酰胞壁酸與N-乙酰葡糖胺之間的β-1.4糖苷鍵能力,它能殺死革蘭氏陽性菌,在體內(nèi)的補(bǔ)體的幫助下對(duì)革蘭氏陰性菌也有一定的抑殺作用,因此具有潛在的藥用價(jià)值。但是天然的人溶菌酶沒有N-糖基化位點(diǎn),理論上用畢赤酵母表達(dá)該蛋白也不會(huì)產(chǎn)生N-糖基化修飾。為獲得甘露糖化修飾的人溶菌酶及對(duì)其相關(guān)特性的研究,本實(shí)驗(yàn)從以下幾個(gè)方面進(jìn)行： 1、利用畢赤酵母表達(dá)甘露糖化修飾的人溶菌酶突變體本研究得到C-端融合2個(gè)N-糖基化位點(diǎn)序列的人溶菌酶突變體的融合基因,并以此構(gòu)建了pPICZaA-hLYZ-myc-His載體,將該載體轉(zhuǎn)化畢赤酵母GS115菌株。得到重組菌分泌表達(dá)的蛋白通過SDS-PAGE分析顯示為一彌散的條帶,彌散蛋白相對(duì)分子質(zhì)量(Mr)位于20-45kDa之間。取其中Mr最小的帶做肽指紋圖譜分析,結(jié)果該重組蛋白的氨基酸序列與人源溶菌酶lysozyme precursor(EC3.2.1.17)的氨基酸重合率達(dá)36%；Western印跡表明彌散的重組蛋白均能與鼠抗人溶菌酶單克隆抗體特異性結(jié)合；重組蛋白經(jīng)過PNGase F酶切分析,SDS-PAGE顯示彌散條帶消失,在理論Mr附近出現(xiàn)一條分子量均一的條帶；用伴刀豆蛋白A(ConA)對(duì)該重組蛋白進(jìn)行檢測,結(jié)果表明該重組蛋白可以與ConA特異性結(jié)合。以上結(jié)果表明得到的重組蛋白為甘露糖化修飾的人溶菌酶。 2、甘露糖化修飾的人溶菌酶可以被巨噬細(xì)胞內(nèi)化通過構(gòu)建人溶菌酶與增強(qiáng)型綠色熒光蛋白融合蛋白的融合基因載體pPICZaA-hLYZ/eGFP,電轉(zhuǎn)GS115構(gòu)建重組菌GS115/pPICZaA-hLYZ/eGFP,該重組菌誘導(dǎo)表達(dá)重組融合蛋白,通過硫酸銨沉淀、G25柱脫鹽、鎳親和層析純化獲得目的重組融合蛋白,脫去咪唑與鹽后凍干濃縮,將該重組融合蛋白與Raw264.7細(xì)胞共孵育,用激光共聚焦顯微鏡觀察,發(fā)現(xiàn)有綠色熒光物質(zhì)進(jìn)入Raw264.7,而在有甘露糖競爭性配體甘露聚糖存在時(shí),觀察不到有綠色熒光物質(zhì)進(jìn)入Raw264.7細(xì)胞。此現(xiàn)象說明甘露糖化人溶菌酶能進(jìn)入巨噬細(xì)胞且這一過程是由甘露糖受體介導(dǎo)的。 3、低甘露糖化糖基酵母工程菌GJK05表達(dá)人溶菌酶及其活性鑒定通過構(gòu)建pPICZaA-hLYZ載體及用作對(duì)照的載體pPICZaA-hLYZ(N/Q),電轉(zhuǎn)化低糖基化酵母工程菌及GS115菌株,分別構(gòu)建重組菌表達(dá)重組蛋白hLYZ及hLYZ(N/Q)。用伴刀豆蛋白A(ConA)檢測重組蛋白的甘露糖化修飾；平板抑菌圈法比較重組人溶菌酶的活性,發(fā)現(xiàn)GS115表達(dá)高甘露糖化的重組人溶菌酶影響了其抑菌的活性。低糖基化酵母工程菌GJK05表達(dá)的重組人溶菌酶與Raw264.7進(jìn)行免疫熒光實(shí)驗(yàn),激光共聚焦顯微鏡可觀察到Raw264.7細(xì)胞內(nèi)有熒光物質(zhì)。 綜上述：本研究獲得了具有活性的甘露糖化的重組人溶菌酶,該重組蛋白具有體外抑菌活性,并且能夠在巨噬細(xì)胞表面的甘露糖受體的介導(dǎo)下進(jìn)入巨噬細(xì)胞,為進(jìn)一步研究甘露糖化人溶菌酶的胞內(nèi)抑菌活性奠定基礎(chǔ)。
[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,
【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R378
，
本文編號(hào)：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,
【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R378
，
本文編號(hào)：2480074
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