大豆铁蛋白吸收铁的途径及体外细胞吸收研究
发布时间：2018-07-14 08:56
【摘要】：铁蛋白是广泛存在于生物体细胞中的一类铁贮藏蛋白,具有调节铁代谢平衡以及去除亚铁离子毒性的双重功能。与动物铁蛋白相比,关于植物铁蛋白的结构和功能的研究甚少。目前已发现豆类植物中90%以上的铁以铁蛋白的形式贮藏在淀粉体中。因此,豆类种子铁蛋白是研究植物铁蛋白的理想模型。植物铁蛋白与动物铁蛋白相比,其在结构上具有三个明显的特征：1、植物铁蛋白N端含有EP肽段,而动物铁蛋白没有。该EP肽段位于植物铁蛋白的外壳表面,参与铁的结合和氧化。2、动物铁蛋白由H和L型亚基组成,而植物铁蛋白只含有H型亚基,H-1和H-2。H-1和H-2型亚基具有-80%的同源性,二者在铁氧化沉淀过程中起着协同作用。在不同来源的植物铁蛋白中,H-1和H-2亚基的组成比例不同。3、植物铁蛋白的三重轴通道和四重轴通道均为亲水性通道,而动物铁蛋白的三重轴通道为亲水性通道,四重轴通道为疏水性通道。铁蛋白的通道是与外界进行物质交换的重要途径。本文以植物铁蛋白(H-1和H-2亚基)为研究对象,一方面,从植物铁蛋白的结构特点出发,研究植物铁蛋白的三重轴和四重轴通道在铁氧化沉淀过程中的作用,揭示为什么植物铁蛋白的三重轴和四重轴通道为亲水性通道?另一方面,从铁蛋白的应用出发,在体外和体内研究铁蛋白的消化稳定性,进而对不同来源的植物铁蛋白的吸收率进行鉴定,阐明其细胞吸收机制。研究成果将为开发高效的功能性补铁制剂奠定理论基础。具体研究结果如下： 1.利用分子克隆、点突变、载体构建、表达及重组蛋白的分离纯化的方法制备了重组大豆铁蛋白rH-1、rH-2、及rH-2的突变体H193AH197A, E165IE167AE171A和E165IE167AE171AH193AH197A,采用柱层析方法纯化得到了电泳纯的rH-2及其突变体。得到了充足的植物铁蛋白,为后续蛋白质的性质研究提供了保障。 2.通过比较重组大豆铁蛋白(rH-2)及其三重轴和四重轴突变体(H193AH197A, E165IE167AE171A, E165IE167AE171AH193AH197A)的铁氧化沉淀活性、聚合特性以及铁释放活性,初步阐明rH-2铁蛋白的亲水性(三重轴和四重轴)通道均为铁离子扩散和氧化沉淀的主要途径。 3.通过建立体外消化模型比较不同亚基组成的植物铁蛋白(rH-1, rH-1:rH-2=1:1, rH-1:rH-2=1:2; rH-1:rH-2=2:1和rH-2)的消化稳定性以及在体外和体内研究加工条件和食品组分对铁蛋白消化稳定性的影响,阐明大部分植物和动物铁蛋白能够抵抗住肠胃的消化作用到达小肠,并且食品组分中脱脂奶粉对铁蛋白具有保护作用。 4.采用同位素59Fe标记不同亚基组成的植物铁蛋白(rH-1,rH-1:rH-2=1:1,rH-1:rH-2=1:2;rH-1:rH-2=2:1和rH-2)和动物铁蛋白,通过Caco-2细胞模型鉴定其铁的吸收率和铁蛋白与细胞表面的相互作用。本文研究发现大豆铁蛋白;rH-l:rH-2=1:1)的吸收率最高,与细胞表面的特异性结合强度最大。因此,大豆铁蛋白是一种潜在的新型高效补铁功能因子。
[Abstract]:Ferritin is a kind of ferric storage protein widely found in biological cells. It has the dual functions of regulating the balance of iron metabolism and removing the toxicity of ferrous ions. Compared with animal ferritin, there are few studies on the structure and function of plant ferritin. At present, more than 90% of iron in legume has been stored in amyloplasts in the form of ferritin. Therefore, soybean seed ferritin is an ideal model for the study of plant ferritin. Compared with animal ferritin, plant ferritin has three distinct structural features: 1: 1. Plant ferritin contains EP peptide in N-terminal, but not in animal ferritin. The EP peptide is located on the outer surface of plant ferritin and is involved in the binding and oxidation of iron. The animal ferritin consists of H and L subunits, while the plant ferritin contains only H-type H-1 and H-2.H-1 and H-2 subunits with -80% homology. They play a synergistic role in the process of iron oxidation and precipitation. The composition of H-1 and H-2 subunits in plant ferritin from different sources is different. The triple axis channel and quadruple axis channel of plant ferritin are hydrophilic channel, while the triplex axis channel of animal ferritin is hydrophilic channel. The quadruple axis channel is hydrophobic channel. The ferritin channel is an important way to exchange substances with the outside world. In this paper, plant ferritin (H-1 and H-2 subunits) was studied. On the one hand, based on the structural characteristics of plant ferritin, the role of triplex and quadruplex channels in the process of iron oxidation was studied. Why triplex and quadruplex channels of plant ferritin are hydrophilic? On the other hand, based on the application of ferritin, the digestion stability of ferritin was studied in vitro and in vivo, and then the absorption rate of plant ferritin from different sources was identified, and the mechanism of cell absorption was elucidated. The research results will lay a theoretical foundation for the development of efficient functional iron supplements. The results are as follows: 1. Using molecular cloning, point mutation, vector construction, expression and separation and purification of recombinant protein, the recombinant soybean ferritin rH-1rH-2, and the mutants of rH-2, H193AH197A, E165IE167AE171A and E165IE167AE171AH193AH197A197A were prepared. The purified rH-2 and its mutants were purified by column chromatography. Sufficient plant ferritin was obtained, which provided a guarantee for further study on the properties of plant ferritin. 2. The iron oxidation precipitation activity, polymerization characteristics and iron release activity of recombinant soybean ferritin (rH-2) and its triple axis and quadruplex axons (H193AH197A, E165IE167AE171A, E165IE167AE177AH19AH197A) were compared. It is shown that the hydrophilic channels of rH-2 ferritin (triplex axis and quadruple axis) are the main pathways of iron ion diffusion and oxidation precipitation. The digestive stability of plant ferritin with different subunits (rH-1, rH-1: rH-2H 1: 1: 1: 1: 1: 2; rH-1: rH-2H 2: 1 and rH-2) was studied in vitro and in vivo by establishing an in vitro digestion model, and the effects of processing conditions and food components on the digestion stability of ferritin were studied in vitro and in vivo. It is shown that most plant and animal ferritin can resist the digestion of stomach to reach the small intestine, and skim milk powder has protective effect on ferritin. 4. The plant ferritin (rH-1rH-1: 1: 1: 1rH-1rH-2) and animal ferritin were labeled with the isotope 59Fe. The iron absorption rate and the interaction between ferritin and cell surface were determined by Caco-2 cell model. It was found that the absorption rate of soybean ferritin was the highest and the specific binding intensity to cell surface was the highest. Therefore, soybean ferritin is a potential new and efficient iron supplement functional factor.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R151
本文编号：2121134
[Abstract]:Ferritin is a kind of ferric storage protein widely found in biological cells. It has the dual functions of regulating the balance of iron metabolism and removing the toxicity of ferrous ions. Compared with animal ferritin, there are few studies on the structure and function of plant ferritin. At present, more than 90% of iron in legume has been stored in amyloplasts in the form of ferritin. Therefore, soybean seed ferritin is an ideal model for the study of plant ferritin. Compared with animal ferritin, plant ferritin has three distinct structural features: 1: 1. Plant ferritin contains EP peptide in N-terminal, but not in animal ferritin. The EP peptide is located on the outer surface of plant ferritin and is involved in the binding and oxidation of iron. The animal ferritin consists of H and L subunits, while the plant ferritin contains only H-type H-1 and H-2.H-1 and H-2 subunits with -80% homology. They play a synergistic role in the process of iron oxidation and precipitation. The composition of H-1 and H-2 subunits in plant ferritin from different sources is different. The triple axis channel and quadruple axis channel of plant ferritin are hydrophilic channel, while the triplex axis channel of animal ferritin is hydrophilic channel. The quadruple axis channel is hydrophobic channel. The ferritin channel is an important way to exchange substances with the outside world. In this paper, plant ferritin (H-1 and H-2 subunits) was studied. On the one hand, based on the structural characteristics of plant ferritin, the role of triplex and quadruplex channels in the process of iron oxidation was studied. Why triplex and quadruplex channels of plant ferritin are hydrophilic? On the other hand, based on the application of ferritin, the digestion stability of ferritin was studied in vitro and in vivo, and then the absorption rate of plant ferritin from different sources was identified, and the mechanism of cell absorption was elucidated. The research results will lay a theoretical foundation for the development of efficient functional iron supplements. The results are as follows: 1. Using molecular cloning, point mutation, vector construction, expression and separation and purification of recombinant protein, the recombinant soybean ferritin rH-1rH-2, and the mutants of rH-2, H193AH197A, E165IE167AE171A and E165IE167AE171AH193AH197A197A were prepared. The purified rH-2 and its mutants were purified by column chromatography. Sufficient plant ferritin was obtained, which provided a guarantee for further study on the properties of plant ferritin. 2. The iron oxidation precipitation activity, polymerization characteristics and iron release activity of recombinant soybean ferritin (rH-2) and its triple axis and quadruplex axons (H193AH197A, E165IE167AE171A, E165IE167AE177AH19AH197A) were compared. It is shown that the hydrophilic channels of rH-2 ferritin (triplex axis and quadruple axis) are the main pathways of iron ion diffusion and oxidation precipitation. The digestive stability of plant ferritin with different subunits (rH-1, rH-1: rH-2H 1: 1: 1: 1: 1: 2; rH-1: rH-2H 2: 1 and rH-2) was studied in vitro and in vivo by establishing an in vitro digestion model, and the effects of processing conditions and food components on the digestion stability of ferritin were studied in vitro and in vivo. It is shown that most plant and animal ferritin can resist the digestion of stomach to reach the small intestine, and skim milk powder has protective effect on ferritin. 4. The plant ferritin (rH-1rH-1: 1: 1: 1rH-1rH-2) and animal ferritin were labeled with the isotope 59Fe. The iron absorption rate and the interaction between ferritin and cell surface were determined by Caco-2 cell model. It was found that the absorption rate of soybean ferritin was the highest and the specific binding intensity to cell surface was the highest. Therefore, soybean ferritin is a potential new and efficient iron supplement functional factor.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R151
【参考文献】
相关期刊论文 前1条
1 胡菊;廖夏云;邓建军;胡小松;赵广华;;黄豆铁蛋白提取新方法及其与豌豆铁蛋白活性比较[J];高等学校化学学报;2009年10期
，本文编号：2121134
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