【作者】 李前勇；

【导师】 鲁成；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2010， 博士

【摘要】 铜蓝蛋白(Ceruloplasmin, EC1.16.3.,缩写为CP)是一种含铜的α2糖蛋白,广泛存在于人和动物体内,是人和动物生命活动中的一种重要金属蛋白酶,在动物体内铜的转运、细胞铁的代谢、胚胎及神经系统的发育、新生毛细血管的形成、自由基的清除等过程中发挥重要作用。铜蓝蛋白可以向机体各种组织细胞快速提供高效可利用的铜,满足各器官组织对铜营养的需要,经母乳在初生动物的早期阶段发挥了重要的营养作用,这些科学结论为将铜蓝蛋白作为畜禽的一种新型生物铜源进行开发利用提供了重要线索。猪是我国畜牧业中饲养最多、分布最广的一种重要家畜,铜不仅是其机体必需的微营养素,而且在一定的范围内高剂量的铜还作为一种重要促生长剂广泛使用,甚至滥用,无机铜的超量使用或滥用已经导致了猪发生铜中毒病、猪肉品质下降、养殖环境土壤板结、水体自洁力下降、铜资源严重浪费及影响人类健康等负面效应。因此,开发安全高效、经济适用、环境友好的畜禽生物铜源有着广阔的应用前景。本研究以我国著名三大优良地方猪种——荣昌猪为试验材料,通过对猪血清铜蓝蛋白的分离纯化、生化特性、抑制动力学、部分功能以及对该蛋白质原基因克隆、生物信息学分析、荧光定量PCR表达量分析等的研究,获得了以下结果：1.采用25%的正丁醇脱脂、35-50%的饱和硫酸铵盐析、截留分子量为10000D的滤膜超滤、DEAE-Sepharose层析、羟基磷灰石柱层析、Superdex-200凝胶柱层析等技术对猪铜蓝蛋白进行了分离纯化,并用邻联大茴香胺检测法监测各纯化步骤溶液中铜蓝蛋白的酶活性,获得了经SDS-PAGE电泳为单带的铜蓝蛋白纯化产物,其酶的比活力提高了217.65倍,回收率达到32%。该蛋白分子量为123.5KD,等电点为5.08,含糖量为7.8%,每分子猪铜蓝蛋白分子中含有铜原子个数为6个,经SDS-PAGE电泳分析,该猪CP为单亚基蛋白质。酶促反应研究得知,所得猪铜蓝蛋白(氧化酶)的最适pH为5.5,pH5-10为其稳定环境；最适温度为55℃。在50～60℃下其酶活力稳定性最好。经对猪铜蓝蛋白(氧化酶)酶促反应常数研究,得出其酶促反应的初速度为24.75gmol／(L·min),Km值为0.95mmol／L,最大反应速度Vmax为0.1124μmol／L。2.研究了效应物对猪铜蓝蛋白酶活力的影响,结果表明猪铜蓝蛋白对胰蛋白酶、胃蛋白酶均有明显的抵抗能力；铜离子、铁离子、锌离子、锰离子和镁离子对铜蓝蛋白的酶活力均有抑制作用,并呈现随着这些金属离子浓度的增加该蛋白质的酶活力逐渐减弱的趋势；氯离子、EDTA、双氧水及叠氮化钠对猪铜蓝蛋白的酶活力也明显的抑制作用,其中叠氮化钠的抑制作用最强,EDTA的抑制作用最弱；柠檬酸、草酸、抗坏血酸对猪铜蓝蛋白的酶活力有抑制作用,并呈现浓度依赖关系,其中抗坏血酸对铜蓝蛋白的酶活力抑制作用为非竞争抑制作用,柠檬酸对铜蓝蛋白的酶活力抑制作用为竞争抑制作用,而尿素对该蛋白质的酶活力基本无明显的抑制作用。3.研究了铜蓝蛋白对猪脑垂体细胞分泌生长激素及对脾细胞、B淋巴细胞、T淋巴细胞和亚群(CD4、CD8)增殖的影响,结果表明铜蓝蛋白可以促进猪脑垂体细胞生长激素的分泌,其中以加入0.4mg／ml的铜蓝蛋白换液培养8h的效果最好；同时,铜蓝蛋白可显著促进脾细胞、T淋巴细胞及亚群(CD4、CD8)、B淋巴细胞的增殖,其中以1.0mg/mL(终浓度为0.5mg/mL)的效果最好。这一结果说明铜蓝蛋白在细胞水平不仅可以促进猪垂体细胞分泌生长激素,还具有有丝分裂原的作用,可以刺激仔猪脾细胞、T淋巴细胞及亚群(CD4、CD8)、B淋巴细胞的分裂、增殖,提高仔猪免疫能力。研究结果丰富了铜蓝蛋白生理功能的内容,同时也为铜蓝蛋白作为一种新型有机生物铜源的开发和应用奠定了基础。4.给试验小鼠补充不同浓度的猪铜蓝蛋白研究了铜蓝蛋白对小鼠机体抗氧化能力及Cu-ATPase的影响,结果显示铜蓝蛋白可明显提高小鼠机体总抗氧化能力、Cu-ZnSOD的活性,提高小鼠肝脏组织中一氧化氮含量,减少机体丙二醛的含量,其中以腹腔注射4mg/只的效果最优,但对小鼠肝脏谷胱甘肽过氧化物酶活力作用不显著：铜蓝蛋白可有效提高小鼠肝脏组织中铜--ATP酶的含量,其中以腹腔注射4mg／只的效果最优。这一研究结果对铜蓝蛋白功能提供了的有益补充。5.通过RT-PCR技术,克隆了荣昌猪铜蓝蛋白基因,序列已登录在GenBank上,登录号为EU714006,基因cDNA长度为3181bp。其最大开放阅读框长度为3180bp,编码1060个氨基酸残基。经与家猪基因组序列比对分析,预测出猪铜蓝蛋白基因位于第13号染色体上,横跨于该染色体基因的42.463Kb-95.199Kb之间,有20个外显子、19个内含子。其推导的氨基酸序列与21个物种CP氨基酸序列比对分析,结果克隆出的猪CP氨基酸序列人CP的同源性最高,达到89%,而与与紫色海胆相似于CP的蛋白的氨基酸的同源性最低,仅为19.2%。经生物信息学软件预测,该蛋白质在19aa--20aa处有较强的信号肽,但无跨膜结构。系统进化树分析表明该基因存在较大的种属差异,但与人、马的亲缘关系较近,反映了在-定程度上该基因受到了人工选择压力的影响。6.通过对铜蓝蛋白基因在荣昌猪脑、心、肺、肝、脾、胃、肠、肾、膀胱及肌肉10种内脏器官组织中的荧光定量PCR表达谱差异比较分析,结果表明在1、15、30、45及60日龄5个不同生长阶段中,铜蓝蛋白基因在同一个器官的表达有差异；而在同一个生长阶段,该蛋白质基因在上述10个器官组织中的表达也有差异。初步揭示了铜蓝蛋白基因在仔猪5个生长阶段中可能具有参与铜的吸收和转运、促进免疫、调节机体铜代谢等功能。本课题获得的猪铜蓝蛋白理化特点及酶学特性、在细胞水平促进垂体生长激素的分泌及免疫细胞的增殖、提高机体CuZn—SOD及Cu—ATP酶活力、该蛋白质的原基因序列及基因在5个生长阶段不同内脏器官中的表达特点等研究结果,为猪铜蓝蛋白作为畜禽的一种新型高效、经济安全、环境友好的生物铜源的开发和利用提供重要的理论依据。更多还原

【Abstract】 Ceruloplasmin （Ceruloplasmin, EC 1.16.3., abbreviated as CP）, a copper-containing a2-glycoprotein widely distributed in human and animals, is an important metalloproteinase for animal and human life. It plays important roles in the processes in animal body, such as the transport of copper, the metabolism of iron in cells, the development of embryo and neural system, the formation of new blood capillaries, and the scavenging of free radicals. Ceruloplasmin can quickly provide efficiently utilizable copper to a variety of tissue cells in animal body to satisfy that the needs of various organs for copper nutrition. And via milk, it also plays an important nutritional function in the early stage of newborn animals. These scientific conclusions provide important clues to the development and utilization of ceruloplasmin as a new type of biological copper source for livestock and poultry. Swine are an important livestock that are bred on the largest scale and distributed most widely in China. Copper is an essential trace element in the organs of swine, and within a certain dose range high doses of copper have been widely used or even abused in pig industry as an important growth promoter. The excessive use or abuse of inorganic copper has led to a variety of negative effects, such as copper poisoning in swine, the decline of pork quality, soil hardening in culture environment, the decline of water’s self-cleaning capability, serious waste of copper resource, and the damage to human health. Hence, there are broad prospects for the exploitation of biological copper sources for livestock and poultry that are safer and more efficient, more economically viable, and environmentally friendlier than inorganic copper. Using one of China’s three famous swine breeds-Rongchang Swine as its experimental material, this paper explores the purification and characterization of ceruloplasmin from swine （sus scrofa）, studies its biological and chemical property, its inhibition kinetics, and its partial functions, and conducts the cloning of such protein’s original gene, the analysis of bioinformatics, and the detection of fluorescent quantitative PCR expression. The main results are as follows:1. Ceruloplasmin of Rongchang swine was separated and purified through the degreasing with 25%Butanol, the salting-out with 35-50%saturated ammonium sulfate, the ultrafiltration with the filter membrane that intercepts the molecular weight of 10000D, the DEAE-sepharose chromatography, the Hydroxyapatite chromatography, and the Superdex-200 gel column chromatography. The enzyme activity of ceruloplasmin in the solutions in the various purification steps was inspected and monitored with o-anisic amine method. The single-band product of ceruloplasmin purification was obtained through SDS-pAGE electrophoresis, and the specific activity of its enzyme was 217.65-fold higher than crude enzyme, and the recovery rate is up to 32%.The molecular weight of swine ceruloplasmin was 123.5KD, and the isoelectric point of this protein was 5.08, and its sugar content was 7.8%. Each molecule of swine ceruloplasmin contained 6 copper atoms. The SDS-pAGE electrophoresis showed that swine ceruloplasmin was a single subunit protein. Enzymatic reaction showed that the optimum pH for swine ceruloplasmin was pH5.5 and pH5-10 was the stable pH environment for it. The optimum temperature for swine ceruloplasmin was 55℃and it had the best stability of enzyme activity within the temperature range of 50℃-60℃. The research into the enzymic reaction constant of swine ceruloplasmin showed that the initial velocity of its enzyme reaction was 24.75μmol/（L-min）, that the Km value was 0.95mmol/L, and that the maximum reaction velocity was 112.4μmol/L.2. The research into the influence of effectors on the enzyme activity of swine ceruloplasmin showed that swine ceruloplasmin had significant resistance against trysin and pepsin, copper, iron, zinc, manganese, and magnesium ions all had inhibitive effect on the enzyme activity of swine ceruloplasmin, and that the enzyme activity of this protein wore off with the increase of the concentration of these metal ions. Chloride ions, EDTA, hydrogen peroxide and sodium azide all had great inhibitive effect on the enzyme activity of swine ceruloplasmin, with sodium azide exerting the strongest inhibition and EDTA the weakest. Citric acid, oxalic acid and ascorbic acid all had an inhibitive effect on the enzyme activity of swine ceruloplasmin, and there existed a correlation between concentration and inhibition. The inhibitive effect of ascorbic acid on the enzyme activity of ceruloplasmin was non-competitive inhibition, and the inhibitive effect of citric acid on the enzyme activity of ceruloplasmin was competitive inhibition. Urea, however, basically had no significant inhibition on the enzyme activity of swine ceruloplasmin.3. Investigations were made into the effects of swine ceruloplasmin on pituitary cells’ secretion of growth hormone, cell proliferation of spleen cell, B lymphocytes, T lymphocytes and subsets （CD₄, CD₈）. The results showed that ceruloplasmin can promote the secretion of growth hormones in porcine pituitary cells, and that, the culture in 0.4mg/ml ceruloplasmin for 8h yielded the best effect. At the same time, swine ceruloplasmin could significantly promote the proliferation of spleen cells, T lymphocytes and subsets （CD₄, CD₈）, and B lymphocyte, and that the concentration of 1.Omg/ml （final concentration 0.5mg/ml） were the best. The results suggest that growth hormone secretion of pig pituitary ceruloplasmin not only were promoted by ceruloplasmin, also the cell proliferation of pig spleen, B lymphocyte, T lymphocyte and subsets （CD₄, CD₈） were increased for the role of mitogene with ceruloplasmin. This conclusion has enriched the physiological function of ceruloplasmin, and also, it had laid the foundation for the development and application of ceruloplasmin as a new source of organic biological copper.4. The experimental mice were administered intraperitoneal injection of porcine ceruloplasmin of different concentrations to explore the effect of porcine ceruloplasmin on mice’s antioxidant capacity and the contents of Cu-ATPase in mice. The results showed that porcine ceruloplasmin could significantly enhance the mice’s total antioxidant capacity and the activity of Cu-ZnSOD, increase the nitric oxide contents in mice’s livers, and decrease the MDA contents in the organism. The intraperitoneal injection of 4 mg for each mouse produced the best effect. However, there were no significant effects on the enzyme activity of mice liver glutathione peroxidase. And porcine ceruloplasmin could remarkably promote the Cu-ATPase content in the mice’s livers. The intraperitoneal injection of 4 mg for each mouse produced the best effect. The result adds favorably to the functions of ceruloplasm.5. The ceruloplasmin genes in Rongchang swine were cloned with RT-PCR technology, and their accession number EU714006 was entered in GenBank. The cDNA length of the gene was 3181bp. Its maximum open reading frame length was 3180bp.1060 amino acid residues were encoded. The comparison between cDNA and sus scrofa genome indicated that swine ceruloplasmin gene was located on the 13^th chromosome, between 42.463Kb-95.199Kb of this chromosome gene, and there were 20 exons and 19 introns. The comparison between the derived amino acid sequence and 21 species amino acid sequences showed the cloned amino acid sequences of swine ceruloplasmin had the highest homology with human CP （89%）, but had the lowest homology with Strongylocentrotus purpuratus （only 19.2%）. Bioinformatics software forecast that there was a strong signal peptide located in 19^th-20^th aa of this protein, but there was no transmembrane domain. Constructed phylogenesis tree indicated that this gene exhibited large difference among different species, but was genetically most related to that of human and horse. This indicated that this ceruloplasmin gene was affected to a certain extent by the pressure of artificial selection.6. The difference of ceruloplasmin gene expression in brain, heart, lung, liver, stomach, intestinal, kidney, bladder and muscle of Rongchang swine were detected by FQ-PCR, and the results showed that ceruloplasmin gene expressions in the same organs were different at the five growth stages of 1^st,15^th,30^th,45^th,60^th days, and that the gene expressions were different in 10 organs at the same growth stage. It is thus initially concluded that ceruloplasmin gene may have been involved in copper intake and transport, in promoting immunization, and in regulating copper metabolism in the organisms at the five growth stages of piglets.The research results of this paper include the physical and chemical characteristics of swine ceruloplasmin and its enzyme properties, its promotion of the growth hormone secretion in pituitary and immune cell proliferation at cellular level, the enhancement of the Cu-ZnSOD and Cu-ATPase activity, original gene sequences of this protein, gene expression characteristics in different organs at the five growth stages of swine. These results provide an important theoretical basis for the development and exploitation of swine ceruloplasmin as a new type of biological copper source for livestock and poultry that is safer and more efficient, more economically viable, and environmentally friendlier than inorganic copper.更多还原