Studies On The Purification, Characterization, Function, Gene Cloning And Expressions Of Ceruloplasmin In Swine

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.