Purification And Function Research Of Active Peptide Form Blood Hydrolyzed By Trypsin

Immunomodulator is some hydrone hydrone that can enhance the immunologic activity of immunologic system, and they are always some peptides, suck as MDP(muramgl dipeptide), Thymic pepeide, Bru-pel and so on. In immunology, immunomodulator is a very important and valuable conception. Scientists had done some immunologic researches fo4r a long time. With the development of science and technology,immunology has become an essential branch of biologic science. It is very obvious to all of us that whether or not the humanbeings'immunologic system ability is strong enough plays a very important role to their normal physiologic activities. If the immunologic system of organism lost their distinguish ability to self and not-self, the physiologtic activities of organism, even their life will be seriously harmed. On the contrary, any activity that can enhance the immunologic activities of organism, will make organism get more safe protection. All these re4sons make immunomodulator become a heat-point in immunologic researches during the last twenty years. On the basis of the immunomodulator important value in scie4nce and application, our research object –a nove peptide with high immunologic enhancement activity also has the important research value. We found the enzymolysis product of pig blood by trypsin hydrolysis is a mixture of some single amino acids, compund amino acids and some peptides, that may obvious enhance the immunologic activities of immunologic ysstem. As a matter of fact, it has been a long time that animal blood is applied to science researches to get some useful biologic materials. Among these animal bloods, the source pig blood is very cheap and rich. The more important reason of the abundant protein content in pig blood, makes them become an important research object. Among these researches of pig blood, using biological purification ways to get some useful single amino acids, compound amino acids or some valuable pepeides is the main aspect. In our research, we used trypsin to hydrolyze fresh pig blood and got the mixture that had ability to enhangce organism immunologic activity, and this mixture is the raw material of our research. Our researchhas two main aspects: 1. We use a series of biological methods, to isolate and purify the aim peptide, with high enhancement immunologic activity, from enzymolysis product of pig blood by trypsin hydrolysis. 2. In order to understand the enhancement immunologic activity of this peptide, we do a lot of immunology and hparmacology experiments in vitro and in vivi, and get some valuable results. During the purification progress, we use Sephadex G-25 chromatography, ion-changechromatograhpy, ultrafiltration to isolate and purify the aim product, and use HPLC to determine the prrity of this peptide. In order to identify the existence of our aim peptide, weconduct T lymphocyte transformation test and E rosette forming test to the product of the progress. The result indicates that our aim product is a kind of thite and easily absorbing water substance. In order to understand the enhangcement immunologic activity of our aim peptide, we conduct some important immunology and pharmacology experiments in vitro and in vivo. In vitro experiments, we mainly research the effect of the aim peptide on T lymphocyte, so we do T lymphocyte transformation test and E rosette forming test. But because in vivo experiments are more important than in vitro experiments in immunology and pharmacology, and the results of in vivo experiments are3 more valuable. In vivo experiments, to three main kind of immunologic cells: T lymphocyte, B lymphocyte, NK natural killer cell, we do T lymphocyte transformation test, B lymphocyte agglutinating titer and NK cell cytotoxic activity test. From all these results, we have a basic understand to the peptide and get five important conclusions about it's enhancement immunologic activity. 1. Whether in vitro or in vivo immunologic experiments, this peptide all show much higher immunologic enhancement activity than control. It indicates that our aim peptide is a positive cellular immunity adjuster. 2. From three tests in vivo related T lymphocyte, B lymphocyte, NK natural killer cell, although this peptide can enhance all three kinds cells immunologic activities, we found it affected the activity of B lymphocyte is muchgreater than T lymphocyte and NK natureal killer cell. The result of the agglutination titer of B lymphocyte (5.0mg/kg . d) is 5.6 (injection) and (enema). Times as much as control. The activity enhanced is all greatly higher than T lymphocyte, NK natural killer cell. 3. To injection groups and enema groups in vitro, although enhancement activity difference between them is not as much as that between T lymphocyte, B lymphocyte, and NK natural killer cell. In all three tests in vitro, injection groups show greater enhancement activity than enema groups, no matter how much dosage level is compared. The reason probaly is, when rats are delivered by enema, their digestive system, especially their stomachs and bowels will be stimulated. This can result in their absorbance ability becoming worse, so the peptide can't fully affect their functions. In the test we also found most rats of enema groups are thinner and with a poorer appetite, it is a also evidence of our judgement. 4. To different dosage of groups, whether injection or enema, the level of enhancement of immunologic activity decreased along with enhancement of dosage. When dosage is 5.0mg/kg . d, the activity of enhancement is much greater than that of other dosage, and when dosage is 10.0mg/kg . d and 20.0mg/kg . d, the enhancement became smaller, when 40.0mg/kg . d, the peptide even...