JP4860109B2 - Composition for enhancing immunity comprising polygamma glutamic acid - Google Patents

Description

  The present invention relates to the use of polygamma glutamic acid having both immunopotentiating and regulatory activities, and more specifically, the use of an adjuvant for vaccines, an immune enhancing composition containing an effective amount of polygamma glutamic acid, a functional food, and It relates to a feed composition.

  Immunity is broadly classified into innate immunity that has been born and acquired immunity that is obtained by adapting to acquired life. Innate immunity, also called “natural immunity”, is a non-specific reaction to an antigen and has no special memory effect. The innate immune system includes skin, mucus tissue, strongly acidic gastric acid, and complement that exists in blood. Examples of the cells include macrophages and polymorphonuclear leukocytes responsible for phagocytosis, and K cells that can kill infected cells. In fact, most infections are protected by this innate immunity.

  Acquired immunity is also called “acquired immunity”, and it can memorize the antigen that invades first, and can react specifically when it invades again and effectively remove the antigen. It is characteristic and serves to reinforce innate immunity. Normal immunity means this. Such acquired immunity is understood as being divided into humoral immunity and cell-mediated immunity for convenience.

  In humoral immunity, B lymphocytes recognize an antigen and then differentiate to secrete an antibody, which mainly functions to remove infected bacteria. Antibodies exist in body fluids and are glycoproteins called immunoglobulins (Igoglobulin: Ig). These include IgG, IgM, IgA, IgD, IgE, etc., each performing a unique function, with some overlapping functions. IgA antibodies are characterized by being transmitted to the fetus through the placenta. Such immunity is called maternal immunity, but it is not so infected for several months after being born.

  Cellular immunity serves to cause T lymphocytes derived from the thymus to recognize the antigen and secrete lymphokine, or to directly kill infected cells. Secreted lymphokines activate macrophages and help phagocytosis. Such cellular immunity primarily functions to remove cells infected by viruses or bacteria that can grow inside the cells. Acquired immunity can be obtained by vaccination with a pathogen or its toxin as an immunogen, and such immunity is referred to as artificial immunity.

  If the immune function is reduced, hypersensitivity reactions (allergy, hypersensitivity) may be caused. However, hypersensitivity reactions such as allergies can be classified into four types based on the expression time and reaction form.

Type I (immediate type) is typical of most allergic reactions. Symptoms include asthma, seasonal or perennial rhinitis, allergic sinusitis, conjunctivitis, food and drug allergies , Atopic dermatitis, urticaria, allergic reaction to bees.
Type II (participation of antibody) causes humoral immune cell lysis reaction and inflammatory reaction. Symptoms include erythrocyte hemolysis due to incompatible blood transfusion and neonatal hemolytic disease.
Type III is caused by antigen-antibody reaction or small molecular weight antigen. Causes acute glomerulonephritis and hypersensitivity pneumonia.
Finally, type IV (delayed type) is a form of cellular immune response by T lymphocytes and macrophages, when tuberculosis tuberculin reaction, transplant rejection reaction, when microorganisms such as viruses invade Causes an important reaction to react.

  Various immune function enhancers and therapeutic agents have been developed and used in order to overcome the problem that the immune function is impaired due to the reasons described above.

  However, when taking a separate therapeutic agent for a long period of time, there is a disadvantage that there is a significant problem of side effects due to differences in immunity among individuals. In particular, prevention of immune diseases is more important than treatment, but currently available therapeutic agents are difficult to take for prevention. Therefore, there is a need for a method that can prevent and treat immune dysfunction without taking a separate immune function enhancer that may have side effects.

In general, mushroom extracts and plant extracts are well known as immunopotentiators known so far, and the effects of such materials have been continuously reported. Typical examples of the mushroom extract include krestin, lentinan, and AHCC. As the plant extract, miltuto is a typical immunity enhancing substance.

  As an existing immunopotentiating substance, there is a polysaccharide Krestin (PSK) extracted from mycelium of Tsuriganetake, which was successfully commercialized in Japan in 1976. This is a substance that increases the activity of macrophages, T cells, and NK cells and enhances immunity by promoting the production of various cytokines such as interferon and interleukin-2. In addition, Lentinan is known by a research report that polysaccharides extracted from shiitake mushrooms have an immunopotentiating effect on cancer at an international conference held in Paris in 1978 by Dr. Goro Amahara of the National Cancer Center of Japan. became. AHCC (Active Hexose Correlated Compound) is an immune function stimulating substance extracted by reacting various types of enzymes after culturing mushroom mycelium. Mistleto, a plant extract, is the English name for mistletoe. It is a semi-parasitic plant that grows around the world and is a host of many types of trees. Has been used as. In Europe, mistletoe is known as a mysterious drug for various diseases like Japan, and is an immunopotentiator that has been traditionally used in the private sector.

  Other substances that enhance immunity include carrots (red ginseng, ginseng, etc.), garlic, bamboo salt, legumes, fermented foods (miso, chili miso, neat soy sauce (like natto), etc. }, Green juice and various vegetables, fruits, organic germanium, various yeast foods, freshwater peas (melanian snail), elm bark, natural cereals (brown rice, germinated brown rice, wheat, pigeons, red beans, mochiawa) Etc.) and various seaweeds (wakame, kelp, etc.).

However, there has been no report to date on the immunopotentiating and / or immunomodulating activity of polygamma glutamate produced from microorganisms.
International Publication No. 02/55671 Pamphlet

  In contrast, the present inventors have demonstrated that polygamma glutamate produced by Bacillus bacteria induces not only macrophage activation but also proliferation, differentiation and activation of T lymphocytes and B lymphocytes. The present inventors have confirmed that polygamma glutamic acid is useful as an immune activity enhancer, and confirmed that the polygamma glutamic acid exhibits the effect of a vaccine adjuvant, thereby completing the present invention.

The main object of the present invention is to provide a use of polygamma glutamic acid that exhibits immune enhancement and regulatory effects.
Another object of the present invention is to provide a feed composition and a functional food comprising the polygamma glutamic acid as an active ingredient.
Still another object of the present invention is to provide a method of using the polygamma glutamic acid as an adjuvant for vaccines.

To achieve the above object, the present invention provides an immunopotentiating composition comprising an effective amount of polygamma glutamic acid and a pharmaceutically acceptable carrier.
The present invention also provides a vaccine adjuvant containing polygamma glutamic acid.
The present invention also provides a functional food having an immunopotentiating effect, which contains an effective amount of polygamma glutamic acid and a pharmaceutically acceptable additive.
The present invention also provides a livestock feed composition having an immune enhancing effect, comprising an effective amount of polygamma glutamic acid.
In addition, the present invention provides an animal immunity enhancing method characterized by administering an effective amount of polygamma glutamic acid to animals that need immunity enhancement except humans.
In the present invention, the polygamma glutamic acid has a molecular weight of 1 to 15000 kDa.

The present invention relates to the use of polygamma glutamic acid having immune enhancing and regulatory activity, and more specifically, an immune enhancing composition, vaccine adjuvant, functional food and feed composition containing an effective amount of polygamma glutamic acid Has the effect of providing.
The composition containing the polygamma glutamic acid of the present invention has immune enhancing and regulatory activities at the same time, and has almost no toxicity and side effects, so that it can be used with confidence even when taken for a long time for the purpose of prevention. Further, it can be used for the prevention and treatment of infectious diseases caused by bacteria or viruses, and enhances immunity against patients whose immunity has been reduced by anti-cancer therapy or whose immunity has been suppressed by AIDS or cancer. In addition, it can be used as a pharmaceutical for regulation and a health functional food, and is also useful as an adjuvant for vaccines.

  The immunopotentiating composition comprising polygamma glutamic acid of the present invention can further comprise suitable excipients and diluents commonly used in the manufacture of pharmaceutical compositions (Remington's Pharmaceutical Science, Mack Publishig Co., Easton PA). In addition, the composition for enhancing immunity containing the polygamma glutamic acid according to the present invention is prepared by oral methods such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It can be used in the form of suppositories and sterile injections.

  Carriers, excipients, and diluents that may be included in compositions containing polygamma glutamic acid include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, acacia gum, alginate, gelatin , Calcium sulfate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

  In the case of formulating, it can be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants and the like that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations contain at least one excipient in the polygamma glutamic acid, such as starch, carbonic acid, etc. It can be prepared by mixing calcium, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, liquids for internal use, emulsions, syrups, etc., but various excipients other than water and liquid paraffin, which are frequently used simple diluents, For example, wetting agents, sweeteners, fragrances, preservatives and the like may be included. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble agents, suspensions, emulsions, lyophilized formulations and suppositories. Examples of water-insoluble preparations and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a suppository base, hard fat (witepsol), macrogol, Tween 61, cacao butter, lauricylva butter, glycerogelatin and the like can be used.

  The amount of the polygamma glutamic acid-containing composition of the present invention may vary depending on the age, sex, and body weight of the patient, but generally the amount is 5 to 500 mg / kg, preferably 100 to 250 mg / kg daily. The administration can be divided into 1 to 3 times, and the dosage can be increased or decreased according to the administration route, the degree of disease, sex, body weight, age, and the like. Therefore, the dosage does not limit the scope of the present invention in any way.

  Since polygamma glutamic acid itself used in the present invention has almost no toxicity and side effects, it can be used with peace of mind when taken for a long period of time for the purpose of prevention.

  In the present invention, polygamma glutamic acid may be added to foods and beverages intended for immunomodulation. At this time, the amount of polygamma glutamic acid in foods and beverages can be generally added in the range of 0.01 to 30% by weight of the total food weight, and the health drink composition is 0.01 based on 100 ml. Can be added in a proportion of -10 g, preferably 0.1-2 g, and can be in the form of powder, granules, tablets, capsules or beverages.

  The health beverage composition of the present invention has no particular limitation on the liquid component except that it contains the polygamma glutamic acid as an essential component in the indicated proportion, and various flavoring agents or natural carbohydrates are used as in a normal beverage. Etc. can be included as additional components. Examples of the natural carbohydrates described above include normal sugars such as monosaccharides (dextrose, fructose, etc.), disaccharides (maltose, sucrose, etc.) and polysaccharides (dextrin, cyclodextrin, etc.), and xylitol, sorbitol, erythritol, etc. Of the sugar alcohol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (rebaudioside A, glycyrrhizine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The amount is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the composition of the present invention.

  In addition to the above, the composition of the present invention comprises various nutrients, vitamins, minerals (electrolytes), minerals, synthetic flavors, natural flavors, colorants, sedatives (cheese, chocolate, etc.), pectic acid and The salts, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may also be included. In addition, each composition of the present invention may include pulp and grains for the production of natural fruit juices and fruit juice drinks, cereal drinks and vegetable drinks. Such components can be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of about 0.01 to 20% by weight per 100% by weight of the total composition of the present invention.

  The polygamma glutamic acid used in the present invention is administered to an animal (for example, cattle, horses, sheep, goats, pigs, dogs, cats, poultry, rats, mice, etc.) in need of immune enhancement by administering an effective amount. Can enhance immunity. In this case, an effective amount of polygamma glutamic acid may be added to the feed and administered as a feed additive. The feed additive of the present invention has a polygamma glutamic acid content of 0.01 to 95% by weight, preferably 0.1 to 80% by weight, based on the total weight of the feed composition in order to enhance immunity against infectious diseases. It may be added within the range.

  Antigenic components that can be used together with the polygamma glutamic acid of the present invention as an adjuvant for vaccines include antigens with poor immunogenicity as peptides, polypeptides, proteins, nucleic acid sequences corresponding to these, or vaccine targets. It may be selected from the group consisting of target cells or lysates thereof, or combinations thereof. The adjuvant for vaccines of the present invention can be used together when administering vaccines by parenteral, mucosal (oral, nasal, lung) and transdermal routes.

  When a microorganism expressing an antigenic protein is used as a vaccine, it is preferable to use the polygamma glutamic acid of the present invention as an adjuvant. In particular, when the lactic acid bacterium expressing the antigenic protein is used as an oral vaccine, it is preferable to use the polygamma glutamic acid of the present invention together with the vaccine adjuvant.

  In addition, the polygamma glutamate of the present invention prevents cancer, particularly prostate, colon, lung, breast, ovary, head and neck, vulva, bladder and brain cancer, glioma, as well as non-infectious chronic diseases. It can be used in addition to the pharmaceutical composition used to treat. Moreover, you may use together with the pharmaceutical composition used in order to prevent and treat a viral and bacterial infectious disease, and the pharmaceutical composition used in order to treat an autoimmune disease.

  Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Production of polygamma glutamic acid Basic medium for production of polygamma glutamic acid (GS medium supplemented with 5% L-glutamic acid; glucose 5%, (NH ₄ ) ₂ SO ₄ 1%, KH ₂ PO ₄ 0.27%, Na ₂ HPO ₄ · 12H ₂ O 0.42%, NaCl 0.05%, MgSO ₄ · 7H ₂ O 0.3%, vitamin solution 1ml / L, pH 6.8) 3L in size The fermenter was inoculated with 1% of a culture solution of Bacillus subtilis var chungkookjang (KCTC 0697BP) and cultured at 37 ° C. for 72 hours at a stirring speed of 150 rpm and an air injection speed of 1 vvm. A sample solution containing polygamma glutamic acid was obtained by adding 2N sulfuric acid solution and adjusting the pH to 3.0.

  The sample solution was allowed to stand at 4 ° C. for 10 hours to remove the polysaccharide in the fermentation broth, and ethanol was added thereto so that the volume was twice that of the fermentation broth, followed by thorough mixing. The mixture was allowed to stand at 4 ° C. for 10 hours, and then centrifuged to obtain a polygamma glutamic acid precipitate. Distilled water is added to the precipitate to dissolve it, and the proteolytic enzyme is added to 100 μg / ml and allowed to react for 6 hours in a 37 ° C. thermostat, and then the extracellular substance present in the polygamma glutamic acid sample. Protein was degraded. This was dialyzed with a sufficient amount of distilled water to remove the free glutamic acid and then concentrated to obtain pure polygamma glutamic acid.

  If necessary depending on the application, the produced polygamma glutamic acid may be cut by an appropriate method to produce a certain molecular weight, or may be separated by an appropriate method and recovered according to a certain molecular weight. The average molecular weight of polygamma glutamic acid used in the following examples was 5 kDa, 10 kDa, 20 kDa, 50 kDa, 1200 kDa and 2000 kDa as a result of GPC analysis.

Induction of TNF-α and IL-1β gene expression from macrophages by polygamma glutamate First, the effect of polygamma glutamate on the activation of macrophages important for the primary immune response of the immune system was examined. At this time, as an index of macrophage activation, the ability to induce gene expression of tumor necrosis factor (TNF-α), which is a main cytokine secreted by macrophages, and proliferation, differentiation and activation of T-lymphocytes and B-lymphocytes The ability of IL-1β involved in the gene expression induction was examined.

For this purpose, a macrophage strain of mouse (Balb / c) (RAW 264.7: ATCC TIB-71) was suspended in DMEM (100 U / ml penicillin-streptomycin, 10% FBS added) medium, and then suspended in a 6-well plate After dispensing at 1 × 10 ⁶ / well and culturing in a CO ₂ incubator for one day, polygamma glutamic acid (γ-PGA: 10 kDa, 50 kDa, 1200 kDa) was added, and 0.1% and 0.01%, respectively. The solution was diluted with DMEM medium not containing FBS to a concentration of 0.001% and cultured for 1 hour.

  At this time, as a positive control group, existing well-known LPS (lipopolysaccharide; Sigma, USA) was used, and stimulation was performed at a concentration of 10 ng / ml. After completion of the culture for stimulation, the RAW 264.7 cell line in each well was removed, and the amount of mRNA that is an indicator of the gene expression inducing ability of TNF-α and IL-1β present in the cells was determined by RT- Confirmed by PCR. After separating mRNA using RNeasy mini kit (Qiagen, Germany), the obtained RNA was dissolved in DEPC-DW, and the concentration and purity were measured using a spectrophotometer.

  Omniscript RT kit (Qiagen, Germany) was used to synthesize cDNA from the extracted RNA. RNA PCR buffer, dNTP 0.5 mM, oligo dT primer 1 μM, RNase inhibitor 0.5 unit, Omniscript reverse transcriptase 0.2 unit were added to 1 μg of mRNA from the total RNA, and reacted at 37 ° C. for 1 hour. Finally, cDNA was obtained by heating at 95 ° C. for 5 minutes.

PCR was performed using the synthesized cDNA as a template. Primers for TNF-α and IL-1β genes were designed based on GenBank Nucleotide Sequence Database. Primers for each gene are as follows.
F primer for TNF-α: 5′-CAg-gCA-ggT-TCT-gTC-CCT-TTC-A-3 ′ (SEQ ID NO: 1)
R primer for TNF-α: 5′-CAC-TTg-gTg-gTT-TgC-TAC-gAC-g-3 ′ (SEQ ID NO: 2)
F primer for IL-1β: 5′-gCT-ACC-TgT-gTC-TTT-CCC-gTg-g-3 ′ (SEQ ID NO: 3)
R primer for IL-1β: 5′-TTg-TCg-TTg-CTT-ggT-TCT-CCT-Tg-3 ′ (SEQ ID NO: 4)

  PCR was carried out by adding 10 μl of 2 × premix buffer (Bioneer) and 10 pmol of each of the above primers to 50 ng of cDNA, and making the total volume 20 μl, followed by predenaturation at 95 ° C. for 40 seconds, and then at 95 ° C. for 40 seconds. It was carried out under conditions of 35 cycles at 55 ° C. for 40 seconds and 72 ° C. for 60 seconds. 5 μl of each PCR product was electrophoresed on a 1% agarose gel (FIG. 1).

  In FIG. 1, A shows β-actin mRNA expressed in all cells as a control group, B shows mouse TNF-α mRNA expression inducing ability, and C shows mouse This shows the ability to induce the expression of IL-1β mRNA. As shown in FIG. 1, in the case of TNF-α mRNA expression inducing ability, an increase in the expression rate was not confirmed as compared with the cells in the control group, but in the case of IL-1β mRNA expression inducing ability, LPS It was possible to confirm an increase in the expression rate similar to cells treated with. From these results, the gene expression of tumor necrosis factor (TNF-α), which is the main cytokine secreted by macrophages, is an indicator of macrophage activation that is important for the primary immune response. It was confirmed that the gene expression induction effect of IL-1β involved in the proliferation, differentiation and activation of T-lymphocytes and B-lymphocytes was exhibited together with the induction effect.

Induction of TNF-α secretion from macrophages by polygamma glutamate In the same manner as in Example 2, polygamma glutamate (γ-PGA: 10 kDa, 50 kDa, 1200 kDa) was added to mouse (Balb / c) macrophage strain RAW 264.7, respectively. After culturing with stimulation at concentrations of 0.1%, 0.01%, and 0.001%, the culture supernatant of each well was removed and TNF-α (Mouse TNF- present in the culture supernatant) was removed. The secretion-inducing ability of α ELISA kit (BD) was measured using ELISA kit.

  In 96 wells prepared (plate coated with anti-mouse monoclonal antibody of TNF-α), 50 μl of TNF-α standard solution and 50 μl of culture supernatant were added and allowed to react at room temperature for 1 hour and 24 hours, respectively. Then, after washing 5 times with a washing buffer (300 μl / well), 100 μl of biotinylated anti-mouse TNF-α polyclonal antibody, which is the primary antibody, was added, reacted at room temperature for 1 hour, and washed with a washing buffer (300 μl / well). ) For 5 times. After that, add 100 μl of avidin-horseradish peroxidase conjugate as a secondary antibody, react for 30 minutes at room temperature, wash 7 times, react with TMB coloring solution for 30 minutes, and then fix the color with 50 μl stop solution. did. And it measured with the ELISA reader at 450 nm, and analyzed the amount of TNF- (alpha). The results are shown in FIG. In FIG. 2, A shows the result of measuring TNF-α secreted into the medium after 1 hour after treating the sample with macrophages, and B is secreted into the medium after 24 hours after treating the sample. It is the result of measuring TNF-α.

  As can be seen from the results shown in FIG. 1 and FIG. 2, it was confirmed that polygamma glutamic acid had an excellent effect as a macrophage stimulant, and showed a tendency depending on the concentration and molecular weight when activating macrophages. The most effective concentration and molecular weight of polygamma glutamic acid were 0.1% and 1200 kDa, respectively.

  Polygamma glutamate was lower in both TNF-α and IL-1β gene expression inducing ability and secretion inducing ability than LPS, a bacterial endotoxin used as a positive control group. It can be said that such an action of polygamma glutamic acid is weaker than LPS is a very preferable phenomenon in the biological immune reaction. This is because, although appropriate secretion of TNF-α and IL-1β is beneficial to the biological defense mechanism, excessive secretion causes a fatal shock to the living body and has great side effects. Eventually, the polygamma glutamic acid according to the present invention is considered to have not only immune enhancement but also an immune regulatory function.

A substance that is injected together with an antigen in order to increase the immunogenicity of antibody production against a viral antigen by polygamma glutamic acid , that is, to enhance a pesticidal response, is called an adjuvant. Freund adjuvant is made by adding a surfactant, Arlacel-A, to a mineral. A soluble antigen is mixed well to form a suspension, which is injected intradermally or subcutaneously to enhance antibody productivity. A tubercle bacillus is added as a complete adjuvant, and a tuberculosis is not added as an incomplete adjuvant. Wax-D in the Mycobacterium tuberculosis component shows the effect of the adjuvant. In addition, alum, aluminum hydroxide, bentonite, pertussis vaccine and the like are also known as having an adjuvant effect.

  As observed in Examples 2 and 3, the polygamma glutamic acid of the present invention also activates macrophages, which are the main antigen-presenting cells, so it is predicted that it can serve as an adjuvant that contributes to antibody production. The study was conducted as follows.

  Whether or not the polygamma glutamic acid of the present invention has a specific immune enhancement effect against an antigen was examined. This experiment was related to an antigen-specific immune response, and in particular, the effect on the humoral immune response by B cells related to antibody production was examined. That is, the infectious gastroenteritis virus (TGE) nucleoprotein (NP) of porcine infectious gastrointestinal disease was used as an antigen, and the rabbit was used as an experimental animal.

  First, as a control group, rabbits injected subcutaneously with TGEN antigen (400 μg / PBS ml) alone were used, and TGEN antigen (400 μg / PBS ml) and polygamma having molecular weights of 5 kDa, 10 kDa, 20 kDa and 50 kDa, respectively. Glutamic acid (γ-PGA) was mixed and divided into experimental groups administered by subcutaneous injection.

  At this time, as a positive control group, an experimental group in which Freund's complete Adjuvant and TGEN antigen (400 μg / PBS ml) were mixed and administered by subcutaneous injection was used. Two weeks after the first subcutaneous injection, the same amount of antigen and the respective polygamma glutamate complex was administered. Rabbit serum was collected every two weeks after the first subcutaneous injection, and the titer of the antibody against the TGEN antigen in the serum was measured by ELISA (Enzyme linked Immunosorbent assay).

  The ELISA was simply performed by coating the plate with TGEN antigen (0.1 μg / ml). After blocking the plates with PBS / 5% fetal bovine serum, the sera of control and experimental rabbits were incubated in various serial dilutions. Thereafter, an anti-rabbit IgG antibody (specific for Fc) to which Horse radish peroxidase was attached was added. All incubations described above were performed for 1 hour at 37 ° C., and after each step described above, washed 3 times with PBS / 0.05% Tween20. ABTS {2,2-azinobis (3-ethylben-zthiazolinesulfonic acid)} 1 mg / ml was added as a substrate to develop the reaction. After 30 minutes, the absorbance at 450 nm was measured with an ELISA reader. The results are shown in FIG.

  As can be seen from the results shown in FIG. 3, the antibody titer against the TGEN antigen in the rabbit injected subcutaneously with the TGEN antigen combined with polygamma glutamic acid according to the molecular weight of the present invention was determined as Freund's complete Adjuvant and Although it was lower than the antibody titer of the rabbit administered in combination with the TGEN antigen, it was higher than the antibody titer against the TGEN antigen in the rabbit injected subcutaneously with only the TGEN antigen. In particular, rabbits using polygamma glutamic acid 50 kDa as a complex had the highest antibody titer. The results showed that the persistence against the increase in antibody titer was significantly increased compared to the control group for at least 6 weeks from the first injection.

  As described above, specific portions of the present invention have been described in detail, and those skilled in the art have ordinary knowledge, and such specific techniques are merely preferred embodiments, and thereby limit the scope of the present invention. Obviously it is not. Accordingly, the substantial scope of the present invention is defined by the appended claims and their equivalents.

It is the photograph which confirmed the gene expression inducing ability of TNF- (alpha) (tumor necrosis factor- (alpha)) and IL-1 (beta) by RT-PCR from the macrophage which is a primary immune reaction cell by the stimulation of polygamma glutamate. It is the graph which confirmed the extracellular secretory expression induction ability of TNF- (alpha) from the macrophage which is a primary immune reaction cell by the stimulation of polygamma glutamate by ELISA. Polygammaglutamic acid and the transmissible gastroenteritis virus (TGE) nucleoprotein antigen complex that induces infectious gastrointestinal diseases in pigs are injected subcutaneously into rabbits and the prescribed period has passed. 2 is a graph showing the induction of an antigen-specific immune reaction by polygamma glutamic acid by measuring the specific IgG antibody titer against nucleoprotein antigen in serum by ELISA.

Claims (2)

A pharmaceutical composition for inducing macrophage activity, T lymphocyte and B lymphocyte proliferative activity, comprising an effective amount of polygamma glutamic acid having a molecular weight of 1 kDa to 15000 kDa and a pharmaceutically acceptable carrier, and not containing an antigen. The pharmaceutical composition according to claim 1, which has a molecular weight of 10 kDa to 1200 kDa.

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