Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/375—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Basidiomycetes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Ganodenorema melesi dam (Ganoderna lucidu ⁇ ) IV 009, which produces a protein polysaccharide (G009) with antitumor ⁇ immune enhancing effect
• FIG. 1 shows a saccharide analysis chromatogram of the protein polysaccharide G 009 of the present invention
• FIG. 2 shows an amino acid chromatogram of the protein polysaccharide G 009 of the present invention
• FIG. 3 shows a chromatogram of the protein polysaccharide G 009 of the present invention.
• FIG. 4 is a microscopic photograph of the mycelium of the strain of the present invention 009
• FIG. 5 is a comparison of the specificity of the strain IY 009 of the present invention.
• the present invention relates to a strain that produces a protein polysaccharide having an anti-vaginal immunity enhancing effect.
• the present inventors collected a large number of basidiomycetes in each region for the purpose of searching for a new polysaccharide having an anti-killing effect and an immune-enhancing effect, and tried to determine the key to the protein polysaccharide produced by the basidiomycetes. did.
• Submerged culture medium Glucose 50 g, peptidase tons 20g, KH 2 P0 4 0.87g, MgS0 4 7H 2 0 0.5g, FeCl 2 .6H 2 0 10ng, MnCl z .4H 2 0 7ag, CuSO ⁇ 5H 2 0 LONG, was 1 beta was added to distilled water ZnCl 2 Amg.
• the ⁇ was adjusted to 5.5 and sterilized with high pressure steam at 121 for 20 minutes.
• the supernatant (400 1) was added to 100 ml of ethanol, centrifuged and separated, and
• G2 (0.4) was obtained by depressurizing and drying the resulting precipitate, and ethanol 800B1 was again added to the remaining supernatant 800B1 after removing G2, and the resulting precipitate was decompressed and dried.
• G3 (l.lg) was obtained.
• Gl (2.1 g) was dissolved in water, applied to Sephadex G-100, and eluted with water. Freeze-dry the tube ⁇ 25-33 to obtain G4 (1.5 g),
• G4 was re-dissolved in water, and the same volume of a mixture of 0.15 M Cetavelon (Cetavelon; Cethyl-trimethyl aBBonius bromide) and 0.1 M Borate buffer (Borate buffer PH 8. ⁇ ) was added. Adjust the pH to 9.0 with 0.5 ⁇ NaOH and remove the precipitate (G7) and supernatant (G6).
• the precipitate obtained here is washed with methanol and acetone and then
• the activity test of the above-obtained fraction on meat type-180 was carried out as follows. 20-25g ICR male mice were transplanted intraperitoneally at weekly intervals and subcultured for meat type -180 (Sarc O » a -180), which was used as a recommended test material. 180 cells of meat type 1 cultured in the mouse peritoneal cavity for 7 days were ascites
• the cell suspension O. IBI was subcutaneously transplanted to the left groin in 10 mice per group. 72 hours after tumor cell transplantation. Purification for 10 consecutive days
• control group received physiological saline
• treatment group received 0.1 ng of protein polysaccharide dissolved in physiological saline at a concentration of 20 ng / kg.
• SUBSTITUTE SHEET The mouse was sacrificed on the 30th day after tumor cell transplantation, the induced solid tumor was excised, and its weight was measured to determine the average tumor weight. was calculated.
• mice 20-25 epsilon was used as the experimental tumor ⁇ cells.
• mice were transplanted into the left inguinal region of the mice, 10 times muscles every other day system, subcutaneous and intraperitoneal injection zong / kg, Shizu
• EAT Ehrlich ascite tunors
• the control group received the same amount of physiological saline at the same time.
• intraperitoneal administration of G 009 and intravenous administration of G 009 showed almost the same level of anticancer effect, and a more prolonged lifespan effect than the control group.
• mice 20-25 g were intraperitoneally transplanted at weekly intervals and passaged
• Injection was performed with saline instead of 15 doses, and the number of mice in the treatment group and the control group was 10 each.
• the mouse was killed 30 days after the tumor transplantation, and the weight of the tumor was measured.
• Example 5 Thymectomy of neonatal mice G 009 administration and antithymic globulin effect after 2 days of age Thymectomy of 2-day-old ICR mice was performed by Sjodin method, and 6 weeks after thymectomy Meat type 180 to IxlO 7 cell
• SUBSTITUTE SHEET The solution was implanted by O. IBI. G 009 was injected intramuscularly at a dose of 20 g / k 10 times every other day at 24 hours after transplantation. To prevent infection of the thymectomized mice, water containing Tetracycline (HC Tetracycline-HIC) was used during the experiment.
• HC Tetracycline-HIC Tetracycline
• antithymic serum was removed by removing the thymus from 2-3-week-old mice, separating thymocytes, and then diluting in phosphate buffered saline to prepare cell suspension S. After the production, 3 rabbits were immunized with 1x10 s cells Z1 thymocytes by intravenous injection at 3 week intervals. One week after the last injection, antithymic serum was collected and inactivated at 561C for 30 minutes.
• igG immunoglobulin G
• Aged mice were injected intraperitoneally with a dose of O.ln] for 10 days, and G 009 was injected intramuscularly 5 times at a dose of 20 »g / kg every other day one day after 180 transplants of meat species.
• A.T.M Antithymic globulin-treated mice
• N.R.G.M Normal rabbit globulin-treated mouse
• Guinea pig serum and human fresh serum are used for complement, red blood cells are used for «sheep erythrocytes, and antibodies are used for anti-sheep hemolysin.
• the activity is the amount of complement disappearance (3 ⁇ 4) of the control group and that of G009. As shown in Table 9, the amount of complement disappearance increases in proportion to the concentration of G009.
• the «sheep erythrocytes 1 X 10 B cells / ml Jodo after immunized by intraperitoneal injection was ⁇ the ⁇ erythrocytes administered 4 days after ⁇ .
• Splenocytes were put to sleep by grinding with a water-cooled balanced salt solution using a blender.
• SUBSTITUTE SHEET This was centrifuged at 2000 rp B for 5 minutes to remove the supernatant, suspended in a 0.832 ammonium chloride solution to remove red blood cells, and left at 37 for 3 minutes.
• the cells were suspended in an ice-cooled balanced salt solution, and the number of spleen cells from which erythrocytes had been removed was measured using a hemocytometer.
• Arcel solution [20.5 g of glucose, 4 g of sodium chloride, and 8.0 g of sodium citrate are dissolved in distilled water of IL, and then passed through a miripore instrument (0.45 ⁇ n).
• the sheep erythrocytes suspended in [Use] were washed four times with a balanced salt solution at 2000 ° C for 5 minutes for 5 minutes, and suspended in a balanced salt solution to a final concentration of 10S !.
• Example 9 Immunostimulatory action Twenty-five weeks old male CR mice of 6 weeks of age were used for 10 animals per group. G 009 is dissolved in saline, and 0.2 ml of each concentration solution is intraperitoneally administered to the mouse. 24 hours after administration 1 B1 peri-drawing ink 17 Black (Perican Drawing Ink 17 Black, manufactured by Gunter-lapner Co., Ltd.) and 3% (V / V) Gelatin
• mice are administered with 0.2 ml of physiological saline.
• Co the content of carbon powder in blood at time o.
• C the content of carbon powder in blood at time t.
• G009 The acute toxicity test for G 009 was performed on mouse and rat rabbits and was measured 14 days after G 009 administration. As shown in [Table]], G009 is a safe substance with extremely low toxicity, since there was no lethality to the administration route and species (species).
• the present inventors searched the anticancer effects of Gano-derma lucjdura ⁇ strains collected in various regions all over the time, and found that the four types showed that the pile cancer effect was deemed to be unbalanced.
• the following experiments were conducted to clarify the phylogenetic differences between the strains and the physiological and genetic differences of the fungal species (available from the Agricultural Technology Agency, Agricultural Technology Agency, Agricultural Technology Agency).
• the strains used in this experiment were four strains of Ganoder-lucidun, which grows naturally in Japan, which are judged to have an excellent anti-cancer effect.
• the collection sources are shown in Table 14.
• the protein under test ( ⁇ bovine serum albumin: BSA) was used as a standard substance, and the BCA protein assay reagent (protein assay reagent) was used.
• the developed gel was immersed in a 0.2 M phosphate buffer (PH 6.5) for 30 minutes. During the immersion, they changed three times with fresh buffer. After adjusting the acidity of the gel after immersion, a coloring solution (a-naphth acetate 20 mg, ethy 1 ene giyco ⁇ nonomethy 1 ether 2 ml, fast blue RR sa ⁇ t 20 mg, 0,2M phosphate buffer 120m]) was added and the color was developed by gradually shaking at 35 ° C for 30 minutes in a dark place.
• a coloring solution a-naphth acetate 20 mg, ethy 1 ene giyco ⁇ nonomethy 1 ether 2 ml, fast blue RR sa ⁇ t 20 mg, 0,2M phosphate buffer 120m
• a coloring solution (102 figCl, 6 ml of solution, fast garnet GBG salt 70 mg, ⁇ -naphtfiyJphosphate 80 mg, ⁇ -nap thythy 1 hospate Omg, 0.1 M acetate buffer lOOml) was added and the color was developed at 37 for 3 minutes.

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• 1990
  • 1990-12-04 KR KR1019900019877A patent/KR920008374B1/ko not_active Expired
• 1991
  • 1991-12-04 CA CA002097821A patent/CA2097821C/en not_active Expired - Fee Related
  • 1991-12-04 AU AU89515/91A patent/AU8951591A/en not_active Abandoned
  • 1991-12-04 EP EP19910920729 patent/EP0563383A4/en not_active Withdrawn
  • 1991-12-04 WO PCT/KR1991/000032 patent/WO1992010562A1/ja not_active Ceased
  • 1991-12-04 JP JP4-500088A patent/JPH084494B2/ja not_active Expired - Lifetime
  • 1991-12-04 RU RU93043871/13A patent/RU2082755C1/ru not_active IP Right Cessation
• 1997
  • 1997-01-20 AU AU12238/97A patent/AU1223897A/en not_active Abandoned

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