Method of producing glucoprotein possessing antitumoral and lectine-like activity

专利摘要:
A glycoprotein suitable for use in treating tumours has a molecular weight of 5,000 to 300,000 as determined by the ultracentrifugation method; the ratio of the weight of its protein moiety, determined by Lowry-Folin's method, to the weight of its saccharide moiety, determined by the phenol - sulfuric acid method, being from 50:50 to 80:20; the amino acid at its N-end being tyrosine, leucine or alanine; the amino acid sequence at its C-end being leucinephenylalanine-valine; its elementary composition being 35.2 to 49.3% of C, 4.8 to 8.0% of H, 4.3 to 12.3% of N, a trace amount to 2.5% of S, a trace amount to 1.2% of P and the balance being 0; its isoelectric point being from pH 2.5 to 5.0; and having a nucleic acid component. The glycoprotein can be obtained from basidiomycetous fungus of genus Coriolus and can be formulated into a pharmaceutical composition with convention carriers or diluents.
公开号:SU1291027A3
申请号:SU823396893
申请日:1982-02-09
公开日:1987-02-15
发明作者:Есикуми Тикао；Фудзи Такаеси；Фурусо Такоо；Мацунага Кенити；Охара Минору；Кобаяси Акира
申请人:Куреха Кагаку Когио Кабусики Кайся (Фирма)；
IPC主号:

专利说明:

The invention relates to medicine and relates to the preparation of a biologically active compound.
The purpose of the invention is to create a method for producing a new glycoprotein with antitumor and lectin-like activity.
The method is illustrated by the following examples.
Example 1. Strain SM-101. Coriolus versicolor (Fr.) Quel (FERM-P2412; ATCC 20547) is cultivated in a nutrient medium containing, wt.%; glucose 5; Penton 0.2; yeast extract 0.3; , 0.1; MgSO 0.1, for 10 days,
The mycelium developed on the surface of the culture medium is homogenized with an aqueous saline solution and a seed culture is obtained.
It is sown in each of the 100 culture vessels with a capacity of 1 liter containing 200 ml of medium of the composition described above each and cultured for 25 days at 25-27 ° C. Obtain 2.0-4.5 g / one vessel germinated mycelium.
100 g of the obtained mycelium is extracted with 3 l of aqueous 0.1N NaOH solution at 97 ° C for 1 hour. The extract is separated, neutralized with acid and concentrated.
The concentrate is desalted and then sequentially subjected to dialysis and ultrafiltration to remove low molecular weight compounds S having a molecular weight less than 5000.
The concentrate (or powdered compound obtained by drying the treated concentrate) is mixed with phosphate buffer to maintain the pH of the mixture at 3.5 and, after neutralizing the supernatant obtained by centrifuging the mixture, is subjected to ultrafiltration to remove salts, and the ultrafiltration is carried out under a pressure of 1.5 kg / cm at 10 ° C with simultaneous cooling and stirring on the apparatus for ultrafilter - 50 purified product, if necessary
tions (desktop HighfIow72000, Ami-con Comnany with diaphragm DM-5). After concentrating the desalted supernatant, phosphate buffer is added to the concentrate to maintain the pH of the mixture at 3, then the mixture is centrifuged and the precipitate is collected.
Fractions precipitated at stabilized pH 3.5, ionic strength 0.30 | U
five
0
five
and the temperature for 2 hours, discarded, and collect only coat; s, precipitated from the remaining solution at pH. 3.0 (isoelectric point in the range of pH 3s0-3.5).
The precipitate is dissolved in water, adjusted to pH 7.0 and purified by desalting, then dried to a powder.
Glycoprotein has the following characteristics.
Molecular weight 5000-300000. The weight ratio of the protein part to its saccharide part is from 50/50 to 80/20. N-terminal acid is mainly tyrosine, leucine or alanine,
The C-terminal amino acid sequence is leucine phenylalanine-valine.
Elemental composition,%: carbon 35.2-49.3; hydrogen 4.8-8.0; nitrogen 4.3–12.3; sulfur from traces to 2.5; phosphorus from trace to 1.2; oxygen else.
The isoelectric point of weight is between 2.5 and 5.0 pH range. .
The nucleic acid is a component of glcoprotein.
According to infrared absorption spectroscopy data, the glycoprotein ig-1 has an absorption maximum in the intervals of 3600-3200 and 1200-1000 cm as well as at 1600 and 1530.
Example 2-18. Instead of strain CM-101 use each of the strains of fungi, presented in table. one,
Mycelium is extracted with an aqueous solvent, such as hot water or aqueous 0.01-2.0 N, alkali solution at 80-100 ° C for 1-8 hours. After removing the extractable residue, the aqueous extract is neutralized with acid and concentrated. . After desalting, the concentrate is dialyzed and / or ultrafiltered, if necessary, to remove a low molecular weight compound (mw less than 5000), obtained
concentrate and dry to a powder. The product is then subjected to fractional wasp f.en.ch. at an isoelectric point with a pH of 255-550, 55 to an ionic strength of 0.1-3.1 / and at a medium temperature of 5-25 ° C5 for more than 0.5 h;
Characteristics of the preparations obtained are given in table. one.
The physical and physiological properties of the proposed compounds in examples 1-18 are given in table 2.
Each of the compounds of examples 1-18 exhibits the following characteristic color reactions of the saccharide and protein;
d-naphthol - sulfuric acid. reaction color is dark red; indole - sulfuric acid reaction - brown color; anthron — sulfuric acid reaction — greenish blue; phenol sulfuric acid reaction - brown color; tryptophan - sulfuric acid reaction - reddish brown color; thioglycolic acid — sulfuric acid; rescdn — greenish brown; OIJCHH — hydrochloric acid reaction — greenish brown; Lowry-Folin reaction - color blue; The ninhydrin reaction of the hydrolysis product of hydrochloric acid glycoprotein is reddish blue.
Table 1
FEKM-P 2412 (ATCC 20547)
FERM-P 2413 (ATCC 20548)
FERM-P 2414 (ATCC 20545)
FERM-P 2415 (ATCC 20549)
FERM-P 2416 (ATCC 20550)
FERM-P 2417 (ATCC 20551)
FERM-P 2418 (ATCC 20552)
FERM-P 2419 (ATCC 20553)
FERM-P 2420 (ATCC 20554)
FERM-P 2421 (ATCC 20555)
FERM-P 2422 (ATCC 20556)
FERM-P 2423 (ATCC 20557)
FERM-P 2424 (ATCC 20558)
versicolor (Fr)
 CM-114
versicolor (Fr)
 CM-115
hirsutus (Fr.)
CM-151
pargemenus (Fr.)
CM - 161
versition (Fr.)
. CM-101
censors (Berk.)
CM-166
Continued tab. I
FERM-P 2425 (ATCC 20559)
FERM-P 2426 (ATCC 20560)
FERM-P 2711 (ATCC 20561)
FERt-I-P 2712 (ATCC 20562)
FERM-P 3686 (ATCC 20564)
FERM-P 988 (ATCC 20565)
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权利要求:
Claims (2)
[1]
1. METHOD FOR PRODUCING AN ANTITUM AND LECTIN-LIKE ACTIVITY Glycoprotein, consisting in the fact that the mycelium of the genus Coriolus is extracted with water or aqueous 0.01-2.0 N. an alkali solution at a temperature of 80-100 ° C for 1-8 hours, low-molecular substances are removed from the obtained extract by dialysis and / or ultrafiltration, the resulting concentrate is subjected to fractional precipitation at an isoelectric point with a pH of 2.5 - 5 at an ionic strength of solution from 0.1 to 3.1 | U at a temperature of 5-25 ° C for 1-8 hours, while maintaining the pH at a predetermined level during the fractionation process, fractions containing the target product are isolated, neutralized, desalted and dried.
[2]
2. The method according to p. ^ Characterized in that the mycelium of the fungus Coriolus versicolor (Fr.) Quel, Coriolus hirsutus (Fr.) Quel, Coriolus pargemenus (Fr.) Pat., Coriolus censors (Berk) Imaz.
SU. ,,, 1291027

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法律状态:

优先权:

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