• 专利附录
  • 专利说明
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    • 专利摘要:
    Novel biologically active substances prepared by dissociating the protein obtained from the cultivation of the microorganism Bordetella and having insulin secretion promoting action as well as glucose tolerance improving action for mammals.
    公开号:SU1012786A3
    申请号:SU782628100
    申请日:1978-06-09
    公开日:1983-04-15
    发明作者:Канбаяси Есинора；Хосода Конти；Ито Акира；Курокава Сигеки；Накамура Цутому；Ногимори Кацуми；Окамото Таира；Терасимо Акио；Такахаси Шаму；Ядума Мотоюки；Томиака Тиканори；Уи Митио
    申请人:Кокеньяку Како Кабусики Кайся (Фирма)；
    IPC主号:
    专利说明:

    Yu

    The invention relates to microbiology and concerns the production of a new complex that can be used in medicine, in the treatment and prevention of various types of diabetes. The purpose of the invention is to obtain a stimulating protein complex. insulin secretion. The goal is achieved by the fact that the method of obtaining a protein complex that stimulates insulin secretion is that pathogenic strains of microorganisms belonging to the species Bordete epertussis are cultured in phase 1 in shaking liquid, the resulting culture liquid is heated the supernatant liquid is passed through a column, the eluir of the target product is phosphate buffer 0.5m NaCe pH 7.0, the resulting eluent is concentrated, purified and the protein complex is separated by step chromatography on components KS-1 with a pier weight. 63000 + 5200, KS-1G with mol. weight. 31000 + 4500 and / or KS-Sh with mol. Weight. 12500 + 1500. The protein complex that stimulates insulin secretion is a protein substance that can be obtained by cultivating microorganisms belonging to the BordeteCCa pertussis microorganisms, which are pathogenic bacteria such as pertussis bacillus, paracultus pertussis and bronchial septic bacteria, and most preferred are BordeteEGa pertussis of the first stage. The ability of pathogenic microorganisms of the species BordeteCpa pertussis on the production of the protein complex is given in the E table. 1. Table Strains Relative yield Pathogenic Mayes according to (control) Tohama 3379 B 18-323 7341 Non-pathogenic Sakairi Miyazaki Insulation and purification of the protein; the culture complex is carried out using known methods, for example, a precipitation method, a chromatography method, a method using molecular sieves, electrophoretic and biological methods, and using either each individually or in appropriate combinations .5 Method using a chromatographic column is convenient for isolating and purifying the complex, while the suprapositive fluid of the culture medium is passed through a column filled with carriers such as hydroxyapatite, CM-sepharose , Coh A sepharose 4 or RAMAsefaroza 6MV. The protein complex is adsorbed onto the column and then eluted from it with a suitable eluting agent, for example, 0.1 M phosphate buffer (pH 7.0) containing 0.5 M NaCC. The purified product is dialyzed to eliminate the unnecessary salts, after which a pure protein complex is obtained. The complex is also present in cultured bacterial cells, and, if necessary, it is isolated by adding NaCt to the suspension to precipitate the complex into the solution. To obtain a protein complex, ammonium sulfate precipitation can be used, in which case solid ammonium sulfate is added to the culture supernatant to a point approaching the saturation solubility, and the pH is adjusted to 6-7 with dilute ammonia water. Then the precipitate is washed with water, and the complex is extracted from 0.1 M tris-buffer (pH 8) containing 0.5 M NaCf. The powder of the protein complex obtained by drying at a temperature below the freezing point, after desalting, does not blur in moist air, white or light brown in color, dissolves in water at room temperature in a concentration of about 3-5 mg / ml. If placed in 6 n.NCE, it forms an insoluble white precipitate. Dissolved in pyidine, sodium dodecyl sulfate, meraptoethanol and cystine solution. When adding a mixture of dry ice with acetone or ethanol, trichloroacetic acid or a solution of tsin chloride or a solution containing some other types of metal ions, a cloudy white precipitate is formed in the cooled state () in a cooled state (). If the rotin complex is placed in a mixed solution of water and chloroform or -butanol, it becomes insoluble and collects near the interface of both liquids. If the aqueous solution of the protein complex is heated to or above, it becomes cloudy white. In the same way, if this complex is dissolved in 0.1 M phosphate buffer (pH 7.0) containing 0.5 M NaC and then dialyzed using distilled water as the external liquid, the complex gradually becomes cloudy-white, however With continued dialysis, the complex again dissolves and the white turbidity disappears. If the high concentration solution is thoroughly dialyzed using 0.01 M acetate buffer (pH 4.5), the complex may turn a light brown color and dissolve. The molecular weight of the protein complex is 77,000 + 6400. The protein content is more than 95% by weight, and the glucid content is approximately 1%, the lipid concentration is less than the lower limit of detection. Composition ratio μM / 100 16 or 24-hour hydrolysis at an IO of 6 n., Aspartic acid 7.5–7.9, threonine 6.8–7.6, serine 5.9–7.6, glutamic acid 9 , 7-10,8, length 5.5-6.4, glycine 8.7-9.6, alanine 9.1-10.8, cystine 1.5-2.6, valine 5.6-6 , 6, methionine 2.5 g-3.3, isoleucine 3.6-4.1, leucine 7.5-8.0, tyrosine 5.1-6.6, phenylalanine-3.33, 9, lysine 3, 1-4,4, histidine 1.41, 6, arginine 6.1-6.6. The protein complex has a stimulating effect on insulin secretion, which lasts from several weeks to several months after administration of a single dose. Acute toxicity LDj is 200 µg / kg of live weight for my baby ddy. Protein complex section. Three components are used by chromatography of the n-column in the presence of known agents for the denaturation of proteins such as 8 M urea and 6 M guanidine hydrochloride. Gel filtration in the presence of 8 M mbchevinn provides for the dissolution of the protein complex in 0.1 phosphate buffer (pH 7.0) containing 8 M urea and O, .5 M HaCB, then the resulting mixture is gelfiltered on a column filled with Sefacryl S- 200 equal to the indicated buffer. As a result, the protein complex is divided into three components with different molecular weights (KC-I with molecular weight. 63000t5200, KC-1I with molecular weight. 31000 + 4500 and KC-I11 with molecular weight. 12500+ 1500) , and additional gel filtration on the same column causes these components to be emitted as separate substances upon disk electrophoresis. Another way to isolate and purify the complex can be chromatography on a column with DEAE-cellulose, the protein complex being dissolved in 0.01 M phosphate buffer (pH 8.0) containing 8 M urea, then passed through a column of DELE balanced cellulose the same sakshm buffer. Accordingly, KC-I and KC-II are adsorbed on this column, KC-1II is not adsorbed, but flows down, KS-1 is eluted with 0.02 M phosphate buffer (pH 7.0) containing 8 M urea, and KC-II elute with 0.05 M phosphate buffer (pH 6.0). In disc electrophoresis, these components appear as separate substances after gel filtration through a Sephacryl S-200 column, equilibrated with 0.1 M phosphate buffer solution containing 4 M urea and 0.5 M sodium chloride. The components of KS-G, KS-11, and KC-I11 obtained in the same manner are more low stimulating secretion of insulin than the original protein complex. Under certain conditions of the separation of the protein EO complex into its components, each of the components can be further divided into smaller protein fragments. For example, the separation of a protein complex using 0.1 M phosphate buffer solution containing 4 M urea, 1% 2-mercaptoethanbaPa and 0.5 M NaC followed by gel filtration through a Sephacryl S-200 column, equilibrated with the specified buffer solution, results 5 protein fragments that contain two additional components in addition to the three components. Separately, substances KS-1, KS-11, KS-Ill have insufficient activity of stimulating secretion, but their connection into certain groups, for example, KS-1 and KS-111, KS-11 and KS-1II and KS-1 and KS -Ill leads to high activity, and the combination of CS-1 and CS-11 is inactive. When the components KS-1 and KS-111, KS-11 and KS-111, KS-1, KS-11 and KC-ill and KS-1 and KS-11 interact in a specific buffer solution, they form separate protein associated compounds, which are called associated substances KSA-1, KQA-1I, KCA-lIi and KSA-1U, respectively. Protein associated substances are obtained as follows. The corresponding combinations of elemental substances, taken in the ratios KS-1: KS-5 5: 1 in the case of KSA-1, KS-11: KS-1 II 2: 1 in the case of KSA-11, are mixed; KS-1: KS-11: KS: 111 5: 2: 2 in the case of KSA-111, and KS-1: KS-11 2: 1 in the case of KSA-1U (ratio by weight of protein), in OD M phosphate A buffer solution (pH 7.0) containing 2 M urea and 0.5 M sodium chloride is then shaken for 24 hours at 37 ° C, and the substances interact with each other in appropriate combinations. Then these mixed solutions are concentrated and gel-filtered on a Sephacryl S-200 column, equilibrated with a 0.1 M phosphate buffer solution (pH 7.0) containing 2 M urea and 0.5 M sodium chloride, or ion exchange chromatographic separation of associated and nonassociated elementary substances, while obtaining separate associated substances KSA-1, KSA-11, KSA-111 and KSA-1U, respectively. The yields of the associated products vary greatly depending on the processing conditions and temperature, pH, time, density, and ionic forces. It was also found that new protein associated substances, although practically equal in the main activity of the protein complex, possess less than 1/50 of leukocytosis activity, less than 1/10 of the activity of increasing sensitivity to histamine, less than 1/25 of actigenicity and less than 1/3 of the LDjQ type compared to with corresponding values for the protein complex. Properties and solubility of elemental and associated substances. The fine ground powders of all desalted and dried at a temperature below the freezing point of the elementary and associated substances are powders, which do not spread in moist air (due to hygroscopicity). KS-111, KSA-11 and KCA-II1 are soluble in water in amounts up to 1 mg / ml at room temperature, while others are poorly soluble in water, but dissolve up to 4 mg / ml in 0.1 M phosphate buffer containing 4 M urea and O, 5 M NaCG. Aqueous solutions of these substances become turbid-white and form a precipitate when ammonium sulfate, dry ice with acetone, and ethanol are added to them. Trichloroacetic acid or ansshocic compounds at 4c. Each of the substances placed in a mixed solution of water and chloroform sludge and butanol is collected around the interface of the liquids. Molecular weight of the corresponding elemental substances, determined by gel filtration on a column (1.5 x 95 cm) filled with sephacryl S-200, equilibrated with 0.1 M phosphate buffer solution (pH 7.0), containing 8 M urea and 0.5 M Nace, the following: KS-1.63000 + 5200; KC-I1 31000 ± 4500g KS-111 12500 + 1500. The molecular weight of the respective associated substances, as determined using a column (1.5 x 95 cm) filled with sephacryl S-200, equilibrated with 0.1. M phosphate buffer solution (pH 7.0), containing 4 M urea and 0, 5 M NaCe, the following: KSA-1 75000 + 8500; KSA-I 35000 + 4500 KSA-I1 85000 ± 13000, KSA-1U 88000 + 6300. The isoelectric pH points of the respective substances, measured using an acrylamide gel electrofocusing technique, are as follows: KS-1 5.6 + 0.3; KS-11 5.4 + 0.4; KS-111 8.3 + 0.3; KSA-1, 6.8 ± 0.4; KSA-11 6,3 0, sT KSA-111 8.2 ± 0.5; KSA-1U 5.6 + 0.4. In tab. 2 shows the composition of the protein complex. Table 2 The amino acid composition of the protein component and the percentage of the composition (µM / 100 µM) are given in Table. 3. Table 3
    : Aspartic acid (Asp) 8.1
    Threonine (Tr) 8.5
    9.3
    5.3 7.7 4.8
    Series (Ser) Glutamic Acid
    Example 1. Preparation and purification of the protein complex.
     Dried at a temperature below the measurement point and stored Bordeta E pertussis bacteria, the Maeo 1 phase strain was cultured and the plates in Bordet-gengon medium for 2 days then the platinum loop with the culture of the indicated bacteria was inoculated into a 500 ml shake flask containing 200 ml of ion-exchange resin, with the addition of a modified, medium Cohen Wheeler (CW-medium), the composition of which is listed in Table. 4, and yes-. those shaken this culture for 20-22 hours at 37 ° C. The concentration of bacteria in the cult-urn solution was determined with a spectrophotometer (wavelength 650 nm), and this solution was added to a two-liter shake flask, into which 1 liter of ion-exchange resin was added with the addition of a CW medium, so that the final bacteria concentration is approximately 0.1x. 10 cells / ml, after which the shaking The tures are continued for 48 h (frequencies | shaking 97 times / min at. The mycological properties of the indicated strain correspond to the data for the whooping cough bacterium stage 1
    Continued table. 3
    9.8
    7/9
    6.2
    Table 4
    | The first acidic potassium phosphate, g
    Soluble starch, g
    0.5% solution of copper sulfate, ml
    1% calcium chloride solution, ml
    4% solution of magnesium chloride, ml
    Polypepton, g
    1% solution of cystine, ml 4 0.5% solution of ferrous sulfate, ml Sodium chloride, g When using a modified liquid medium, it is added with distilled water, brought to 1000 ml and, after adjusting the pH to 7.2, with 20% NaOH solution to the medium solution Another 3 g of anion exchange resin (Diaion SA-20 AP, Mit-subishi Kasei Co.) is added and then subjected to high-pressure steam sterilization at 15 minutes. The resulting 48-hour shaking culture solution is heated at 5 ° C for 30 minutes and then centrifuged (15,000 rpm) at 4 ° C in order to separate the aqueous fluid and bacteria cells. The supernatant thus obtained from the solution of the culture is used as a starting material for purification and more efficient treatment of the target protein complex. 10 liters of the supernatant thus obtained, after adjusting the pH to 6.0 with 1 n, were passed through a column (2.5x4 cm), which was filled with oxyapatite at a flow rate of 200 ml / h as the first purification step. Most of the protein passes through the column, not absorbed on it. The concentration of protein, determined by the method of Lowry, is given in Table. 5 To determine the absorbed substance, the column was first washed with 0.01 M phosphate buffer solution (pH 6.0), and then, after increasing the molar concentration of phosphate buffer solution to 0.1 at pH 7.0, the absorbed proteins were effectively eluted from the column. However, under these conditions, the protein complex from the column was not eluted. Therefore, an additional elution is carried out with a phosphate buffer solution of the same composition, but containing 0.5 M NaCC. Under these conditions, the target protein complex can be isolated with a high degree of activity in accordance with the eluted protein. The protein complex thus obtained is concentrated and, after placing it in the membrane for dialysis, with a maximum permeability to mol. weight 8000, twice subjected to dialysis (within 12 hours) with distilled water and twice (within 12 hours) with 0.01 M phosphate buffer solution (pH 6.0). For further purification, the solution containing the protein complex is passed through a column (1.5x10 cm) filled with carboxymethyl sepharic C8-6B, equilibrated with 0.01 M phosphate buffer solution (pH 6.0). Material that has not been absorbed in this column is inactive. After increasing the molar concentration to the pH of the phosphate buffer solution to 0.1 and 7.0, respectively, a similar elution is carried out by adding 0.5 M saline, after which the protein complex is obtained in accordance with the eluted protein. Since the obtained product still contains a small amount of impurities when determined by the method of disk electrophoresis, this protein complex is concentrated further and, after being placed in the dialysis membrane, twice subjected to dialysis (for a total of 12 hours) with distilled water and twice (for a total of 12 hours ) with 0.01 M phosphate buffer solution (pH 7.0). The sample obtained after dialysis is passed through a column (1.58 cm) filled with Con A-Sepharose 4b, which is equilibrated with 0.01 M phosphate buffer solution (pH 7.0). When treated with the same buffer solution, trace amounts of protein are eluted, but this protein fraction does not possess activity. Upon further elution with 0.1 M phosphate HtJM buffer solution (pH 7.0), containing 0.5 M Naig, a protein complex is obtained in accordance with the eluted protein. Since this portion of the protein still contains the smallest amount of impurities when determined using disk electrophoresis, it is collected, concentrated and dialyzed with 0.01 M phosphate buffer solution (pH 7.0) containing 0.5 M NaCE. After dialysis, the sample is subjected to gel filtration on a column (2.8–60 cm) filled with R-100 biogel, equilibrated with the same buffer solution. As a result, a pure active factor is obtained, corresponding to a protein fraction, giving a peak molecular weight of about 80,000. The efficiency of isolation, purification, and others, the data obtained at this purification stage, are given in table. 5. As a standard, bovine serum albumin is used. The purity of the substance is determined according to polyacrylamide gel electrophoresis data (polyacrylamide concentration of 7.5%, 1N. KOH — ice on acetic acid as a buffer solution (pH 4.3). The amount of sample per gel is 30 μg (in the form of protein). The experiment was carried out by applying 4 MA for 2 h, it showed 10 amide-amidecter and 7% solution of acetic acid was decolorized. The sample obtained at this final stage is a separate substance, practically free from impurities, and stimulating insulin secretion. good This is consistent with the activity for this isolated substance. To determine the structure of the subunit of this substance, it is then subjected to disk electrophoresis with SDS (dodecyl sulfate-sodium) according to the Chapilo technique. 50 µg of the sample of the substance (based on protein) is added to the mixture 1% SDS 1% mercaptoethanol and 4 M urea After 2 hours of incubation, the mixture is applied onto a 10% polyacrylamide gel containing 1% SDS, then a current of 8 mA / gel is passed through, the Coomsie is blue and discolored 7.5 Acetic Acid. The results are shown in table. Dried at a temperature below freezing and stored bacteria of the whooping cough Bordete ea stage 1, strain Tohama-cultured. In the medium of Bordet gendon (Bg-medium) containing 20% defibrinized horse blood for 3 days, then for 20-24 hours cultured a. In a scoop table, and then a platinum culture loop of the bacteria is inoculated into a .500 mm flask, in which 200 ml of ion-exchange RESIN is previously placed, to which half a polysin is added. Tetic liquid nutrient medium (modified medium Cohen-WheeKeg), the composition of which is given in table. b, after which the culture is shaken for 20-24 hours at 37 ° C. The concentration of bacteria in the culture solution is determined using a spectrophotometer (wavelength 650 nm), and the resulting solution is added to a two-liter shake flask, into which 1 l of said CW medium is placed, so that the final concentration of bacteria reaches 0.07 to 0.1510 cells / ml. After this, the culture is shaken for 4 hours at 37 ° C (shaking frequency 100-120 times / min). The agitated solution thus obtained is heated at 30 minutes, then centrifuged (at a speed of 15,000 rpm) at 4 ° C in order to separate the supernatant from the bacterial cells. Table 6 Quantity Composition of modified medium Casamic acid, g10 Yeast extract, g1 Primary acidic phosphorus, potassium oxide, g0.5 Soluble starch, g2 0.5% solution of copper sulfate, ml1 Continuation of the table. 1% calcium chloride solution, ml 4% magnesium chloride solution, ml Polypeptone, g 1% cystine solution, ml 0.5% ferrous sulfate solution, ml Sodium chloride, g When using this liquid medium it is diluted with distilled water to obtain a total amount of 1000 ml, and after the pH has been adjusted to pH 7.2 with a 20% NaOH solution, 3 g of anion exchange resin (Diaion A-20AP) is added to it, and then sterilized 1520 min high pressure steam at 21 ° C. 10 liters of the culture media thus obtained on the annoying liquid are passed through a column (5-2 cm, flow rate 60 ml / h), filled with oceanapatite, washed with 100 ml of 0.01 M phosphate buffer solution (pH 6.0). Thereafter, the column is washed with 300 ml of O, 1 M phosphate buffer solution (pH 7.0), and 0.1 phosphate buffer solution (pH 7, containing 0.5 M sodium chloride, at a rate of 15 ml / h to elute the target active factor The protein complex thus obtained is concentrated to 15 ml with polyethylene glycol (average mol. Weight 20,000) and dialyzed four times in 24 hours with 2 liters of distilled water, then four times dialyzed in 24 hours from 1 liter of 0.01 M phosphate buffer solution (pH 6.0) to equilibrium. Then the resulting product is passed through to a jar (1.5-10 cm) filled with Asp Tr Ser Glu Pro Gly Ala Cis / 2 Blow it out to you oo Note: Hydrolysis in 6N HC bauximelsulfarose CL-BV, equilibrated with the same buffer solution as previously, the material that was not absorbed in the column does not have activity.After increasing the molar concentration and the pH of the phosphate buffer solution to 0.1 M and 7.0, respectively, a similar treatment is performed by adding 0.5 M sodium chloride and a protein complex corresponding to the eluted block is obtained. This protein complex is concentrated to 10 ml and dialyzed 4 times in 24 hours with 2 liters of distilled water, after which 4-fold dialysis is carried out for 24 hours in equilibrium with 1 liter of 0.01 M phosphate buffer (pH 4.5 a solution containing 0.1 M sodium chloride, or pH 4.5 at a content of 0.1 M lithium chloride — hydrochloric acid). This solution is passed at a rate of 5 ml / h through a column (1.28 cm) filled with P-acetoximercuraniline-Sepharose 6MB, equilibrated with the indicated buffer solution, followed by thorough washing with them (about 200 ml). The absorbed material was eluted with the same buffer solution, to which 0.01 M g-cystine was added. In the unabsorbed fraction, there is no protein complex, it is all in the final eluate. This protein complex is dialyzed six times for 48 hours with 2 liters of distilled water, and dried at a temperature below the freezing point, resulting in a yield of 7.3 mg of light brown powder. The resulting product gives a separate band upon polyacrylamide gel electrophoresis (a gel with a pH of 4.3 is used) and its isoelectric point of pH is 7.8 + 0.5. The product contains, wt%: protein about 92, glucid 1.8-5.6 and lipids 0-2. The molecular weight of this substance according to gel filtration (using the P-150 biogel on a column with a size / rum of 1.8-95 cm and 0.01 M acetate buffer solution with a pH of 4.5) is 510001. 4300. In the table. 7 shows the composition of amioic acids obtained Table 7 Mem Il Leu Tir Fe Liz Gis Apr at 110 ° C for 16 h. Efficacy of administration, purity and other data obtained at this stimulating insulin secretion activity is 1.349 U μg, acute toxicity of LDcQ is 232 μg / kg for male GFpay and 174 µg / kg for female mice. Example 2. Preparation and purification of components. 1.20 mg of purified insulin secretion stimulating protein complex is dissolved in 3 ml of 0.1 M phosphate buffer solution (pH 7.0), containing 8 M urea and 0.5 M sodium chloride, and after allowing the mixture to stand for 2 hours 37 ° C. It is subjected to gel filtration on a column (1.595 cm) filled with sephacryl S-200, equilibrated with the indicated buffer solution. In this way, the protein complex is divided into three components with different molecular weights (KC-I, KS-G1 and KS-Ill in order of increasing molecular weight). In order to purify these components further, their fractions are collected, concentrated and again subjected to gel filtration on the same column. 2 .20 mg of purified insulin secretion stimulating complex of the protein is dissolved in 3 ml of 0.01 M phosphate buffer solution (pH 8.5) containing 8 M urea and, after standing for 2 hours, the mixture is passed through a column (1 , cm), filled with DEAE-cellulose, balanced with the specified buffer solution. KS-11 passes through the column without being absorbed on it, and KS-1 and KC-1I are absorbed in the column. KS-1 is eluted with 0.02 M phosphate buffer solution (pH 7.0) containing 8 M urea, and KS-11 with 0.05 M phosphate buffer (pH 6.0). For further purification of this final stage, see table. 8. Table 8 of these materials, they are concentrated and then subjected to gel filtration on a column (1.5-95 cm) filled with Concentrated S-200, equilibrated with 0.1 M phosphate buffer (pH 7.0) containing 8 M urea and O, 5 M sodium chloride. KS-1 has two 1; 1 subgroups on the high molecular side of the protein complex, KS-II corresponds to a subgroup of the intermediate molecular weight of the protein complex, and KS-111 to a subgroup of low molecular weight. Example 3. Obtaining and cleaning components. The components KS-1, KS-11 and KS-111, obtained in example 2, are associated in appropriate combinations, as a result of which new components are obtained that stimulate insulin secretion called KSA-1 (KS-1 and KS-111 association), KSA-11 (KS-11 and KS-111 associations), KCA-I11 (KS-1, KS-11 and KS-111 associations) and KSA-1U (KS-1 and KKS-11 associations). The optimal conditions for the preparation of these substances KSA-1, KSA-11, KSA-G11 and KSA-1U are as follows. In the case of KSA-1, KS-1 and KS-111 are mixed in a 5: 1 ratio in an aqueous solution, and after adjusting the pH to 7.0, the resulting mixture is shaken for more than 24 hours. To obtain KSA-11 - KS-11. and KS-111 is mixed in a 2: 1 ratio and, after adjusting the pH to 7.0, the resulting mixture is agitated for more than 1 hour. To obtain KCA-1II - KS-1, KS-11 and KS-111 are mixed in a ratio ( based on protein) 5: 2: 2, the pH value is set at 7., 6 and shaken for 24 hours at, to obtain KSA-1U - KS-1 and KS-11, mix in a 2: 1 ratio, then set the pH 7.0 and shake for 24 hours at. Under these conditions, all these substances can be obtained with a yield of more than 95%; however, in order to eliminate unassociated substances, the resulting products are subjected to a distance gel filtration through a column (1.5-95 cm) filled with Sephacryl S-200, balanced 0. , 1 M phosphate buffered saline (pH 7.0), containing 2 M urea and 0.5 M sodium chloride. The components thus obtained are manifested by separate electrophoresis as separate compounds. Example 4. Pharmacological action. 1. Determination of insulin stimulating secretion. The stimulating insulin secretion of each of the components can be determined by measuring the response of animals to various types of insulin secretagogues, usually glucose is used as a stimulant for these purposes. Male Wistar rats (weighing 130-140 g) are used. Test method. Components of varying strength are dissolved in physiological saline and 0.2 ml of each of these solutions is injected intravenously with anesthesia with ether into the femoral vein of experimental rats. After three days, stimulating insulin secretion was determined for each of the substances. Before the experiment, the rats go hungry for 18-20 hours. To determine the stimulating secretion, about 1 ml of blood is taken from the tail vein of each rat, immediately after this intraperitoneal injection of 30% glucose solution is carried out in an amount of 1 ml per 100 g of weight, and after 15 minutes 0.1 ml of blood is taken again in the same way. Stimulating insulin secretion is determined by the difference in blood sugar level and insulin concentration in the blood before and after glucose administration. The blood sugar level is determined by the glucose oxidase method to the insulin concentration by the double antibody method. The potential immunoactivity of the components can be determined using various experimental systems. Test animals: female Wistar rats. (Body weight 200 + 10 g) and
    thirty
    ten
    COP-1
    sixteen
    10 male rats of the Wistar strain (body weight 250 ± 20 g). The test procedure is as follows. Dinitrophenyl ascaris (DIF-AS), obtained by binding the nitrophenyl group to a protein obtained from Ascaris suura, is used as an antigen. 1 ml of this antigen and various active substances are dissolved in 0.5 ml of physiological saline, the solutions thus obtained are injected subcutaneously into the soles of both the front and rear extremities of male rats under ether anesthesia for primary immunization. For control, 1 mg of DNP-AS is dissolved in 0.5 ml of physiological saline containing 10 dead bacteria cells. B. pertussis and similarly sensitized to effect primary and secondary binding. 5 days after the initial immunization, 0.5 ml of a physiological solution containing 0.5 mg of DNP-AS is injected into the rat dorsal muscle under ether anesthesia, and after 3 days the level of anti-DNP-LS antibodies in the serum is determined. This definition is carried out as follows. 0.1 ml of blood obtained from the rat tail vein is placed in a test tube with 0.2 ml of saline containing heparin, and after separation in a centrifuge at a rotation speed of 3000 rpm at 4 ° C for 15 minutes, the supernatant liquid for preparing five-fold diluted serum. The level of antibodies in the serum thus obtained is determined using a passive anaphylactic skin reaction according to the method of Thad. The level of antibodies in serum is expressed in terms of the maximum dilution of serum, in which blue spots appear more than 5 mm, and the activity of promotion of the release of antibodies (force) of each preparation is expressed in units of activity, and the activity corresponding to lCl -24-fold antibody levels are referred to as 1000 units. The specific activity is determined by dividing the unit of force by weight. G Stimulating insulin secretion and potential immunoactivity are presented in Table. 9. T a b, l and c a 9 1AP - protein complex The protein complex that stimulates insulin secretion, isolated and purified in accordance with the invention, not only acts as a stimulating insulin secretion agent in mammals and supports blood sugar levels within normal limits. but is also suitable for stimulating the secretion of antibodies and for enhancing cellular immunity. Since this protein complex provides these actions in extremely small doses, it can be used as a therapeutic or prophylactic drug for various types of diabetes, or as a therapeutic drug against diseases resulting from impaired immuno-functions (for example, malignant tumors, aplastic anemia, rheumatoid arthritis etc.). The protein complex that stimulates insulin secretion, its constituent components KC-I, KS-11 and KC-1I1, and their associated substances KSA-1, KCA-I1, KSA-Sh and KSA-1U have activity towards stimulating the secretion of insulin and potential immunoactivity. The components of the protein complex, each separately, are characterized by a weak stimulating activity of insulin secretion, and when combined with each other - by high activity. In particular, all substances without exception, including KS-111, have a high stimulating insulin secretion; therefore, it is this component of KS-I1 that plays an important role in the development of stimulating insulin secretion. Side effects and toxicity. The protein complex, which stimulates insulin secretion, is isolated and purified, has a high stimulating activity of insulin secretion in extremely small doses and therefore can be used with high efficiency for the treatment and prevention of various types of diabetes. However, this protein complex also has a number of side effects.
    Continued table. 9 stimulating insulin secretion, such as increased white blood cell count and histamine susceptibility. jKpoMe addition, marked by the strong antigenicity of the complex. This antigenicity is so high that it can cause anaphylactic shock during the second and subsequent administrations. The substances KSA-1, KSA-11, KCA-1II and KSA-1U, obtained as a result of the formation of protein complex from already separated components, have markedly reduced side effects. In particular, CSA-1, although practically corresponds to the protein complex for stimulating insulin secretion, is 50 times less active in terms of increasing white blood cell count, 10 times less active in terms of increasing histamine sensitivity and 30 times less antigenicity than complex. The acute toxicity of the LDjQ products from these substances decreases accordingly. The protein complex that stimulates the secretion of insulin causes a change in histamine sensitivity and the activity and promotion of leukocytosis, as well as high antigenicity in extremely small doses. The components of KS-G, KS-11 and KS-P1 protein complex usually have more than 10 times lower level of side effects than the complex itself. In particular, KC-I and KS-11 practically do not give side effects, however, KS-11 to a noticeable extent provokes these side effects. Associated substances are usually much weaker in terms of side effects than the protein complex. In particular, KSA-G does not have side effects at all, even when administered in a tenfold dose, which is due to the absence of KS-11 in it, which is responsible for the side effects. Acute toxicity LDjQ (µg / kg live weight) of the protein complex, stimulating insulin secretion.
    its components KS-1, KS-11 and KS-I1 and their associated substances KSA-1, KSA-1, characterized by high activity of promoting insulin secretion, has other useful effects in pharmacology, such as improved glucose tolerance and increased secretion insulin contributes to the treatment of diabetes caused by streptozotocin and improves the stability of glucose tolerance in hereditary diabetes. These activities are maintained for several weeks to several months after a single dose of the substance. The proposed substances can be applied as a therapeutic drug for d: isabetics. Currently, the treatment of diabetes is based on insulin injections or oral antidiabetic drugs, but they are a simple remedy for treating symptoms. In addition, patients must visit insulin injections hospitals every day, taking antibiotic medications involves the risk of an abnormal drop in blood sugar levels. Protein complex
    KSA-I, KCA-ill and KSA-1U are given in table 10.
    Table 10 stimulates insulin secretion, but also has the ability to increase the insulin concentration in the blood only when the level of sugar in the blood already rises under the influence of various conditions (glucose accumulation under conditions of elevated levels of sugar in the blood, such as during a meal) , and quickly reduces high blood sugar to normal levels. In addition, the proposed substance retains activity for several weeks to several months after a single administration. Therefore, when the insulin secretion response to blood sugar levels decreases, the intake of this substance causes the normal activity of insulin secretion. Due to these properties, the substance finds a wide scope for use, i.e. it is not only useful as a therapeutic drug for diabetes, but is also effective in its use in the pre-diabetic stage of the disease, or as a preventative. treatment, therapeutic or diagnostic product for the initial stage of diabetes.
    权利要求:
    Claims (1)
    [1]
    METHOD FOR PRODUCING PROTEIN COMPLEX STIMULATING SECRET
    CICI INSULIN, consisting in the fact that pathogenic strains of microorganisms belonging to the species BordeteEEa pertussis are cultured in the first phase in a liquid nutrient medium with shaking, the resulting culture fluid is heated, the supernatant is separated, it is passed through the column, and the target product is eluted with phosphate buffer with 0.5 M NaC8 pH 7.0, the resulting eluate is concentrated, purified and the protein complex is separated by step chromatography into components KS-I with a molecular weight. 63000 + 5200, KC-II with mol.weight. 31000 ± 4500 and / or KC-ΪΙΙ with mol.weight. 12500 + 1500. : §
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    同族专利:
    公开号 | 公开日
    DE2825464A1|1978-12-21|
    BE868022A|1978-12-11|
    JPS545020A|1979-01-16|
    GB1577700A|1980-10-29|
    FR2393809A1|1979-01-05|
    FR2393809B1|1982-07-02|
    JPS6019733B2|1985-05-17|
    CA1113412A|1981-12-01|
    US4234570A|1980-11-18|
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    JPS59206316A|1983-05-11|1984-11-22|Kaken Pharmaceut Co Ltd|Active substance derivative for enhancing secretion of insulin and production thereof|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP52069154A|JPS6019733B2|1977-06-10|1977-06-10|
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