• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    A method is provided for producing an enzyme having fibrinolytic activity per se and/or activity as a plasminogen activator which comprises culturing non-cancerous epithelial cells and isolating an enzyme-containing fraction from the culture. The enzymes may also be characterised in terms of their molecular weights and amino acid complements.
    公开号:SU1507204A3
    申请号:SU833664932
    申请日:1983-11-04
    公开日:1989-09-07
    发明作者:Аткинсон Энтони；Электриквала Эсгар；Брайан Гриффитс Джон；Латтер Эми；Энтони Райли Патрик；Морган Саттон Петер
    申请人:Паблик Хелт Лаборатори Сервис Борд (Фирма)；Юниверсити Колледж (Фирма)；
    IPC主号:
    专利说明:

    This invention relates to medicine, in particular to methods for producing fibrinolytic enzymes.
    The aim of the invention is to increase the pharmacological purity of the target product.
    Figure 1 shows the fibrinolytic activity obtained after elution of the fraction; in Figure 2, the peak of the active enzyme coincides with a small protein peak.
    Example. In the preparation process, Cytodex-3 (Cytodex-3) manufactured by Pharmacia Fine Chemicals at a concentration of 10 g / L is used as a microcarrier. The culture apparatus is a 5 L Biocul type fermenter manufactured by L.H. The variable Std, with a working capacity of 4.5 liters, is connected to a spray tank containing 2 liters of medium. The ratio between the surface area and the beating medium is more than 4 times higher than in the bottles installed in the roller using the same level of the end and the tip of the microcarriers, and the cells could be maintained viable and active only by carefully controlling the oxygen concentration indicator, nutrient concentrations and high irrigation rates. For cultivation, GPK (cucmi-222) or WEB (cucmi-221) cell lines are used, which are deposited at the Pasteur Institute.
    A field for cells containing 2,
    cells are obtained in a culture in a roller bottle and added to a culture containing Eagles (MEM) growth medium with the addition of 10% fetal bovine serum, 0.001% Tween-80 and HEPES buffer solution. After 72-96 hours at 37 ° C, the cells grow in a sufficient amount and cover about 70% of the amount of substrate (typically, about 1.4 10 cells). The medium is drained, the cells and the carriers are washed twice with medium containing no serum and the plants are filled with the medium containing no growth medium (MEM). Next, incubate at 37 ° C for at least 60 hours so.
    to ensure further cell growth and enzyme secretion.
    At the end of the incubation period, the top layer is drained and rotated for 10 minutes, in the amount of 450 g, in order to remove any cells present in the suspension. The upper layer is then rotated for 30 minutes at, at 40,000 g. A 50 ml aliquot share is separated for fibrin analysis on the plate, and the upper layer is subjected to freeze-drying and transferred into a bottle and stored in a refrigerator with liquid nitrogen. Alternatively, the upper layer may be subjected to concentration by ultrafiltration.
    Co-centered upper culture layer is partially purified by chromatography on a column filled with zinc chelate agarose (4.4 x 12 cm), previously equilibrated with 0.02 M tris-HC1 pH 7.5, containing 1 M sodium chloride and 0 , 01% Tween-80. 5 liters of conditioned medium 1 is fed to this pot; the feed rate is 120 ml / h. After washing the column with 2 liters of equilibration buffer solution, the combined proteins are eluted with a linear gradient from 0 to 0.05 M imidazole (total volume 1 l) in the equilibration buffer. The eluant is collected in an 8 ml fraction. The ultraviolet absorption for each fraction is determined at a wavelength of 280 nm, and fibrin-lytic activity is determined using an fibrin plate assay. The active fractions are combined and their characteristic activity is determined according to the method of lysis of a fibrin thrombus.
    In the next step, the fractions merged together were injected into a 2.2 x 15 cm column filled with concanavalin-A-agarose, urap) 1 hung with 0.01 M sodium phosphate buffer solution (pH 7.5) containing 1 M sodium chloride and 0.01% Tween-80. This column is washed with a balancing buffer solution with a feed rate of 10 ml / h until the ultraviolet absorption of the eluant at a wavelength of 280 nm is below 0.15. Linear 1 radiator. balancing buffer
    solution (200 ml) to 0.01 M sodium phosphate buffer solution (pH 7.5) containing 0, 6 M alpha-B-methyl mannoside, 2 M KSCN and 0.01% Tween-80, HC- was used for elution absorbed material. Fractions of a volume of 5 ml are collected for the purpose of measuring ultraviolet absorption and fibrinolytic activity, as in Figure 1. The active fractions are pooled together, and the concentration of KSCN is increased to 1.6 M by the addition of solid KSCN. The solution thus obtained is further concentrated by dialysis with solid polyethylene glycol having a mol.m. 15000-20000 daltonov.
    The concentrated solution (5-8 ml) is subjected to centrifugation and gel filtration on a 2 cm column filled with Sephadex G-150 (ultra fine) in a 0.01 M sodium sulfate buffer solution (pH 7.5) containing 1.6 M KSCH and 0.01% Tween-80. Fractions with a volume of 3 ml are collected at a feed rate equal to. The active enzyme eluted as the only peak that coincides with the small protein peak (Figure 2). The fractions containing the active enzyme are poured together, concentrated by dialysis with respect to polyethylene glycol and stored at -80 ° C. Alternatively, the eluant obtained from a column filled with cancavalin-A-agarose may be further
    purified by affinity chromatography on lysine-sepharose or fibrin-sepharose. The results obtained during the purification of the enzyme produced from marine keratocytes of pigs are summarized in Table 1.
    The fibrinolytic activity of exams obtained from keratocytes of swine filth and human breast epithelium was compared with the activity of paciBopa standard urokinase. The standard portion (bottle) of urokinase was dissolved in 50 mM sodium diethylbarbiturate buffer solution containing 0.1 M sodium chloride and 0.25% w / v gelatin (pH 7.8). Several solutions of standard urokinase and a fibrinolytic enzyme were prepared in the indicated buffer solution. All other reagents were diluted in the same buffer solution and kept cold before mixing 0.2 ml of diluted urokinase or solutions / enzyme were placed in a series of tubes of mm size. Next, 0.05 ml of human plasminogen (3 mg / ml), 0.5 ml of human fibrinogen (0.25% w / v) and 0.05 ml of thrombin (40 NIH units / ml) were injected into the tubes. The contents of the tubes were subjected to rapid mixing and then the tubes were placed in a water bath having a temperature of 37.5 C. A stopwatch was immediately started and the prom, corresponding to the end of lysis, was fixed with a gentle inversion of the tube. The calibration graph was obtained in logarithmic coordinates for both lysis time (in minutes) and urokinase content.
    in a blood clot. The dilution factor of the enzyme solution, giving the same lysis time as the standard urokinase solution at 0.5 IU / ml, was used to calculate the activity of the undiluted enzyme solution. The fibrinolytic activity of the enzyme was thus contracted in international units (international units).
    The average characteristic activity (fibrinolytic activity / mg of protein) of cleaner 1 enzyme obtained from guinea pig keratocytes was approximately 12,500 IU / mg. Control. One portion volume
    670 mg of freeze-dried material was obtained in accordance with the proposed method, but without the presence of cells in it. This material gave negative results during the fibrin platelet. The upper layers of several other cell lines were also analyzed for fibrinolytic activity and also gave either a negative result or showed only traces of fibrinolytic activity.
    The results are presented in table 2.
    The molecular weight of the purified enzyme obtained from guinea pig keratocytes was approximately 56,000,000,000 under non-reducing conditions, and about 62,000,113,000 under reducing conditions (by adding
    Leia 2-mer 11Toethiol), which allows to conclude about the presence of disulfide bridges in the enzyme molecule. The molecular weight is in good agreement with an approximate molecular weight of 65000t3000, as assessed using gel filtration on a column filled with Sepdex G-1ZO. Isoelectric points (pi) of purified enzymes derived from guinea pig keratocytes and epithelial cells human mammary gland, equal to 4.6 units.
    In Table 3, it follows that the guinea pig keratocyte and human breast epithelial enzymes are basically the same in composition, but with a few differences that can be expected since they are two different types of animals (human and guinea pig). Both the guinea pig keratocytes and the epithelium of the human mammary gland have an amino acid composition different from the amino acid composition published for HEPA.
    The ability of a guinea pig keratocyte to attach to the complete bloodstream was experimentally tested in a test tube using a closed Chandler-type circulation system. Experimental protection was carried out in a 3 mm inner diameter polyethylene tube (which was pre-washed with 0.01% - a solution of Tween) by mixing citrated whole human blood (1 ml) with 50 ml of 0.02 M, and 25 ml of thrombin (100 NIH V / ml). After incubation for 1 ripii 37 ° C while rotating at 30 rpm, the thrombus was poured into a Petri dish, washed twice with a buffer solution of 0.05 M sodium diethyl barbiturate (pH 7.8) containing 0.25 wt.% Gelatin and 0.01 M sodium chloride. This thrombus was then transferred by an aspirator back to the tube along with 0.9 ml of autologous plasma or buffer solution. Then 100 ml of an enzyme produced from guinea pig keratocytes (approximately 500 international units) was added. The circulating system was again placed on the rotating table and incubated for 2 hours at 37 ° C. The resulting clots were then removed, buffered.
    solution, as mentioned above, and then extracted with 1 ml of a 0.05 M sodium diethyl barbiturate buffer solution, characterized by a pH of 7.8 pH units and containing FM KSCN for 1 hour at 4 C. Aliquot fractions extract and the top layer of the thrombus, with a volume of 50 ml, from the tube was then applied to fibrin plates containing plasminogen, and were incubated for 18 h at.
    The results show that the fibrinolytic activity of the thrombus extract, and consequently, the amount of the enzyme combined, was significantly higher than in the upper layer of the thrombus. Similar results
    were obtained from experiments that were repeated using
    enzyme labeled
    isotope
    In.
    15 20 25

    thirty
    35
    40
    45
    50
    55
    From the point of view of these results, the radiolabeled enzyme can be used for diagnostic purposes as an effective means of localizing the thrombus in the body. I
    Thus, the use of a stable non-cankerogenic cell line to produce an enzyme characterized by fibrinolytic activity alone or as a plasmogenic activator allows to obtain a substance with a higher pharmacological purity. Known methods for producing such enzymes with similar activity either use carcinogenic cells or unstable cell lines. The use of a carcinogenic cell line is unacceptable due to the danger of using harmful material. Ps stable cell lines cannot be used in an industrial way due to their limited lifetimes.
    权利要求:
    Claims (1)
    [1]
    Invention Formula
    The method of obtaining a substance with fibrinolytic activity and / or activity of plasmogenogen stevator by culture of cell culture dependent on blood serum, selection of culture medium, its centrifugation followed by chromatographic
    purification of the target product, about 10 tons and so that, in order to increase the pharmacological purity of the target product, the GPK cell line (cue mi-222) or BEB (cucmi-221) is used for cultivation, cultivation is carried out in Minimum Essential Medium Eagle containing 10% fetal bovine serum, 0.001% Tween-80 and 20 mM HEPES buffer solution to a concentration of 1, Ax10 cells, then the medium is removed, the cells are filled with serum free and cultured again, the selected medium is cleared from the cells and centrifuged for 30 min. About g, the sunarnatant is concentrated, passed through a column filled with zinc chelate agarose and previously equilibrated with a 0.02 M solution of tris-hydrochloric acid pI 7.5, containing 1 M sodium chloride and 0.01% Tween-80, then eluted proteins
    with a linear gradient from 0 to 0.05 M imidazole, the active fractions are selected, injected into a column filled with concanavalin-A-agarose and equilibrated with a 0.01 M sodium phosphate solution pH 7.5, containing 1 M sodium chloride and 0 , 01% Tween-80, again eluted with a linear gradient of 0.01 M sodium phosphate solution containing 0.6 M alpha-O-methylmannoside, 2 M potassium rhodanide, 0.01% Tween-80, and the active fractions, potassium rhodanide is added to them to a concentration of 1.6 M, the resulting solution is added g through solid polyethylene glycol, then centrifuged, the supernatant the athant is passed through a column filled with Sephadex G-150 in a buffer solution of 0.01 M sodium sulfate pH 7.5, containing 1.6 M potassium rohanide and 0.01% 1 eip-80, then the target product is eluted and concentrated by dialysis on polyethylene glycol ,
    Table 1
    F1 0rinolysis LINE cells
    table 2
    Table 3
    The composition of amino acids (expressed as the number remaining in percent)
    inova
    that
    He was determined
    9.8
    5.4
    9.2 13.1
    7.1 10.4
    6,6
    4.1
    0.9.
    3.0
    8.1
    4.0
    3.7
    3.3
    5.5
    5.9
    Continuation of table 2
    0.9 5.1 6.4
    0.1 6.1 8.4 7.8 6.0 2.4 4.8 9.7 3.5 4.5 2.8 6.5 4.8
    2.1 9.8 4.6 6.4 11.5 4.3
    7.1 8.3 6.7 1.0 3.5 13.9 2.2 3.6 2.6 7.1 5.1
    100 200 (ml)
    2
    (nl) fi-g
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    引用文献:
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