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    • 专利摘要:
    Double-stranded RNA synthesized using native human DNA as template is found to be an excellent interferon inducer with low toxicity in spite of homologous native to host cell. It is produced by reacting ATP, GTP, CTP and UTP with one another in the presence of a native human DNA as template by the catalytic action of an active RNA polymerase to form RNA and subjecting the resulting RNA to annealing by heating it at a temperature of 70 DEG to 100 DEG C. and gradually cooling to room temperature or below to form double-stranded regions between their molecules.
    公开号:SU933001A3
    申请号:SU792793202
    申请日:1979-07-26
    公开日:1982-05-30
    发明作者:Аримура Хирофуми；Нагаи Масанори；Ямаути Такеси；Китагава Цутому；Суяма Тадаказу
    申请人:Дзе Грин Кросс Корпорейшн (Фирма)；
    IPC主号:
    专利说明:

    39 of the Lymean DNA through the catalytic activity of the active polyme.  R. RNA basics to produce RNA, followed by annealing of the resulting RNA by heating at 70-100 ° C and gradually cooling to room temperature or below to form two-stranded zones between its molecules.  Natural human DNA is obtained by extraction from the tissues of the human body in the usual known manner, in particular by extraction with phenol, sphenol saturated with water, or with phenol saturated with buffer solution.  For example, DNA is secreted from the che (placenta in accordance with the method of Parish).  Frozen at the human placenta is homogenized in the presence of 60 p-aminosalicylate sodium and a phenol-cresol mixture to effect nucleic acid extraction and deproteinization.  The deproteinization of the surface-rising liquid obtained after centrifugation with the phenol-cutting mixture is carried out once more.  Well, the lead acid precipitates out of the liquid that comes to the surface after tsintrifugation.  The precipitate is dissolved in a weak ionic solution and highly concentrated sodium chloride is added to salve out the RNA.  After removal of the RNA by centrifugation, the PH residue and highly aggregated DNA are removed.  Glycogen is then removed with an ultracentrifuge.  The DNA obtained from the supernatant fraction is precipitated and dissolved in a weak ionic solution.  This solution is dialyzed from distilled to, before, and dialysate is lyophilized to obtain natural DNA as a lyophilized product.  The DNA thus obtained is used as a template for the polymerization of ribonucleic triphosphates under the catalytic action of RNA polymerase to synthesize RN RNA polymerase usually means DIC-dependent RNA polymerase and is an enzyme catalyzing the synthesis of RNC by polymerizing ribonucleoid triphosphates through a double ester bond q using DNA as a template.  This enzyme is specific, t. e.  in quality. Matrix is valid only DNA extracted from the same source as the enzyme.  However, the active RNA polymerase used as an enzyme has a low specificity of the template, so that it is possible to use human DNA as a template for RNA synthesis.  To isolate and purify active RNA polymerase, for example, when it is obtained from Micrococcus lysodeikticus, the original Nakamoto method is used, modified by part of the improved Weiss method.  For this, cells are harvested at the last α-log phase of their growth, stored until used in a frozen state, and when used, they are washed and lyophilized.  Streptomycin sulfate extract is added to this extract for - concentration of a complex of nucleic acids and nucleopotins.  and after elution of the RNA polymerase from this complex, by adding phosphate buffer solution, streptimicin sulfate is again added to precipitate selectively only nucleic acid.  This precipitate is removed by centrifugation, and the membrane components and the ribosomal fraction are removed in an ultracentrifuge.  Protamine sulfate is then added to form the protamine polymerase RNA complex, and RNA polymerase is eluted from this complex with a phosphate buffer solution.  The RNA polymerase thus eluted is reacted with protamine sulfate to create another complex, re-eluted from the complex with phosphate buffered saline, and the eluate is subjected to fractionation with ammonium sulfate.  The fraction precipitated with a saturation of 30-50 is collected and processed with cation exchange resin to obtain purified RNA polymerase.  The activity of the thus obtained polymerase RNA in the range of 50-10,000 units / mg protein.  RNA synthesis is then carried out using RNA polymerase and human DNA as a template.  The mixture of reactants for RNA synthesis contains u / ml RNA polymerase, 100–1000 μg / ml DNA, 0.2–2 mM ATP, GTP, CTP, and VTP, respectively, as the basis, mM MnClg 0 mM spermidine hypochloride, and 0.01– 0.1 M tri-HC (pH 7.0-8.0). ZO-itO C reaction temperature, reaction time 1-10 h. Good results are obtained when the reaction time is h.  The reaction is completed by cooling and additional processing steps: dehydrogenation of the DNA polymerase with deoxyribonuclease, removal of proteins with phenol treatment, purification with dialysis.  The dialysate is then concentrated by ultrafiltration, and the concentrate is centrifuged in CsCl in a CTI incubator using the Glisin method to isolate RNA.  The RNA obtained in this way is purified by precipitation with ethanol, and the resultant product is subjected to a subsequent temperature treatment reaction.  This reaction, the purpose of which is to obtain double-stranded RNA, is carried out in accordance with the method of Robinson et al.  The reaction is carried out in such a way that RNA is dissolved in double concentrated SSC standard salt citrate, 0.15 M.  NaCl, 0.015 M sodium citrate) at a pH of 6.5-7.5, the resulting pa thief is heated to 70-100 ° C, then the temperature is gradually lowered to room temperature to terminate the reaction.  In this case, it is desirable to heat the solution to 70-100 ° C for 3 to 10 minutes, quickly cool to room temperature, then repeat to heat up to 70-10 ° C, after which the solution gradually gradually cools to room temperature.  At this stage, it is desirable to keep the temperature between high and room temperature for a long time.  Upon completion of the reaction, the RNA is reduced by precipitation from ethyl alcohol or similarly, the resulting precipitate is centrifuged and washed, then dissolved in an aqueous solution of low-concentration sodium chloride, dialyzed in water at a low temperature for 10-30 hours, at must be sterilized by filtration, then lyophilized to obtain a preparation.  The interferon inducer thus obtained in the form of double-stranded RNA synthesized with natural human DNA as a matrix is white, odorless, tasteless, contains at least about% (normally 10-15) double-stranded parts in the molecule.  The specified interferon inducer in the form of double-stranded RNA has the following physical and chemical properties.  Molecular Weight.  The sedimentation coefficient is determined by the density gradient of sucrose by centrifugation (in the rotor at 38000 rpm, 3.5 hours) using the mouse L-cell ribosomal RNA as a standard.  The sample includes 11-13 components.  Solubility.  Soluble in distilled water, in a buffer solution of 0.01 M tri-HC (7.5) ,. a double concentrated solution of SSG, in an SSC solution with a concentration of O, T (pH 7.0), and so on. d.  Insoluble in ethanol and acetone.  Ultraviolet absorption spectrum.  0.02 mg / ml aqueous solution. The sample is given an ultraviolet absorption spectrum typical of nucleic acid.  The maximum absorptive capacity is 2 bO nm, and the minimum is 230 nm.  Color reaction.  The reaction is positive when tested by Orsinol and is negative for diphenylamine and indol, corresponds to the characteristic of the RNA color reaction.  Precipitation reaction.  A solution of ethanol twice the volume is added to 0.5 mg / ml of the aqueous solution of the sample and allowed to stand for more than one hour at -20 ° C.  Almost 100% of the material is precipitated.  Dabavleniya doloviny about. A unit of cooled 505; $ tay (trichloroacetic acid) k-0.1 mg / ml of an aqueous solution of the sample translates into an insoluble form 80-90 RNA-TCA, which can be collected using a filter (0.5 μm).  Dehydrogenation with ribonuclease.  The sample is dissolved in a SSC double-concentrated solution (pH 7.0) and processed together with bovine pancreatic ribonuclease (10 µg / ml) and -T ribonuclease (1 µg / ml) for 30 minutes at.  As a result, approximately 11-13 samples remain unabsorbed.  On the other hand, the sample is dissolved in an SSC solution (pH 7.0) with a concentration of 0.3, heated, then rapidly cooled and processed together with the above-mentioned ribonuclease after transferring the solution to a double concentration SSC.  As a result, 2-3 remain unabsorbed.  Deoxyribonuclease uptake.  The sample is dissolved in 0.01 M tri-HC buffer solution (pH 7,) and treated with deoxyribonuclease (50 µg / m) for 30 minutes.  The resulting spin of the insoluble TSA fraction is the same as with the above treatment, and the increase in absorbing capacity at 2bO is not noticeable to them, either before or after processing.  Alkaline hydrolysis sensitivity.  The sample is dissolved in 0.3 M potassium hydroxide and kept at 37 for 18 hours.  The sample is fully hydrolyzed.  Analysis of the hydrolysis product using thin-layer homatography shows only individual spots of adenylic acid, amidic acid, cytidinoic acid and Iuridinovry acid.  Composition Analysis of the composition is performed by thin layer chromatography on an alkaline hydrolysis product.  As a result of the analysis, it was established that the molar ratio of each nucleotide is in the range of 27,, O-30.5 adenylic acid, 20.6-24.7% amidic acid, T6.8 2.3 cytidic acid, and 27.832, 7 uridic acid.  , The density of the floating sample in ".  The density of the floating sample in.  measured using an ultracentrifuge (Spinco SW 50.1 rotor, 31500 rpm 72 h).  It is equal to 16351640.  .  Thermal denaturation of ribonucle ase - resistance of RNA.  The sample is dissolved in a SSC solution (pH 7.0) with a concentration of 0.1, the temperature is melted / (determined according to method K.  Colby and D, D, Dyusberg, It was found to be equal to 71 ° C. Spectral absorption capacity increases with increasing temperature.  The sample is dissolved in an SSC solution (pH 7.0) with a concentration of 0.1, then the increase in absorbance at 2 bO nm is determined with increasing temperature.  An increase of about 20% in the range of | temperatures of 25-90 C.  Purity.  The sample purity assessment is performed as follows.  The amount of protein is determined by the method of Folin-Louni, and the amount of DNA is determined by the SST method modified by Mitsuno et al.  No protein and DNA contamination is observed.  Observations under the electron microscope.  Observations under an electron microscope show that most RNA molecules are linear, 0.1-3 to 3 microns in length.  Double-stranded RNA, which has the physical and chemical properties described above, can be used as an interferon for the reason that it induces interferon and has extremely low toxicity.  In the manufacture of various medical drugs, stabilizers, among which may be human albumin, mannitol and.  others  agents of increased solubility, glycine, and substances such as sorbitol — can be added to the solution before lyophilization, and the solution is already. contains active material.  When using, the medicament prepared in this way is dissolved (in saline, sterilized water, sterilized isotonic solution for injections, t t. d. ) and prescribed to patients as an interferon inducer by intravenous, muscle or subcutaneous injection.  An effective dose of 1 to 2 mg per kilogram of body weight per day, if necessary, the dose can be increased, the drug is effective as a medicine, even in crude form, t. e.  In the mixed state with single-stranded RNA, k% or more of double-stranded RNA are sufficiently effective.  The biological properties of the drug are described in detail in examples 1-7.  Example 1 Antiviral activity is determined by measuring viral productivity in accordance with a partially modified method.  T. e.  0,0; 1.0; 10 and 50 µg / ml samples of an interferon inducer (a preparation of the present invention and Poly 1: C as a control), 50 µg / ml dextran DEAE (molecular weight 50x10) and 3 ml of Eagle's minimal medium (hereinafter referred to as MEMIGL ), supplemented with 0.5% uterine bovine serum, are added to monolayer cells (2.5-3x10 cells / flask) in 50-ml culture flasks, then kept at 37 ° C for 20 h.  After the incubation period, the Eagle MEM medium containing the interferon pathogen is removed, and the cells are washed three times with Hanks' brine, then Vesicular stomatitis viruses are added (V. S. V. ) with frequency of infection from 2 to 3 plakoobrazuyuschih units per cell.  Viruses adsorb when for 1 h.  After adsorption to remove free virus V. S. V.  the cells are washed five times and 5 ml of new medium is added (Eagle's MEM medium containing 1 uterine bovine serum).  After an incubation period of 20 h at 3 / C, the cells are cooled in a refrigerator at.  Cooling and thawing are repeated twice, then the cells are sent to a centrifuge (2000 rpm, for 10 minutes).  The virus titer in the supernatant was determined by analyzing FL cell cultures.  Viruses from cells treated with MEM Eagle medium without an interferon pathogen (containing serum or DEAE - dextran) are used as control cells.  The cells used belong to the two species established.  human amniotic FL cell lines and RK-13i rabbit kidney cells; and human foreskin diploid germ cells (HFS) and the human embryo, outgoing kidney NK cells.  Heteroploid cells of NK cells were initially seated approximately 130 times.  These cells are grown in Needle MEM medium supplemented with 10 uterine calf serum, 100 μg / ml penicillin G, 100 μg / ml streptocytic sulfate and 60 μg / ml kanamycin.  Upon completion of the incubation of the viose, the cells are cultured in a medium in which the amount of serum is reduced to 1.  Cells are cultured in an incubator containing C0 „(CO).  Used as a control substance Poly 1: C, examined in phosphate buffered saline. pH /, 0} with a concentration of 2.5 mg / ml, stored at -30 ° C, and diluted when used.  The results are shown in Table.  1, whence it is clear that although there is a slight difference between the cells, the antiviral activity of the preparation is the same as that of Poly 1: C.  Table 1 - blooming unit, Example 2.  The interferon induction activity is checked by the addition of the proposed drug to cultured cells.  This test is carried out using the Wilzeck method, while 2 ml of Needle MEM medium (without -serum) containing 5-50 µg / ml of interferon pathogen and 100 µg / ml of cyclohemixide are added to the HFS cells that form the overlay in a plastic Petri dish. diameter 6 mm.  Cells incubated in a thermostat for 5 h at 37 C.  0.5 ml of actinomycin is added to the medium. so that the final concentration becomes 1 µg / ml.  Then continue for 1 h at 37 incubation.  After the incubation is complete, the cells are washed with Hanks' brine five times, then 5 mg of MEM Needles with 0.5% serum is added.  After 20 hours of incubation at 37 ° C, the bank transfer is collected and centrifuged (1000 rpm) for 5 minutes to obtain an interferon-containing substance.  The interferon test is carried out using PL cells.  The interferon test is carried out according to a modified Wilzeck method.  M, on-layer cell cultures in a plastic Petri dish are washed and taken in two twice-diluted interferon samples in Eagle's MEM medium containing the uterine calf syrup, then placed in a 20 C thermostat at.  After washing the cell layers, they are exposed to about 100 per cup of viruses y. S. V. , and the obtained plates are counted after two or three days incubation.  The titer of interferon is expressed as the reciprocal of the dilution of the sample, which reduced the number of plaques by 501 from the control that was applied to HEM Needles instead of the sample.  The titer of interferon samples of human cells is determined by the simultaneous determination of standard human leukocyte interferon to transform into international units (1 U).  Poly 1: C is used as a control.  The proposed drug exhibits a higher interferon induction activity as compared with Poly 1: C, EXAMPLE 3.  The action of the interfer on a living organism. .  Male rabbits weighing 2 „0-2.5 kg.  administered an intravenous injection of 10,100 µg / kg of the drug or Poly 1: C as a control.  Immediately before the injection, as well as after, 1, 2, k, 8, 11 and 2 hours after the injection, blood is taken from the rabbits.  The interferon induction activity is determined by analyzing the interferon content of the resulting serum.  The method of T | 1t interferon is similar to the aforementioned method of Vilcek et al.  with the exception that instead of FL cells, RK-13 cells are used. The proposed preparation exhibits the activity of inducing interferon in vivo. body equal to or greater than the activity of Poly 1: C.  Example.  Toxicity test.  The effect of the proposed drug and RoGu1: C as a control agent on cultured cells is being studied. At the same time, j ml of a suspension of human amniotic cells FL (60 X U cells per ml) and 1 ml of MEM Needles medium containing 5.50 and 250 μg / ml (finally dissolved 5 times) of the preparation are simultaneously placed in a flask of 5 ml.  The cells are cultured for 6 days. .  After 2 and 6 days of cultivation, two flasks are taken from each group, and they are washed with phosphate buffer solution to remove floating cells.  The remaining cells are separated from the glass surface with 0.02 ethylenediamine tetraacetate (EDTA) and trypsin (0.25%) to count their number.  Then the number of viable cells is counted using the erythrocin B coloring substance and hemocytomer.  The control drug Poly 1: C is to some extent toxic even at a concentration of 1 μg / ml, at 10 μg / ml, toxicity is substantially noticeable, and at 50 μg / ml, cell multiplication was not observed.  In contrast to the control, the resulting preparation does not detect toxicity at a concentration of 10 µg / ml or less, cell multiplication is suppressed only very slightly even at a concentration of 50 µg / ml.  .  It can be seen from the above experiments that the obtained preparation is a means of stimulating interferon, has antiviral activity that is approximately equal to or greater than the activity of Poly 1: C, and is less toxic compared to it.  PRI me R 5.  The acute toxicity of the resulting preparation was tested using a male mouse (UjBL, according to method. Lidic and Wilcoxon.  The result showed that intraperitoneal administration of the drug at the level of 50 mg / kg body weight or intravenous infusion at the level of 25 mg / kg did not result in the death of the mice.  For mice, the dose for subcutaneous administration of the drug is at least 50 mg / kg, with intravenous administration at least 25 mg / kg.  The production of synthetic double-stitch RNA is illustrated by the following examples.  PRI me R 6.  Isolation and purification of the DNA of the human placenta.  The placenta, frozen immediately after childbirth and stored at, is crushed into pieces up to 60 g, 15 volumes of a 6% aqueous solution of aminosalicylate and 15 volumes of a mixture of phenol and cresol are added to it (composition: phenol 500 g, m-cresol 70 ml water 55 ml and 8-hydroxylquinoline 0.5. The mixture obtained is homogenized at room temperature for one minute in a mixer at maximum speed.  The suspension thus obtained is stirred at room temperature for 20 minutes, then centrifuged for 30 minutes at 6000 X g to remove the denatured protein and phenol.  Sodium chloride is added to the supernatant so that its final concentration is 3 and also half the volume of the phenol-cresol supernatant.  The mixture is stirred at room temperature for 20 minutes, then centrifuged for 10 minutes at 5 ° C and 8000 X g.  To the supernatant, add two volumes of cooled ethyl alcohol-cresol mixture (volume ratio 9: 1) and gently mix to form a precipitate.  After the fibrous sediment is wound on a glass rod, the remaining liquid is left to stand for 1 hour, then the remaining precipitate is recovered by centrifugation. The total precipitate is thoroughly washed in a sufficient volume of 75% ethanol containing 2% acetate. sodium and completely dissolved in. 200 ml 0. 1 M sodium acetate containing 5 mM sodium fluoride.  Sodium chloride with a final concentration of 3 M is added to this solution.  This solution is settled for 15 hours at (-2) - (- 5) s and centrifuged for 1 ° C at 12000 X g.  To the supernatant liquid, add a water volume of the cooled ethyl cellulose and gently mix until a white fibrous precipitate is obtained.  After settling in a water bath at 0 s for 20 minutes, the fibrous precipitate is removed using a glass rod, dispersed and dissolved at room temperature in 300 ml of 3 M sodium acetate buffer solution (pH 6.0) containing 5 mM sodium fluoride .  When the dissolution is almost complete, the solution is centrifuged at 5 ° C for 10 minutes at 2000 X g to remove residual PWK and highly aggregated DNA as precipitate.  An equal volume of cooled ethyl cellulose is added to the supernatant, and the mixture is left to stand for 20 minutes in a water bath at.  The fibrous precipitate obtained is wound on a glass rod, dispersed and dissolved at room temperature in 200 ml of a 0.1 M sodium acetate buffer solution (pH 6.0) containing 5 mM sodium fluoride.  Koi-pa dissolution is almost complete, the solution is centrifuged for 90 minutes at and 60,000 X ig to remove glycogen as sediment.  Sodium acetate with a final concentration of 0, 3 M is added to the supernatant and mixed with an equal volume of chilled ethylcellulose.  After settling in a water bath for 20 minutes, a fibrous precipitate is collected using a glass rod and dissolved in 16 ml of 0.1 M sodium acetate buffer solution (pH 6.0) containing 5 mM sodium fluoride.  When the dissolution is complete, the solution is transferred to the dialysis tubing and dialyzed for tS h with 200 ml of distilled water five times. , change the external solution.  The dialysate is centrifuged to remove insoluble materials, and the DNA concentration is determined by measuring the absorbency ratio of 2bO of the diluted material to the surface.   The concentration calculation is carried out taking into account the fact that the coefficient of spectral absorption capacity at 260 nm of 1 mg / ml DNA is 20.  Solutions containing 6.5 mg DN are placed in 10 ml tubes and lyophilized.  After these procedures, an amount of iiO kg of chemo DNA is obtained. To estimate the mystic of the DNA preparation, the amount of protein is determined by the improved Lowry method. The amount of RNA is determined by the Mitsuno method.  The protein was Q, k% in the form of albumin human placenta.  RNA was C, 8% of the total number of nucleotides.  Purification of RNA polymerase.  Microcx-eCUS lysodeiktiois is grown on nutrient broth using Jar Fermenter.  Upon reaching the last logarithmic growth stage, they are collected and stored — for example.  kOQ grams of frozen cells are dispersed in 2 liters of 0.05 M tri-HC1 buffer solution and centrifuged to remove washed cells, which are then dispersed in 0.01 M tri-HC1 buffer solution (pH 8.0) containing -0, 2 M sucrose to obtain a final volume of 2 liters.  To this suspension, 600 mg of white lysozyme chicken eggs are added, and the suspension is kept in an incubator at.  After 15 minutes of incubation, 6 ml of 0.1 M MgClg is added and kept in a thermostat for another 5 minutes to destroy the cell walls.  Then 18 ml of 0.1 M MgCla are added.  and a ml of water cooled to.  The mixture is vigorously stirred.  All procedures are carried out in a water bath at 0 ° C.  After 10 minutes, 600 ml of 10% streptomycin sulfate was added, and after another 10 minutes, the resulting precipitate was centrifuged for 10 minutes at 20,000 X g.  The precipitated pellet is suspended in a BOO ml of 0.01 M tri-HC1 buffer solution (pH 8.0) containing 0.2 M sucrose, 0.1% streptomycin sulfate and 0.00035 M MgCl.  After 10 min, the solution is centrifuged for 10 min at 20,000 X gjc. The precipitate is homogenized in 720 ml {Solution prepared by mixing In ml of 1 M potassium phosphate buffer solution (pH 7.5) jB ml of 0.1 M MgCl2. and BO ml of 2 M sucrose, diluting the mixture with distilled water to 720 ml using a Teflon homogenizer from Potter-Elweichem.  32 are added to this homogenate.  ml, M potassium phosphate buffer solution (pH 7.5) and after another ten minutes -. 70 ml of 10% sulfate. After 10 minutes of stirring, the resulting solution was separated by centrifugation for 30 minutes at 5 ° C and 30,000 x g.  and the resulting supernatant is further centrifuged for 2 hours at 105,000 x g to remove the membrane components and the ribosomal fraction.  110 ml of a 1 M potassium phosphate buffer solution (pH 7.5) and then 160 ml of 2.5% non-neutralized protamine sulfate are added to a BOO ml of ultracentrifuged supernatant to precipitate the complex compound with protamine sulfate .  After 10 minutes of stirring, the precipitate is collected by centrifuging for 10 minutes at 5 ° C and 20,000 x g.  The pellet is homogenized in IBO ml of 0.2 and sodium phosphate buffer solution (pH 7.5) containing 0.2 M sucrose for elution of RNA polymerase.  After t0 minute stirring, the suspension is centrifuged for.  10 min at and 30,000 x fi.  A BIO of a 0.1% sulfate solution of the non-neutralized protamine is added to the supernatant, and the RNA polymerase again precipitates as a complex compound with protamine sulfate.  After 10 minutes of stirring, the suspension is centrifuged for 15 minutes at and 20,000 x g.  The pellet is homogenized in 50 ml of a 0.1 M potassium phosphate buffer solution (pH 7.5) containing 0.2 M sucrose, and the homogenate is centrifuged for 10 minutes at 5 ° C and 30,000 X g.  The collected precipitate is homogenized in 30 ml of a 0.2 M potassium phosphate buffer solution (pH 7.5) containing 0.2 M sucrose to elute RNA polymerase.  After 10 minutes: stirring the suspension is centrifuged for 15 minutes at and 30,000 xg. 7.27 g of ammonia 1 a was added to the supernatant (30 ml) in order to obtain a 0% saturation to effect ammonia sulphate fractionation.  15 minutes after the addition of ammonium sulfate, the suspension is centrifuged for 10 minutes at 5 ° C and 30,000 x g. The precipitate is dissolved in 5 ml of 0.02 M buffer. a solution of tri-HC1 (pH 7.5) f containing 0.3 M sulfate. ammonia, and add an equal volume of glycerol.  The solution is diluted 2.5 times.  A 0.01 M buffer solution of triHCl (pH 7.5) and passed through a CM cellulose column in a 17 glass pan, prepared using 2, k g CM cellulose to remove ribonuclease.  The column is washed with 0.01 M buffer solution tr, i-HC1 (pH 7.5), containing 20% glycerol and 0.06 M ammonium sulfate, and the eluate is passed through a column of CM-cellulose, prepared using 0.8 g CM cellulose.  After washing the column, 22.2 ammonium sulfate was added to the total 80 ml of eluate to precipitate the ammonium sulfate.  After 15 min, the precipitate obtained is collected by centrifugation for 15 minutes at 30000 X g, and the precipitated substance thus obtained is dissolved in J ml of 0.02 M tri-HC1 buffer solution (H 7.5) containing 0.3 M sulfate ammonia, and then add an equal volume of glycerol and before use stored at.  From “GO of frozen cells, 3 mg of RNA polymerase preparation with a specific activity of 190 units is obtained.  per mg of protein (total activity 8200 units) and the ratio of the spectral absorption capacity at 280: 260 nm 1.55.  One unit of enzyme activity is defined as the amount catalyzing the introduction of 1 n mol 1A C - ATP insoluble TCA material during 10 minutes of incubation.  Each reagent is dissolved in 0.1% tri-HC1 buffer solution (pH 7.5). Table.  2 shows the reagents used in the synthesis of RNA, the total amount of 500 ml.  Table 10,000 100 m 200 18, pdr} table maintenance  2 Non-neutralized spermidine trihydrochloride 2 Tri-HC, pH 7.5 The above reagents are thoroughly mixed and 1a kept in a thermostat for 3 m in a water bath.  This mixture is then cooled in a water bath until the end of the reaction and heated for 5 minutes at 65 ° C to inactivate the RNA polymerase.  The reaction mixture was cooled to approximately flow through water, until it was added, then 5b ml of O, 1 M MgCl 2 was added to it.  to bring the solution to 10 mM. Then, 5.6 mg of deoxyribonuclease (without ribonuclease) is added and placed in a thermostat for 30 minutes at 37 ° C to dehydrogenate the DNA.  8 for 3 minutes, the reaction mixture is heated to inactivate the deoxyribonuclease, then cool with running water to room temperature. .  After that, 5bO ml of double concentrated SSC (pH 7.0} and 1120 ml of phenol-cresol mixture (composition: phenol 500 g, t-m-cresol 70 ml, water 55 ml, 8-hydroxyquinoline 0.5 g ).  The reaction mixture is shaken for 5 minutes, then centrifuged for 10 minutes at 5,000 x g.  Separated from the phenol layer and the denatured protein layer, the supernatant receives an additive of half the volume of the above-mentioned phenol-cresol mixture, then it is shaken at room temperature for 5 minutes.  The supernatant obtained by centrifugation was dialyzed again with a 5 M SSC solution with a double concentration (pH 7.0).  containing 0.05% sodium dodecyl sulfate (hereinafter referred to as SDS), 1 hour at room temperature and another 1 hour after replacing the external solution.  Thereafter, the external solution is replaced with 5 liters of 0.01 SSC concentration (M 7.0)) and is continued overnight at 2-5 ° C.
    The dialysate (890 ml) is ultrafiltered with G using a hollow fiber membrane filter until thickened to 19 ml.
    19 g are dissolved in a 19 nl ultrafiltered concentrate. Then the total volume was adjusted to 26 ml in do-h with a 0.01 M triN1 buffer solution (pH 7.5) containing DjCl in the ratio of 1 g per 1 ml of buffer solution. This solution (2b ml) is applied to 7.8 ml of 5.7 AND CgCl (pH 6.5) containing 0.1 M EDTA, then 1.3 m of 0.1 M of a three-HC1 buffer solution (rL 7.5) and all this is centrifuged. Centrifugation is carried out on a rotor at 27,000 rpm (130,000 x g) for 15 m at 15 ° C.
    After centrifugation, the supernatant was removed and the RNA in the form of bright granules was dissolved in 30 ml of a 0.1 M tri-HC1 buffer solution (pH 7.5). The resulting solution is cooled in a bath at. And two volumes of cooled ethyl alcohol are added to it, all this is settled for 1 hour at -20 ° C. Then, the resulting precipitate is collected by means of high-speed centrifugation. Floated substance. removed and the precipitate re-dissolved in a 0.01 M tri-HC1 buffer solution (pH 7.5) ..
    The yield of purified RNA is 8.6 mg in accordance with the calculation from the A2bO 22 ratio, where A2bO is the spectral absorption capacity at. 260 nm 1 mg / ml RNA solution.
    When adding 50 TCA, about 80% of purified RNA is precipitated as the insoluble fraction of TCA. The sedimentation coefficient of the purified RNA, which is measured by gradient centrifugation of sucrose density using b-cell ribosomal RNA as the standard for the cell, is about 12 s.
    Annealing RNA. 3 mg of purified RNA is dissolved in 4.8 ml of 0.01 M three-He1 buffer solution (pH 7.5) and 1.2 ml of SSC 10-fold concentration (pH 7.0) is added to the solution in order to the concentration of RNA at twice the SSC concentration was 500 µg / ml. The solution obtained in this way is placed in a glass test tube with a plug, heated for 5 minutes at 10 (PC, quickly heated in a water bath at, then placed in a water bath at 85 ° C. The temperature of the water bath is gradually reduced over 2 hours. h before, and the recovery occurs k h at. Then the water bath heater is turned off, and the solution is settled overnight with gradual cooling to room temperature.
    The sedimentation coefficient of annealed RNA is determined by the method of gradient centrifugation of sucrose density. A peak is observed at 12 s, and the result obtained is practically no different from what was obtained before annealing, however, a small new peak is observed in the region of approximately 28 s.
    The stability of ribonuclease annealed RNA (the number of RNA-hybrid RNA; the number of double-stranded parts) is determined by the Heppert method. 30 μg of annealed RNA are treated together with 20 μg of bovine pancreas ribonuclease and 2 μg of ribonuclease jT in a double concentration SSC solution (pH 7.0) for 30 min. 13.3 insoluble are obtained using filtrate (0.5 μm) TCA fractions without traces of dehydrogenation,
    On the other hand, 30 μg of the sample, heated at 0.3 SSC and rapidly cooled in a water bath at 0 ° C, is subjected to double-concentration ribonuclease treatment at the SSC, as mentioned above, as a result, non-dehydrated 2.3 insoluble TSA fraction is obtained .
    Annealed RNA was regenerated using ethanol precipitate according to a typical method. The precipitate is dialyzed with distilled water, dialysate is sterilized by filtering with separation into test tubes and subjected to lyophimation. The preparations thus obtained are interferon and interferon and have its physical, chemical and biological properties, jPr and MER 7. Isolation and purification of DNA human placenta.
    The process is carried out in the same manner as in Example 6.
    权利要求:
    Claims (1)
    [1]
    Purification of Ercoli RNA polymerase. The strain E. coll is grown in nutrient broth using a Jar Fermenter, harvested at the last stage and logrification growth phase and stored at. 250 g of these cells are suspended in 750 ml of Grinding buffer solution (0.05 M tri-HC buffer solution, pH 7.9) containing 5 glycerol, 2 mM EDTA, 0.1 mM dithiothreitol, 1 mM 2-mercaptoethanol, 0.233 MNaCl, 130 μg / m of white lysozyme chicken eggs and 23 μg / ml of fluoride phenylmethanesulfonyl) and set aside for 20 minutes at. Then, 0.0125 vol 4 DOS (sodium deoxycholate) is added and the mixture is stirred for 39 seconds. After settling for 20 minutes, it is stirred again for 30 seconds. Then 1000 ml of TCEA + 0.2 M NaCl (TCEH 0, 01 M of a three-HC1 buffer solution (pH 7.9) containing 5 glycerol, 0.1 mM EDTA and 0.1 mM dithiothreitol, TCEA + 0.2 M NaCl — TCRED with a content of 0.2 M NaCl) were added to the solution and stirred vigorously for 5 min. The suspension is centrifuged with kS min at k C and 100.00 X g. 0.075 volumes of Polymin P (pH 7.9) was added to the resulting supernatant. After 5 minutes, the resulting precipitate was collected by centrifugation for 15 minutes at C and 6000 x g. The pellet is homogenized in a TCEA + 0.5 M NaCl (TCEA with a content of 0.5 M NaGl), the homogenate is stirred for 10 minutes at, and then centrifuged for 30 minutes at 4 ° C and 6000 x g. The precipitate obtained is homogenized in a TCEA of + 1.0 M MaSH (a TCEA with a content of 1.0 M NaCl) and stirred for 10 minutes. The homogenate is centrifuged for 30 min at C ° C and booO X g.K ammonium sulfate is added to the resulting material in a proportion of 35 g per 100 ml, the whole is stirred for 30 min at k C. Then the suspension is centrifuged for kS min and at 10,000 x g. The resulting precipitate was dissolved in 700 ml of TCEA. This solution was passed through a column of agarose and the DNA of a calving goiter (2.6 x 15 cm), prepared according to the Shalar method, to absorb polymerase, RNA. The column is washed with 500 ml of TCEA + + 0.15 M NaCl (TCEA with a content of 0.15 M), then with 500 ml of TCEA- + j + 0.3 M NaCl (TCEA with a content of 0.3 NaCl). After that, they pass through the column 500 ml of TCEA + Column are passed with 500 ml of TCEA + + 1.2 M NaCl to elute polymerase RNA. Ammonia sulphate was added to the eluate in a proportion of 35 g per 100 ml and stirred for 30 minutes at tfC. The resulting suspension is centrifuged for 30 minutes at C and 10,000 x g. The result is a precipitate-solution of 7 ml of TCEA + 0.5 M NaCl + 30% glycerol (TCEED + 0.5 M NaCl with the difference that 301 glycerol is contained instead of 5 ° 4). The solution is divided into 2-3 equal parts, and each part is subjected to filtration through a gel through a column with Bio Gel A 5 M (Polyacrylamide gel), balanced by TCEA -t-0.5 M I-IaCl. The elution is carried out with a TCEA + +0.5 NaCl. Fractions with the activity of polymerase, RNA are extracted from the eluate and used to synthesize RNA. The specific activity of the RNA polymerase thus obtained is equal to 320 units / mg of protein as determined by the Burgess method using the calf goiter gland DNA as a template. The unit of enzyme activity is defined as the amount catalyzing the introduction of 1 n volume of k C-ATP into the material insoluble for 10 min. The spectral absorption coefficient between 280 and 2bOnm was found to be 1.86. The total activity of RNA polymerase obtained from 250 g of frozen cells was equal to 30,000 units, and the total amount of protein was 96 mg. Synthesis, isolation and purification of RNA. Reagents for RNA synthesis are listed in Table. 3; (only 150 ml). Each reagent was dissolved in 0.04 N triS1 buffer solution (pH 7.9). T 1 - T a b l and c a 3. Polymerase 20 units / ml 3000 units of NK human plate 200 µg / ml 23, Continued table. 3 0.5 toto ethanol T | zi-HC1, M pH 7.9 These reagents are thoroughly mixed and placed for 3 m in a thermostat at 37 ° C. The reaction mixture is then cooled in a water bath at the end of the reaction, heated for 5 minutes at the time of inactivation of the RNA polymerase, and cooled with straight water at (Rx 37 ° C, then 16.7 ml of 0.1 M MgClj is added. to bring the solution to 10. After this, 1.7 mg of deoxyribone lease (without ribonuclease admixture) is added to a thermostat for 30 minutes at 37 ° C for dehydrogenation, DNA. The reaction mixture is heated to (100 ° C for 3 minutes for inactivating deoxyribonucleases, then cooled with cork water to room temperature. Then add 170 m l S-double concentration (pH 7.0} and ml of phenol-cresol mixture (composition: phenol 500 g, m-cresol 70 ml water 55 ml, hydroxyquinoline 0.5 g) Shake the reaction mixture for 5 min at room temperature , then centrifuged for 10 minutes at and 5,000 x g. Half the volume of the phenol-cresol mixture is added to the surface of the denatured protein and to the supernatant, and shaken at room temperature for 5 minutes. / The emulsion is centrifuged and after floating up the substance is dialyzed from 5 l. SSC double concentration (pH 7.5), containing 0.05% Sre, for 1 h at room temperature, and dialyzed again for 1 h after replacing the external solution. Then, the external solution is replaced with 5 liters of concentration 0.01 (pH 7.0) and dialysis 01 is carried out overnight at. The dialysate (310 ml) is subjected to ultrafiltration using a fiber membrane filter to obtain a 19 ml concentrate, 19 ml of the concentrate obtained by ultrafiltration, I19 g dissolve. Then the total volume was adjusted to 26 ml with the addition of 0.01 M 6v (a beDnogo solution of tri-HC1 (pH 7.5) containing CgCl in the proportion of 1 g per 1 ml of buffer solution. The solution (26 ml) was applied to 7.8 ml 5 , 7 M С $ C1 (pH 6.5), containing 0.1 M EDTA, and then another 1.3 ml of 0.1 M 0.1 M triCl1 buffer solution (pH 7.5) is applied and centrifuged. Centrifugation is carried out in a rotor for 15 h at and .2700 rpm (, 130,000 X g). After centrifugation, supernatant is removed and RNA in a clean granule is dissolved in 3 ml of 0.01 M tri-HCl buffer solution (pH 7.5). .,.,. The resulting solution is cooled ft - "- - the bottom of the bath n At the OS, add two volumes of cooled ethanol and allow to stand for 1 hour. The resulting precipitate is collected by low-speed centering of the fugging. The supernatant is removed and the precipitate is redissolved in a 0.01 M tri-HC1 buffer solution. (pH 7.5). The yield of purified RNA is 9.0 mg. When adding 501 TCA, approximately 90% of purified RNA as an insoluble fraction of TCA precipitated. The sedation coefficient is purified. . measured by centrifugation to determine the sucrose density gradient with the use of mouse ribosomal RNA as a standard, is equal to 11 seconds. Annealing RNA. 3 mg of purified RNA p is dissolved in 12.0 ml of 0.01 M tri-HC1 buffer solution (pH 7.5), 3.0 ml of SSC with 10-koat concentration of fpH 7.0) is added so that RNA at twice the SSC concentration was 200 μg / ml. The resulting solution was placed in a glass test tube with a stopper, heated for 5 min at. rapidly cooled in a water bath, then, then placed in a water bath at 85 ° C. The temperature of the water bath is gradually lowered to 2 hours, and during the reaction A more hours pass. After -: the heater of the water bath is turned off, and overnight the solution is allowed to settle, gradually cooling. to room temperature. The stability of ribonuclease annealed RNA (the number of hybrid RNA-PH number of the double-stranded portion) is determined by the method of Hempert and others. 30 µg of annealed RNA are treated together with 20 µg of bovine pancreatic ribonuclease and 2 µg of ribonuclease-1 in 2 SSC of double concentration (pH 7 , 0) when for 30 min, the resulting 12.0% insoluble fraction of TCA dehydrogenation was collected with a millior filter (0 μm). On the other hand, 30 µg of the sample, heated at 33 ° C of 0.3 concentration for 5 minutes with and rapidly cooled in a water bath, were subjected to ribonuclease treatment, as mentioned above, as a result, 3.3% of the insoluble fractions were collected. TCA using a filter. Annealed RNA was regenerated by precipitating 1 and ethanol according to a typical procedure. The precipitate is dialyzed with distilled water, the dialysate is sterilized by filtration, separated in ampoules and subjected to lyophilization. The purified RNA contains,%: adenylic acid 27.3, amidic acid 21.5, cytidine acid 18.5, uridine acid 32, 7 The proposed method allows to increase the interferon-inducing activity of the target product. The method of obtaining a synthetic double-stranded RNA having interferon-inducing activity of a human crystal pattern as a template for enzymatic synthesis, is distinguished by the fact that, in order to increase the interferon-inducing activity of the target product, adenosine triphosphate, uridine triphosphate, triphosphorus, crystal, and a crystal body, a crystal crystal, a crystal sphere placenta in the presence of active RNA polymerase of Mictococcus lysodeiktiais or Eseherichia coli for 2 hours at t in the trisol socks scrapping the buffer solution at pH 7.0-8.0, the resulting RNA is separated from the protein in the usual way, separated from the protein, and the RNA is centrifuged using CgCl as a shock absorber, the target product is precipitated with ethanol and kept in a thermostat at t 70100 C, then rapidly cooled to room temperature or below. Sources of information taken into account in the examination 1. Japanese Patent ff 2008 / 75t Cl, 113 E 4, 1975.
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    同族专利:
    公开号 | 公开日
    AT370131B|1983-03-10|
    IT1117755B|1986-02-24|
    FI792346A|1980-01-29|
    IT7949883D0|1979-07-27|
    JPS5519239A|1980-02-09|
    ES482856A1|1980-04-01|
    FI69868B|1985-12-31|
    DE2930604C2|1984-08-02|
    FI69868C|1986-05-26|
    DE2930604A1|1980-02-07|
    CH651317A5|1985-09-13|
    FR2433535A1|1980-03-14|
    FR2433535B1|1982-10-08|
    ATA517079A|1982-07-15|
    GB2027033B|1982-11-17|
    SE7906425L|1980-01-29|
    JPS5648520B2|1981-11-16|
    GB2027033A|1980-02-13|
    US4313938A|1982-02-02|
    CA1136562A|1982-11-30|
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    US4082613A|1976-04-23|1978-04-04|The Regents Of The University Of Minnesota|Process for the production of insulin by genetically transformed fungal cells|US6835557B1|1980-01-08|2004-12-28|Biogen, Inc.|DNA sequences, recombinant DNA molecules and processes for producing human interferon-like polypeptides|
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    CA2929600A1|2013-11-28|2015-06-04|Bavarian Nordic A/S|Compositions and methods vectors for inducing an enhanced immune response using poxvirus vectors|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP9221278A|JPS5648520B2|1978-07-28|1978-07-28|
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