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    • 专利摘要:
    An aqueous solution containing a physiologically active substance, which is produced by recombinant DNA technique and which has cytotoxic activity against L-M cells and is capable of inducing hemorrhagic necrosis of transplanted Meth A sarcoma in the BALB/c mouse, can be effectively, efficiently purified by column chromatography using a column packed with a dye-bonded crosslinked agarose gel.
    公开号:SU1630602A3
    申请号:SU853985474
    申请日:1985-11-22
    公开日:1991-02-23
    发明作者:Кадзихара Юнити；Киета Такао；Хаяси Хироси
    申请人:Асахи Касей Когио Кабусики Кайся (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to biochemistry, in particular, to a method for purifying a physiologically active substance, wherein said physiologically active substance is a substance obtained by recombinant DNA technology using recombinant DNA containing a DNA encoding substance that has cytotoxic activity against LM cells and is capable of induce hemorrhagic necrosis of MetA transplanted sarcoma in BaL B / s mice.
    Human tumor necrosis factor is a substance obtained by the traditional method with recombinant DNA containing DNA encoding full names. The human full name exhibits cytotoxic activity against L-M cells and induces hemorrhagic necrosis of MetA sarcoma transplanted in BaL B / s mice. The human full name is a polypeptide having the following amino acid sequence:
    (L
    O
    CO
    Ser Ser Ser-Arg Thr Pro Ser Asp LysPro Val Ala His Val Val
    Ala Asn Pro Ain Glu Gly Gin LeuGin Trp Lou Asn Arg Arg
    Ala Asn Ala Leu Leu Ala Asn Gly ValGlu Leu Arg Asp Asn Gin
    Leu val val pro ser glu glü leu tyr leu tyr ser gin val
    Lnu Phe Lyo Gly Gin Gly Cyfi Pro Ser Thr Hi in Vnl Lr.u Lou Thr
    S
    His Thr lie Ser Arg lie Ala Val Ser Tyr Gin Thr Lys Val Asri
    LysSer Pro Cys GinArg GluThrPro Glu
    Gly Ala Glu Ala Lys ProTrp Tyr Glu Prolie TyrLeuGly Gly
    Val Phe Gin Leu Glu LysGly Asp Arg LeuSer AlaGlulie Asn
    Arg Pro Asp Tyr Le A AspPhe Ala Glu SerGly GinValTyr Phe Gly lie He Ala Leu.
    Human TNF containing specified plasmid encoding TNF
    a new amino acid sequence, with the following nucleotide sequence, is obtained as a result of cultivation:
    virovi strains .i, transfer-i
    TCA TST TST CGA ACC CCG AGT GAG AAG CCT CTA GCC CAT GTT CTA GCA AAC CST CAA GCT GAG GAG CAG CTC CAG CG TGQ AAG AAC CGC CGG GCC AAT GCC GTC CTG ASC GAT AGC GGC. AAC CAG CTG GTG GTG CCA TCA GAG GGC CTG TAG CTC ATC TAG TCC CAG GTC CTC TTC AAG GGC CAA GGC TGC CCC TCC ACC CAT GTG CTC CAC ACC PBX AGC CGC ATC GCC GTC TCC TAG CAG ACC AAG GTC AAC CTC CTC TCT GCC ACC AAG AGC CCC TGC CAG AGG GAG ACC CCA GAG GGG GCT GAG AAG CCC ATP CGTG TAG GAG CCC ATC TAG CTG GGA GGG CTC TTC CAG CTG GAG AAG CGT GAG CGA CTC AGC GCT GAG ATC AAT CGG CCC GAC TAT CAG TTT GATG GAT TAG CAG CTT GATG GTG GTC CAG GTC TAG TTT GGG PBX ATT GCC CTG,
    Human TNF and the indicated DNA-infecting phage containing rDNA,
    get the following way0 and get the specified genomic set
    Use the genomic set (bibliot or man
    current) of bacteriophage propilin and the TNF cDNA genome of the rabbit met. m t P-labeled
    ny set of human bacteriophage Tissue insert
    rabbit or human, for example, tissueEach of the genomic sets of the rabbit-pancreas gland, orriophage / rabbit and bacteriophage / human
    human, crushed and processed, placed on plates for actual
    to digest DNA and protein masses of bacteria and screen
    during the deposition of high-DOD hybridization with P-labeled cDNA
    rabbit sokomolecular DNA or che-TNF rabbit.,
    Lovekao High molecular DNA of the hour-From suitable clones are isolated sotically digested with the corresponding DNA by endonucleases, the resulting DNA fragments are determined by fractionation-friction maps and analyzed by size, obtaining the Southern-hybridization fragment. Restrictions from 15 to 20 k "Ps, t", and clone, ration fragments containing genes
    using the vector Charon AL fgas of the Vek-fzo rabbit or human, subcliators are packed in vitro into particles into vectors and then sequenced.,
    516
    The main rabbit cDNA sequence is compared with the rabbit's full name gene to determine the rabbit exons and introns of the rabbit's full name. The basic human TNF gene sequence is compared to the main rabbit gene sequence to determine the exons and introns of the human FULLo gene. The rabbit's full name, which is calculated from the main rabbit gene follower
    obtained by deletion of the introns of the rabbit TNF gene and a combination of its azenes are aligned with the amino acid sequence of the main cDNA of the rabbit TNF. The human amino acid sequence is then determined from the main DNA sequence coding for human TNF obtained by deleting the introns of the gene encoding human TNF and combining it exon Encode human TNF into an appropriate expression vector to form recombinant DNA containing Recombinant DNA is used to transform the corresponding host cell, which allows the desired human FN00 to grow in culture and express the resulting human TNF has 155 amino acid residues in its
    mature, starting with serine
    An aqueous solution containing crude human TNF is subjected to column chromatography using a column filled with an agar gel crosslinked with a dye. Before carrying out column chromatography, an aqueous solution containing crude human TNF is partially purified using one or a combination of traditional biochemical techniques to obtain an aqueous solution. containing partially purified human TNF as a suitable biochemical methods for the partial purification of human TNF using comfort desalting procedure is used in which ammonium sulfate, ion-exchange chromatography to remove protein contaminants, in which is used an anion exchange resin such as DEAE-Sepharose, aqueous processing solution Polymin P to remove proteinaceous contaminants, methods of gel filtration, electrophoresis and
     The solution containing the physiologically active substance is subjected to column chromatography using a column filled with cross-linked agarose heparum dye. The aqueous solution containing the physiologically active substance includes as mentioned aqueous solutions containing unpurified human TNF.
    and aqueous solutions containing partially purified human full name
    A crosslinked agarose gel bound with a dye is prepared by co-valent binding of a dye as a lignin with a crosslinked agarose gel. Any crosslinked agarose gel can be used as a crosslinked agarose gel, for example Sepharose, - B
    Zibacron blue F3GA, procyon red NEZV, Green A0 are used as the dye.
    Crosslinked agarose gels bound to a dye are commercially available.
    5 available ,, Blue Safarosa CL-bV, Affigel Blue, Matrex Gel Blue A and t0n are used as Fiba Cyan F3GA associated with crosslinked agarose gel.
    0 on red NEZV bound to the agarose gel / nourished gel, use Matrex Gel Red A and t0p0 Matrex Gel is used as Green A bound to a cross-linked agar gel.
    5 Green A0
    The dye-linked cross-linked agarose gel indicated is filled into a column and used for column chromatography. Column chromatography of Phium is carried out as follows.
    A column filled with a dye-coupled agarose gel is equilibrated with buffer containing 0.1 M NaCl (ph 5.5-6.5) 0 As
    5 buffers use phosphate buffer and so forth. An aqueous solution of the physiologically active substance is balanced in such a way that the column is applied. Preferably, the pH of the aqueous solution of the raw physiologically active substance is set to 5.5-6.5. Then the column is washed with the same buffer. After this, elution is carried out using a buffer having a higher concentration of sodium chloride as eluent than in the buffer used to balance the column, for example, buffer containing 0.5 M or more of NaCl.
    Lining is also carried out using a buffer having a pH of 8.0 or higher as eluent. Thus, a fraction containing a human full name is obtained.
    According to the proposed method, a human full name is obtained in pure form with a high yield “
    The dye-coupled agarose gel used in the invention is resistant to an aqueous alkaline solution and to heat and, therefore, can be subjected to treatment with an aqueous alkaline solution in order to easily remove pyrogens that are undesirable impurities for pharmaceutical use. and can be autoclaved sterilized
    Thus purified, the full name has antitumor activity, while it does not have a toxic effect on normal cells. In the in vivo experiment, Met A sarcoma transplanted to mice is used, with 300 units of purified human name given to the mouse, which is obtained according to the invention, its activity evaluated as (+) o Also, significant growth inhibitory or regression of cancer is observed after the administration of purified human full name compared to the control - a mouse that was physiologically injected This solution, in mice that have been transplanted, has Kolen carcinoma 260. The cytotoxic activity against various cancer cells of purified human name is assessed as in an in vitro experiment, with the exception that PC-8 asenocarcinoma cells (lung cancer) and normal cells (kidney cells of a newborn person), and cells of the foreskin of a newborn person as a control instead of LM cells, the cells are incubated at 37 ° C for 72 hours instead of 37 ° C for 48 hours
    The received human full name possesses an excellent anti-tumor ak
    Tivnostyo Therefore, the received human full name is particularly useful in the clinical application of a human full name as an anticancer drug.
    0
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    An analytical method is used to evaluate the antitumor activity of human TNF.
    Method for in vivo assessment of TNF activity. Met A sarcoma cells (2 10 cells) subcutaneously with each BAL mouse / s mouse are transplanted. After 7 days, mice with tumors with a diameter of 7-8 mm without hemorrhagic necrosis and with good vascularization are taken for evaluation0 A full name (0.5 ml), diluted with saline, is injected through the tail vein of each mouse. Sample activity is assessed after 24 hours according to the following criteria: (-) there are no changes; (+) weak hemorrhagic necrosis; (+): moderate hemorrhagic necrosis (central necrosis extending to about 50% of the surface of a transplanted tumor); (+): significant hemorrhagic necrosis (massive decrosis in the center of the transplanted tumor, constituting a small viable rim along the periphery of the tumor) 0
    In vitro evaluation method (analytical method using L-M cells)
    A sample (0.1 ml) of a human name, serially diluted with medium, and a suspension of LM cells (0.1 ml, 1.105 cells / ml) were added to each well of the G-chamber plate of a micro titrator. containing 10% volume / volume of newborn calf serum. The plates are incubated at 37 ° C for 48 hours in an atmosphere containing 5% carbon dioxide. At the end of the culture period, 20 µl of a 20% aqueous solution of glutaraldehyde is added to fix the cells. After fixation, the plates are washed and allowed to dry and added 0.1 ml of a 0.05% solution of methylene blue for staining viable cells. Plates are washed thoroughly to remove excess methylene blue and allowed to dry. Then 3% hydrochloric acid is added to each well in order to extract methylene blue from the stained cells. The absorbance of each cell at 665 nmo is measured. Absorption is proportional to the number of viable cells. Dilution of a human full name, which corresponds to an absorption of a substance equal to 50% of the absorption of the control group, to which a sample of a human full name was not added, is obtained graphically or by calculating, Dilution is defined as one unit ml0
    Example 1 About Rabbit females, weighing 2.0-3.0 kg, are injected with 50 mg (killed by formalin Propionibacterium iacnes (Corynebacterium parvum)); venous ear After 8 days, again injected through the ear vein with 100 µg of endotoxin. (Lipopolysaccharide from Escheripia coli 026: 36), and after 2 hours, whole blood is collected from the heart. To the collected blood, sodium heparinate is added in an amount of 100 units per 100 ml. Then the blood is centrifuged while cooling at 5000 rpm for 30 minutes to remove blood cells and insoluble solids. products0 The result is plasma (2.4 L), which has cytotoxic a ciency serum Name U / ml, from about .40 rabbits
    24 g of cellite was added to 2.4 liters of plasma. The resulting mixture was stirred for 1 hour, and then filtered. The filtrate was mixed with 1.2 liters of 0.04 M trans-HCl buffer (pH 7.8) and then applied to the column. with DEAE-Sepharose CL-6B, equilibrated with 0.04 M trans HC1-buffer (pH 7.8). The column was washed with 0.04 M Tris-HCl buffer containing 0.1 M. NaaCl, and the adsorbed FIO was eluted using 0.04 M Tris-HCl buffer (pH 7.2) containing 0.1 8 M NaCl. Fractions possessing cytotoxic activity against L-cells. Are concentrated by ultrafiltration. The concentrate thus obtained is applied to a Sephacryl S-200 column, equilibrated with 5 mM phosphate buffer, and the gel is filtered 9 using the same buffer. The active fractions are concentrated by ultrafiltration, resulting in a purified full name, having an activity of 3.5 HO6 units. and specific activity of 19 x X10 e units / mg0
    Partially purified full name from rabbit serum is mixed with Freund's complete adjuvant (1: 1), and then subcutaneously, 1 but injected into the back for 12 weeks. male BaL b / c0 mice Repeat. Sanny operation after 2 and 4 weeks after the first injection. One week after the last injection, whole blood is collected. Serum is obtained from the collected blood.
    0
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    The serum thus obtained is added to the culture medium to evaluate the cytotoxic activity of the full name against L-cells in such an amount that it is diluted 500 times in final concentration. The cytotoxic activity of the full name of the rabbit serum is not assessed. The full name of the rabbit serum is not cytotoxic activity against L-cell From the above result, it is concluded that the mouse serum obtained at this stage contains the antibody and the full name of the rabbit serum
    Formalin-killed cells of Propionibacterium acnes (Corynebacterium parvum) were injected intravenously into female rabbits. Seven days later, the rabbit was subjected to troteotomy and washed with a physiological saline solution, resulting in a floating cell0 % volume / / volume of the serum of the newborn calf, the cells are incubated at 37 ° C in an atmosphere containing 5% carbon dioxide. The cell culture is divided into 3 groups and one of them is added dissolved endotoxin, derived from Escherichia coli (lipopolysaccharide from Escherichia coli 026: B6) at a concentration of 10 ug / ml. The same amount of sterile water is added to the other. The supernatant of the cell culture, to which endotoxin is added, exhibits cytotoxic activity against L-cells, and the activity reaches a maximum value in 7 hours. Such activity is suppressed by anti-TNF antibody, but not suppressed. normal mouse serum
    On the other hand, the supernatant of the cell culture, to which no endotoxin was added, does not show cytotoxic activity against L-cells.
    The cell culture, to which endotoxin is added, is then added with radioactive L- (S) methionine (13000 Ci / mmol) (1 mCi / ml). 0 The supernatant layer is analyzed by SDS-polyacrylamide gel electrophoresis. The concentration of the gel is set to 12.5 wt.% . After electrophoresis, the gel is treated with ESHA-MSE and, after staining, exposed to X-ray film B, the supernatant layer of the cell culture in the presence of endoxin is observed to form a substance having about 175,000 milk.
    Next, the supernatant layer of each obtained cell culture was subjected to electrophoresis on SDS-polyacryl. The resulting RNA solution was heated at 68 ° C for 2 minutes, then quickly cooled. To the solution was added 500 µl twice concentration of 10 mM Tris-EDTA buffer (pH 7.4), containing 1 mM EDTA, 0.1 wt.% / volume SDS and 0.5 M lithium chloride), the mixture is applied to 200 mg of oligo-T-cellulose,
    amide gel. After that, the gel was built in - 1Q in a column of washes with 10 ml of the same sachet for 1 hour in 2.5% NP40V buffer (single concentration)
    and then in water for 2 hours. After shaking, each migration line is cut by cutting and cut into 2 mm wide sections in a direction perpendicular to the direction of migration. Each strip is cultured with cells and the cytotoxic activity against L-cells is evaluated. In the strip,
    on which the supernatant was cultured 2Q and applied to a column with oligo-T-celler cells containing endotoxin, pro LULOSE, fractions were collected, adsorbing cytotoxic activity against L-cells in the position corresponding to MolO 175000 V no cytotoxic state is observed in other positions. 25
    NOSTIO
    The cell culture is incubated for 2 hours after the addition of endotoxin,
    then centrifuged to collect sucrose
    current Extraction of the cytoplasm-- n -rn / -. "cin
      ., „„ „„ „ISCO 570 gadienter, using tris-buffer solutions (25 mM Tris-HCl content, pH 7.2, 2 mM ETA and 1 wt.% / / Volume of SDS), containing respectively 5 and 25% sucrose
    Ultracentrifugation was performed using a Beckman Sn41 rotor at 40,000 rpm for 12 hours at 4 ° C, fractions were collected every 400 µl with
    , Using a device for collecting fractions, 2.5 ml of 5.7 M hydrochloride cells was placed in advance, and then this “OLOMO Osago cesium and O, 1 ml of ELTA solution was precipitated, and the fractions were centrifuged and the column was eluted with 2 ml of elution buffer containing 10 ml mM Tris-HCl buffer (pH 7.4), 1 mM EDTA 15 and O, 1 wt% / volume SDS. 0.05 volumes of sodium acetate solution and 2.5 volumes of ethanol are added to the eluate, the mixture is cooled to -20 ° C to precipitate. The precipitates are collected by centrifugation.
    oligo-c T cellulose. 85 μg of mRNA are extracted, as determined by UV spectral analysis
    880 µg of mRNA is dissolved in 250 µl of water and the resulting solution is poured into 10 ml of a 5–25% linear sucrose gradient of density, плот Gradient of ratchet RNA from harvested cells and extraction of mRNA from cytoplasmic RNA, 4 ml of 4 M solution is added 3–10 cells, the mixture is sprayed with a homogenizer, the residue is removed by centrifugation and 2.4 g of cesium chloride are dissolved. The mixture is carefully poured into a full-length tube, in which 35
    (pH 7.5) and then centrifuged at 30,000 rpm for 12 hours at 20 ° C using a Neman rotor.
    After removing the supernatant, the pellet is dissolved in 1 ml of 10 mM Tris-HCl buffer containing 5 mM EDTA and 1 wt% v CDS0. The resulting solution is extracted 4: 1 by volume with a mixture of chloroform and 1-butanol. 0.05 is added to the aqueous phase. a volume of 2 M sodium acetate and 2.5 volumes of ethanol, settled at -20 ° C for 2 hours or more, as a result of which RNA precipitates. The precipitate is collected by centrifugation, dried, and then dissolved in 500 µl of sterile water. As a result, cytoplasmic RNA0 is obtained.
    shutter in sterile water
    The mRNA is translated using,. In Xenopu.s laeves societies, fractionated mRNA is dissolved in sterile water to a concentration of 1 µg / µl, the solution is injected into the societies in as small as 500 ml per cell. The cells are then cultured for 24 hours in Bart's solution (containing 7, 5 mM Tris-HCl pH 7.6, 88 mM NaCl, 1 mM potassium chloride, 0.33 mM calcium nitrate, 0.41 mM calcium chloride, 0.82 mM magnesium sulfate, 2.4 mM sodium bicarbonate, 13 I / penicillin G and 18 µg / ml of streptomycin), which contains 1 mg / ml of serum bovine50
    Ј5
    The resulting RNA solution is heated at 68 ° C for 2 minutes, then rapidly cooled. To the solution was added 500 µl of a two-fold concentration of 10 mM Tris-EDTA buffer (pH 7.4) containing 1 mM EDTA, 0.1% by weight / volume of SDS and 0.5 M lithium chloride), the mixture is applied to 200 mg of oligo-T-cellulose
    in the column washes 10 ml of the same buffer (single concentration)
    and applied to a column with oligo-T-cellulose, fractions collected, adsorbed
    The column material was eluted with 2 ml of elution buffer containing 10 mM Tris-HCl buffer (pH 7.4), 1 mM EDTA and O, 1 wt% / vol SDS. 0.05 volumes of sodium acetate solution and 2.5 volumes of ethanol are added to the eluate, the mixture is cooled to -20 ° C to precipitate. The precipitates are collected by centrifugation.
    and applied to a column with oligo-T-cellulose, fractions collected, adsorbed
    oligo-c T cellulose. 85 μg of mRNA are extracted, as determined by UV spectral analysis
    Dissolve 880 µg of mRNA in 250 µl of water and pour the resulting solution into 10 ml of a 5–25% linear sucrose gradient in density, Гра Gradient to dissolve in sterile water.
    The mRNA is translated using,. In Xenopu.s laeves societies, fractionated mRNA is dissolved in sterile water to a concentration of 1 µg / µl, the solution is injected into the societies in as small as 500 ml per cell. The cells are then cultured for 24 hours in Bart's solution (containing 7, 5 mM Tris-HCl pH 7.6, 88 mM NaCl, 1 mM potassium chloride, 0.33 mM calcium nitrate, 0.41 mM calcium chloride, 0.82 mM magnesium sulfate, 2.4 mM sodium bicarbonate, 13 I / ml penicillin G and 18 μg / ml streptomycin), which contains 1 mg / ml serum bovine0
    five
    go albumin. Oscites are placed in a liquid culture with a glass rod, the liquid culture is then centrifuged and the supernatant layer is evaluated for cytotoxic activity against L-. mRNA cells that need to be translated to produce a polypeptide having maximum activity will sediment as 16S in size. This activity is removed by anti-TNF antibody, but not removed by normal mouse serum.
    Using 5 μg fractionated
    mRNA, preparing double stranded DNA. Double-stranded DNA is fractionated on a 3.5% polyacrylamide gel, 330 ng are obtained, from about 100 to 200 base pairs, 7 ng of this fraction are increased by deoxy-residues using terminal deoxyluylotyl diltransferase and annealed 56 ng of plasmid pBR 322, which was digested with Pst 1 and increased by deoxy-G-residue
    The resulting mixture was inserted K-12 (strain HB 101, ATCC, 33964) to transform the strain. The result is 12,000 transformants.
    Rabbit TNF (activity: 510 units are subjected to electrophoresis on a SDS polyacrylamide gel “A part of the gel is stained with Kumasi Brillant blue about a band in a position corresponding to 17500, cut out of the gel and extracted with 1% ammonium bicarbonate o Remove about 180mg of TNF as a protein
    150 µg of the extracted TNF is dissolved in 75 µl of 1% ammonium bicarbonate, then 3 µg of TRISC trypsin is added. The mixture is incubated at 37 ° C for 4 h. The mixture is then fractionated by high performance liquid chromatography on a column filled with Cosmosil 503 as packed of the material to result in trypsino fragments
    High-purity TNF and its trypsin-digested fragments: then desalted onto a Seph-dex G-25 column and dried with freezing. Purified TNF and trypsin-digested fragments each are decomposed by Edman from the N-terminal of PTH-released at each stage of the PTH amino acid
    ,,
    uh
    ,, t - пJQ th
    15
    20
    0 to - 25
    i jq
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    . , a- 0
    40
    50
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    by high performance liquid chromatography, the model SP 8100 detects that TNF has the following N-terminal amino acid–. sequence: Ser-Ala-Ser-Arg-Ala-Leu-j-Ser-Asp-Lys-Pro-Leu-Ala-Hes-Val-Val-Ala-Ala-Asn-Pro-Glu-Val- - Glu-Gly-Gln-Leu-Glu.
    One of the trypsin-digested fragments has the following N-terminal amino acid sequence: Glu-Thr-Pro Glu Glu Ala Glu Pro Met Ala0
    Oligodeoxynucleotides, complementary to the main mRNA sequence, are synthesized according to the improved phospho-triester method. When obtaining oligodeoxynucleotides 128, oligodeoxynucleotides determined from the amino acid sequence of rabbit TNF, are classified into five groups, namely groups 16,16,32,32 and 32, and synthesized as mixtures of oligodeoxynucleotides of the corresponding groups Protect the obtained oligodeoxynucleotides of the corresponding groups according to the traditional method and purify the column chromatography using Sephadex G-50 and electrophoresis on a 20% polyacrylamide gel containing 7 M urea „
    The oligodeoxynucleotides of the corresponding groups thus obtained are dialyzed against 0.1 mM Tris-EDTA buffer solution,
    Each of the purified oligodeoxynucleotides of the respective groups is labeled using Tl polynucleotides and AND gP adenosine triphosphate, and then purified by column chromatography. The radioactive material is included in each of the nucleotides of the respective groups in an amount of about 3 x ppm / µo Oligodeoxynucleotide probes, each received in the form of a mixture of the corresponding group, indicated as shown in Table 1
     i
    Part of the amino acid sequence of rabbit TNF, the main sequence of mRNA, determined from the amino acid sequence of rabbit TNF, and the main sequences of the synthetic oligonucleotide probes of the respective groups are given in Table.
    The mRNA of the cells that produce the full name is treated with a solution containing 1 M glyoxal, 10 mM and 50 vol% dimethyl sulfoxide at 50 ° C for 60 min, and then subjected to fractionation using electrophoresis on a 1.1% agarose gel0 Fractionated The mRNA is transferred to an electrophoretic transfer device filter. The mRNA on the device filter is then treated with 5 x Denhardt with a solution containing 5xSSC solution and 150 μg / ml of denatured salmon spermatozoa at 65 ° C for 2 h, and then treated with 5 X Dencardt with a solution containing 1.10 cpm / ml , labeled, oligodeoxynucleotides and 5 x SSC solution, at 50 ° C for 2 h0. The filter cake is washed with 6 X SSC solution four times at room temperature, at 40, 50 and 60 Co. Irradiated filters are exposed to x-ray capturing
    ku xar-5. As a result, oligodeoxynucleotides, designated as probe MJ, are found to more strongly hybridize with mRNA.
    Transformants are transferred onto a cellulose filter and hybridize DNA with labeled oligodeoxynucleotide (probe MJ) under the same conditions as in Example 12 "49 colonies are selected, which strongly hybridize with labeled oligodeoxynucleotides (probe III), and then fixed on another nitrocellulose filter ,, Then, using 49 colonies, further hybridization is carried out to select nine colonies that more strongly hybridize with labeled oligodeoxynucleotides (probe MJ) 0
    Approximately 5 µg of plasmid is obtained from each of the nine colonies. Each of the obtained plasmids is digested using Pst I, Tag I, Rsa I and Pvu II restriction enzymes, then electrophoresis is performed on a 1% agarose gel and the fragments obtained when cleaved with appropriate restriction enzymes, along their length,
    The results confirm that all nine strains of the corresponding ninth colonies contain a fragment obtained by cleaving Pvu II and Rsa I, 50 np, and that most
    g you 15 20
    25
    30 35 40
    45
    50
    55
    of the nine strains have a fragment obtained by splitting Rsal, of size 200 Poo oo. In other words, the results confirm that the nine strains have partially common main sequences
    Seven strains containing plasmids listed in Table 02 are cultured separately in 2 ml of LB medium containing 10 µg / ml of tetracycline until the optical density of the solutions reaches a given value, then centrifuged to obtain the corresponding strain,
    Each of the obtained strains was separately added to 2 ml of physiological solution and disrupted by sonication. The resulting solutions were centrifuged and the cytotoxic activity against the L-cells of the obtained supernatants was determined. As a control test, the same procedure was repeated using strains containing the plasmid pBJA 3220
    Suppress cytotoxic activity against L-cells with anti-TNF antibody.
    E0coli strains containing plasmids pRR2-7 and pBR 18 are cultured in 1 l of M9 medium containing 10 μg / ml tetracycline a „
    The basic sequence of insertion of each plasmid is determined according to the Maxam-Diilberth chemical procedure. The basic sequence thus determined is in agreement with the partial amino acid sequences defined in Example 90.
    At this stage, the plasmid is constructed using the recombinant plasmid pR 12 to obtain direct expression of the full name in Eocoli, using 1a as a promoter. First, 10 µg of the plasmid pR 12 Yued.Ap is digested at 37 ° C for 2 h, then the wire t electrophoresis on 4% polyacrylamide gel to isolate fragments 630 p „o0 Isolate about 1 μg of gel from the gel by electroelution; two oligodeoxy nucleotide are synthesized: GATCCATGTCAGCTTCTCGGGCC-3 and 5 -CGAGAAGCTGACATG-3; - synucleosides (about 100 mmol), using PT polynucleotide kinase. After completion of the reaction, the reaction mixture is extracted with phenol, and then
    17
    chloroform. Then, the resulting synthetic oligomers are mixed with 0.5 μg of Ara with the fragment of LZO p, o, and precipitated with ethanol. The fragment is ligated with synthetic oligomers at 4 ° C using 10 units of Td DNA ligaseOI. After completion of the reaction, the reaction mixture is precipitated with etheiol and stirred with 20 units of BamI at 3 / ° C for 3 hours, then electrophoresis is performed on 4% polyacrylamide gel for electroelution extraction of the 670p0o fragment I digest μg of the pUC-8 plasmid with VagoH and extracted with pheno163
    )
    scrap, and then chloroform, then precipitated with ethanol to obtain a vector, bind 0.5 µg of the obtained vector with the obtained fragment having
    BamHI plot at both ends, containing 20 pR 17 and precipitated with ethanol0 Frag25
    transient Eocoli using the resulting vector and cultured on agar medium containing 1 mM IITG and 0.004 wt0% / volume X-gal to obtain about 200 white colonies. DNA plasmid from 100 such transformants and digested with BamHI. As a result, 15 plasmids were found to contain the indicated BamHI fragment (about 670 Poo „). In order to investigate the direction of insertion, more than 15 EcoRI plasmids with only one recognizable region are digested. on its pUC-8, and 11 plasmids having Only one recognizable region in its fragment region (approximately 670 Poo0), electrophoresis is carried out on a 6% polyacrylamide gel. As a result, 7 plasmids have the indicated fragment consisting of approximately 140 p00, and that the transcription direction of the lac promoter on pUC-8 is in accordance with the clock for oligodeoxynucleotides encoding FIO
    DNA sequence analysis shows that these seven plasmids have
    35
    40
    45
    The mentality is ligated with synthetic oligomers at 4 ° C overnight, using 10 eDOTs. After completion of the reaction, the reaction mixture is precipitated with ethanol and 20 eDoEr-ORI are digested at 7 ° C for 3 hours. Then electrophoresis is carried out on 4% polyacryl amide gel to extract a fragment (about 670 p0, oo) using electric elution
    The plasmid pOP9b-1b is obtained.
    1 μg of pOP95-15 is digested with EcoRI and extracted with phenol and then chloroform, then precipitated with ethanol to obtain a vector,
    Using the hcdnc ligase, bind 0.5 µg of the vector with the fragment (about 670 bp) obtained by binding the synthetic oligonucleotide to the oligonucleotide encoding the TNF „trans form E, coliJM101 (ATCC 33876), using the resulting vector, and cultured on medium containing 1 mM IPTG and 0.004 May0% / volume X-gal to obtain approximately 150 white colonies o
    A plasmid of DNA was prepared from 100 of these colonies and digested with EcoRI. As a result, 12 plasmids were found to contain the specified EcoRI fragment (about 670 PoO0). To check the insertion direction, more than 12 plasmids of PVUH nPstl were digested and electrophoresis was carried out for 1, 5% agarose gel0 As a result, it was determined that four plasmids have the desired fragments (about 1280 p „o„ and about 2600 Poo „) and that the direction of transcription of the lac UV5 promoter
    the same sequence and have the desired nucleotide sequence when compared to the Lac promoter of synthetic DNA and cDNA0
    Construction of other plasmids was carried out using the pR 17 plasmid to obtain direct expression of TNFα in EoColi using lacUV-5 as a promoter. First, 10 µg of the pR 1710 units of plasmid was digested.
    g
    63060218
    Ara at 57 ° C for 2 hours, electrophoresis was performed on a 4% polyacrylamide gel to isolate a fragment consisting of about 630 p00. About 1 µg of the fragment was electrically electrolyzed. In the same way as in stage 10, two oligodeoxucleotides are synthesized: L -ATCATGTCAGCTTCTCGGGCC-3 and 5g-CCAASSTASATS-3. Then, each L1 -end of both oligodeoxynucleotides (about 100 pmoles) is phosphorylated using the TC of polynucleotide kinase
    After completion of the reaction, the reaction mixture is extracted with phenol and then chloroform. Then the synthetic oligomers are mixed with 0, мк μg of the previously prepared Ara fragment (about 630 PoOa) obtained from the plasmid
    ten
    25
    zo
    35
    40
    45
    0
    The mentality is ligated with synthetic oligomers at 4 ° C overnight, using 10 eDOTs. After completion of the reaction, the reaction mixture is precipitated with ethanol and 20 eDoEr-ORI are digested at 7 ° C for 3 hours. Then electrophoresis is performed on a 4% polyacrylamide gel to extract a fragment (about 670 p0, oo) using electric elution
    The plasmid pOP9b-1b is obtained.
    1 μg of pOP95-15 is digested with EcoRI and extracted with phenol and then chloroform, then precipitated with ethanol to obtain a vector,
    Using the hcdnc ligase, bind 0.5 µg of the vector with the fragment (about 670 bp) obtained by binding the synthetic oligonucleotide to the oligonucleotide encoding the TNF „Transform E, coliJM101 (ATCC 33876), using the resulting vector, and cultured on medium containing 1 mM IPTG and 0.004 May0% / volume X-gal to obtain approximately 150 white colonies o
    A plasmid of DNA was prepared from 100 of these colonies and digested with EcoRI. As a result, 12 plasmids were found to contain the specified EcoRI fragment (about 670 PoO0). To check the insertion direction, more than 12 plasmids of PVUH nPstl were digested and electrophoresis was carried out for 1, 5% agarose gel0 As a result, it was determined that four plasmids have the desired fragments (about 1280 p „o„ and about 2600 Poo „) and that the direction of transcription of the lac UV5 promoter
    consistent with that of oligodeoxynucleotides encoding FIO
    The analysis of the main sequence showed that these four plasmids have the same sequence as the lac UV5 promoter, the synthetic oligodeoxynucleotide and cDNA are properly combined with each other. The resulting plasmids are called pTNF - lac UV.b-Io
    The E0coli strains containing the plasmids obtained in Example 16 are cultured in 50 ml of LB medium containing ampicillin overnight. The strains are then transferred to 5 liters of LB medium containing 100 µg / ml ampicillin and continue to be cultured at 37 ° C in for 3 h0 Isopropyl-D-thiogalactopyrano is added to the number of mice that have completely disposed of.
     2T 2SZ525S
    Dose removal used in the test
    The results are shown in the tabLoZ Example 2 of the Colony EoColi K-12 strain MC1061 transform each of pR18, pB 2-7, pH 2-2 plasmids. In particular, colonies E, coli K12 strain MC1061 are cultured in LB medium & until the optical density of the culture broth reaches 0.3 at 550 mm. Collect 50 ml of the grown E0 coli culture, wash with 2 Ь ml of a mixture containing 10 mM MOPS (pH 7.0) and 10 mM KCl1 and again suspended in a mixture containing 0.1 M MOPS (pH 6.5), 50 msMaC and 10 mMKVS. The resulting suspension is cooled on ice for 30 minutes, centrifuged and suspended in 2 ml of a mixture containing 0.1 M MOPS (pH 6.5), 50 mM CaClЈ and 10 mM YavCl and 30 μl DMS00 K 200 μl (aliquot The resulting suspension was separately added with 10 µl of the DNA solution of each plasmid. Each of the mixtures was cooled on ice for 3 D and then heated for 60 s at
    44 C Immediately after this, 5 ml of LB medium preheated to 37 ° C is added to each of the heated mixtures, then incubated at 37 C for 1 hour. The resulting culture broth is centrifuged to form balls of cells. The supernatant is discarded, LB- is added. medium and stirred to re-suspend each of the cell beads.
    zid to a final concentration of 1 mM „Continue cultivation for another 6 h, then cool. Then, the strains are collected by centrifugation. Strains are introduced into 5 liters of 0.04 m Tris HC1-buffer solution (pH 7.8) and destroyed by sonication in order to obtain a solution of the strain of strain of the strain obtained. Thief has a cytotoxic activity against L-cells 5- U7 unit / l0 The solution is purified to obtain 1.2 - 10 ° unit / VDO. The specific activity of the full name is 6, unit 0 / mg0.
    Sample (0.2 ml) name is evaluated in the analysis of in vivo
    Twenty days after the injection of the sample, the development of the tumor is monitored and the extraction dose is determined according to the following equation:

    Each of the suspensions obtained is inoculated onto a plate with LB agar containing 30 μg / ml of tetracycline, then incubated overnight at 37 ° Co. The result is a colony of tetracycline-resistant transformants, each of which is transformed

    miro plasmid pB2-20
    pR 18, pB 2-7 or
     , ,
    0
    five
    Each of the transformants transformed with plasmids pB 2-7 or pR 18 is grown and amplification of the plasmid is collected, the transformant is in the lead and the plasmid DNA is dried0 Illustratively, it is established that each of the transformants is inoculated into LB medium containing 30 μg / ml tetracycline and incubated at 37 ° C with vigorous shaking. This step is repeated to achieve growth of the transformant and amplification of the plasmid. The cultured transformant is harvested by centrifugation at 4000g for 10 minutes at 4 ° C. The supernatant is discarded. The resulting bead is washed in 100 ml of ice-cold GTE (0.1 M NaCl, 10 mM Tris-HCl pH 7.8 and 1 mM EDTA), lysis is carried out at boiling in a solution of 20 mg / ml lysozyme in 10 mM Tris -HCl PH 8.0. The viscous product is transferred to an ultracentrifuge tube and centrifuged at
    21
    25,000 rpm for 30 minutes at 4 ° C to obtain a DNA solution. Measure the volume of the DNA solution. For each 1 ml, exactly 1 g of solid cesium chloride is added and thoroughly mixed until all the salt has dissolved. 0.8 ml of ethidium bromide solution (10 mg / ml) is added for every 10 ml of cesium chloride solution. The final solution density is 1.55 g / ml, and the ethidium bromide concentration is approximately 600 µg / mlLo Solution cesium chloride is transferred to a tube suitable for centrifugation, and the rest of the tube is filled with a light steam with an Athens oil. Centrifugation is carried out at 45,000 rpm for 36 hours at 20 ° C to obtain two DNA bands (the upper band consists of linear bacterial DNA and a nicker circulating plasmid DNA, and the lower band consists of closed circular plasmid DNA ) “The lower DNA band is collected in a glass tube through a hypodermic needle inserted into the side wall of the tube. Ethidium bromide is removed and the aqueous phase is dialyzed against TE buffer. The plasmid DNA solution is treated with PBK-ase and extracted with an equal volume of balanced phenol. The aqueous phase is layered on a Bio-Gel A-150 column with a balanced TAE (pH 8.0) and 0.1% SDS, the DNA in the column is washed and used to collect fractions of TE with 0.1% SDS Fractions precipitate this




    nol to get pure plasmid DNA about
    When performing these procedures, get 250 μg of pure DNA plasmids rV 2-7 and 134 μg of pure DNA plasmids pR 180
    Plasmid pB 2-7 plasmid 40 µg was taken, digested with a P3Hretraction enzyme and subjected to electrophoresis on a 4% acrylamide gel, After DNA electrophoresis, stained and cut out the desired division line „
    Using 500 mg of isolated PstI insert, nick translation is carried out. For the nick translation, 80 pmol of radioactive dOTP and 25 µl of the reaction system (at 400 Ci / mmol) are added to a mixture consisting of 2.5 µl of solution A (dHTP solution), 2.5 μl of solution B (500 ng of the test DNA, namely PstI inserts),
    22
    Q 5 0 5 0,.
    0
    five
    0
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    1.3 µl of cold dCTP (65 pmol, 50 pmol / µl dCTP) and 11.2 µl of solution E (H20) were added 2.5 ml of solution L (DNAase 1, DNA polymerase I) and the reaction was carried out within 60 min. Then a solution of D (stop buffer) in the reaction mixture is added to stop the reaction, then the tRNA carrier is added, precipitated twice with ethanol and dissolved in 500 µl of water. The specific activity per 1 µg of DNA is 9.3 cpm.
    80 µg of plasmid pR 18 DNA is digested with RSal restriction enzyme and electrophoresis is performed on a 4% polyacrylamide gel. The following target insertion bands are cut and purified using a column: about 640 Po00 - 3.77 µg (52% recovery); about 175 PoO, - 1.77 µg (50% recovery) o
    Insert above about 640 p0o. denoted by a 3-fragment pR (meaning the 3-untranslated pR 18 region), and the insertion is approximately 175 p0Oo denoted pR 18-s (denotes the pR 18 coding region) o
    In addition, the above procedures are repeated using PstI and Ms til restriction enzymes instead of the restriction enzyme Psal to obtain the following band: about 450 p0. 3.65 µg (60% recovery) “The insert shown is referred to as 5-fragment pR 180
    57
    P-labeled pB 2-7 plasmid insert was used as a hybrid probe to screen 10 plaques of the bacteriophage Charon 4A / human genomic set obtained by inserting a hybridization method into the Charon 4A binding site of EcoRI fragments of digested human DNA. blu
    Since not all bacteriophages in the original culture contain the necessary genetic material for the preparation of human TNF, a probe is used that has a basic sequence that is complementary to the rabbit TNF gene. DNA phage plaques that have the desired genetic material, including a radioactive probe, are identical in their radio activity. From the kit, nine hybridized plaques are isolated
    The procedures and conditions are as follows.
    1 „Number of blinkers (4ilO blinkers on a 150 mm K 25 plate).
    2c Transfer to nitrocellulose filters
    Zo Hybridization: add probe insert 1, ppm / ml pB 2-7,
    42 ° C, 19.5 h. I
    4. Rinsing: 2 x SSC - 0.1% VTS at room temperature, immersion 10 min./4, 1 SSC - 0.1% VTS at 50 ° С, immersion 30 min 2 „
    5 „Exposure: XAR-5, -80 ° С, 39 h.
    In the above screening, 12 candidate strains are obtained. Second screening is carried out. Next, using these strains, a third screening is performed in the same manner as mentioned to obtain nine strains containing the desired fragment. Using the obtained strains, the fourth screening is performed to confirm that nine strains contain the desired fragment. The resulting nine bacteriophages containing the desired fragment are designated HG-1 -HG-9, respectively.
    10 plaques of bacteriophage Charon 4A are used (the genome of the rabbit which is obtained using digested rabbit DNA instead of digested human DNA, 6,740 plaques of bacteriophage Charon 4A (the genome of the rabbit) are used instead of 10 plaques of bacteriophage Charon 4A (genome of humans) in this way , receive two bacteriophage strains (RG-1 and RG-2) containing the genomic set of TNF rabbit gene0
    HG-3, HG-b, HG-7 DNA is obtained from each bacteriophage.
    B110 E0 coli LE 392 cells are suspended in 18 ml of CM and 3,109 pF and bacteriophage are added and infected with EoColi at 37 ° C for 20 min. Then the resulting mixture is added to 3 L of broth and shaken the culture at 37 ° C for 23 60 ml of CHC1 $ are added to the mixture and shaken for another 30 minutes. Sodium chloride is added to the mixture to a final concentration of 1 M, the mixture is left to stand for 15 minutes, then centrifuged to obtain a supernatant. Then polyethylene glycol is added to the mixture (mol0m0 6000 so that the concentration of polyethylene glycol is 10% by weight,%, and left to stand at 4 C for 22 h Bacteriophages
    0
    five
    0
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    „
    collected by centrifugation. The obtained bacteriophages were suspended in 28 ml of CM and an equal volume of CHClja was added. After stirring for 30 s, the mixture was centrifuged to obtain the aqueous phase. The CM was added to the aqueous phase to give a total amount of 30 ml. Add 26.4 g of SLCC to the obtained mixtures and carefully dissolved, then ultracentrifugation is performed (45000 rpm for 20 h) to obtain bacteriophages in the form of a strip. The resulting mixture containing bacteriophages is dialyzed against 10 mM NaCl - 50 mM Tris (pH 8) - 10 mM MgClz Then EDTA is added to the mixture. Proteinase K and SDS, so that their concentrations were respectively 20 mM, 50 μg / ml and 0.5 wt.% / volume. The mixture is then treated at 65 ° C for 1 hour and extracted with phenol, a mixture of phenol and CHC1e (1: 1 by volume), and then the resulting aqueous phase is dialyzed against 10 mM Tris (pH 8) - 1 mM EDTA. Absorption of the aqueous phase in UV -spectrum shows that bacteriophage HG-3 pure DNA was obtained
    Almost the same procedure as described for the production of bacteriophage HG-3 DNA and DNA of bacteriophage HG-6 and HG-7 ° is repeated.
    Sau-blot analysis was performed. The terns of the DNAs obtained. The procedures and conditions are as follows.
    U DNA: HG-3 825 ng each; HG-6 935 ng each; HG-7 685 ng each
    20 Digestion by various restriction enzymes: 10 units0 Vagani- + 10 units “EcoRI; 10 items Vahan + Ued. EcoRI; 10 food Hindlll; 10 units „Hindlll + 10 units„ EcoRI; 10 units of PVU II; 37 ° C, 3 h
    30 Electrophoresis: 0.8% agarose 5 gel, TAE, 28 v, 15.5 h
    0
    4o Transfer to nitrocellulose filters
    5 o Pre-hybridization: 30 ml PDSO, 42 ° C, 6
    6 "Hybridization: 5-fragment (1 -U5 kpm / ml) pG18, 42 ° С, 14 h"
    7 ° Flushing: 2X SSC - 0.1% VTS at room temperature, immersion 10 min M, 1 XSSC - 0.1% VTS at 50 ° C, immersion 30 min x 2.
    8 ° Exposure: XAR-5, -80 ° C, 2 intensified screening, 14 h
    The results of hybridization are given in tabl040
    A Southern blot analysis was performed. As a result, it was found that the 5 fragment of pG 18 hybridizes with one fragment band from the fragments obtained by splitting RG-1 and RG-2 with each Bam HI, EcoRI, Hindlll, BglHI and Waga HI + EcoRI0
    Use the Landy method. 33 microns of HG-3 EcoRI DNA are digested at 37 ° C for 3 hours. The digest is subjected to electrophoresis on a 1% low-melting agarose gel (conditions; 1 TAE, 20 at 14.5 hours) o Strip 2.9 is isolated from the agarose gel 65 ° C for 15 min. Remove from the molten EcoRI gel - split HG-3, having a length of 2.9 p. O (hereinafter referred to as HG EcoRI 2.9 p. O fragment) by extraction with phenol three times, and then extraction with ether, then precipitated with ethanol containing ammonium acetate. “In this way, 637 ng (yield about 30%) of HG 3 EcoRI, 2.9% of the fragment that
    255 ng of the obtained fragment are bound to 56.5 ng of the digested EcoRI, using 2.5 units of Т T ligase at 4 ° C for 20 h
    Transform E0coli K 12 strain JM83 using the obtained binding product o Specifically cultured Eocoli K 12 JM83 strain on LB medium until the optical density of the culture broth reaches 0.3 at 550 nm "Collect 50 ml of the growing culture E0 coli K 12 strain JM83, washed with 25 ml of 10 mM MOPS (pH 7.0) - 10 mM NaCl and resuspended in 25 ml of 0.1 M MOPS (pH 6.5) - 50 mM CaCl - 10 mM RsCIo The suspension is cooled on ice for 30 min, centrifuged and resuspended in a mixture of 2 ml O, 1 M MOPS (pH 6.50) - 50 mM CaC12 - UmM RsCL and 30 µl DMSOo. Added to 203 µl of suspension 10 µl in of the ligation product containing 10 kg of the ligation product. The mixture is cooled on ice for 30 minutes and then heated at 40 ° C for 60 Co. Immediately after this, 5 ml of LB broth preheated to 37 ° C are added to the heated mixture. ,behind
    iQ
    15 0 5 0
    they are incubated at 37 ° C for 1 hour. The obtained culture broth is centrifuged and the supernatant layer is discarded. LB medium is added to the resulting cell bead and then inoculated with LB plate containing 30 μg / ml ampicillin and 40 μg / ml X- GALO Colonies containing the E0 coli K 12 strain JM83, which are transformed with plasmids having an insert, are white, while the colonies containing the K 12 strain JM83, which are transformed only with the plasmid, are blue. The resulting white colonies are again inoculated with an LB plate containing 30 µg / ml ampicillin and 40 µg / ml X-gal to confirm
    Ten colonies (bacterial clones) are selected from the obtained white colonies and screened using mini-preparative techniques.
    In particular, each colony is cultured overnight on LB medium containing 30 μg / ml ampicillin. Growing cells are harvested and suspended in a solution containing 2 mg / ml lysozyme, 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl (pH 8.0). The suspension is allowed to stand at room temperature for 5 minutes, then 200 µl of 0.2 N is added. NaOH-1% VTS. After slow stirring, the suspension is left to stand at room temperature for 2 minutes. After this, 150 µl of 3 M sodium acetate (pH 5.2) is added, left to stand at -20 C for 10 minutes, then centrifuged for 15 minutes 900 μl of cold ethanol is added to the supernatant layer to recover the supernatant obtained, then centrifuged for 5 minutes to obtain precipitate. The resulting precipitate is washed with 70%
    ethanol and dried to obtain plasmid DNA. In the method described, ten plasmid DNA is obtained
    Each plasmid is dissolved in 10 mM
    0 Tris-0.1 mM EDTA (pH 8.0), mixed with EcoRI and subjected to electrophoresis for restriction analysis. The conditions for digestion and electrophoresis are as follows.
    Digestion: DNA plasmid solution, one fifth of the amount obtained above; EcoRI, 3 units; 37 C; 1.5 hours
    Electrophoresis: 1% agarose gel; 1 X TAE; 120 in; 2h
    35
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    five
    The restriction analysis indicated that eight of the ten clones are positive. The eight clones have 2.9 bp. a fragment. Of the eight positive clones, one clone is selected and designated as its Eocoli K 12 strain JM83 (pHGE) (ATCC 39656) „
    1.89 mg of pHGE DNA is obtained, with the exception that E0 coli is used.
    K 12 strain JM 83 (pHGE) instead of E. coli pH 2-7 and pR 18o
    30 μg of RG-1 are digested with the help of EcoRIo. A fragment having a length of about 3.5 Pooo is extracted from the obtained mixture of fragments, but with the exception that the prepared mixture of fragments and 0.8% low melting agarose gel are used0 1.0 μg of RG is obtained -1 EcoRI-cleaved fragment (about 3.5 p00) 0 Link the resulting EcoRI-cleaved RG-1 fragment (3.5 p000) to pUC13 digested EcoRI, use the resulting EcoRI-cleaved fragment (3.5 p0 o) to replace the split EcoRI fragment HG-3 (2.9 p0o „) 0
    The K 12 strain JM83 is transformed and the bacterial clones are screened0. The resulting clone is designated as E0 coli K 12 strain JM 83 (pRGE) (ATC-39655),
    1.70 mg of pRGE DNA is obtained, with the exception that E0coli K 12 strain JM83 (pRGE) is used instead of pH 2-7 and pR 18 „
    PHGE restriction enzyme analysis was performed
    Procedures and conditions are as follows,
    1 about Digestion of pHGE DNA with EcoRI: 18, b μg DNA pHGE, 64 units, EcoRI, 37 ° C, 2 h,
    2 ° ethanol precipitation: precipitates
    3, Addition of distilled water to precipitate: preparation of solution I µg / ml of EcoRI-digested pHGEo
    4 o Digestion with various restriction enzymes: 1 μg pHGE / EcoRI.
    Restriction enzymes: 5 units „Pvu 11,5 efl.Pvu 11 + lOefloPsal, Ued Psal, 4 units. Mst 11.3 units „Aval, 9 units0 PstI 37 ° С, 2 h0
    5 ° Electrophoresis: 2% agarose gel, 1 X TAE, 28 V, 14.5 h „
    6о Transfer to nitrocellulose filter.

    0
    five
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    e
    7, First prehybridization: 30 ml FDSS, 42 ° C, 6 h
    8 "First hybridization: 5-fragment (5-10 cpm / ml) pR 18 (obtained at stage 4), 42 ° C, 14 h
    9, Washing: 2xSCC - 0.1% VTS at room temperature, immersion 10 min X 4, 1 SSC - 0.1% VTS at 50 ° С, immersion 30 min X 2 „
    10 ° Exposure: XAR-5, -80 ° C, 2 intensive screenings, 17.5 hours,
    11. Washing: 0.5 M NaOH 1.5 M NaCl (immersion 1 min), 0.5 M Tris - 1.5 M NaCl (immersion 1 min), (immersion 1 min).
    120 Exposure; exposure time is 19h0
    13 Second prehybridization, 140 Second hybridization: pB 2-7 insert (obtained in Step 3), 43 ° C, 16.5 h0
    15o Flushing, 160 Exposure: 19.5 hours. 17 Washout 18o Exposure: 20 hours.
    19, Third prehybridization: s - 0 fragment (4,540 kpm / ml), pR 18, 42 ° С, 15 hs
    21 "Flushing, 22. Expositions
    Example 300 Restriction enzyme analysis of the DNA plasmid pRGE0 A restriction enzyme analysis of the DNA plasmid pRGE was performed.
    Eocoli K 12 strain JM83 (pHGE) and E. coli K 12 strain JM83 (pRGE) was used instead of E. coli K 12 MS strain MC01061, having pB 2-7, l E. coli K 12 strain MC1061, having pR 18. Thus 150 pg of each of the pRGE DNA plasmids and the pHGE DNA plasmids are obtained.
    five
    0
    exposure time
    exposure time
    Main sequences of pRGE and pHRGE are determined.
    The pR 18 sequence is compared to that of the pRGE to clarify the structure, including the exon and intron, of the rabbit TNF gene. Then the main pRGE sequence is compared to that of pHGE to study the homology and the consistent sequence around the border between the nitron and the exono. The structure, including the exon and intron, of the human TNFα gene.
    The sequence encoding the name of the rabbit and the name of the person is shown below. In the main sequences, the upper row shows the main RCT GCT TCT CGG GCC CTG ACT GAG AAG CCT CTA-GCC CAC CTA CTA
    NTSA TCT TCT CGA ACC CCG AGT GAG AAG CCT CTA GCC CAT GTT CTA
    RGCA AAC CCG CAA GTG GAG GGC CAG CTC CAG TGG CTG AGC CAG CGT
    HGCA AAC CCT CAA GCT GAG GGG CAG CTC CAG TGG CTG AAC CGC CGG
    RGCG AAC GCC CTG CTG CGC AAC GGC ATG AAG CTC ACG GAC AAC CAG
    HGCC AAT GCC CTC CTG GCC AAT GGC GTG GAG CTG AGA GAT AAC CAG
    RCTG GTG GTG CCG GCC GAC GGG CTG TAG CTC PBX TAC TCC CAG GTT
    HCTG GTG GTG CCA TCA GAG GGC CTG TAC CTC ATC TAC TCC CAG GTC
    RCTC TTC AGC GGT CAA GGC TGC CGC TCC - - - TAC GTG CTC CTC ACT
    HCTC TTC AAG GGC CAA GGC TGC CCC TCC ACC CAT GTG CTC CTC ACC
    RCAC ACT GTC AGC CGC TTC GCC GTC TCC TAC CCG AAC AAG GTC AAC
    HCAC ACC PBX AGC CGC PBX GCC GTC TCC TAC CAG ACC AAG GTC AAC
    RCTC CTC TCT GCC PBX AAG AGC CCC TGC CAC CGG GAG ACC CCC GAG
    HCTC CTC TCT GCC PBX AAG AGC CCC TGC CAG AGG GAG ACC CCA GAG
    RGAG GCT GAG CCC ATG GCC TGG TAC GAG CCC ATC TAC CTG GGC GGC
    HGGG GCT GAG GCC AAG CCC TGG TAG GAG CCC ATC TAT CTG GGA GGG
    t
    RGTC TTC CAG TTG GAG AAG GGT GAC CGG CTC AGC ACC GAG GTC AAC
    H-GTC TTC CAG CTG GAG AAG GGT GAC CGA CTC AGC GCT GAG PBX AAT
    RCAG-CCT GAG TAC CTG GAC CTT GCC GAG TCC GGG CAG GTC TAC TTT
    HCGG CCC GAC TAT CTC GAC TTT GCC GAG TCT GGG CAG GTC TAC TTT
    RGGG PBX ATT GCC CTG
    H GGG PBX ATT GCC CTG
    In a 500 µl stainless steel reaction reactor with stainless steel filters, 20 mg of polystyrene resin is placed at each end of the resin, which is bound to a 2.0 µM nucleoside bond with a succinate bond. The resin is treated with zinc bromide (l M) in a mixture of methylene chloride and isopropanol ( 85:15) to remove the dimethoxytrityl (DMT) protecting group, washed with dimethylformamide, pyridine and acetonitrile, dried in a stream of nitrogen,. Add a solution to the dried resin.
    the sequence encoding the BUT of the rabbit (R), and the bottom p d - the main sequence encoding the name of the person (H):
    20 µM DMT-nucleotide and 60 µM mesitylenesulfonyl nitrotriazole in 200 µl of pyridine Conduct the coupling reaction at 45 ° C for 20 minutes. This cycle of deprotection and coupling is repeated for consecutive nucleotides until the desired oligodeoxynucleotide is on the resin. The resin is then processed to remove the oligodeoxynucleotide from it and purify
    I
    Thus, the following oligodeoxynucleotides are obtained:
    5 -MTTCAGTCATCTTCTCGAACCCCGAGTGAGAA-3; (1)
    3 -GTACAGTAGAAGAGCTTGGGGCTCACTGTTCGG-5; (2)
    5 -GCCTGTAGGCCATGTTGTAGCAAACCCTCAAGC-3; (3)
    3 -ACATCGGCTACAACATCGTTTGGGAGTTCGACT-5 „(4)
    Digested W µg plasmid pHGE 20 units. Eco 10 After electrophoresis on a 1% low-melting agarose gel, a fragment of 2.9 gio0 is eluted. This fragment is inserted into the Eco I fragment from the replicative form M13mp9 phage M13mp9 phage is selected because it is especially suitable for the preparation of DNA fragments. The product is transformed into E. coli M103 BRL „
    Single strand DNA M13mp9-HGEo prepared
    Oligoleoxynucleotide (4) - W -ACATCGGGTACAACATCCTTTTGGGTTTGAC
    51, obtained in stage 14, is used as an delonetor for intron. Zo Delotor for intron 3 is designated
    The delotor YOZ-4 has a main sequence that is complementary to the main base sequence before (Exon 3) and after (Exon 4) Intron 3 to be deposited. Intron 3 is deleted as follows
    Phosphorylated 164 ng (15 pmol) E3-4 using TC.-kinase and add 3 mM ATP to have the template M13mp9-HCE (1.65 µg, 0.5 pmol) 0 The reaction mixture is heated at 6 ° C, cooled to room temperature temperatures for 5 minutes and finally cooled in ice water. To dATP, dCTP, dGTP, dTTP and ATP (0.4 mM) add 5 units of Klenow fragment, 10 units of T ligase in Hinbuffer, 10 mM Tris-HCl (pH 7.2 2 mM MoCl1. and 1 mM V-mercaptoethanol, the reaction mixture (final volume 50 μl) is incubated for 30 min at 4 ° C and for 30 min at room temperature. DNA from the primary reaction oligonucleotides are used for Transfections of Eocoli JM103. The plugs obtained in this way dive onto UT plates. The resulting colonies are hybridized at 55 ° C for 2 hours with f5-labeled E2-4. At this stage, the separator is used as a probe for the identical
    sequencing of DNA sequences that have an appropriate complementary main sequence after it has been deleted
    five
    P
    5 Q s
    0
    five
    trono phage are isolated from these colonies that hybridize with deltoretor0
    The resulting phage is placed on a plate and plaques are marked on the UT plate. The clones are allowed to hybridize at 55 ° C for 2 hours with P-labeled EZ-4o. Positive clones are obtained and the amino acid sequence of the DNA phage is determined to select the phage from which intron 3 is completely delineated. One such phage is designated Mp9-HGE D3-1a
    The replicative form of ir9- HGE DZ-1 is digested with EcoRI. An EcoRI fragment is isolated and cloned into EcoBR32 in rB 327 digested to obtain the pHGE plasmid D 3-1.
    Construction of another plasmid was carried out using the pHGEU.3-1 plasmid to obtain such a plasmid that would directly express the full name in E. coli, using 1ac UV 5 as a promoter. First, 10 µg of the plasmid rNSEDZ-1 10 udo Aval and EcoRI are digested at 57 ° C for 2 hours and electrophoresis is carried out on a 4% polyacrylamide gel to isolate the fragments, Electroelution, about 1 µg of the gel is isolated. Two oligodeoxynucleotide 5 -AATTCATGTCATCTTCTCGAAC-3 and 5-TCGGGGTTCGAGAGAAGATGACATG-3 are synthesized according to the procedure of stage 14.
    Each 5 -end of both oligodeoxynucleotides (about 100 pmoles) is then phosphorylated using a full nucleotide kinase. After completion of the reaction, the reaction mixture is extracted with phenol, then chloroform. 1 and precipitated with ethanol. These fragments are bound at 4 ° C overnight using 10 U of T-ligase
    After completion of the reaction, the mixture is precipitated with ethanol, then electrophoresis is carried out on a 4% polyacrylamide gel to extract the fragment by electrofusion.
    Plasmid pOP95-150 is constructed
    1 µg of pOP95-15 EcoRI is digested and extracted with phenol and then chloroform, then precipitated with ethanol to obtain a vector. Bound to 0.5 µg of the obtained vector using T-DNA ligase with the obtained fragment. According to the procedure of E.coli Ml01 ( ATCC 33876) is transformed using the obtained vector and cultured on agar medium containing 1 mM IPTG in 0.004 wt0% / / volume to obtain about 100 white colonies,
    KPC plasmids are obtained from these transformers and digested with EcoRI to identify these plasmids containing the target EcoRI fragment "In order to investigate the insertion direction, these plasmids are digested with PvuII and PstI and electrophoresed on a 1.5% agarose gel to select plasmids with a fragment of about 1280 base pairs and about 260 base pairs, indicating that the direction of the lacUVS promoter transcription is consistent with the direction of the olyrooxynucleotides encoding the full name
    The analysis of the main sequence shows that these two plasmids have the same sequence and that lacUVS-npoMOTop and the synthesized oligodeoxynucleotides and DNA
    properly combined with each other. The resulting plasmid is designated pHTNF-lacUV5-2.
    E. coli containing pHTNF-lacUV5-2 is cultivated on a traditional nutrient medium. Bioanalysis of the product for TNF-activity shows almost the same activity as that obtained with the plasmid pTNT-lacUV5-1 5-1, containing the gene name of the rabbit, under the control of the lac promoter,
    Example 35o Using the pHGE plasmid and oligodeoxynucleotides (1) - (4), pHTNFlacUV5-lo is prepared
    Example 3.1. Cultured with the usual E0 coli method, containing the pHTFlacUVS-I0 plasmid. To obtain the desired human FIO, 1 mM IPTG was added to the resulting culture to induce, and then the culture was incubated to obtain Eoso11 cells containing the specified human FIO. Cells are harvested by centrifugation, then sonicated in 1 L of 0.04 M
    0 5
    0 5
    0
    Q with
    five
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    five
    Tris-PS1 buffer (pH 7.8), resulting in a cell extract containing the specified human full name
    The cell extract has an activity of 4.5 105 and / or a specific activity of 3.0. 104 U / mg
    Cell extract was applied to a column with DEAE-Separate CL-6B, sufficiently balanced with 0.04 M Tris-HCl buffer (pH 8.0). The column was washed with 0.04 M Tris-HC1 buffer (pH 8.0 ), and then eluted using 0.04 M Tris HC1 buffer (pH 8.0) containing 0.1 M NaCl as eluent. The fraction possessing cytotoxic activity against L-cells is concentrated by ultrafiltration to obtain a crude solution having a specific activity of 4.0 "105 U / mGo
    The crude solution is applied to a Sephacryl 6-200 column equilibrated with 5 mM phosphate buffer (pH 7.4) containing 0.15 M NaClo. Gel filtration is carried out by adding the same buffer solution to the column. The fraction possessing cytotoxic activity against L-cells is concentrated by ultrafiltration to obtain a purified solution having cytotoxic activity against L-cells 2.0 "I O5 C / ml and containing a human full name, having a specific activity of 7.5.10 C / mg.
    The purified solution is mixed with a different volume of Freund’s complete adjuvant and emulsified to form an emulsion. The emulsion thus prepared is injected subcutaneously with BAL B / s to male mice 3 times at 2-week intervals to immunize the mouse. The amount of emulsion injected is 0.2 ml injection-mouse After 4 weeks from the third injection, 0.5 ml of purified solution to complete the immunization.
    one
    3 days after the final
    immunization mice are sacrificed and the mouse spleen is crushed. The spleen is cut into pieces, then filtered under pressure using a stainless steel mesh to obtain spleen cells. The cells are then suspended in a minimum basic medium. Needle to obtain a suspension of cells of the village35.
    Zenki in MBM, Spleen cells and misloma cells of mice (P) X63-Ag3Ul were each washed with MEM 3 times and mixed in a 4: 1 ratio, then centrifuges; - Filled at 800 rpm for 15 minutes to obtain a precipitate. 2 ml of a 44% v / v solution of polyethylene glycol 2000 in MEM is gradually added to the sediments in the centrifuge tube. Za1630602 on
    ten
    A 10,000-fold diluted solution of anti-mouse IgG, labeled with periksadozy, in an amount of 0 ml / dimple was added to the washed and left for 1 h at room temperature. The cells were washed with physiological saline containing 0.1% serum bovine albumin. substrate solution (30 mg of o-phenylenediamine, 7 μl of 30% aqueous hydrogen peroxide, 10 ml of 0.1 M citric acid and 10 ml of 0.2 M disodium phosphate) in an amount of 0.15 mg / carbon to the mixture was added. 1 ml of MEM and honey-5 killing 30 minutes later, absorption in each corner is measured lenii at 492 nm to establish depressions containing cells producing antibody for
    The centrifugal tube containing the mixture is gently rotated. The MEM is then added to the mixture at a ratio of 2 ml / min in such an amount that the total volume of the resulting mixture becomes 10 ml. The mixture is then centrifuged at 600 rpm for 5 minutes to obtain a precipitate. The precipitate is suspended in Rozevel Park Memorial Institute (hereinafter referred to as RPMI), containing 10% of the serum of the newborn calf, to obtain a suspension containing cells in the number of myeloma cells / ml. The suspension is inoculated into each well of the plate of a microfiter with 96 wells in an amount of 0.1 ml / hole.
    A day later, 0.1 ml of RPMI 1640-10% CHT medium containing GAT (b-x-xanthine, 410 M aminopterin and 1.6 10 M thymidine) (hereinafter referred to as GAT-environment) are added to each well. After that, every 3 or 4 days in each recess, half of the medium is replaced with fresh HAT medium. 7 days after the addition of RPMI 1640-10% of CHT medium containing GAT, hybridoma cell growth is observed in several recesses and 2 or 3 weeks later, growth is observed hybridoma cells in almost all the grooves.
    About 1 ml of the culture supernatant in the well, in which the growth of hybridoma cells is observed, is placed in each well of the microtiter plate with 96 holes, in which the human name is recorded, and the microtiter plate is left for 1 hour at room temperature. each well is washed with saline containing 0.1% bovine albumin
    0602 on
    36
    ten
    A 10,000-fold diluted solution of anti-mouse IgG, labeled with periksadozy, in an amount of 0 ml / dimple was added to the washed and left for 1 h at room temperature. The cells were washed with physiological saline containing 0.1% serum bovine albumin. Substrate solution (30 mg of o-phenylenediamine, 7 μl of 30% aqueous hydrogen peroxide, 10 ml of 0.1 M citric acid and 10 ml of 0.2 M of disodium phosphate) in an amount of 0.15 mg / ug lubeniye for 30 minutes later measured by 0
    absorption in each well at 492 nm to establish grooves containing cells that produce antibody o
    Cells in each well that show high anti-activity are selected using a glass capillary and cloned to produce clones. Clones are screened.
    5 using, as a criterion, the activity of the antibody, in the same manner as described, in order to obtain 2 clones that possess high antibody activity, namely
    0 hybrid cell line H117C and hybrid cell line NSh2R „
    Each of the clones obtained is cultivated in RPMI 1640 medium containing 10% OHT, to multiply the cells clone. Then the cells of the clones are harvested and suspended in RPMI 1640 medium containing 15% CHT and 10% dimethylsulfoxide.
    five
    40
    the suspension is stored in liquid nitrogen. Each 10 cells of both hybrids
    home cells obtained in stage 4, are inoculated into the abdominal cavity of a BAL B / c mouse, which was previously injected with 0.2 ml of a bailiff (2,6,10,14-tetrames of tilpenotecan) intraperitopeally, to obtain ascites fluid ml of ascites fluid collected from mouse
    To 10 ml of the ascites fluid obtained in step 5 was added 2.66 g of ammonium sulfate (35% of saturation) and the mixture thus obtained was stirred at 4 ° C overnight, resulting in a precipitate.
    The precipitate was separated by centrifugation and applied on a DEAE-Safarose CL-bB column, equilibrated with 0.01 M phosphate buffer (pH 8.0), the Column was washed with the same buffer, and then
    37lb
    eluted using 0.01 M phosphate buffer (pH 8.0) containing 0.2 M NaCl as eluent. The fractions obtained from the bottom of the column are subjected to polyacrylamide gel electrophoresis to determine fractions containing monoclonal antibody0 97 mg of monoclonal antibody are obtained. antibodies produced by the hybrid cell line H117C, and 199 mg of monoclonal anti



    body produced by the hybrid cell line NU 2F.
    The definition of the subclass is carried out with
    using anti-mouse IgG (sodium carbonate, dialy20 added
    25
    Both Both monoclonal bodies H117C and NSh 2F belong to the class IgG ,,,
    The purified solution of the human name is diluted with MEM culture medium containing 10% CHT to obtain solutions containing concentrations of 20 and 200 C / ml. The two monoclonal antibodies obtained are diluted with the same culture medium to obtain solutions of appropriate concentrations. Both solutions are placed in each well of a 96-well microtiter plate in a quantity of 0.05 ml / hole. After incubating for 10 hours at 37 ° C for 1 hour, 0.1 ml of suspension containing 10 L- is added to each hole. cells / ml of the same culture medium. After this, almost the same procedure as described for the in vitro experiment to determine the cytotoxic activity against the L-cell is repeated. At the same time, a study is also carried out in which neither human name nor monoclonal antibodies, and research in which no monoclonal antibody is added, but a human full name is added.
    The results are shown in Table 05, where the dose of activity removal is indicated in magnitude, calculated from the equation
    35
    40
    45
    Caused antibody solution to 50 ml on bulk Sepharose CL-4B activated with cyanogen bromide Mixture is gently stirred at 4 ° C overnight for reaction After the completion of the reaction, the resin is washed well with an aqueous solution containing 0.5 M NaCl and 0.1 M sodium carbonate. The resin is then mixed with 50 ml of 1 M aqueous ethanolamine, gently stirred at room temperature for 2 hours, as a result of which unreacted active groups are protected. Then the resins are well washed with 8 M aqueous urea and physiological saline to obtain the resins associated with the monoclonal antibody H117C for use as an adsorbent for affinity chromatography I
    Almost the same procedure as described for the preparation of resins associated with the monoclonal antibody HIII2F, is repeated.
    Resins bound with monoclonal antibody H117C serve for packing the column (2.5x8 cm).
    Example 302o Eocoli containing the plasmid pHTNF - lacW 5-1 s is cultured in the traditional manner and the cells are harvested. The cells are lysed in 2 liters of 0.02 M Tris-HCl buffer (pH 7.8), resulting in a cell extract containing a human full name. . The extract has an activity of 5.2 Ц 10 C / ml, and the human full name contained in it has a specific activity of 4.2-44 C / ml
    Both monoclonal antibodies completely remove non-human full name activity at high concentrations. At low concentrations, the HUI2F monoclonal antibody possesses more than 50
    Example 302o Eocoli containing the plasmid pHTNF - lacW 5-1 s is cultured in the traditional manner and the cells are harvested. The cells are lysed in 2 liters of 0.02 M Tris-HCl buffer (pH 7.8), resulting in a cell extract containing a human full name. . The extract has an activity of 5.2 Ц 10 C / ml, and the human full name contained in it has a specific activity of 4.2-44 C / ml
    A higher dose of removal than mono-55cin was added to the extract in such an amount that the semi-basic H117 antibody should be added to a final concentration of 0.7 wt.% /
    A thin / bulk device is used to obtain a precipitate of nucleicle gel isoelectric foic acids. After removal of sediment
    38
    kusirovanie to measure the isoelectric points of both monoclonal antibodies. Measurements are carried out using a pharmacyl pHZ-10
    Monoclonal antibody H117C and monoclonal antibody NSh2R were found to have isoelectric points of 6.7-7.0 and 6.2-fc, 5, respectively
    50 ml of an aqueous solution containing 150 mg of monoclonal antibody H117C against an aqueous solution containing 0.5 M NaCl and 0.1 M are dialyzed
    20
    25
    -jo
    five
    0
    five
    0
    An antibody solution to 50 ml of swollen Sepharose CL-4B resin, activated with cyanogen bromide. The mixture is gently stirred at 4 ° C overnight to conduct the reaction. After the reaction is complete, the resin is washed well with an aqueous solution containing 0.5 M NaCl and 0.1 M sodium carbonate. . The resin is then mixed with 50 ml of 1 M aqueous ethanolamine and gently stirred at room temperature for 2 hours, resulting in the protection of unreacted active groups. Then the resins are well washed with 8 M aqueous urea and physiological saline to obtain the resins associated with monoclonal antibody H117C for use as an adsorbent for affinity chromatography I
    Almost the same procedure as described for the preparation of resins associated with the monoclonal antibody HIII2F, is repeated.
    Resins bound with monoclonal antibody H117C serve for packing the column (2.5x8 cm).
    Example 302o Eocoli containing the plasmid pHTNF - lacW 5-1 s is cultured in the traditional manner and the cells are harvested. The cells are lysed in 2 liters of 0.02 M Tris-HCl buffer (pH 7.8), resulting in a cell extract containing a human full name. . The extract has an activity of 5.2 Ц 10 C / ml, and the human full name contained in it has a specific activity of 4.2-44 C / ml
    Steptomycin5 is added to the extract in such a quantity that half
    by centrifugation, the supernatant layer was applied to a DEAC-Sepharose CL-E column, equilibrated with 0.02 M Tris-HCl buffer (pH 8.0). The column was washed with the same buffer and then eluted using 10 mM phosphate buffer (pH 7.5) containing 0.1 M sodium chloride as eluant to obtain a crude solution (A) having a specific activity. 3.90 X K 10% / mgo
    After adjusting the pH to 6 with hydrochloric acid, the crude solution A is applied to a Blue Sepharose CL-6B column, equilibrated with 10 mM phosphate buffer (pH 6.0) containing 0.1 M sodium chloride o the Column is washed sufficiently and this was eluted using 10 mM phosphate buffer (pH Y, 0) containing 0.5 M sodium chloride as eluent to obtain a fraction containing purified human FN00 As regards the extract, in the supernatant obtained in removal of nucleic acids, raw solution A and fraction, gender By means of column chromatography according to the invention, the specific activity, the degree of extraction and purification are determined in accordance with the methods given.
    Example 4. Crude solution A is purified using a monoclonal antibody column, which is a column filled with resin bound to a monoclonal antibody, a column equilibrated with 50 mM phosphate buffer (pH 7.5) containing 0.15 M sodium chloride, and put t
    TCA TCT TCT CGA ACC CCG ACT GAG AAG CST CTA GCC CAT GTT CTA GCA AAC CST CAA-GCT GAG GGG CAG CTC CAG CGG AG CGGG GCC AAG GCC CG CGGG AAG CGC
    CTG; TG GTG CCA UCA USA G.AS; cir; e C: TG TAG hundred ATC tle tss CAG GTC
    CTC TTC AAG GGC CAC GGC TGC CCC ACC CAT GTG CTC CTC ACC ACC ATC AGC CGC ATC GCC GTC TAG CAG CAG ACC AAG GTC AAC CTC CTC TCG GTC ATC AAG AGC CCC TGC A CAG AGG GAG ACC CCA GAG GAG GCT GAG GCC CCC TGG TAT GAG CCC ATC TAT CTG GGA GGG
    0
    0
    0
    five
    0
    five
    on the column, the crude solution of A0. After sufficient washing, is eluted with 0.1 M glycine / sodium chloride (pH 10.0) as eluent to obtain a fraction containing purified human FULL.
    In the fraction thus obtained, the specific activity, degree of extraction and purification are determined. According to the mentioned procedures.
    The results are shown in Table 07, together with the results obtained in Example 10.
    Example 3 Almost the same procedure as in Example 1 is repeated, with the exception that Matrixes are used separately. Gel Red A and Matrix Hep Red B instead of Blue (Blue) Sepharose CL-6B result in: what are fractions containing purified human name.
    In the fractions thus obtained, the specific activity, the degree of extraction and purification are determined according to the methods given,
    The results are shown in Table 8 together with the results obtained in Example 1o.
    权利要求:
    Claims (1)
    [1]
    1. The method of purification of recombinant tumor necrosis factor, including the destruction of Escherichia coli bacteria transformed with recombinant DNA with a gene encoding tumor necrosis factor and having the following nucleotide sequence:
    411630602
    GTC TTC CAG CTG GAG AAG GGT GAG CGA CTC AGC GCT GAG ATC AAT
    CGG CCC GAG TAT CTC GAG TTT GCC GAG TCT GGG CAG GTC TAG TTT
     GGG PBX ATT GCC CTG,
    by centrifuging and ultrasonic treatment in 2 l of 0.02 M tris-HC with a pH of 7.8, application of the cell extract onto a column with DEAE-Sepharose equilibrated against 0.02 M tris-HCl buffer, washing with the same buffer at pH 8 , 0, elution with 10 mmol phosphate buffer containing 0.1 M NaCl, at pH 7.5, applying the solution without additional purification on a column packed with a cross-linked agarose gel with a covalently immobilized dye using as
    Lys pro val ala val val
    Ser Ser Ser. Arg Thr Pro Ser Asp
    i
    Ala Asparagle Alu Asus Alaglu Alaglu Gly Cys Pro Ser Thr NEP Vnl Leu Lou Thr i His Thr Lie Ser Arg Lie Ala Val Ser Tyr Gin Thr Lys Val Asn
    Leu Le Ser Ser Ala He Lys Ser Pro Cys Gin Arg Glu Thr Pro Glu
    Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro He Tyr Leu Gly Gly
    s
    Glu Lys Gly Asp Arg Le Glu Lys Gly Asp Arg Le Glu Lys Aly Glu
    Note. X is a ribonucleic acid residue A, Ct G or U; 7 - rnbocucleic acid residue A or G; M - deoxycarbonucleic acid residue T Ilk C; H is the deoxymbonucleic acid residue A or G; Z - deoxyribonucleic acid residue A, C, SilT.
    Green A ligand, Cibacron, Blue 13vA, Procion Red HF3s, equilibrated against 10 mmol phosphate buffer with pH 6.0, containing 0.1 M NaCl, conducting gel filtration with the addition of the same buffer solution and concentrating the fraction having cytotoxic activity against L-cells using ultrafiltration.
    2 "Method according to claim 01, characterized in that the tumor necrosis factor is a polypeptide having the following amino acid sequence:
    Lys pro val ala val val
    Table 1
    table 2
    Table 3
    most hybridized fragment
    45
    1630602 Table 5
    Crude solution A 3.9 "10 After treatment with Blue Sepharose
    CL-6B1,5 U
    After treatment on a column with monoclonal antibody1,4 -10
    46
    47
    Crude solution A
    After processing
    Blue sepharose
    CL-6B
    After processing
    Mattsix Gel
    Ked A
    After processing
    Matrix Gel
    Green A
    J63060248
    Table 8
    3.9 Yu
    1001.0
    1.5. S4
    90
    3.8
    1.4 10
    65
    3.6
    1.4 -10e
    80
    3.6
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    同族专利:
    公开号 | 公开日
    DD240216A5|1986-10-22|
    EP0183198A1|1986-06-04|
    CA1265650A|1990-02-06|
    IL77101A|1991-01-31|
    KR940000542B1|1994-01-24|
    AT50794T|1990-03-15|
    AU5024885A|1986-05-29|
    EP0183198B1|1990-03-07|
    KR860004149A|1986-06-18|
    AU591879B2|1989-12-21|
    US4880915A|1989-11-14|
    JPH0229317B2|1990-06-28|
    DE3576362D1|1990-04-12|
    JPS61124392A|1986-06-12|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US4879226A|1984-04-06|1989-11-07|Asahi Kasei Kogyo Kabushiki Kaisha|Novel human physiologically active polypeptide|
    US4677064A|1984-11-09|1987-06-30|Cetus Corporation|Human tumor necrosis factor|
    US4677063A|1985-05-02|1987-06-30|Cetus Corporation|Human tumor necrosis factor|
    US4770995A|1985-08-29|1988-09-13|New York Blood Center, Inc|Detection of the sensitivity of cells to the effects of tumor necrosis factor and lymphotoxin|
    US4677197A|1985-10-30|1987-06-30|Cetus Corporation|Purification method for tumor necrosis factor|DE3582183D1|1984-03-06|1991-04-25|Dainippon Pharmaceutical Co|DNS ENCODING THE HUMAN TUMORNIC CROSIS FACTOR AND THE HUMAN TUMORNIC CRISIS FACTOR POLYPEPTIDE.|
    US5288852A|1984-03-06|1994-02-22|Dainippon Pharmaceutical Co., Ltd.|Human tumor necrosis factor polypeptides|
    US4879226A|1984-04-06|1989-11-07|Asahi Kasei Kogyo Kabushiki Kaisha|Novel human physiologically active polypeptide|
    US6686455B1|1984-07-05|2004-02-03|Genentech, Inc.|Tumor necrosis factor|
    IL73883A|1984-12-20|1990-12-23|Yeda Res & Dev|Monoclonal antibodies against tnf-alpha,hybridomas producing them and method for the purification of tnf-alpha|
    AU601675B2|1984-12-21|1990-09-20|Biogen, Inc.|Purification, production and use of tumor necrosis factors|
    JPS61155330A|1984-12-28|1986-07-15|Denichi Mizuno|Protein drug|
    US4870163A|1985-08-29|1989-09-26|New York Blood Center, Inc.|Preparation of pure human tumor necrosis factor and hybridomas producing monoclonal antibodies to human tumor necrosis factor|
    US4777242A|1986-10-10|1988-10-11|Phillips Petroleum Company|Purification of recombinant tumor necrosis factor|
    AU632460B2|1987-07-16|1993-01-07|Schering Corporation|Purification of gm-csf|
    US5391706A|1987-07-16|1995-02-21|Schering Plough Corporation|Purification of GM-CSF|
    US5597485A|1988-05-13|1997-01-28|Vilmax S.A.|Process for separating proteins|
    DE4331358A1|1992-10-12|1994-04-14|Braun Melsungen Ag|Process for the quantitative selective removal or preparation of tumor necrosis factorand / or lipopolysaccharidesfrom aqueous liquids|
    DE19515554C2|1995-04-27|1999-06-17|Braun Melsungen Ag|Use of an agent and device for the simultaneous extracorporeal elimination of tumor necrosis factor alpha and bacterial lipopolysaccharides from whole blood and / or blood plasma|
    ES2545526T3|2001-02-01|2015-09-11|Sigma-Aldrich Co. Llc|Enhanced affinity matrices with enhanced visibility for molecular drag and immunoprecipitation applications|
    MXPA05005921A|2002-12-02|2005-10-19|Abgenix Inc|Antibodies directed to tumor necrosis factor and uses thereof.|
    US20050287515A1|2004-06-24|2005-12-29|Reinhold Deppisch|Removal of bacterial DNA from therapeutic fluid formulations|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP59246184A|JPH0229317B2|1984-11-22|1984-11-22|
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