Method of producing recombinant plasmide dna encoding human tumor necrosis factor

专利摘要:
PURPOSE:To obtain the titled factor, by cloning cDNA coding human necrotic factor with a cloned DNA coding a rabbit cancer necrotic factor as a probe, integrating the resultant cloned cDNA in a plasmid, transforming a microorganism with the plasmid, and using the transformant microorganism. CONSTITUTION:A human macrophage is collected from an air cell, blood or abdominal cavity, etc. and cultivated in an inducer, e.g. endotoxin, to separate a fraction containing a human cancer nectrotic factor mRNA from the above-mentioned cell. A single-stranded cDNA is prepared from the above-mentioned mRNA with a reverse transcriptase, and converted into a double-stranded cDNA, which is inserted into a vector. The resultant vector is then transformed to a host to prepare a cDNA library. A cDNA coding a human cancer necrotic factor is cloned from the above-mentioned cDNA library with a DNA coding a rabbit cancer necrotic factor as a probe, and a microorganism transformed by a plasmid having the integrated cloned DNA is used to give the aimed factor, e.g. a polypeptide having amino acid sequence expressed by the formula.
公开号:SU1614765A3
申请号:SU853869300
申请日:1985-03-05
公开日:1990-12-15
发明作者:Ямада Масааки；Фурутани Ясуджи；Нотаке Мицие；Ямагиси Юнити
申请人:Дайниппон Фармасьютикал Ко., Лтд (Фирма)；
IPC主号:

专利说明:

The invention relates to genetic engineering, in particular to the construction of recombinant plasmid DNA, which encodes a human tumor necrosis factor.
The method consists in constructing plasmid DNA as a result of culturing human macrophages with an inducer, separating the fraction containing the human RNF from the full name of the induced cells, obtaining single DNA from l-RNA using reverse transcriptase with subsequent day conversion. DNA, introduction of day-DNA into a vector, transformations with a recombinant vector of the host organism and obtaining clones, subcloning to-DNA encoding the human TNFα polypeptide or its main part from the selected clones and recombining nt plasmid DNA encoding human TNF.
The data, which is proposed method, are given in table. 1-1, 1-2, 2-1, 2-2, 3-10. : PRI i MER 1. Macrophaps are obtained. Humans The resulting macrophages are seeded into a dish with a cell density of approximately 2-10 to 1-10 cells per cm, and pre-cultured at 35 -38 ° C, preferably at 37 ° C, in an atmosphere of 100%. wet; containing 5% carbon dioxide for about 30 minutes to 2 hours.
An endotoxin obtained from Gram-negative bacteria, for example, Li Oi, is then added as an inducer.
3 vl
Oi Sp
I
popolysaccharide derived from Escheri-colia coli; Pseudomonas aeruginosa or Salmonella tyhi, and cycloheximide is added as a protein synthesis inhibitor. Cultivation is continued for another 3-8 hours to accumulate human RNA RNA in macrophages. The amount of endotoxin is usually about 1-100 μg / ml. As an inductor, you can additionally add phorbol ester, such as phorbol-12-myristate-13-acetate, phorbol-12, 13-didecanoate or phorbol--12, 13-dibenzoate in an amount of about 1-2000 ng / ml. The amount of a protein synthesis inhibitor varies depending on its type. For example, in the case of cycloheximide, it is J 0.1–50 µg / ml. As the culture medium, various culture media suitable for the cultivation of mammalian cells can be used (RPMI-1640, MEM medium Eagle and modification of Dulbecco MEM medium), In a preferred embodiment, in the culture
animal serum was added to the medium; (such as fetal bovine serum or calf serum) in an amount of about 1-20%; After cultivation, total RNA is extracted from the cells, and then oligo (dT) -7 split or poly (U) safarose is affinity column chromatographed and the fraction containing the field is separated (A-i-RNA, Fraction enriched with i-RNA a human full name is obtained by treatment of the first fraction with poly (A) and RNA by electrophoresis on an acid-urea agarose gel or by centrifugation in a rpa density of ca:; arose.
To confirm that the mRNA fraction encoding the human full name is obtained, the mRNA is translated into protein and its biological activity is tested. Introduce mRNA into Xenopus laevis oocytes and analyze the in vitro cytotoxic activity against large L translated protein cells.
Example 2, Poly (A). -N-RNA 1ShI enriched with i-RNA fraction is used as the matrix, and (dT) as the seed for the synthesis of single-DNA using reverse transcriptase (nanpiiMep, obtained from avian myeloblastosis virus in the presence of dATP, dPTD, dGTP and dTTP), Oid-DNA is used as a matrix and synthesis
to J5 20 25
ZO, Q
ds
50
five
p.sToT day-DNA using reverse transcriptase or DNA polymerase E, coli.
The resulting dna DNA is introduced into. plasmid pBR 322, cleaved with Pst restriction endonuclease. The resulting recombinant plasmids are introduced into a host cell, such as E, coliXl776, to obtain a set of cDNA clones by selecting tetracycline resistant colonies.
The k-DNA kit is hybridized using a P-labeled rabbit IT DNA fragment, target clones containing recombinant plasmids, having a human TNFα coding DNA encoding, are selected.
32
P-Labeled c-DNA is synthesized using a fraction of poly (A) -and-RNA or a fraction enriched in human r-RNA. TNF, as a matrix. Separately, the mRNA fraction obtained by a similar method is used as a matrix, except that uninfected macrophages are used as Cf and R-labeled c-DNA is synthesized. P-labeled c-DNA is used as an inducing negative sample. Plasmid clones that strongly hybridize with the inductive positive sample but do not hybridize with the negative induction sample are taken above the cDNA kit.
The operation described below is carried out in order to confirm that the resulting clones contain an insert to the DNA encoding the human TNFα polypeptide. The plasmid DNA is extracted from the indicated clones, converted into single-stranded DNA by heating or alkaline treatment and fixed on nitrocellulose filters. The mRNA fraction containing the human TNFα nRNA is added to the filters for hybridization with fixed DNA. The isolated mRNA is introduced into Xenopus laevis oocytes to determine whether the human TNFα encoded the mRNA,

Using the above methods, transformed systems are obtained that have recombinant plasmids containing a DNA fragment having 1gk; baseline concentration, xc -hyg: m; amber human RNA.
If the obtained cloned DNA | does not contain a whole region that encodes a polypeptide of a human full name, then larger DNA samples are taken by selection from the c-DNA kit using cloned TNF fragments of c-DNA from the transformed systems as a sample.
The cloned cDNA encoding a polypeptide containing the amino acid sequence of the human TNF polypeptide is analyzed by determining the base sequences of some cloned cDNA fragments, detecting the base sequence, homologous base sequence of the ctn of TNF and selecting those for DNA
full name with rabbit shshzm name. This indicates that the complete amino acid sequence of the human TNF polypeptide is not always necessary for the expression of the activity; the partially modified human TNFα polypeptide also has activity because it contains an active,
0 H1NY full poly-peptide TNF,
II p r i me r 3. DNA modification is performed by cleaving the DNA with suitable endonuclease restriction and cleaving one or more codons with the appropriate AND
J5 exrnucleases and / or endonucleases, taken 11 individually or in combination, followed by substitution with degenerative or other codoies, for example, chemically synthesized codons
25
contain a sequence of bases, 20 according to the phosphothermal method or a flap corresponding to the integral coding region for the human TNF polypeptide.
The homology between the base sequence encoding the rabbit TNF and the base sequence coding for the human TNF polypeptide, as well as the homology between the established amino acid sequences of the rabbit TNF polypeptide and the human TNF polypeptide, are presented respectively in Table 2. 5 and 6.
From this homology and from the established N-terminal and C-terminal amino acid sequences of rabbit plasma TNF, 35
codons.
PRI me R 4o Obtaining polypeptide human TNF.
A plasmid encoding a human TNFα polypeptide is obtained by introducing the cloned cDNA encoding a human TNFα polypeptide into a suitable vector. All vectors proliferating in transformed microorganisms can be used.
A plasmid to produce an unfolded polypeptide is constructed by linking a DNA fragment containing the base sequence encoding the human TNF polypeptide, where the ATC initiation codon is added to the end and the stop codon (TAA, TAG or TGA) is at the 3-terminus, with that the K-DNA of a human full name encodes a TNF precursor polypeptide containing 233 amino acid residues, and the human TNF polypeptide itself is a polypeptide corresponding to 155 amino acid residues from the carboxy terminus of the preceding nick "
The presence of baseline homology and established amino acid sequences of human TNF and rabbit TNF indicates that they are genetically derived from the same gene, and indicate that a significant portion of the normal regions is the sequence required for the expression of biological activity . However, as shown below, the complete human TNF polypeptide does not immunologically intersect with the full name of the rabbit shshzm. This indicates that the complete amino acid sequence of the human TNF polypeptide is not always necessary for the expression of the activity; the partially modified human TNFα polypeptide also has activity because it contains an active,
HL1 center of full polypeptide TNF,
II p r i me r 3. DNA modification is performed by cleaving the DNA with suitable endonuclease restriction and cleaving one or more codons with the appropriate AND
exrnucleases and / or endonucleases, taken 11 individually or in combination, followed by replacement with degenerative or other codoies, for example, chemically synthesized codons
according to the phosphotisphyr method or
codons.
PRI me R 4o Obtaining polypeptide human TNF.
A plasmid encoding a human TNFα polypeptide is obtained by introducing the cloned cDNA encoding a human TNFα polypeptide into a suitable vector. All vectors proliferating in transformed microorganisms can be used.
A plasmid to produce an unfolded polypeptide is constructed by linking a DNA fragment containing the base sequence encoding a human TNF polypeptide, where the ATC initiation codon is added to the termination and the stop codon (TAA, TAG or TGA) is at the 3-terminus, with a DNA fragment, encoding the promoter and sequence of Shine-Dalgarno. The plasmid is constructed by introducing a c-DNA fragment having a BASE sequence coding for
the human TNFα polypeptide, where a tearing codon is added to the 3-terminus, into the vector, with the result that the translational reading structure corresponds to the structure in the structural gene. The method for producing a human TNFα polypeptide in the form of a fusion polypeptide has the advantage of minimizing the decomposition of the product in transformed host cells. The complete human TNF polypeptide corresponding to the amino acid sequence from serine at the 2 position of a leucine in 236 half-lines, in the top lines of the table. 6, not with
ten
holds methionine. Consequently, a plasmid / engineered introduction of a human FU name to the DNA fragment associated with the 3-terminus of the base sequence of the structural gene to be fused through the methionine codon (ATC) allows the human full name as a fusion product by a methionine peptide bond splitting, for example by treatment with bromine cyan, Transformed organisms are obtained by introducing an expressive vector into a host organism; - such as (a skin organism, for example, E.coli, After this, the cultivation of transformed organisms produces a human polypeptide or a polypeptide containing 1-1 methionine in the N-terminus. The product can accumulate both in the cytoplasm and in the B cell periplasm depending on the method of constructing the expression vector, DP TorOj, in order to induce nojranen-25 type in the periplasm, construct a plasmid using a gene encoding a secretory protein, such as an alkaline phosphatase gene (phoA) or a protein gene.
15
20
ka, phosphorus bonding (phoS), in re- 0, arginine, caffeine, P15ocaine,
by binding the DNA encoding the human TNFα polypeptide in the correct translational reading structure to the above gene in a suitable place after the DNA region encoding the signal peptide.
The resulting transformed organisms are cultivated under suitable conditions until the complete production of the target polypeptide. After that, the polyceptic is extracted from the cult of the phy. After the accumulated polypeptide E C -: the host host's plasma is subjected to destruction by lysozyme treatment, freezing and thawing or ultrasonic dispersion, or using a French press, then centrifuged or filtered and collected the extract,
The polypeptide thus obtained is obtained by conventional protein purification techniques, for example, by a combination of extraction, ultrafiltration, dialysis, ion exchange, gel filtration, electrophoresis, affinity chromatography, and so on.
I
In the manner described above, a human TNF polypeptide is obtained according to an icon
40
45
50
55
hydrochloric acid, gluconic acid, etc.
The modification of the human TNFα polypeptide or its allelic mutant polypeptide is carried out according to known methods.
Chemical and physico-chemical properties of the polypeptide,
{molecules of weight,
The molecular weight of rf-TNF was measured by gel filtration analysis on a G 3000 SW T 8K gel column in accordance with high pressure liquid chromatography. Whose pressure, using 0.2 And phosphate (pH 7) buffer with or without 8 M urea and 0.5% 2-mercaptoethanol as a solvent, bovine serum albumin is used as a protein marker of molecular weight. (MW 66000), rabbit triosephosphoisomerase (MW 53000), egg albumin (MW 45000), porcine pepsin (MW 32700), soy protein trypsin inhibitor (MW 20500), horse myoglobin (MW 17800), and horse cytochrome C (MW 12400)
As a result, it is established that rF-TNF has a molecular weight of 45,000 ± ± 5,000 and 18,000 + 3,000 daltons, respectively
five .
five
0
sttenigo and / or polypeptide with methionine at the N-terminus of the polypeptide.
Example 5: By modified human FULL polypeptides, polypeptides derived from allelic mutants of DNA encode a human TNF (an allelic mutant polypeptide) human coding polypeptide; they are obtained by adding an amino acid or peptide (consisting of two or more amino acids) to N- OKOH-C or termination of the human TNF polypeptide or allelic mutant polypeptide, or by removing one or more amino acids from the human TNF polypeptide or allelic mutant polypeptide iodine (e.g., removal of 4 amino acids from the N-terminal of the human TNF – TNF), as well as:: aqueous, such as esters, acyl derivatives or acid amides, formed using the functional group in the molecule, the amine residue at the N-terminus or -carboxylic residue at the C-terminus, and their salts formed by amino residues or carboxyl residues, for example, with sodium hydroxide, hydroxide
five
0
five
0
five
hydrochloric acid, gluconic acid, etc.
The modification of the human TNFα polypeptide or its allelic mutant polypeptide is carried out according to known methods.
Chemical and physico-chemical properties of the polypeptide,
{molecules of weight,
The molecular weight of rf-TNF was measured by gel filtration analysis on a G 3000 SW T 8K gel column in accordance with high pressure liquid chromatography. Whose pressure, using 0.2 And phosphate (pH 7) buffer with or without 8 M urea and 0.5% 2-mercaptoethanol as a solvent, bovine serum albumin is used as a protein marker of molecular weight. (MW 66000), rabbit triosephosphoisomerase (MW 53000), egg albumin (MW 45000), porcine pepsin (MW 32700), soy protein trypsin inhibitor (MW 20500), horse myoglobin (MW 17800), and horse cytochrome C (MW 12400)
As a result, it is established that rF-TNF has a molecular weight of 45000 ± ± 5000 and 18000 + 3000 daltons, respectively, in the absence and presence of urea and 2-mercaptoethanol,
k-DNA, introduced into the expression plasmid pHTR 91, encodes 155 amino acid residues (except for methionine originating from the initiating ATC codon). From the established amino acid sequence, rn-weight molecules of rF-TNF JQ (17097 daltons) are calculated. the weight is consistent with the value determined in the presence of urea and 2-mercaptoethanol.
This fact indicates that 5 rF-TNF is present as a monomer (subunit) in the presence of urea and 2-mercaptoethanol, but exists as an aggregate, for example in the form of a trimer, in the absence of denaturing 20 agents,
Isoelectric point
The isoelectric point is determined by iso-electrophoresis gel electrophoresis at 3 W for 3 h with 25 using a plate of 5% polyacrylamide gel, and a pH gradient from 4.0 to 6.5,
Protein is stained with Coomassie brilliant blue. Separately, the gel is cut into 3-mm wide ZO strips and soaked in 20 mM Tris-HCl (pH 7.8) buffer for elution of the protein. Cytotoxic resistance is clearly observed in the eluate of the gel cut from the site corresponding to the position of the protein determined by staining.
As established, the isoelectricity of the pF-TNF point is 5.9 + 0.3.
Amino acid composition 40
The amino acid composition of rF-TNF is determined using an amino acid microanalyzer by a fluorometric method using orthophthalic aldehyde after hydrolysis of the sample with hydrochloric acid. /
50 μg of rf-TNF is hydrolyzed in bn.HC at 11. Amino acid composition is calculated with correction based on the values determined in each sample hydrolyzed 24.48 and 72 hours. Cystine and cysteine are determined as cysteic acid converted by peroximuracy oxidation; acid. Tryptophan is determined by the fluorometric method.
The results are summarized in table. 7
The amino acid composition is in good agreement with the composition calculated from
base sequences, coding-yupdih polypeptide of human full name.
Determination of the N-terminal amino acid sequence.
The N-terminal amino acid sequence of rF-TNF is determined by the Edman decomposition procedure.
Phenylthiohydroaminoamino acid, obtained from the N-terminal amino acid in the Edman decomposition method, is identified by high-pressure liquid chromatography using a column (4.6 x 250 mm) with TSK-gel DDS 120A. These operations are replicated many times to determine the new N-formed trailer amino acids.
It is found successively that the N-terminal amino acid sequence of the pF-TNF is as follows;
NH-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp
Some polypeptides obtained in microorganisms using recombinant DNA technology have a methionine residue at their N-termini, derived from a start codon (ATC),
Determination of the C-terminal amino acid sequence.
The C-terminal amino acid sequence of rF-TNF is enzymatically determined using carboxypeptidazo
rF-TNF is cleaved with carboxypeptidase-A and carboxypeptidase Y at molar ratios of xbstra enzyme. 1, 25 and 1: 1000, respectively. Free amino acids released from the C-terminus of rF-TNF as a result of double cleavage are identified on the amino acid microanalyst: lysator at appropriate intervals from 2 to 180 minutes after cleavage
The C-terminal amino acid sequence of the rF-TNF sequence is determined to be as follows:
 Tyr-Phe-Cly-Ile-Ile-Ala-Ieu-COOH,
Decomposition of RF-TNF trypsin.
500 μg of rF-TNF are decomposed with 20 μg of TRNC-treated trippe at room temperature for 3 hours.
The decomposed product is subjected to preparative and electrofocal gel electrophoresis. Proteins stain Coomassie with brilliant blue. In a separate experiment, the gel is cut into strips 3 mm wide and cut into # 1 gel soaked in 20 mM Tris-HC1 (pH) buffer for elgoproan protein.
As a result, it is determined that the decomposition product eluted from the excised gel, corresponding to a pH zone of about 0.3 lower than the isoelectric point of rF-TNF, has cytotoxic activity.
As a result, it is determined that the partial amino acid sequence of the decomposition product is as follows:
N-terminal: .JIHj -Thr-Pro-Ser-Asp-
C-terminal; --Ile-Ala-Leu-COOH.
As shown by the amino acid sequence, the degradation product is a polypeptide resulting from cleavage of four K-coicitic amino acids (Ser-Ser-Ar-Arg) from rF-TNF, and has the above cytotoxic activity. This means that at least four N-terminal amino acids in do not play a significant role in its biological activity.
Biological activity.
Cytotoxic activity against mouse L-Mo cell A sample (0, 1 ml) was serially diluted with the medium indicated below and Oj1 ml of the suspension of mouse L-M cells (100,000 cells per 1 ml) was added to each well of a multiwell plate. Use the minimum nutrient medium, Eagle, containing 1% fetal bovine moron. The plate is incubated at 3T C for 8 hours B floor with a humid atmosphere containing 5% carbon dioxide. After incubation, 20 µl of 25% glutavalantae are added to fix viable cells. After fixation, the plate is crushed and dried. After that, 0.1 ml of a 0.05% solution of methylene blue is added for staining of fixed cells. The excess methylene blue is washed and the plates are dried. Methylene blue
bout, swtmmed with fixed cells, eluted with 200 μl 0.36 n. HC1 and its absorption is measured at 665. Absorption is proportional
Q 5
Q
5 o
Q 5
five
the number of viable cells. The concentration of biological activity required to kill 50% of L-M cells is defined as 1 U / ml. Cytotoxic activity with respect to L-M cells, as determined in the indicated conditions, is expressed as units (LM) in order to distinguish from cytotoxic activity with respect to L-929 microbial cells as target cells.
Protein content is determined.
As a result, it is established that rF-TNF has a specific activity equal to 2-10 units (LM) and more per 1 mg of protein.
The antitumor effect on sarcoma Meth A transplanted to mice about
The antitumor effect on mice with Meth A sarcoma is assessed as follows.
BALB / C mash weighing about 23 g is intracutaneously transplanted with 200,000 cells of Meth A sarcoma. In the skin of the peritoneum and after 7 days, mice are taken from which the tumor was 6-7 mm in diameter. On the seventh day after tumor transplantation, rf-TNF was injected into a tumor mass or intravenously. The endotoxin content in the rf-TNF formulation was less than 0.01 ng per 1-10 units (LM) of cytotoxic activity.
When injected into the tumor mass, necrosis of the tumor graft is observed in all mice that have been given rF-TNF in doses of 110, 3-10 and 1-10 units (LM) per mouse for 24 hours after injection, and the tumor completely regresses in the amount of 3/5, 5/5 and 5/5, respectively, at each indicated above dose. With intravenous administration, necrosis is observed in all mice that received an injection of rF-TNF at a dose of 3 S and 1-S units (LM) per mouse, and the proportion of total regression is 3/5 and 4/5, respectively.
Inhibit the effect of rf-TNF on the growth of human tumor cells in vitro. The inhibitory effect of rf-TNF on the growth of human tumor cells and normal cells is assessed in vitro under the following conditions. Human tumor cells or normal cells are seeded at 10,000 cells per well R 1 ml of Eagle minimum nutrient medium containing 10% fetal bovine serum using a 24-well plate. RF-TNF was added at a resultant concentration of 1 OU units (LM) / ml and then cultured at 37 ° C for 4 days in a fully humid atmosphere containing 5% carbon dioxide. 4 days before the end of the culture, 1 µl of H-thymidine was added to each well. After cultivation, the cells are washed with phosphate-saline buffer and lysable with 0.5% sodium dodecyl sulfate. The amount of H-thymidine introduced into the cells is determined by measuring. p radioactivity of lysate. five
Inhibitory effect as a fraction of growth inhibition calculated by the following equation:
Growth inhibition ratio (%) (a - b) (a -100), where a and b represented
are radioactivity injected into cells, respectively, in the absence of RF-TNF and in the presence of RF-TNF,
The results, summarized in Tables 8, show that rF-TNF significantly inhibits the growth of human tumor cells, but does not affect normal cells. This observation indicates that rf-TNF selectively attacks tumor cells.
/
immunological properties. A solution of rF-TNF (100 units (LM) / mp) is mixed with an equal volume of 100 times diluted purified antibody against rabbit name. After incubation at 37 ° C for 2 hours, the cytotoxic activity of the reaction mixture is measured.
Table -I-I
-TSA GCT TST CGG GCC CTO AGT GAC AAG CTA GCC CAC CTA GTA GCA AAC CCG C A SAG GGC CAG CTC CAG TGG CTG AGC CAG
; . "
GCG AAC GCC CTG CTG GCC AAC GGC ATG CTC ACG GAC AAC CAG CTG GTG GTG CCG GAC GGG CTG TAC CTC ATC TAC TCC CAG CTC TTC AGC GGT CAA GGC TGC CGC TCC GTG CTC CTC ACT CAC ACT GTC C C C C C C C C ACT AAC AAG GTC AAC JTS TST GCC ATC AAG AGC CCC TGC SAC GAG ACC CCC GAG GAG GCT GAG CCC ATG TGG TAC GAG CCC ATC TAC CTG GGC GGC TGC CGG CGG AAG GG GTC CGG CTC CGG CTC ACC GAG GTC ATC CAG CG CAG CG CTC CGG CGG ATP CAG CGG AAG GAG CGG GTC CGG GTC CGG GTC CGG GTC CGG GTC CGG GTC CGG CAG ASC GAG GCC GAG TCC GGG CAG GTC TAC TTT ATC ATT GCC CTG- (3)
1-2 table
Ala Ser Arg Ala Leu Ser Asp Lys Pro Alaggle Ala Le Tyr ser gin val
 5161476516
. Table 1 Continuation 1
Le Gyme Gly Gly Gly Gly Cys ArgGr TyrGaGy Trp Tyr Glu Pro He Tyr Leu Gly, Gly Val Phe Gin Leu Gly Lys Gly Asp Arg Leu Gly
Table 2-1
(5) -AGC ACT GAG AGT ATC PBX CGG GAC GTC GAG CTG GCG GAG GGG CCG CTC CCC AAG AAG GCA GQG GGG CCC CAG GGC TCC AAG CGC TGC CTC TGC TCG CTC GTG CTC TTC TGC CTG CAC TTC AGG GTG ATC GGC CCT C.AG GAG / VA GAG CAG TCC CCA AAC AAC CTC CAT CTA GTC AAC CCT GTG GCC CAG ATG GTC ACC CTC CAC GTA GTA GCA AAC CCG CAG GTG GAG CGC CAG CAG CG CG CGTG AAG CAG CGT CGG AAC GTC A CG TGG ASC AAC GGC ATPG AAG CG ATS CG AAC CAG ATS CG CTG GTG ASC GG ASC GAC GG GGG ASC CTC ATC ATS CATC AUC AGC GGC TGC CGC TGC GTG CTC CTC CTC ACT GTC TGC AGC CGC TGC GTC GTC TGC ASC AAG AAG GTC AAC CTC ATC CTC TCG GTC ATC AAG GAG CCC PBX TAC.CTG GGC GGC GTC GTC GTC AAG CAG CGG AAG GGTG CGG CTC CGG AAC GAG CGG AAC CAG CG TAG GAC TG GG CTG GTC TG GTC GGG CAG GTC TAC TTT GGG ATG ATT GCC CTG- (34
1614763 and
..Tabzhtsa 2-2
Thr Glu Ser MetHeArgAsp Val Glu
Ala Glu Gly ProLeuProLys Lys Ala
Gly Pro Gin GlySerLysArg Cys Leu
Leu Ser Leu PheSerPheLeu Leu Val
Gly Ala Thr ThrLeuPheCys Leu Leu
Phe Arg Val HeGlyProGin Glu Glu
Gin Ser Pro AsnAsnLeuHis Leu Val
Pro Val Ala GinMetValThr Leu Arg
Ala Ser Arg AlaLeuSerAsp Lys Pro
Ala His Val ValAlaAsnPro Gin Val
Gly Gin Leu GinTrpLeuSer Gin Arg
Asn. Ala Leu LeuAlaAsnGly Met Lys
Thr Asp Asn GinLeuValVal Pro Ala
Gly Leu Tyr LeuHeTyrSer Gin Val
Phe Ser Gly GinGlyCysArg Ser Tyr
Leu Leu Thr HisThrValSer Arg Phe
Val Ser Tyr ProAsnLysVal Asn Leu
Ser Ala He LysSerProCys His Airg
Thr Pro Glu GluAlaGluPro Met Ala
Tyr Glu Pro HeTyrLeuGly Gly Val
Gin Leu Glu LysGlyAsp Arg Leu Ser
Glu Va.l Asn GinProGluTyr Leu Asp
Ala Glu Ser GlyGinValTyr Phe Gly, He Ala Leu
10 20 30
GGGGGGGGGGGGGGGCCCTCTGGAGAGAGCGdCATGAGCACTGAGAGTATGATCCGGGAC
CCCCCCCCCCCCCCCGGGAGACCTCTCTlCGCGGTACTCG GACTCTCATACTAGGCCCTG
70 80 90 100 110 120
GTCGAGCTGGCGGAGGGGCCGCTCCCCAAGAAGGCAGGGGGCCCCAGGGCTCCAAGCGCJ CAGCTCGACCGCCTCCCCGGCGAGGGGTTCTTGGTCCCCGGGTTGGGGTtiCGCG
. . "H,
Haell 130 140 150 160 170. 180
TGCCTCTGCCTCAGCCTCTTCTCTTTCCTGCTCGTGGCTGGAGCCACCACGCTCTTCTGC ACGGAGACGGAGTCGGAGAAGAGAAAGGACGAGCACGACCTCGGGGTGGAGAAGACG
 , Eabdatsa.
Haell 40 50 60
1 0200210220230240
CTGCTGCACTTCAGGGTGATCG.GCCCTCAGGAGGAAGAGCAGTCCCCAAACAACCTCCAT GACGACGTGAAGTCCCACTAGCCGGGTTTGTTTGTCGTCGGGTTTGTTGGAGGTA
250 260270280 | AvaI2-90300
STftSTHfi; i5SSTSTS5SES ™ TCAcicTCAGATCAGCTT4cGGGCCCTGAGTGAC
: GGGACTCACTG
GATCAGTTGGGACACCGGGTCTACCAGTGGGAGTCTAGTCGAAGAGCC
310 320 330 340 350 360
SSSSS C GTGGAGGGCCAGCTCCAGTGGCTGAGC TTCGGAGATCGGGTGCATCATCGTTTGGGCGTTCACCTCCCGGTCGAGGTCACCGACTCG
370 380 390 400 410 420
CAGCGTGCGAACGCCCTGCTGGCCAACGGCATGAAGCTCACGGACAACCAGCTGGTGGTG GTCGCACGGTTGGGGACGACCGGTTGCCGTACTTCGGTGCCTGTTGGTCGACCCCAC
430 440 450 460 470. 480
CCGGCCGACGGGCTGTACCTCATCTACTCCCAGGTTCTCTTCAGCGGTCAAGGCTGCCGC GGGCGGCTGCCCGACATGGAGTAGATGAGGGTCCAAGAGAAGTCGCCAGTTCCGACGGCG
490 500 510 520 530 540
TSSTiSS T CGCTTCGCCGtCTCCTACCCGAACAAGGTC AGGATGCACGAGGAGTGAGTGTGACAGTCGGCGAAGCGGCAGAGGATGGGCTTGTTCCAG
550 560 570 580, 590 600
f SSSTSSr CC GACCclcCGAGGAGGCTGAGCCC iTGGAGGAGAGACGGTAGTTCTCGGGGGGTGGCCCTCTGGGGCljCCTCCGACTCGGG
610 620. 630 640
Aval
650 660
..I t -x / - J J
ATGGCCTGGTACGAGCCCATCTACCTGGGCGGCGTCTTCCAGTTGGAGAAGGGTGACCGG TACCGGACCATGCTCGGGTAGATGGACCCGCCGCAGAAGGTCAACCTTCCCACTGGGCC
  , 680 690 700 710 720
CTCAGCACCGAGGTCAACCAGCCTGAGTACCTGGACCTTGCCGAGTCCGGGCAGGTCTAG GAGTCGTGGCTCCAGTTGGTCGGACTCATGGACCTGGAACGGCTqAGCCCGTCCAGATG
730 740 750. 760 770 780
TTTGGGATCATTGCCCTGTGAGGGGACTGACCACCACTCCTCCCCCTCTGCCACCCCAGC AAACCCTAGTAACGGGACACTCCCCTGACTGGTGGTGAGGAGGGGGGGGGGGGGTCG
790 800
CCCCTCACTCTGGGCGCCCTCAG GGGGAGTGAGACCCGCGGGAGTC
Continuation of table 3
: GGGACTCACTG
Aval
650 660
t -x / - J J
 1614765
  Table
GACCCACGG
-30-20-10-1
I
STSSASSSTSTSTSSSSSTOOLLLLASSASS
1 10 20. ZO I I I I
ATGAGCACTGAAAGCATGATCCGGGACGTG HetSerThrGluSerKetlleArgAspVal
40 50 60
GAGCTGGCCGAGGAGGCGCTCCCCAAGAAG GluLeuAlaGluGluAlaLeuProLysLys
70 80 90
ACAGGGGGGCCCCAGGGCTCCAGGCGGTGC ThrGlyGlyProGlnGlySerArgArgCys
100 110 120
TTGTTCCTCAGCCTCTTCTCCTTCCTGATC LeuPheLeuSerLeuPheSerPheLeuIle
130 140 150
GTGGCAGGCGCCACCACGCTCTTCTGCCTG ValAlaGlyAlaThrThrLeuPheCysLeu
160 170 180
CTGCACTTTGGAGTGATCGGCCCCCAGAGG LeuHisPheGlyVallleGlyProGlnArg
190 200 210 I I t
GAAGAGTTCCCCAGGGACCTCTCTCTAATC GliiGluPheProArgAspLeuSerLeuIle
220 230 240
AGCCCTCTGGCCCAGGCAGTCAGApCATCT SerProLeuAlaGlnAlaValArgSerSer
250 260 270
TCTCGAACCCCGAGTGACAAGCCTGTAGCC SerArgThrProSerAspLysProValAla
280 290 300
CATGTTGTAGCAAACCCTCAAGCTGAGGGGG HisValValAlaAsnProGlnAlaGluGly
310 320 330
CAGCTCCAGTGGCTGAACCGCCGGGCCAAT GlnLeuGlnTrpLeuAsnArgArgAlaAsn
340 350 360
GCCCTCCTGGGCAATGGCGTGGAGCTGAGA AlaLeuLeuAlaAsnGlyVaXGluLeuArg
231614765
Continuation of that
370380390
GATAACGAGCTGGTGGTGCCATCAG / GGGC AspAsnGlnLeuValValProSerGltiGly
400 410 420
CTGTACCTCATCTACTCCCAGGTCCTCTTC LeQTryLeuIleTyrSerGlnValLeuPhe
430 440 450
AAGGGCCAAGGCTGCCCCTCCACCCATGTG LysGlyGlnGlyCysProSerThrHisVal
460 470 480
CTCCTCACCCACACCATCAGCCGCATCGCC LeuLeuThrHisThrlleSerArglleAla
490 500 510
GTCTCCTACCAGACCAAGGTCAACCTCCTC ValSerTyrGlnThrLysValAsnLeuLeu
520 530 540
TCTGCCATCAAGAGCCCCTCCCAGAGGGAG SerAlalleLysSerProCysGlnArgGlu
550 560 570
ACCCCAGAGGGGGCTGAGGCCAAGCCCTGG ThrProGluGlyAlaGluAlaLysProTrp
580 590 600
TATGAGCCCATGTATCTGGGAGGGGTCTTC TyrGluProIleTyrLeuGlyGlyValPhe
610 620. 630
CAGCTGGAGAAGGGTGACCGACTCAGCGCT GlnLeuGluLysGlyAspArgLeuSerAla
640 650 660
GAGATCAATCGGCCCGACTATCTCGACTTT GlullcAsnArgProAspTyrLeuAspPhe.
670 680 690
GCCGAGTCTGGGCAGGTCTACTTTGGGATC AlaGluSerGlyGlnValTyrPheGlylle
700 710 720
ATTGCCCTGfrGAGGAGGACGAACATCCAAC lleAlaLo j)
730740
CTTCCCAAACGCCTCCCCTGC
WGAGCACTG / i LTSLSSASTO
GAGCTGGCCGAGG .VqGpGCTCCCCAAGAAG
; AGCTGGCGGAGG
: GCTCCCCAAGAAG
bfflllCCTCAGCCTCTTCT
Sh
: CTCAGCCTCTTCTCritrTCCTGldrC
150
GTGGd C
GTGG
CyGCACTT CTGCACTT
.GAAGAqШ1ССС
GAAGACSCAGfflGCCC
DGSYSYUSS SGSDYSSS
qrGGCCCAGj (
GJTGGCCCAGJ TGGTCJ
AGATCA AGATCJ
ft TU F GROWGD D
ri "LN JL X 4 J. v v: arVr
  U grotogl1l tl l L. J. i. C X CUvjVj
CTTCTCG {G (
ClpAGGQGCAGCTCC
jAGGQC CAGCTCCAGTGGCTGAGbgAG
GA KG
ZLT ACCAGCTGGTGGTTGdA 3AC ACCAGCTGGTGGTGCC
Table 5
/ i
F
Qq TGULTCCGGGACG IG GIV TGATCCGGGACG1C
: GCTCCCCAAGAAG
Sh
TTCCT
CritrTCCT
3CCACCACGCTCTTCTGCCTG 3CCACCACGCTCTTCTGCCTG
; TGATCGGCCCCpAGlAi
; TGATCGGCCC IICAGJG
CCCOGAGTGACAAG: CCI | GAGTGACAAG
Sh
ITAGCAAACCdTCAA TAGCAAACCCKCAA
27
G CCTCTTCAlAq G JT
:: TCTTC / feriG (3ccAAGGCTGcqcjc Ststlsl) aff rdAossstf | s
ASSSYG
GTGCTCCTCACCpACAQCATCAGC
 fnf f y-J-rJj i V 4 ".HC -" - "
TGCTCCTCAQllCACACTGTCAGC
CGqApCGCCGTCTCCTACq fel
CGqrlrcGCCGTCTCCTAca: b /
540
ACCTCCTCTCTGCCATCAAGAGCCCCTG C AACCTCCTCTCTGCCATCAAGAGCCCCTGC
570
CWGA
GGAGACCCCJb GGGGGCTGAQG
CAiCfGGAGACCCqqЗAGGAGGCTGAGf
600
JQCCTGGTA TCA GCCCATCTA / TCTGGdA .fifllqG {CCTGGT / j AGCCCATCTA | qCTG C
630
Fjij
TCTTCCAGOTGGAGAAGGGTGACCGA iTCTTCCAGJIJTCGAGAAGGGTGACCGJG
660
: TCAGCP: rcAG
21
SAOTfTTGCCGAGTOTlGGGCAGGTCTAC and 4S15CCGAGT CJGGGCAGGTCTA, C
TTTGGGATCATTGCCCTGTGA TTTGGGATCATTGCCCTGTGA
Note. Top lines; sequence of bases
coding human TNF-1 precursor.
Bottom line: the base sequence coding for the rabbit TNF precursor.
The areas enclosed in the rectangle are
homologous plot.
The designation shows the removal of the codon.
The designation xxx shows a breaking codon.
1614765
Continuation table 5
420
28
510
A
: AAGGTC: AAGGTC
29
Met Ser The Glu Ser 5th He Ser Thr GIU Ser Met He
Leu leu
Ala ala
Glu glu
Thr ala
Gly Gly Pro Gin Gly Ser Gly Gly Pro Gin Gly Ser

Phe cys
E
eu ser leu phe ser eu ser leu phe ser
Val Ala Gly Ala Thr Thr Leu Phe Cys
Gl glu
i
Gin
Phe ser
He ser val asn
Arg Ser Ser
Arg Ala Asn Ala Leu Ala Asn Gly
I
sp asn gin leu sp asn gin leu
1614765
thirty
Table b 10
Arg asp val arg asp val
Glu Ala Gly Pro
Leu Pro Lys Lys .eu Pro Ly.s Lys
Arg lys
Lgd cys rg cys
Phe leuj he phe leg leu
Arg asp
Asn asn
Leii ser led his
LeiP Leu
Ala
Val thr
Val leu
Thr pro ala leu
Ser Asp Lys Ser Asp Lys
110
Asn arg ser gin
120
Val met
130
 Val proj val val pro
31
predecessor of the human name, bottom line: the predecessor of the rabbit name. The sections encircled by a rectangle are a homologous region. The designation shows the removal of the amino acid.
16i4765. | 2
Continuation of table 6
140
33
bladder 1 (ATCC CRL 1500) carcinoma
mammary gland (ATCC CRL 1543) osteogenic sarcoma (ATCC CCL 218) colon adenocarcinoma (ATCC NTV 22) mammary adenocarcinoma (ATCC CRL 1440) renal
Leiomyblastoma 1 (ATCC CRL 1469) epitheloid carcinoma (ATCC CCL 2) epitheloid
carcinoma
161A765
Table
34
7
Table 8
49 97 47 37 69 98 59 31
1614765
 Table
10 20 30 GGGGGGGGGGGGGGGCCCTCTGGAGAGAGC 40 50 60
GCCATGAGCACTGAGAGTATGATCCGGGAC MetSerThrGluSerHetlleArgAsp
70 80 90 I I I
GTCGAGCTGGCGGAGGGGCCGCTCCCCAAG ValGluLeuAlaGluGlyProLeuProLys100 110 120 I I (
AAGGCAGGGGGGCCCCAGGGCTCCAAGCGC LysAlaGlyGlyProGlnGlySerLysArg
130 140 150 t I I
TGCCTCTGCCTCAGCCTCTTCTCTTTCCTG CysLeuCysLeuSerLeuPheSerPheLeu
160 170 180
CTCGTGGCTGGAGCCACCACGCTCTTCTGC LeuValAlaGlyAlaThrThrLeuPheCys
190 200 210
CTGCTGCACTTCAGGGTGATCGGCCCTCAG LeuLeuHisPheArgVallleGlyProGln
220 230 240
GAGGAAGAGCAGTCCCCAAACAACCTCCAT GlUGluGluGlnSerProAsnAsnLeuHis
250 260 270
CTAGTCAACCCTGTGGCCCAGATGGTCACC LeuValAsnProValAlaGlnMetValThr
280 290 300
CTCAGATCAGCTTCTCGGGCCCTGAGTGAC LeuArgSerAlaSerArgAlaLeuSerAsp
310 320 330
AAGCCTCTAGCCCACGTAGTAGCAAACCCG LysProLeuAlaHisValValAlaAsn Pro
340 350 360
CAAGTGGAGGGCCAGCTCCAGTGGCTGAGC GlnValGluGlyGlnLeuGlnTrpLeuSer
370 380 390
CAGCGTGCGAACGCCCTGCTGGCCAACGGC GlnArgAlaAsnAlaLeuLcuAlaAsnGly
 I61a76538
IIIKLL (: I, Kenya Table 9
400 410 420 I t
ATGAAGCTCACGGACAACCAGCTGGTGGTG MetLysLeuThrAspAsnGinLeuValVal
430 440 450
CCOGCCGACGGGCTGTACCTCATCTACTCC ProAlaAspGlyLeuTyrLeuIleTytSer
460 470 480
CAGGTTCTCTTCAGCGGTCAAGGCTGCCGC GlnValLeuPheSerGlyGlnGlyCysArg
490 500 510
  I TCCTACGTGCTCCTCACTCACACTGTCAGC SerTyrValLeuLeuThrHisThrValSer
520 530 540 t t
CGCTTCGCCGTGTGCTACCCGAACAAGGTC ArgPheAlaValSerTyrProAsnLysVal
550 560 570 I I I
AACCTCCTCTCTGGCATCAAGAGCCCCTGG AsnLeuLeuSerAlalleLysSerProCys
580 590 600
CACCGGGAGACCGCCGAGGAGGCTGAGCCC TiisArgGluThrProGluGluAlaGluPro
610 620 630 I II
ATGGCCTGGTACGAGGCCATCTACGTGGGC MetAlaTrpTyrGluProIleTyrLeuGly.
640 650 660
GGCGTCTTCCAGTTGGAGAAGGGTGACCGG GlyValPheGlnLeuGluLysGlyAspArg
670 680. 690
CTCAGCACCGAGGTCAACCAGCCTGAGTAC LeuSerThrGluValAsnGlnProGluTyr
700 710 720
CTGGACCTTGCCGAGTCCGGGCAGGTCTAC LeuAspLeuAlaGluSerGlyGlnValTyr
730 740 750
TTTGGGATCATTGCCCTGTGAGGGGACTGA PhcGlyllelleAlaLeu
760 770 780 CCACCACTCCTCCCCCTCTCCCACCCCAGC
790 800 CCCCTCACTCTGGGCGCGCAG
Preparation of i-RNAs of name from human alveolar macrophages.
Human alveolar macrophages are harvested by broncho-alveolar washing with phosphate buffered saline. Alveolar macrophages, 6.3-10 cells, are suspended in RPMI-1640 medium containing 10% fetal bovine serum, and seeded in petri dishes (8 cm in diameter) at a cell concentration of cells per dish. They are pre-cultured at 37 ° C in a fully humid atmosphere containing 5% carbon dioxide. After 1 hour of cultivation, endotoxin (a lipopolysaccharide derived from E. coli), TPA (forebol-12-myristate-13-acetate) and cycloheximide (synthesis inhibitor protein) is added to the plates in such a way that their resulting steel concentrations are 10 µg / ml, 10; ng / ml and 1 μg / ml, respectively. After that, the cultivation is continued for 4-4.5 hours (total time 5-5.4 hours). The culture medium is removed by suction, and macrophages adhered to the plates are lysi-pyjoT and homogenized in a 5 M guanidyl cyanate solution containing 0.6% N-lauroyl sarcosinate sodium and 6 mM 1 of sodium sludge. A pp; kaK T homogenate in a 5.7 M cesium chloride solution containing 0.1 M EDUC is centrifuged for 20 hours at 26,500 rpm using an ultratrifuge to obtain a total fraction of RNA in the form of granules. The granules are dissolved in a small amount of a 7 M urea solution containing 0.35 M NaCl, 20 mM Tris-HC (pI 7.4) and 20 mM EDTA, and separated by precipitation from ethanol. I get 159 hasg of total RNA,
The total RNA fraction is dissolved in 1-110L of HGL Tris-HCl (pH 7.4), buffer., Containing 1 mM EDTA (indicated as TK solution), and the solution is heated for 5 minutes. A solution of sodium chloride is added to a resultant concentration of 0.5 M, and the solution is applied to a column with oligo (lT) -cellulose equilibrium state with respect to the TE-solution containing 0.5 M chloris6 µg of poly (A) -i-RNA dissolve 40 µl of bufgra 50 mM Tris-HC (pH8 containing 10 mM MgClg, 10 mM dith
sodium, Poly (A) and RNA are eluted from 55 treitol, 4 mM sodium pyrophosphate.
columns using a TE-solution and hemlock 8 M1SG „
Poly (A) -i-RNA is dissolved at a concentration of 1, -9 ng / nl in distilled1,25 mM each of three deoxyrib nucleotide triphosphates, dGTP, dATP and dTTP, 0.5 mM dCTP, 167 nM alpha-dCTP (specific radioactivity
water and the solution is injected into Xenopus laevis oocytes at a dose of approximately. 50 nl per ooi; and microinjection method. Ten oocytes are incubated in 100 µl of Barth medium at 22 ° C for 24 hours. The oocytes are homogenized and centrifuged at 10,000 rpm for 10 minutes. The supernatant is analyzed for FIO activity by determining cytotoxic activity by cell ratio: mouse L-929.
Method for measuring cytotoxic activity with respect to cells
L-929 is as follows.
A sample (O, 1 ml) was serially diluted with the medium indicated below, and O, 1 ml of L-929 cell suspension was added to each well of a 96-well plate.
(cells / ml) containing actinomycin D (2 μg / ml). Use the minimum nutrient medium Eagle containing 1% fetal bovine serum. The plate is incubated at
38.5 ° C for 18 h completely. a humid atmosphere containing 5% carbon dioxide,
Methods for determining the number of viable L-929 cells and evaluating biological activity are the same as methods for analyzing cytotoxic activity using. L-M cells as target cells, Cytotoxic activity against L-929 cells, as determined under the above conditions, is expressed as units (L-929) in order to distinguish it from cytotoxic activity with respect to murine L-M cells,
The supernatant obtained as described above has a cytotoxic activity of 6.6 units (L-929) per ml. This indicates that the sample of poly (A) -and-RNA contains i-RNA name,
Synthesis of c-DNA
Complementary DNA is synthesized using 50 using the obtained poly (A) -i-RIC as a matrix,
6 µg of poly (A) -and-RNA is dissolved in 40 µl of buffer, 50 mM Tris-HC (pH8.3), containing 10 mM MgClg, 10 mM dithio5 treitol, 4 mM sodium pyrophosphate.
treitol, 4 mM sodium pyrophosphate.
1.25 mM of each of the three deoxyribonucleotide triphosphates, dGTP, dATP and dTTP, 0.5 mM dCTP, 167 nM alpha R-dCTP (specific radioactivity
3000 Ci (NtMonb), - 4 µg of oligo (dT) and 120 units of reverse transcriptase, obtained from the avian myeloblastosis of birds (HS-i), and incubated at 43 C for 30 minutes. After this, the reaction is stopped by adding EDTUK. The reaction mixture is extracted with phenol-chloroform (1: 1) and ammonium acetate is added to the aqueous phase to a resulting concentration of 2.5 M. The resulting hybrid of c-DNA-and -RNA is separated from the water phase by precipitation from ethanol. DNA-RNA is dissolved in 10 µl of 20 mM buffer-15 dinene to the end of dcc-DNA (for Tris-HCl (pH 7.5) containing 5 Mf
MgClI, 10 mM ammonium sulfate, 100 mM
potassium chloride, 0.15 mM betanicotinamide dine dinucleotide, 5 μg
bovine serum albumin,
0.04 mM of each of the four deoxyribon5psleotid triphosphates, dHGP, dATP,
dTTP and dCTP, 0.9 units, ribonuclease N.
; E. so11 and 23 units. DNA polymerase I,
by the fact that it contains 80 units of terminal deoxynucleotide-diltransferase and H-dGTP instead of P-dCTP) and incubated at 374 for 20 hours 20 minutes, attaching approximately 10-15 desoaxiguanidic acid residues (dG) to the 3-end m. The reaction mixture is exchanged by a mixture of phenol-chloroform and DN
| E, coli, and incubated at 12 ° C for 25 pBR 322, terminating in oligo (dG)
35
60 min, and then another 60 min at 22 ° C for the synthesis of dic-DNA. The reaction is stopped by adding EDTA, DNA-DNA is extracted with phenol-chloroform and separated by precipitation of 30 from ethanol.
Preparation of c-DNA with oligo (dC) -end
DNA-DNA was dissolved in 100 µl of 100 mM sodium cacodylate buffer solution (pH 7.2) containing 2 mM CoCl2, 0.2 mM dithiothreitol, 0.1 mM alpha P-dCTP (specific radioactivity 3 Ci (mmol) and 10 units of terminal deoxynucleotidyltransferase-40zy, and incubated for 30 min to achieve the attachment of oligo (dC) termini to the 3 termini of DNA-DNA,
The reaction is stopped by the addition of 45 EDTA DNA-DNA terminating oligo (dC),
 extracted with phenol-chloroform and isolated by precipitation from ethanol of DNA-DNA, terminating in oligo (dC),
dissolved in 10 mM Tris-HCl 50 CpH 7.4) buffer containing 1 mM EDTA and 100 mM sodium chloride to give the resulting dna-DNA concentration terminated in oligo (dC) equal to 2 µg per 1 ml
is isolated from the aqueous phase by precipitation with ethanol. The resulting pBR 322 DNA with the attached termini is dissolved in the same buffer that was used to dissolve the oligo (dC) —end DNA-DNA so that the resulting concentration of the pBR 322 DNA with the attached termini was 20 μg / ml .
Constructing recombinant plasmids,
120 µl of oligo (dC) -terminal c-DNA solution is mixed with an equal volume of oligo (dG) -terminal DNA solution pBR32 and the mixture is incubated sequentially at 65 ° C for 5 minutes and at 57 s for 120 min to achieve renaturation and construct recombinant plasmids.
Selection of host organisms
Using the recombinant plasmids obtained above, the E. coli X 1776 strain is transformed.
E, coli X 1766 is cultivated at 37 ° C in 20 MP b-brooia (composition: 10 g tryptone, 5 g yeast extract, 5 g sodium chloride
and 1 g of glucose
55
on 1 l; pH 7.2), containing 100 µg / m of diaminopimelic acid and 40 µg / ml of thymylin, until the turbidity, measured at 600 nm, reaches 0.5. The cots are harvested by centrifugation at 10 ml of 10 mM Tris-IS1 buffer (pH 7.3) containing
Obtaining pBR 322 DNA terminating in oligo (dG).
10 µg of pBR 322 DNA was dissolved in 100 µl of 20 MiM Tris-HCl buffer (pH
7.4) containing 10 mM magnesium chloride, 50 mM ammonium sulfate and; 10 kg of bovine serum albumin, and 15 Pst I restriction endonucleases are added. The mixture is incubated at 37 ° C for 1 hour. After completion of the reaction, the reaction mixture is extracted with phenol-chloroform and the resulting DNA is separated from the aqueous phase by precipitation from ethanol, The obtained DNA is dissolved in 200 µl of the same buffer solution, which was used above, to attach to the end of dkk-DNA (except that it contains 80 units of terminal deoxynucleotide-transferase and H-dGTP instead of P-dCTP) and incubated at 374 in about 20 minutes, attaching approximately 10 to 15 dezoaxiguanidic acid (dG) residues to 3-termini. The reaction mixture is extracted with a mixture of phenol-chloroform and DNApBR 322 ending in oligo (dG),
five
0
0
five
0
is isolated from the aqueous phase by precipitation with ethanol. The resulting pBR 322 DNA with the attached termini is dissolved in the same buffer that was used to dissolve the oligo (dC) - terminal DNA-DNA so that the resulting concentration of the pBR 322 DNA with the attached termini is 20 µg / ml.
Constructing recombinant plasmids,
120 µl of oligo (dC) -terminal c-DNA solution is mixed with an equal volume of oligo (dG) -terminal DNA solution pBR322 and the mixture is incubated sequentially at 65 ° C for 5 minutes and at 57 s for 120 min to achieve renaturation and construct recombinant plasmids.
Selection of host organisms
Using the recombinant plasmids obtained above, the E. coli X 1776 strain is transformed.
E, coli X 1766 is cultivated at 37 ° C in 20 MP b-brooia (composition: 10 g tryptone, 5 g yeast extract, 5 g sodium chloride
and 1 g of glucose
five
on 1 l; pH 7.2), containing 100 µg / ml of diaminopimelic acid and 40 µg / ml of timylin, until the turbidity, measured at 600, reaches. 0.5 The cots are collected by centrifugation at 10 μm — 10 mM Tris-IS1 buffer (pH 7.3) containing
50 mM calcium chloride. The cells are resuspended in 2 ml of the same buffer solution and left to stand for 5 minutes. To the 0.2 ml of suspension, O, 1 ml of the solution of recombinant plasmids is added. The mixture was left to stand for 15 NOTH and then held at 42 ° C for 2 mn. Then feed 0.5 ml of L-broth with additives, cultivated by shaking for 1 hour. An aliquot of the culture was removed. , applied to an agar plate with L-broth containing supplements containing tetracycline at a concentration of 15 µg Ump, and cultured at JUS for approximately 12 hours. The k-DPC kit is obtained by selecting transformed cells resistant to tetracycline,
Cloning k-dick of human TNF
Transformed cells incorporating recombinant plasmids containing c-DNA encoding a human TNF polypeptide are selected from the c-DNA kit by hybridization analysis using DNA fragments derived from cloned c-DNA that encodes rabbit TNF as samples.
The cDNA encoding rabbit TNF was isolated from recombinant pK TNF 802 plasmid. The base sequence is presented in Table. 3. Cleaved with restriction endonuclease Na I or Naa I, Split DNA fragments were precipitated from ethanol. They are subjected to polyacrylamide gel electrophoresis to isolate target DNA fragments.
The DNA fragment (299 PoO.), Corresponding to bases from 285 to 583, is shown in Table. 3, was obtained by digesting the Ava I restriction endonuclease (designated as Ava 1 fragment) by a DNA fragment (88 bp), corresponding to bases from 33 to 120, as shown in Table 2. 3, is obtained by cleavage with the restriction endonuclease Haye II (denoted as Hay 11 fragment). Ava 1 fragment and Nae 11 fragment are labeled with a radioactive label P. These labeled DNA fragments are used as a test to check for the c-DNA kit and select transformed cells that have a plasmid containing c-DNA
the human TNFα polypeptide, a columnar hybridization assay according to the method of Hanahan and Meselson.
lou
using a P-labeled Ava 1 fragment, 43 clones are selected from approximately 20,000 clones as a sample for the first selection. In addition, the 43 selected clones are subjected to a second selection using
32.
using a Na-labeled 11-fragment as a probe.
Using these analyzes, 6 clones are selected that have; Combinational plasmids strongly hybridizing to DNA using both fragments of the rabbit TNF.
Express.
The recombinant plasmid DNA was isolated from 6 transformed organisms, namely: plasmid No. pHTNF 1, pHTNF 4, pHTNF 5, pHTNF 13, pHTNF 22 and pHTNF 26, respectively. Each of the recombinant plasmids was introduced into E. coli 11 HB101 to obtain transformed organisms containing recombinant plasmids.
Transformed cells are cultivated in 50 ml of LB-broth (composition: 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride per liter; pH 7.5) until the turbidity of the culture, measured at 600 them, reaches approximately 0.8 .. After that, collect approximately 3-5 x10 cells. Cells are lysed with small modifications of the procedure. Cells are suspended in 1 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 0.1% lysozyme and 30 mM NaCl. After standing for 30 minutes in ice water, the cells are lysed by sixfold freeze-thawing. Cell residues of y are yielded by centrifugation and a clear lysate is obtained. The lysate obtained from each transformed organism is analyzed for cytotoxic activity against L-929 cells.
It was found that the lysate obtained from a transformed organism containing pHaznip pHTNF 13 has a cytotoxic activity of 186.1 units (b-929) / ml.
Determination of base sequence in cloned cDNA.
The recombinant plasmid pHTNF 13 was isolated as described above. .
the mid DNA is cleaved with the aid of the Pst I restriction eidonuclease oo and the cloned cDNA inserted into the vector is isolated. Thereafter, the cloned cDNA fragment is cleaved with various restriction endonucleases, and the base sequences of the resulting 16 fragments are determined by the Maxam-Gilbert method using L-929 cells as target cells. Rabbit plasma TNF was prediluted with phosphate buffered saline and the diluted solution was used as a control sample of TNF. As the presented results show (see Table 10), the cytotoxic activity of the polypeptide — human TNF — is not neutralized by the antibody.
Table 10
bratsy
Antibody against rabbit plasma TNF
Cytotoxicity activity units (L-929) / ml
25
thirty
35
Lysate from tran- Not added - 186.1
formed elk
th organism
containing
plasmid
pHTNF 13 Added 191.0
KROLICH SH
plasma
RNOH not added 572.3
Added CO, 1
 The cytotoxic activity is shown as the activity in the solution of the original sample used for the test. PRIME 5.
Production of human TNF polypeptide in Escherichia coli, -
Expression under the control of the tac promoter.
The cloned kDNA was isolated from the recombinant pHTNF 13 plasmid. 50 kDNA was then cleaved with the restriction endonuclease Eco RI to remove a portion of the non-coding region below the TNF-encoding region. The resulting DNA fragment (approximately 55.1 tons, p.o.) is inserted into the larger DNA fragment obtained from the plasmid pBR 322 by restriction digestion.
15
20
l
25
thirty
five


0 5
endonyclease Pst I and Eco R 1 and cocht the recombinant plasmid, which is inserted into the TNF DNA DNA and the tetradiclin y resistance gene called pHT 113.
K-DNA isolated from pHT 113 is digested with Ava I and Hind III restriction endonucleases and the resulting DNA fragment (578 bp), which includes the majority of the cd-dividing region of the full human TNFα polypeptide (called CFNF fragment) are isolated by polyacrylamide gel electrophoresis.
The DNA fragment comprising the tac promoter region is isolated as follows. Plasmid DNA (300 µg / pDR 540) is dissolved in 2 ml of 10 MiM Tris-HCl buffer (pH 7.5) containing 50 mM NaCl, 6 mM MgCl2 and bMM 2-mercaptoethanol, and is digested with Eco RI restriction endonucleases. and Vaga HI incubation at 37 ° C for 60 min. After addition of sodium chloride to the resulting concentration of 0.3 M, the cleaved DNA fragments are extracted from ethanol, the DNA fragment, including the promoter region. tac, are separated by polyacrylate gel electrophoresis with a yield of 8.3 µg.
A fragment of the tac promoter is associated with a chemically synthesized oligodeoxyribonucleotide adapter represented by the following formula:
5 -GATCCATGTCATCTTCTCGAACC 3 -GTACAGTAGAAGAGCTT ;; GGGCT
This adapter includes the initiating ATG codon and the corresponding base sequence. 5 is the terminal portion of the base sequence that encodes the complete polypeptide of human TNF, and has Wat HI and Ava I co-ends. The resulting DNA fragment is called a tac - promoter-adapter fragment.
Separately, a larger DNA fragment (approximately 4.3 kb), which includes the ampicillin resistance gene (called the pBR 322-Amp fragment, is cut out of the plasmid pBR 322 by cleavage with restriction endonucleases Hind III and Eco RI and isolated gel electrophoresis using agarose (0.7%) with a low thermal softening.
1 µg of cfnof-fragment and 6 µg of pBR 322 - / - 1mp fragment are dissolved in 66 mM
Tris-HCl buffer (pH 7.6) containing 6.6 mM magnesium hporst and incubated at 55 s for 10 min. After that, ATP and dithiothreitol are added, respectively, to concentrations of 1 and 10 mM, 168 units of T DNA ligases are added and the mixture is incubated at 22 ° C for 120 minutes. The resulting DNA fragment was extracted by pheno extraction; scrap and get about 4 micrograms. The DNA fragment (0.8 µg) is bound to the tac-promoter-adapter fragment (0.3 µg) under the same conditions as described above, except that. use 63 units of T DIC-ligase.
The reaction mixture is diluted 6 times.
: distilled water and dares to
 equal volume of cell suspension
lE.coli IM103 / P-1i, Calcined Enlightened Lysate.
Transformed 1M103 / pHTT26 cultivations in LB broth at
s (0.5 MP) are seeded in 5 she LB and isopropyltozide is added at 37 ° C for 1 minute to result
10 1 mM and culture for 4 hours. The cells are pensed in 1 ml of 50 m (pH 8.0) containing both 30 mM NaCl and oc-j av
15 for 30 min. dry ice is repeated six times over the mixture - this is live at, the glue is removed by centrifugals
: this is the icy way. The mixture is subsequently incubated in ice water; for 20 minutes, with with for; 1 min and at room temperature for 10 min and add LB broth. The mixture is shaken at 37 ° C for 60 pm. An aliquot of the resultant cell suspension is applied to LB-ara. The lysate has a cytotoxic activity of 99,000 units (L-929) / ml. This cytotoxic activity is not neutralized by the antibody against 25 plasma TNF. This confirms that the human TNF polypeptide immunologically does not intersect with rabbit plasma full name. Expression under promoter control is degraded with restriction endonucleases Ava I and Sal I for cleavage into 3 fragments (size approximate plates containing ampicillin at a concentration of 25 µg / ml, and culture of ra trp.
tiviruk T overnight at 37 ° C, OT-recombinant plasmid pHT 113 collect ampicillin-resistant colonies.
One of the transformed organisms capable of producing a polypeptively 0.8, 1.3 and 2.6 kp.). Fragtide of human TNF, was named ment 1.3 1.3 k.po. - DNA comprising in MM103 / RITT26 „most of the region encoding the complete human polypeptide
The calcium-treated E. coli phn and a portion of the tet-IM103 resistance gene are prepared as follows, the racycline is allocated (denoted as E. coli IM103 cells are deposited in Ava I-Sal I fragment) Ava I-Sal I-cH and cultured at 37 ° C, the fragment is bound to the next, the night. 1 ml of the resultant culture of the synthesized oligodeoxy is sown in 100 ml of LB broth using a N. dopolrybonucleotide adapter. This adaptive culture is cultivated at 27 ° C until it is designated as an adapter 1 until the turbidity of the culture, defined by 5 -CGATATGTCATCTTCTCGAACC, at 650 nm, reaches 0.6, 3 - j: ATACAGTAGAAGACCTTGGGGCT After standing for 30 min in ice water, cells are harvested by centrifuge. The resultant DNA fragment is designated as the CFNO adapter fragment. 50
girovaniya and suspended in 50 ml. A 50 mM calcium chloride solution is then held at 0 ° C. for 60 minutes, the cells are collected by centrifugation and resuspended in 10 ml of a 50 mM calcium chloride solution containing 20% glycerol, and this suspension is used as a calcium-treated cell suspension E .coli IM103.
55
Separately, a DNA fragment (35 p, o,), including a portion of the trp promoter region, is cut off from the PDR 720 / P-L receptor by cleavage with restriction endonucleases Eco R I and Hp. I, and the DNA fragment is bound to a chemically synthesized adapter having the following formula:
Transformed organism 1M103 / pHTT26 cultured overnight in LB broth at 37 ° C, Culture
(0.5 MP) are sown in 5 ml of fresh LB broth and further cultured at 37 ° C for 1 hour. Isopropyl beta-B-thiogalactoside is then added until the resulting concentration is reached.
1 mM and cultivation is continued for another 4 hours. Cells are harvested and suspended in 1 ml of 50 mM Tris-HC buffer (pH 8.0) containing 0.1% lysozyme and 30 mM NaCl, and the oc-j is allowed to stand at
 within 30 min. Thereafter, freezing in a dry ice-ethanol mixture is repeated six times and thawing at, cellular residues are removed by centrifugation to obtain
The lysate has a cytotoxic activity of 99,000 units (L-929) / ml. This cytotoxic activity is not neutralized by the antibody against CRH plasma TNF. This confirms the fact that the human TNF polypeptide does not immunologically intersect with rabbit plasma name, Expression under control trp promoter.
decompose with Ava I and Sal I restriction endonucleases for cleavage into 3 fragments (approximate size Recombinant plasmid pHT 113
clearly 0.8, 1.3 and 2.6 kp. o.). Fragment ment 1.3 k.po. - DNA, comprising most of the coding region
Separately, a DNA fragment (35 p, o,), including a portion of the trp promoter region, is cut off from the PDR 720 / P-L receptor by cleavage with restriction endonucleases Eco R I and Hp. I, and the DNA fragment is bound to a chemically synthesized adapter having the following formula:
5 -AACTAGTAC (; CMGTT (; ACGTAAAAAGGGTTMT 3 -TTGATCATGCGTTCAAGTGCATTTTTATATATAG
Bound DNA pht is designated as a fragment of the trp, - promoter.
Plasmid pBR 322 was cleaved with restriction endonucleases Eco RI and Sal I and the larger DNA fragment was isolated (approximately 3.7 kb). By successively linking these three DNA fragments, the CFNO adapter fragment, the trp promoter fragment, and the larger pBR 322 fragment, an expressional plasmid pHTR 91 is constructed. The expressive plasmid is introduced into E. coli HB101 cells in a manner and one of the transformed organisms is named HBIOI / pHTR 91, TpaHcmopNMpOBaHHM organism HB101 / pHTP 91 is cultured overnight at 37 ° C in modified medium M-9 (composition: O, 7% 1 2H20; 0.3% 0.05% NaCl; 0.1% 2 mg / ml of vitamin 0.5% casamic acid, 1 mM O, 1 mM CaClg and 0.5% glucose). The culture (0.05 ml) is sown in 5 ml of the same medium and cultured at 37 ° C for 1 hour. After that, 3-beta-indole-acrylic acid and another resulting concentration of 20 µg / ml is added and the cultivation is continued for 4 h. Cells are harvested and processed according to the described method and receive an enlightened lysate.
The lysate has a cytotoxic activity of 1.01-10 units (L-929) per 1 ml.
Expression under the control of the phoS promoter.
The obtained Ava I – Sal I fragment is associated with a chemically synthesized deoxyribonucleotide jpoM adapto, having the following sequence:
5 -СATSSATSTSATSTTSTSSAASS
Z -STLASASASAASASSTTSSSSSSST
The bound DNA fragment was cleaved with the restriction endonuclease Wat H I, and a DNA fragment was obtained that had cohesive ends of Vaga H I at both ends (designated as CSFN-Tet Vaga H I fragment).
Separately, plasmid pSN 5182, containing the phoS gene ,; digested with a hpA I and Eco R1 reetri- cation endonuclease to obtain a DNA fragment (approximately 4 kp), including
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and the phos promoter region and the Shine-Dalgarno sequence, the DNA rtoc sequence, the lean signal peptide and the N-terminal part of the phosphate-binding protein, and the tetracycline resistance gene.
This DNA fragment is designated as the Hpa I –Eco R, I fragment, the Hpa I – Eco RI fragment is linked to a chemically synthesized binder oligodeoxyribonucleotide having the following sequence: 5 -CCCGGATCCGOG -3 -GGGCCTAGGCCC After that, the linked DNA fragment are restriction endo. luclase Wat H 1. The resulting DNA fragment (approximately 3.6 k.Po o), which has cohesive ends of Vaga H I at both ends, is designated as a fragment of the Phos promoter Vaga N I, Tet,
The IFNO-Tet fragment, Wat HI, is linked to the Phos promoter - Vaga H I, Tet fragment, and an expression plasmid is constructed to produce a human TNFα polypeptide linked to a phosphate-binding protein, which is called pHTS 115, an expression plasmid is introduced into E. coli HB-IOI according to the method described above.
One of the transformed organisms (HB101 / PHTS 115) is cultured in 5 ml of TG medium (composition: 120 mM Tris-HCl buffer, pH 7.4, containing 0.2% glucose, 80 mM NaCl, 20 mM KCl, 20 1.W , 3 mM Ma., 1 mM MgCi ,, 0.2 mM CaC, 200 nM FeCl, 20 mg / l leucine, 20 mg / l proline and 10 mg / l vitamin B), which is present in KH 14) 4 at a concentration of 0.64 mM, at 37 ° C for 20 hours, with shaking. The cells are harvested by centrifugation, resuspended in 2 ml of TG medium containing 0.064 mM in concentration, and cultured for 6 hours. The cells are collected by centrifugation and rinsed with 1 ml of 10 mM Tris-HCl buffer (pI 7.2) containing 30 mM NaCl. After that, they are again suspended in 0.2 m 33 mM TRIS-HCl buffer (pH 7.2) and mixed with an equal volume of 33 mM Tris-HCl buffer (pH 7-, 2) containing O, 1 mM EDTA and 40% sucrose. After incubation at 37 ° C for 10 min, the cells are harvested, resuspended in 0.4 ml of ochol 51, and a 0.5 mM solution
MgCl 2 and allowed to stand in ice water for 10 minutes with occasional shaking. The cell residues are removed by centrifugation and a clear extract is obtained (hereinafter referred to as a periplasmic extract).
The periplasmic extract has a toxic activity of 1.3440 units (L-929) per ml.
The molecular weight of a polypeptide having a cytotoxic activity, as determined by polyacrylamide gel electrophoresis (12.5%) with sodium dodecyl sulfate, is J9000 dapton, which indicates that the polypeptide is obtained as a combined protein.
Expression under the control of the PhoS promoter.
The recombinant plasmid DNA (pHTNF 13) is cleaved with a restriction: Pst I endonuclease for cutting .1 DNA fragment (approximately 1.2 tons, bp), including human F-DNA. The DNA fragment is then cleaved with the restriction eubonuclease BbE1 and a DNA fragment is obtained (approximately 0.9 ppb; then 060C is meant as the Bblue I – Pst I fragment) 6 μg of the Ble I fragment — Pst I is dissolved in 80 μl of 20 mM buffer (pH 8.0) containing i2 mM CaC, 12 mM MgCl., 0.2 M NaCl, 1 mM EDTA and 0.02 units of Bal 31 exopuclease, and incubated at 30 ° C for 30 min for cleaving approximately 50-150 base pairs from each end of the DNA fragment. Then the ends of the fragment are again increased by incubating at 37 ° C for 60 minutes with 10 units of IEcoli DNA polymerase (large fragment) in the presence of each of the four deoxyribonucleotide triphosphates, dGTP, dTTP, dCTP and dTTP taken at a concentration of Oj1 mM and 50 µg / ml bovine serum albumin. The DNA fragment with extension ends is cleaved with the restriction endonuclease ECO RI and the resulting DNA fragment (approximately 750 i.e., having a blunt end, and Eco RI is a thin horse isolated with a yield of approximately 3 µg (denoted as Bal 31 - Eco R 1 fragment) .
Bal 31 - Eco R 1 fragment is bound to Hpa I fragment - Eco R I
Mr. JQ
s
20
25 30 Q Q
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(approximately 4 kb), obtained from pSN 5182, and construct an expressive plasmid containing the phoS promoter region, the Shine-Dalgano sequence, the DNA sequence encoding the signal peptide and the N-terminal part of the phosphate binding protein, and the DNA sequence which is the common full polypeptide of human TNF and a part of its polypeptide, which is a procession.
The expression plasmid is called pHTS 37 and introduced into E. coli HB101 to produce a transformed organism, which is called HBIOI / pHTS 37. The transformed organism HB101 / / pHTS 37 is cultured and the periplasmic extract is obtained in the same conditions.
The obtained periplasmic extract has a cytotoxic activity of 4.93 "10 units (L-929) per 1 ml.
The extract is subjected to polyacrylamide gel electrophoresis (12.5%) with sodium dodecyl sulfate. Electrophoresis was carried out at 35 V for 12 hours using tris-glycine (pH 8.3) buffer containing 0.1% sodium dodecyl sulfate. Protein is stained with Coomassie brilliant blue. Two protein bands not observed in E. coli HB101 extract are observed at positions corresponding to 22,000 and 16,500 daltons. Separately, the gel is cut into pieces of 2 mm width and the calceous piece of gel is shaken in Eagle's minimal nutrient medium containing 1% fetal bovine serum, overnight to elute the protein from the gel. Carbohydrate eluate is used to determine cytotoxic activity against L-929 microbial cells. As a result, it is determined that strong cytotoxic activity is observed in the eluate obtained from a gel cut from a position corresponding to a protein stained with Coomassie brilliant blue and having an established molecular weight equal to 16,500 daltons.
Based on the structure of the expressive pHshazhid pHTS 37, it is concluded that the human TNF polypeptide produced in the transformed organism should be a fusion protein that includes part of the phosphate binding protein and polypeptide
TID of a human full name with a part of his predestination. The theoretical molecular weight of the fusion protein is calculated to be approximately 23,000 daltons. The molecular weight of the total nolipeptide of the human name is equal to 17097 dal. This study suggests that the resulting protein can be converted into the full polypeptide of the human name in the host cell by limited hydrolysis , possibly at a specific site (Arg-Ser), connecting him with a full psi-1 peptide of a human full name with a part of his predecessor
If the periplasmic extract is incubated with 5 µg / mp of trypsin at 37 ° C for 1 hour and the reaction mixture is subjected to electrophoresis on polyacryl amide gel with sodium dodecyl sulfate under the conditions described above, then a protein band corresponding to a molecular weight of about 22,000 daltons is observed, which represents em a protein that disappears under the cleavage action of trypsin
PRI me R 7, Obtaining a modified polypeptide of a human full name.
An expressive plasmid, pHT RD 4, containing DNA — a coding polypeptide resulting from the cleavage of four amino acids from the N-terminus of the full polypeptide of a human full name, is constructed in the same way as the expression plasmid pHTR 91, except that instead of using adapter 1, following synthetic adapter: 5 -CGATATGACC G -TATACTGGGGCT
Using this synthetic adapter, a base sequence followed by an initiation codon (ATG) encodes an amino acid sequence consisting of 151 amino acid residues resulting from cleavage of four amino acids (Ser-Ser-Ser-Arg) from the N-terminus of the complete polypeptide human name.
The expressive plasmid pHTRD4 was introduced in E. coli HB101, and the transfused harvested organism was cultured overnight at 37 ° C with the modified mean M-9. The culture (0.05 ml) is sown in 5 MP of the medium itself and cultured for 1 hour. After
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3-beta-1 pdol-acrylo-BOCh acid is added to this in a resultant concentration of 20 µg / mp, and the cultivation is continued overnight. The cells are harvested by centrifugation and treated according to the same procedure as described in Example 2, and an enlightened lysate is obtained.
Cytotoxic activity lysate 1.1-10 units (L-929) per 1 ml.
PRI me R 8. Obtaining polypep-T1-1da-predecessor of the human full name in Escherichia coli,
 An expressive plasmid pHTR RE3 containing a cDNA encoding a polypeptide precursor of a human full name is constructed as follows. The cloned cDNA was digested from the recombinant pHT 113 plasmid by double digestion with restriction endonucleases Pst I and Hind III, and the c-DNA fragment was then further partially digested with Agi AI restriction nucleotide (DNA) (approximately 820 b.p.) comprising a base sequence encoding most of the precursor polypeptide. The resulting DNA fragment is bound by a chemically synthesized oligodeoxyribonteotide fragment having the following formula: 5 -CGATATGAGCA 3 -TATAS
The bound DNA fragment is called the Pre-TNF fragment.
Separately, a DNA fragment containing a portion of the trp promoter region is cut out with the PI3 plasmid pUR 720 by cleavage with the restriction restriction enzyme Eco R I and Ira I, and the DNA fragment is bound to the following chemically synthesized. / nv-i adapter
.CTAGTACGC /: AGTTCACGTAAAAAGGGTAAT 3 -TT (; ATCATGCGTTCAAGTGCATTTTTATATATAGC
The resulting DNA fragment is bound to the Rge-TNF fragment and the associated DNA fragment is combined with a DNA fragment (approximately 4.3 kb), it has 111; it has an ampicillin resistance gene isolated from the plasmid pBR 322 cleavage with restriction endonucleases Eco K 1 and Hind III.
An expressive n-ia Mn / ty pHTR RH was constructed using a technique that was introduced in K. coli. HB101, and trans-- formed ort.-Igplem cultivate
overnight when cultured in modified medium M-9, Culture (0.05 ml) is sown in 5 ml of the same medium and cultured at 37 ° C for 1 h. After that, 3-beta-indole-acrylic acid is added to obtain a resultant concentration of 20 µg / ml and the cultivation is continued overnight. The cells are harvested by centrifugation by | Q
The lysate has a cytotoxic activity of 1.8-10 units (L-929) per 1 ml of
II p Imper 9, Preparation and purification 15 The described operation is repeated, Elupolypeptide of human full name.
Getting to the Escherichia cell.
The transformed organism HB101 / / pHT 91 is used to obtain polyates with cytotoxic activity, combined and concentrated using ultrafiltration as described above. And, at last, the concentrate of a nappeptide of a human full name. Transform-2o was carried on a column (0.7x25 cm) with BiOGel P-6 ;-( B10-RAD) and desalted.
The resulting desalted sample, having a cytotoxic a; resistance, is homogeneous from the point of view of electrophoretic analysis.
The organism is cultivated in Tei (part of the night at 3 ° C in e) ulon LB; containing tetracycline at a concentration of 12.5 "kg / mpa Culture culture is sown in 10 volumes of modified; Wednesday
M-9 and cultured at 37 ° C for 1 hour. After the addition of 3-beta-indole-acrylic acid in the resulting concentration of 20 µg / ml, the incubation is continued for 4 hours. DOR cells are harvested, processed and prepared and obtained. bright lysate. Lysate is used in dp polypeptide purification chelveichesko-. first name
. , 35
Cleaning; polypeptide human name.
The polypeptide of the human name is purified from the lysate according to the following procedure. A column (5x20 cm) filled with DEAE-Sep-40 harose CL 6B, previously brought to equilibrium with respect to 20 mM Tris-HC1 buffer (pH 7.8), was injected with 1030 ml of lysate. The column is washed with 3 liters of the same. the buffer itself, and 45 are then eluted with a linear gradient of Nad from zero to 0.3 M in the same buffer with a flow rate of 133 MP / h. The eluate is fractionated on an acne polyacrylamide gel with sodium sulfate dod, and it is used as a purified polypeptide full name for carrying out a
Example 10. The method of the semi-lyophilized polypeptide of the full name.
The solution of the purified polypept of the full name is used for the lyophilized polypeptide of the human full name.
10 ml of a solution of purified human FULL NAME, having a toxic activity of 2, nits (LM) are mixed with 0.1 volume of NaCl containing 10% human serum albumin and 20% nit. After adjusting the pH of the solution to 6.8, the resulting solution is filtered off through a roflow membrane. 5 ml of sterile solution are placed in a glass vessel and dried out of the container. Each suck keeps 10 units (LM) clean
qi m 10 ml. Fractions possessing cyto-50 ° ™ peptide of a human full name.
toxic activity, collected and combined,
The cytotoxic activity at this stage is 53%, the specific activity is increased approximately 9 times.
The combined active fractions are desalted and concentrated to 1/10 vol. Example 11. Getting to the rabbit name.
Production of i-RNA name from rabbits of alveolar macrophages.
Rabbits (weighing about 2.5 kg) were intravenously injected with Propioni bacterium gages at a dose of 100 mg per rabbit, and the rabbits were slaughtered during the black days. Lungs
by ultrafiltration with Diaflo - using a UM10 membrane.
The concentrate is subjected to preparative isoelectrofocusing gel electrophoresis. An aliquot of the concentrate was applied to a plate of polyacrylamide gel (0.2x10x10 cm) with a pH gradient from 5.6 to 6.1, created with Imraobiline. The electrophoresis is carried out at a voltage of 2400 V for 16 hours at. The gel is cut into pieces 10 mm wide and the protein eluted with 20 mM Tris-HCl buffer (pH 7.8). Vyata, which have cytotoxic activity, are combined and concentrated using ultrafiltration as described above. And finally, a concentrate of sodium dodecyl sulfate nana polyacrylamide gel, and it is used as a purified polypeptide of a human full name for analysis.
Example 10. A method for preparing a lyophilized polypeptide of a human full name.
A solution of the purified polypeptide of a human full name is used to prepare a lyophilized polypeptide of a human full name.
10 ml of a solution of purified polypeptide of a human full name, having a cytotoxic activity of 2, units (LM) are mixed with 0.1 volumes of 8% NaCl containing 10% human serum albumin and 20% D-mannitol. After adjusting the pH of the solution to 6.8, the resulting solution is sterilized by filtration through a microflow membrane. 5 ml of sterile solution is placed in glass vessels and dried out. Each vessel contains 10 units (LM) of purified
° ™ peptide human name.
Example 11. Getting to the DNA of the rabbit name.
Production of i-RNAs of name from rabbit alveolar macrophages.
Rabbits (weighing approximately 2.5 kg) were intravenously injected with dead dry cells of Propioni bacterium acnes at a dose of 100 mg per rabbit, and after 8 days the rabbits were slaughtered. Light nes57
washed several times with phosphate buffered saline through a tube, injected into the animal's trachea, and alveolar macrophages collected. Of the 12 rabbits, approximately three alveolar macrophages are obtained,
Alveolar macrophages are suspended in R PM1-164O medium R, containing 10% fetal bovine serum, and seeded in Petri dishes (8 cm in diameter) with a cell density of cells per dish. They are pre-cultured at 37 ° C in a fully humid atmosphere containing 5% carbon dioxide. After pre-culture for 1 hour, endotoxin (lipopolysaccharide obtained from E. coli), TPA (foreball-12-myristate-13-acetate) and cycloheximide (protein synthesis inhibitor) are added so that their resulting concentrations were equal to 10 µg / ml, 10 ng / ml and 1, respectively. Cultivation was continued for another 4–4.5 h (total culture time was 5–5.5 h), the culture medium was removed by suction, and macrophages, adhered to the cups, are lysed: and homogenized in 5 M solution of guanidyl cyanate containing. 0.6% N-lauroylsacrosinate sodium and 6 mM sodium citrate. The homogenate is dissolved in a 5.7 M solution of cesium chloride containing 0.1 M EDTA. and centrifuged for 20 hours at 26,500 rpm using an ultracentrifuge to obtain a fraction of total RNA in the form of granules. The granules are dissolved in a small amount of a 7 M urea solution containing 0.35 M NaCl, 20 mM Tris-HCl (pH 7.4) and 20 mM EDTA, and are separated by precipitation from ethanol. Of the 12 rabbits receive 5.2 kg of total RNA. .
The total RNA fraction is dissolved in 2 ml of TE solution, the solution is heated at 65 ° C for 5 minutes. A solution of NaCl is added to the resulting concentration of 0.5 M of 0.5 M, and the solution is applied on a column of oligo (dT) -cellulose, previously equilibrated with a TE solution containing 0.5 M NaCl. Poly (A) -and-RNA is eluted from the column with a TE solution in 314 μg yield. The resulting poly (A) -and-RNA was subjected to agarose gel electrophoresis (gel concentration 1%, in the presence of 6 M urea, pI 4), and fractionated into 7 fractions according to
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molecular size, the Poly (L) -and-RNA is extracted and each fractionation TroBel gel is melted
g at 70 ° C for 10 minutes, followed by sequential extraction with phenol and chloroform and precipitation from ethanol. The content of rabbit i-RICs in poly (A) -and-RNA isolated from each
Q fragments are determined by i-RNA translational analysis using Xenopus leavis oocytes,
A higher concentration of the n-RNA of rabbit TNF is isolated from the fraction
15 corresponding to a molecular size of 1.6–2.7 kV (abbreviated to enriched rabbit TNF i-RNA),
The fraction enriched in the rabbit TNF i-RNA is obtained in this way
20 used in subsequent experiments.
Synthesis of c-DNA.
K-DPK is synthesized under the following conditions. 4 μg fraction enriched..and RNA
25 rabbit TNF was dissolved in 100 µl of 50 mM Tris-HCl buffer (pH 8.3) containing 10 mM MgCl2, 70 mM KCl, 1 mM dithiothreitol, 0.5 mM concentration of each of the following deoxyribonucleotide triphosphates: dTTP, dCTP, dATP and DPGP (dSTF mett, specific activity 4.440 disintegrations per minute nanomolar), 3 µg oligo (dT); (2 -8 units of reverse transcriptase derived from avian myeloblastosis virus, and incubated at 43 ° C in for more than 90 yn. After this, the reaction is stopped by the addition, the resulting hybrid to the extra DNA-and-RNA extr40, is ruled with phenol-chloroform (1: 1) and Highlight removed from the aqueous phase by precipitation from ethanol-Matrix and RNA is removed by alkali treatment at 65-70 C. The synthetic DNA vzdel one-by-niem osazhde45 from ethanol.
The single DNA precipitate is dissolved in 40 µl of 0.1 M HepeS buffer (pH 6.9) containing each of four deoxyribonucleotide triphosphates, dATP, dTTP, 50 dGTP, and dCTP, at a concentration of 0.5, 70 mM KCl, 5 mM MgCl, 1.5 W 2-mercaptoethanol and 8 units of E, coli DICK-ps-limerase 1 (large fragment) and incubated at 15 ° C for 20 h for
HS synthesis of DNA-DNA. The reaction is stopped by the addition of sodium dodecyl sulfate. DNA is extracted with a mixture of phenol-chloroform and precipitated from ethanol.
The resultant dna-DIC is dissolved in 10.0 μl of a 50 mM sodium acetate solution (pH 4.5) containing 1 mM ZnSO, 200 mM NaCl, 0.5% glycerol, and 0.5 single nuclease S1, and incubated for 20 min for splitting the structure. The reaction is stopped by adding EDTA, the reaction mixture is extracted with phenol-chloroform, and then with diethyl ether, the dns-DNA is isolated by precipitation from ethanol.
Preparation of oligo (dC) -terminal c-DNA. The above-obtained dna-DNA is dissolved in 100 μp of a buffer of 130 mM sodium cacodylate -30 mM Tris-HSl (pH 6.8) containing 1 mM CoCl, 0.1 mM dithiothreacy tol, 0.2 µg poly (A), 0.1 mM H-dCTP (specific activity of 5400 disintegrations per minute at picamb71) and 10 units of terminal desoaxynucleotidyl transferase, and incubated at 3 for 20 min to attach the ends of the oligo ( dC) to the 3 ends of dna-DNA. Reaki 1yu stopped by adding EDTUK. The reaction mixture was extracted with phenol-chloroform and then with diethyl ether, and 6 (dC) -terminal dna-DNA was isolated by precipitation from ethanol. Oligo (dC) -terminal dns-DNA was dissolved in 10 1 Tris-HCl buffer (pH 7., 4) containing 1 mM EDTA and 100 mM NaCl, so that the concentration of oligo (dC) -terminal dns-DNA was equal to 0.2 mcg / mp.
Obtaining oligo (dG) -terminal DNA of ttazshshchy pBR 322 (10 μg).
Dissolve in 100 µl of 20 mM Tris-HCl buffer (pH 7.4) containing 10 mM MgCl, 50 mM (KNz.) And 10 µg of bovine serum albumen, and 15 units of Pst I restriction endonuclease are incubated. 37 ° C for 1 hour. After stopping the reaction, the reaction mixture is extracted with a phenol-chloroform mixture, and the resulting DNA is isolated from the aqueous phase by precipitation from ethanol. The obtained DNA is dissolved in 200 µl of the same reaction buffer that was used above to attach to the ends of the dna-DNA (except that it contains 80 units of terminal deoxynucleotidyl transferase and H-D1TP instead of H-dCTP) and incubated at 20 min. For attachment to the end of about 10-15 residues dG, the reaction mixture
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extracted with phenol-chloroform and oligo (dG) -terminal DNA of plasmid pBR 322 was isolated from the aqueous phase by ethanol precipitation. The resulting terminal plasmid DNA was dissolved in the same buffer that was used by dp dissolving the oligo (dC) -terminal dna DNA, so that the concentration of the terminal plasmid DNA was 2 µg / ml.
Construction of recombinant p-lasmids.
50 μl of oligo (dC) -terminal 5 k-DNA solution is mixed with 10 μl of oligo (dG) -terminal solution of pBR 322, and the mixture is sequentially incubated at 65 s for 10 min, for 120 mrp1, at 45 ° C for 60 minutes, at 35 ° C for 60 minutes and at room temperature for 60 minutes to achieve binding and construct a recombinant plasmid.
Selection of transformed organisms.
The strain E. coli X 1776 transform using the above recombinant plasmid.
0 E. coli X 1776 is cultured with 20 ml of L broth containing 100 µg / ml of diaminopimelic acid and 40 µg / mp of thymidine until the turbidity measured at 600 mji reaches 0.5. Cells are harvested by centrifugation at and washed with 10 ivM Tris-HCl buffer (pH 7.3) containing 50 mM CaC. The cells are resuspended in 2 mp of the same buffer and allowed to stand for 5 minutes. O, 1 ml of the lighter recombinant plasmid solution was added to 0.2 MP of the suspension. The mixture is left to stand for
5 15 min and then hold for 2 min at. Thereafter, 0.5 ml of L broth with additives added above was added and the culture was carried out with shaking for
Q 1 h. An aliquot of the culture is taken and applied to the agar plate with the additive-containing broth L containing 15 µg / ml of tetrashchklin, and cultured for approximately
g 12h, Select the transformed op 7
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tetracycline resistant canines that are used as a cDNA kit,
Hybridization analysis.
Analysis using a colony gnbndisc is carried out using a P-labeled k-DNA sample to select those transformed organisms that have a plasmid containing k-DNA, encoded rabbit name, in the k-DIK kit. Inductive plus and minus samples of P-labeled opc-DNA are synthesized according to the procedure described previously using n-RNA obtained from inductive plus and minus alveolar macrophages, except that R-dCTP with high specific radioactivity is used, This trial selects colonies of transformed organisms containing: recombinant plasmids, strongly hybridized by inductive plus-probe, but not hybridized by inductive-minus-probe. Of the 20,000 colonies, 50 are selected.
20 of the selected 50 colonies are then subjected to u-RNA-hybridization-retranslation analysis. Plasmid DNA is extracted from each transformed organism and fixed on nitrocellulose filters after thermal denaturation. A fraction of poly (A) -and-RNA containing rabbit N-containing rabbit name is added to the filter and incubated at 50 ° C for 3 hours to achieve hybridization. Hybridized i-RNA is isolated and introduced into oocytes to determine whether it is a complete mRNA and RNA of a rabbit name. As a result of this test, three colonies having plasmids containing
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PST I's SOY and their dimensions are determined by polyacrylamide / yum gel electrophoresis. 17 colonies of transformed organisms are selected, containing K-DNA inserts with a size of at least 1 kb. From a transformed organism containing the largest c-DNA (transformed organism 5Q i X 1 776 / pR TNF 802; plasmid pRTNF 802) the cloned c-DNA is isolated and its base sequence is determined as described below.
Determining the sequence of the os-
The transformed organism A1776 / PR TNF 802 is cultivated in boulevard L containing diaminopimelin
1 SLOT and thymidine. The cells are processed and 20 receive plasmid DNA. The DNA DNA was cleaved with Pst I restriction endonuclease, purified, and the cloned cDNA was obtained. The fragment of the cloned cDNA is then cleaved with various restriction restriction enzymes.
35
mi, the base sequences of the fragments cleaved by the gami restriction endonuclease approach are determined.
The defined sequence of bases is presented in table. 9. It is established that the base sequence from 277 to 738 base is the region encoding the full rabbit name in accordance with the H-terminal and C-terminal amino acid sequences of TNF purified from rabbit plasma 4efi. Bases 34 through 276 are assumed to be a sequence of DNA strongly hybridized with the basis of the coding of the rabbit TNF polyRNA. K-DNA fragments are obtained from a plasmid containing the largest K-DNA (about 750 bp), digested with restriction endonuclease Dde I, and used as a sample for further selection. These DNA fragments are labeled P.C. Using these samples, a set of K-DNA samples are selected for analysis.
peptide required for the formation of the precursor of the rabbit TNF.
Example T2. Generation and purification of rabbit plasma TNF.
Rabbits (2.5-3.0 kg body weight) are injected with intravenous injection of 50 mg of sacrificed Progionibacterium acnes dry cells. After 8 days
  -.-п- X --- .. iiu. d „v rabbit is injected intravenously.
Identification of colonies of the transform-50 ° colony of endotoxin (lipopolysaccharized organisms, which have plasmid-P VDa, obtained from E. coli). Through
dyns containing c-DNA, hybrids, 2 h from each rabbit take blood
labeled samples. In this heartfelt puncture. Blood mixing results in a positive result, with 100 units of heparinate cat- 98 colonies of approximately 60,000 co-55 P are centrifuged at
loni Plasma 5000 rpm for 30 min in the case of ohmid DNA and inserts to DNA are cut out by cooling, removing blood from the blood and not from these recombinant plasmids, soluble compounds. 400 rabbits
splitting restriction endonuclease-get 24 l of plasma.
-
ZO
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Zoya Pst I and their dimensions are determined by polyacrylamide / yum gel electrophoresis. 17 colonies of transformed organisms are selected, containing K-DNA inserts with a size of at least 1 kb. From a transformed organism containing the largest c-DNA (transformed organism 5Q i X 1 776 / pR TNF 802; plasmid pRTNF 802), the cloned c-DNA is isolated and its base sequence is determined as described below.
Determining the sequence of the os-
The transformed organism A1776 / PR TNF 802 is cultivated in boulevard L containing diaminopimelin
1 SLOT and thymidine. The cells are processed and 20 receive plasmid DNA. The DNA DNA was cleaved with Pst I restriction endonuclease, purified, and the cloned cDNA was obtained. The fragment of the cloned cDNA is then cleaved with various restriction restriction enzymes.
35
mi, the base sequences of the fragments cleaved by the gami restriction endonuclease approach are determined.
The defined sequence of bases is presented in table. 9. It is established that the base sequence from 277 to 738 base is the region encoding the full rabbit name in accordance with the H-terminal and C-terminal amino acid sequences of TNF purified from rabbit plasma 4efi. Bases 34 through 276 are assumed to be sequences of bases encoding the fullness of bases encoding poly
peptide required for the formation of the precursor of the rabbit TNF.
Example T2. Generation and purification of rabbit plasma TNF.
Rabbits (2.5-3.0 kg body weight) are injected with intravenous injection of 50 mg of sacrificed Progionibacterium acnes dry cells. After 8 days
631
24 liters of plasma are added with EDTA (; 24 g) and 240 g of zeolite, and the mixture is heated for 1 hour, then sequentially filtered through; three filters with pore sizes of 3, 1, and 0.2 µm.
; 12 liters of d, 04 M Tris-HCl buffer (pH 7.8) and cJMecb are applied to a column (27x45 cm) d DEAE-Sepharose CL-bV, equilibrated against 0.04 M tris buffer, to 24 liters of filtrate. HC1 (| pH 7.8), containing 0.1 M NaCl, Pos. This column is washed with 75 L of Tris-HC buffer (pH 7.8), containing Oi, 1 M NaCl and then 50 liters of 0.04 M and Tris-HC buffer (pH 7.8), containing 0 |, 15 M NaCl, then eluted with Oj, 04 M with Tris-HC buffer (pH 7.2), mixed with 0.18 M NaC, Eluate is fractionated into 8 l fractions, and fractions possessing cytotoxic a) are collected. ktivnost. The active fractions are combined with HJ5iOT and diluted with an equal volume of Tris-HC buffer (pH 7.8). The diluted solution is applied to a column (| lGx13 cm) with DEAE-Sepharose-CL-6B.Kol- 5nku 1 liter of 0.04 M buffer t |) IC-HC (pH 7.8) containing 0.1 M SchS, and then elute with 5 liters of 0.04 M butyl-HC (pH 7.2) containing M NaC. The eluate fractionated sweat into fractions of 250 mp and fractions possessing U1 (more and 1totoxic activity, co-CijipaioT and combined,
i The active fraction is heated at 6lj) C for 30 minutes and rapidly cooled to 4 ° C. The cold solution is concentrated by ultrafiltration,
The resulting concentrate was loaded with a column (5x80 cm) with Sephacry S-200, U1) suspended against 0.005 M phosphate buffer (pH 7.4) containing 0.1 M NaC, and eluted with the same c-KbjM buffer. The eluate is fractionated into 40 ml fractions, and the active fractions are collected, combined and concentrated by a U4 filter.
The active fraction concentrate, obtained by gel filtration, is applied to a column with Zn-halate sepharose, as described below. The column (1.6x x2 | 0 cm) is filled with chelating sepharose (a sol with a fixed iminodiacetic acid), washed with 120 ml of rast- acja zinc chloride (1 mg / ml) and equilibrated with respect to the 0.05 M phosphate booster ( pH 7.4), containing 0.1 M NaC, then in a column of
476564
d t the concentrate obtained in the previous step and elute with the same buffer. Collect non-adsorbable fractions on the column. Cytotoxic activity is almost completely excreted in these fractions.
The active fractions obtained at the previous stage are concentrated and concentrated. 10 are worn per column (1.5 x cm x) with Togo pear HW-55, fully balanced with respect to 0.005 M and phosphate buffer (pH 7.4) containing 0.15 M NaC. The column was eluted with the same 15 buffer and the active fractions were collected. This sample is a purified rabbit plasma TNF.
Example 13
Definition of N-terminal and C-terminal amino acid sequences of rabbit plasma TNF.
Purified rabbit TNF is used and the N-terminal and C-terminal amino acid sequences are determined.
The N-terminal amino acid sequence is determined by Edman decomposition. The resulting phenylthiohydantoinamic acid is identified by high pressure liquid chromatography using a Lorbax ODS column.
The C-terminal amino acid sequence is determined enzymatically using carboxy peptidase. Purified rabbit plasma TNF is cleaved by carboxypeptidases A and Y with a molar ratio of enzyme to substrate equal to 0: 1:25 and 1: 1000, respectively. Free amino acids released from the C-terminus of rabbit plasma TNF are duplicated by means of amino acid micro analyzers at appropriate intervals of 2-180 minutes after cleavage.
It has been consistently established that the N-terminal and C-cocate amino acid sequences of rabbit 0 plasma TNF are as follows.
N-termination: 8Г-А а-8Г-АГ§-А at ... С-termination: ... - Val-Tyr-Phee-C y-I e-e-A a-Ieu
PRI and MER 14. Obtaining antibodies against rabbit plasma TNF.
A solution of TNF containing 1.9.10 units (L-929) of purified rabbit plasma TNF is emulsified with
the volume of the C-raind full stimulator, and the emulsion was injected with I117. In addition, after 8 weeks, the same amount of purified rabbit plasma name is entered intraperitoneally injected with aluminum hydroxide gel. Whole blood is collected by cardiac puncture 9 weeks after the first immunization, centrifuged. And.- get antiserum containing antibodies: against rabbit plasma TNF.
Antiserum is passed through a Sepharose 4B column equipped with the whey protein components of normal rabbits. Wire operation times, get purified anti-AA, specific to the heme of plasma TNF. Immunoele10
The main reason is that the wasp: cultivating chapovera macrophage of DOM together with a lipopolysaccharide obtained from Kscherichia coli and forelbol-12-syristate-13-acetate in the presence of cycloheximide, from the macpnarop B-fraction, containing the fragments of the polyphenol antioxidant, is used in the presence of cycloheximide; using a column with oligo (dT) -cellulose, synthesis on the mRNA fraction of a single-stranded cDNA using reverse transcriptase obtained from avian myeloblastosis of birds, in the presence of dGTP, dTTP, dTTP, conversion into a dyunite: cDNA using DNA- polymerase I and ribonuclease H, obtained from E. coli, in the presence of dGTP, dATP, dTDTF, dTTP, preparation of 20 double-stranded cDNA with an oligo (dC) tail, using a deoxynuleotide transferase in the presence of dCTP, sample tjoTKy plasmid p2R2322322p2R2322322322322322R2322322R and adding oli 15
cytophoresis and double diffusion techniques 25 that (dG) tails to split Pst I
through the gel, it is confirmed that this antibody forms a single band of sediment with purified rabbit plasma TNF,
This antibody solution, diluted approximately 60,000 times, possesses an ability to neutralize 50% of 500 units (L-929). the cytotoxic activity of rabbit plasma TNF, and diluted 1000 times can completely neutralize 50 units (LM), the cytotoxic activity of rabbit plasma name,
Plasmind pBR 322 DNA using terminal deoxynucleotidyltransferses in the presence of dGTP units, combining double-stranded cDNA with oligo (dC) tails with split Pst I DNA pBR 322 with oligo-SdH) tails, transformed with derived templates, with the help of the same templates, which are derived by recombinant templates, which are derived by recombinant samples, transformed by derived recombinant samples, and by means of the same samples that are derived by recombinant samples. recombinant plasm1-shchu pHTN F-13, by hybridization using Ava I or Hae II fragments as a recombinant plasmid pRTN F802, which encodes the tumor necrosis factor, and the plasmid pPTN F-13, which tumor necrosis factor with a the following nucleotide i-amino acid sequence;

权利要求:
Claims (1)
[1]
Invention Formula
The method of obtaining recombinant plasmid DNA pHTN 713, encoding a human tumor necrosis factor,
1102030
ATGAdCACTGAAACCATGATCCGGGACGTG HetScrThcGluSqrHehllcArgAspyal
40 I
50 I
60 I
GAGCTGGCCGAGGAGGCGCTCCCCAAGAAG GlbLcuAlaGluGluAIaLcuProLysLys
80
90
70
ACAGGGGGGCCCCAGGGCTCCAGGCGGTGC ThrGlyGlyProGlnGlySerArgArgCys
0
The main reason is that the wasp: cultivating the cultivation of chapoveal macrophages, DOM, together with the lipopolysaccharide obtained from Kscherichia coli and forelbol-12-myristate-13-acetate in the presence of cycloheximide, factor from the macroparobB fraction, containing mRNA of the factor factor Factor; using an oligo (dT) -cellulose column, synthesizing a single-stranded cDNA into mRNA fractions using reverse transcriptase derived from avian myeloblastosis, in the presence of dGTP, dTTP, dCTP, turning into a dyunitic one: cDNA using DNA polymerase I and ribonuclease H obtained from E. coli, in the presence of dGTP, dATP, dTDTP, dTTP, obtaining a 20 double-stranded cDNA with an oligo (dC) tail with plasmid shRT-transferase in the presence of dCTP, an image of the plasmid pRTRTRTR the restriction enzyme Pst I and the addition oli5
about (dG) tails to split Pst I
Plasmind pBR 322 DNA using terminal deoxynucleotidyl transferase in the presence of a variety of tpmnas recombinant plasma pH1-pHTN F-13; hybridization using Ava I or Hae II fragments as a probe of recombinant plasmid pRTN F802 encoding a rabbit tumor necrosis factor, the plasmid pPTN F-13 encoding a tumor necrosis factor whether, includes the following nucleotide and amino acid sequence;
50 I
60 I
80
90
161476568
100 110 120
  I
TTGTTCCTCAGCCTCTTCTCCTTCCTGATC LcuPheLeuSerLeoPheSerPheLeulle
130 140 ISO
GTGGCAGGCGCCDOCACG CTCTTCTGCCTG
ValAlaGlyAlaThrThrLcuPheCysLeu
160 170 180
“I) i
CTGCACTrrnGACTGATCGGCCCCCAGAGG LounicPhcGlyVnlllcGlyProGlnArg
190. 200 210
. GAAGAGTTCCCCAGGGACCTCTCTCTAATC GluGluPhcProArgAspLouSerLcuIle
220 230 240
AGCCCTCTGGCCCAGGCAGTCAGAtrCATCT SerProLcuAlaGlnTilaVdlArgSerScr
250 260. 270
TCTCGAJVCCCCGAGTGACAAGCCTGTAGCC ScrAirgThrProScrAspLysProValAla
280 290 300
CATGTTGTAGCAAACCCTCAAGCTGAGGGGG HisValValAlaAsnProGlnAlaGluGly
310 320 330
CAGCTCCAGTGGCTGAj-VCCGCCGGGCCAAT GlnLeuGlnTrpLeuAsnArgArgAlaAsn
34 (G 350,360
GCCCTCCTGGCCAA7GGCGTGGAGCTGAGA lilaLeuLeuAlaAsi lyValGluLeuAtg
370 300 390
GATAACCAGCTGGTGGTGCCATCAGXCGGC AspAsnGlnLeuValValProSerGluGly
400 410 420
CTGTACCTCATCTACTCCCAGGTCCTCTTC LeuT yLeulleTyBSerGlnValLcuPhe
430 440 450
AAGGGCCAAGGCTGCCCCTCCACCCATGTG LysGlyGlnGlyCysProSerThrHisVal
4GO 470 4PO
CTCCTCACCCACACCATCAGCGGCATCGCC LeuLeuThrHisThiTlleSerArglleAla
161476570
490 500 510 I. I
GTCTCCTACCAGACCAAGGTCAACCTCCTC ValSerTyrGlnThrLysValAsnLeuLou
520 530 540 t
TCTGCCATCAAGAGCCCCTGCCAGAGGGAG SerAlalleLyGSerProCysGlnArgGlu
550 560 570 I
ACCCCAGAGGGGGCTGAGGCCAAGCCCTGG
ThrProGluGlyAlaGluAl "itysProTrp
580 590 600 - "I TATGAGCCCATCTATCTGGGAGGGGTCTTC TyrGluProIlcTyrLcuGlyGlyValPhc
610 G20 630
CAGCTGGAGAAGGGTGACCGACTCAGCGCT GlnLcuGluLysGlyAspArgLcuSerAl.a
. 640 650 660
  I GAGATCAATCGGCCCGACTATCTCGACTTT GlulleAsnArgProAspTyrLeuAspPhe
670 680 690 I I. I
CCCGAGTCTGGGCAGGTCTACTTTGG.GATC AlaGluSerGlyGlnValTyrPheGlyllc
ATTGCCCTG IIcAlaLcu
40 50 60
 "". GAGCTGGCCGAGGACGCGCTCCCC / vAGAAG GluLeuAlaGluGluAlaLeuProLysLys
70 80 90
ACAGGGGGGCCCCAGGGCTCCAGGCGGTGC ThrGlyGlyProGlnGlyScrArgArgCys
. 100 110 120
 IrTGTTCCTCAGCCTCTTCTCCTTCCTGATC LeuPhcLcuScrLcnPheSerPheLeuIle
130 140 150
GTGGCAGGCGCCACCACGCTCTTCTGCCTG ValAlaGlyAlaThrThtLeuPheCysLeu
16.0 170 180
CTGCACTTTGGAGTGATCGGCCCCCAGAGG LcuHisPhcGlyVallleGlyProGlnArg
1614765
190. 200 210
GAAGAGTTCCCCAGGGACCTCTCTCTAATC GluGluPhcPrbArgAspLcuScirLcuIle
220 230 240
AGCCCTCTGGCCCAGGCAGTCAGATCATCT ScrProLcuAlaGlnAlaValArgScrSer
250 260 270
TCTCGAACCCCGAGTGACAAGCCTGTAGCC ScrArgThrrroScrA jpI.ynrroVnlAla
200290300
. CATGTTGTAGCAAACCCTCAAGCTGAGGGGG HisValValAlaAsnProGlnAlaGluGly
310 320 330
CAGCTCCAGTGGCTGAACCGCCGGGCCAAT GlnLcuGlnTrpLcuAsnArgArgAlaAcn
340 350 -360
GCCCTCCTGGCCAATGGCGTGGAGCTGAGA AlaLcuLeuAlaAsnGlyValGluLcuArg
1 370 300 390
GATAACCAGCTGGTGGTGCCATCAGAGGGC AspAsnGlnLcuValValProScrG luGly
400 410 420
CTGTACCTCATCTACTCCCAGGTCCTCTTC LeuTryLcuIleTyoSerGlnValLouPhe
430 440 450
AAGGGCCAAGGCTGCCCCTCCACCCATGTG LysGlyGlnGlyCysProSerThrHisVal
460 470 480
CTCCTCACCCACACCATCAGCCGCATCGCC LeuLcuThrHisThrlleSerArglleAla
490 500 510
GTCTCCTACCAGACCAAGGTCAACCTCCTC ValSerTyrGlnThrLysValAsnLcuL u
520 530 540
TCTGCCATCAAGAGCCCGTGCCAGAGGGAG SecAlallcLysSerProCysGlnArgGlu
550 5GO 570 I
ACCCCAGAGGGGGCTGAGGCCAAUCCCTGG ThrProGluGlyAlaGluAlnLycProTrp
5PO 590 COO
  I TATGAGCCCATCTATCTGGGAGGGGTCTTC TyrGluProIleTyrLeuGlyGlyValPhe
 31b1a7h5
, 610 620 630
CAGCTGGAGAAGGGTGACCGACTCAGCGCT GlnLcuGluLysGlyAspArgLeuSorAla
.640 650 660
GAGATCAATCGGCCCG ACTATCTCGACTTT GluIlcAsnArgProAspTyrLcuAspPhe
670. 6QO 690
GCCGAGTCTGGGCAGGTCTACTTTGGGATC AlaGluSerGlyGlnValTyrPheGlylle
ATTGCCCTG IleMaLeu

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同族专利:

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公开号 | 公开日

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JPH0157955B2|1989-12-08|

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JPS60185799A|1985-09-21|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

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US4879226A|1984-04-06|1989-11-07|Asahi Kasei Kogyo Kabushiki Kaisha|Novel human physiologically active polypeptide|

---

GR851626B|1984-07-05|1985-11-26|Genentech Inc|

---

JP2675294B2|1984-10-15|1997-11-12|カイロンコーポレイション|Human tumor necrosis factor|

---

AU601675B2|1984-12-21|1990-09-20|Biogen, Inc.|Purification, production and use of tumor necrosis factors|

---

WO1990003395A1|1988-09-22|1990-04-05|Teijin Limited|Novel physiologically active polypeptide, recombinant plasmid, recombinant microbial cells, medicinal composition, and process for recovering purified polypeptide|

---

JPH0242986A|1989-06-28|1990-02-13|Asahi Chem Ind Co Ltd|Dna coding novel polypeptide having physiological activity to human|

---

JPH07101993A|1993-10-01|1995-04-18|Akira Kaji|New anti-tumor peptide|

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EP3763729A1|2010-11-25|2021-01-13|Imnate Sarl|Immunogenic peptides for use in the prevention and/or treatment of infectious diseases, autoimmune diseases, immune responses to allofactors, allergic diseases, tumors, graft rejection and immune responses against viral vectors used for gene therapy or gene vaccination|

法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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JP59043617A|JPH0157955B2|1984-03-06|1984-03-06|

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