A method of obtaining a factor that stimulates the formation of colonies of granulocytes

 

(57) Abstract:

Usage: the invention relates to biotechnology, in particular the production of factors that stimulate the formation of colonies of granulocytes using recombinant DNA technology. The essence of the invention: a method of obtaining a polypeptide or glycoprotein having the activity of human factor stimulating the formation of colonies of granulocytes, which provides for double-CDNA complementary to the RNA /mRNA/ that encodes a polypeptide having the specified activity, and chromosomal human gene encoding a polypeptide having the activity of a catalyst for the formation of colonies of granulocytes, the introduction of gene into the vector, the transformation of the obtained recombinant DNA recipient cells, isolation and purification of the target product. 40 Il., 17 table.

The invention relates to a method for producing a polypeptide or glycoprotein having the activity factor stimulate the formation of colonies of granulocytes (G-CSF using the technology of recombinant DNA molecules.

Biological effects of G-SF, is their ability to induce differentiation of leukemic cells in the bone marrow and Akamai is promising. Attempts to isolate and purify the G-SF from the supernatant of cell culture, but homogeneous G-SF was not produced in large quantities, because it is produced in low concentrations and after the cleanup is obtained in trace amounts from a large volume of culture fluid.

In Fig.1 presents the sequence of the various probes, IWQ, and LC; Fig.2 nucleotide sequence of the insert pHCS-1; Fig. 3-5 nucleotide sequence of the CDNA insert in pBRG4; Fig.6 amino acid sequence of the precursor of human G-CSF, derived on BRG4 CDNA; Fig.7 amino acid sequence of human G-CSF is derived from BRG4 CDNA; Fig. 8-10 nucleotide sequence of the CDNA insert in BRV2; Fig.11 amino acid sequence of the precursor of human G-CSF is derived from pBRV2 CDNA, Fig.12 amino acid sequence of human G-CSF is derived from pBRV2 CDNA; Fig.13-17 nucleotide sequence of the human chromosomal gene encoding G-CSF; Fig.18 sites of cleavage by restrictases pBRG4 or PBRV2 CDNA of human G-CSF; Fig. 19 sites of cleavage by restrictases human chromosomal gene, code the teachings of the PL-promoter-containing vector (+VSE line); Fig.23 the method of obtaining trpthe promoter-containing vector (+VSE line); Fig.24 part of the method of obtaining the tac-promoter-containing vector (VSE line); Fig. 25-26 way to obtain the PL-promoteroperator vector (-ANY line), Fig.27 the method of obtaining trpthe promoter-containing vector (-ANY line); Fig.28 schematically the structure GA410; Fig.29 methods of constructing recombinant expression vectors N-G4, pN-G4VA and N-G4VA in Fig.30-31 are two ways of constructing pH GG4-dhfr; Fig.32 ways of constructing pG4DR1 and pGDR2; Fig.33 schematically the structure of the pH GV2; Fig.34 methods construction of recombinant expression vectors N-V2, N-VA and N-VA in Fig. 35-36 two ways of constructing a recombinant expression vector GV2-dhfr; Fig. 37 ways of constructing V2DR1 and pV2DR 2; Fig.38 schematically the structure of pML cèze, Fig.39 schematically the structure pN cèze, Fig.40 schematically by structure pD26SVCE and pDRCE .

The gene encoding the polypeptide having the activity of human G-CSF (G-CSF) is a DNA (CDNA) which is complementary and the messenger RNA (MRNA), which is obtained in the form of fractions 15-17S by zentrifugenbau in the gradient raft is whether two lines CDNA.

CDNA one line contains the complete sequence or part of the gene encoding the polypeptide of I or II (Fig.6 and 7). More specifically, this CDNA has a nucleotide sequence that goes from the ATG at nucleotide positions 32-34, counting from the 5'-end (Fig.3-5), to ZZZ in positions of nucleotides 650-652, either from ATT in the provisions 122-124 to ZZZ in the provisions 650-652. Alternative CDNA has a nucleotide sequence or part of it, CDNA of this line here below called CDNA (+ANY).

CDNA another line has all or part of the gene encoding the polypeptide of I or II shown in Fig. 6-7. More specifically, this CDNA has a nucleotide sequence that goes from the ATG at nucleotide positions 31-33, counting from the 5' end [see Fig. 8-10] to ZZZ and nucleotide positions 640-642, either from ATT in the provisions 121-123 to ZZZ in the provisions 640-642. Alternative CDNA can have the nucleotide sequence shown in Fig. 8-10, or part of the CDNA of this line here below called CDNA (-ANY).

The above-described gene can be obtained in the following ways: first, get MRNA encoding G-CSF, cells of the mammal animal or other host cells with the ability to produce the polypeptide having the activity is eating subjected to directed selection by known methods.

The gene of the present invention also includes a human chromosomal gene that encodes a polypeptide having the activity of G-CSF. This gene contains all or part of the nucleotide sequence shown in Fig. 13-17.

The human chromosomal gene can be obtained from any type of human cells, such as cells, extracted from the liver or the kidneys, or of the malignant cells. Library of the human chromosomal gene can be obtained from human cells by any known method [see Maniatis et all, Cell, 15, 687(1978); and Manidtis et al. Molecular Cloning, old Spring Harbor Laboratory, RV 269, ff. (1982)] which is illustrated below:

extracted human chromosomal DNA from sources such as the liver of a human embryo, phenol, or other suitable reagents, partially or fully digest extracted DNA with a suitable restriction enzyme to obtain a DNA fragment of an appropriate length, insert the DNA fragment into a vector a DNA fragment of phage using T4 DNA ligase or other suitable ligase, with optional attaching a linker containing a restrictive site for a suitable enzyme, such as RI, then get particle-phage way laying in vitro and KL transform is against using as a vector in the above methods, include Charon 4A and MBL-3 and MBL-4.

The cell of a mammal, which can be used as a source of MRNA, is a strain of cells, for example extracted from the cells of cancer of the oral cavity of the person, SNI-2 deposited in the National collection of cultures of microorganisms or C. N. C. M. under inventory number I-483). Obtaining and RNA can be performed using one of the methods which have been proposed for gene cloning of a number of other physiologically active proteins: for example, all RNA receive by first machining surface-active agent and a phenol in the presence of ribonuclease inhibitor such as complex vanadia of ribonucleoside [see Berger and Birkenmeier, Biochemistry, 18 - 5143(1979)] or by centrifugation in CsCl density gradient with subsequent processing guanidinylation [see Chirgwin et al. Biochemistry, 18,5294(1979)] then get the poly(A+) RNA(MRNA), exposing the entire RNA periodic adsorption or affinity chromatography on a column of oligo (dt) cellulose or poly-U-sepharose, when using sepharose 2B as a carrier. Poly(A+) RNA can be further fractionate suitable means, such as centrifugation in density gradient of sucrose. The ability received such education is EP, MRNA broadcast in protein and verify its physiological activity, alternative, determine the identity of this protein using antibodies anti-G-CSF. More specifically, mRNA injected oocytes enopus Laevis for broadcasting [see Gurdon et al. Nature, 233, 177(1972)] or you can spend translational reaction with rabbit reticulocytes or wheat germ [Schleif and Wensink, "Practical Methods in Molecular Biology". Springer-Verlag, NY (1981)] the Activity of G-CSF, you can try applying, applying the method of cultivation in soft agar using bone marrow cells, and methodology for this method already described [Metcalf, "Hemopoietic Colonies", Springer-Verlag, Berlin, idelberg, NY 1977)]

Single-stranded CDNA synthesized using the thus obtained mRNA, which is used as a matrix, then this single-stranded CDNA synthesized double-strand CDNA and double-strand CDNA is inserted into a suitable vector DNA, to obtain a recombinant plasmid. This recombinant plasmid can be used to transform a suitable host, for example scherichia coli, in order to obtain a CDNA library.

Double-strand CDNA get from mRNA using one of the following two methods: mRNA treated with reverse transcriptase with oligo(dt), which is to the which corresponds to part of the amino acid sequence of the protein is G-CSF, and synthesize CDNA which is complementary to MRNA, by processing the reverse scriptase, and used as the seed of the synthesized oligonucleotide. Double-strand CDNA can also be obtained in the following ways: mRNA is decomposed and removed by treatment with alkali, and the resulting single-stranded CDNA is treated first by reversetranscriptase or DNA polymerase I (for example, slice Klenow(Klenow), then S1 nuclease, alternative MRNA can be directly processed PH Asai H and DNA polymerase (e.g., E. coli polymerase I). For more information, see Maniatis et al. "Molecular Clonig", old Spring Harbor Laboramory (1982); and after Gubler and offman, Gene, 25, 263 (1983).

Thus obtained double-strand CDNA is inserted into a suitable vector, such as for example, one plasmid vectors EC-type, typical representatives of which are pS CIOI, DF 41, Col E1, RMV, pBR322, pBR327 and rsus, or one of the phage vectors, typical examples of which include gt, c, gt 10 and gtWS, and then the recombinant vector is used for transformation of E. coli strain (e.g., H, NW, DN or S) in order to obtain a cDNA library (see, for example, "Molecular Cloning", above).

Double-strand CDNA can be attached to the following vector SP is Kim's way of DNA fragment, and the vector DNA, which was derived by restriction enzyme, attached to the specified CDNA by treatment with DNA ligase phase T4 in the presence of ATP. Alternative attach links dC, dG or dt, DW-links) respectively by double-strand cDNA and the vector DNA, which is cleaved by the restriction enzyme, and subjected to renaturation solution containing both DNA (see above "Molecular Cloning").

The host cell can be transformed thus obtained recombinant DNA by any known method. If the host cell is E. Coli, it is possible to use the method developed anahan [J. Mol. Biol. 166, 557(1983)] where recombinant DNA added to the competent cells, obtained in the presence of CaCl2, MgCl2or RbCl.

Directed the selection of cells carrying the desired gene can be accomplished using several methods, which include: plus-minus method used for CDNA cloning of interferon [aniguchi et al. Proc. Jpn. d. 55, ser. Century, 464 (1979)] the method of analyzing the hybridization broadcast [Nagata et al. Nature, 284, 316(1980)] and the method of hybridization of colonies or patches using oligonucleotide probe synthesized by a chemical image based on the amino acid sequence of a protein having the activity of human G-CSF [gene encoding a polypeptide having the activity of human G-CSF, was incorporated into a suitable vector DNA for transformation of other prokaryotic or eukaryotic host cells (Proc. Natl. Acad. Sei, USA82, July 1985, PP 4360-4364) (prototype).

E. coli can be transformed BR 322, which is a vector capable of replication in E. Li [see Bolivar, Gene, 2, 95 (1975)] This vector contains genes that are resistant to both ampicillin and tetracycline, and any of the properties can be used to identify transformed cells. Examples of the promoter that is necessary for the genetic expression in prokaryotic hosts include the promoter-lactamase gene [hang et al. Nature 275, 615 (1978)] the lactose promoter [see Goeddel et al. Nature 281, page 544(1979)] and tryptophanyl promoter [see Goeddel et al. Nucleic Acid Res. 8, 4057 (1980)], etc.

Any of these promoters can be used to obtain polypeptide having the activity of human G-CSF of the present invention.

Eukaryotic microorganism, such as Saccharoms erevisiae, it is possible to transform a vector, such as plasmid YRp7 [see Stinchcomb et al. Nature, 282, 39, (1979)] This plasmid has the gene TCR as a selection marker for yeast strains lacking the ability of p. the action of the promoter, which can be used for gene expression include acidic fosfatazy the promoter of the gene [Miyanohara et al. Proc. Natl. Acad. Sci. USA, 80,1 (1983)] and alcoholdehydrogenase the promoter of the gene [Valenzuela et al. Nature, 298, 347 (1982)]

For transformation of mammalian cells, such as cells OS, the cells of the Chinese hamster ovary (Cho) cells With a-127 and Hela cells can be used, for example pSV2-gpt [see Milligan and Berg; Proc. Natl. d. Sci. USA, 78, 2072(1981)] Vectors used for transformation of these cells contain the site of initiation, a selective marker, a promoter, a position which precedes the position Expressivo gene, a polyadenylation signal, etc.) For gene expression in mammalian cells can be used the promoter of retrovirus, virus polyoma, adenovirus, simian virus 40 (SV 40), etc. When using promoter, SV 40, the desired gene expression can easily be done according to the method of Mulligan with TCS. (Mulligan et al. described in Nature, 277,108(1979).

Sites initiating that you can use, derived from SV 40, Polyanovo virus, adenovirus, virus, bovine papillomavirus (BPV), etc. Used selective markers include gene fortransfer (ARN (3') II or I, gene timedancing, gene E. coli anthine guaninephosphoribosyltransferase (Ecogpt) gene dihydrofolate ilovecosmo interferon [see Nishi et al. J. Biochem. 97,153 (1985)] or a change in the nucleotide sequence does not lead to any change in amino acid sequence, or changes in the amino acid sequence do not affect the functional activity of the protein.

The activity of G-GCS may also possess a polypeptide with a deletion, addition or substitution of one or more amino acids in the sequence shown in Fig.6, 7 and 11 and 19.

Below is described a method of obtaining a factor in accordance with the invention.

(I) Obtaining a probe.

Homogeneous protein of human CSF was obtained by purification of the surface layer of the culture of tumor cells lines SNI-2 and determined its amino acid sequence from the N-type. The fragments obtained by digesting promotional and treatment with trypsin, and amino acid sequences of these fragments were also identified [example 3(I), (II) and (III)]

On the basis of certain amino acid sequences were synthesized three nucleic acid probe (A), (LC) and (IWQ), having the sequence shown in Fig.1 (example 4). The probe (A) was mixed type and consisted of 14 consecutive nucleotides. Probe (IWQ) consisted of 30 follower is ilovecosmo cholecystokinin gene [kahashi et al. Proc. Natl. d. Sci. USA 82, 1931 (1985)] Probe (LC) was a 24-nucleotidyl probe, which was synthesized from nucleotides in positions 32-39 from the N-terminal amino acid sequence shown in example 3(I), on the basis of the nucleotide sequence shown in Fig.3-5.

Chemical synthesis of nucleotides can be accomplished through the use of upgraded phosphocreatine method for solid-phase method, and it was considered by Narang (Narang) [Tetrahedron, 39,3-22(1983)]

You can also use the probes based on the amino acid sequences at positions which are different from those used in the above-mentioned probes.

(2) Construction of CDNA library.

Cells SNI-2 were homogenized after adding guanidinoacetate solution, and the whole RNA was obtained by centrifugation in a density gradient, CsCl.

Poly (A+) RNA was isolated from total RNA using column chromatography on oligo (dt)-cellulose. After that, the synthesized single-stranded CDNA using reversetranscriptase, and added a ribonuclease H and E. cli DNA polymerase I, to obtain the double-strand cDNA. The dC circuit was added to the obtained defnitely cDNA, which was attached to the vector RV R 322 to which li H, and built a library of cDNA RV R 322-line (examples 5 and 6).

In a similar way by double-strand CDNA was attached to the vector gt 10 using the linker Eco RI and constructed a CDNA library line-phage (example 7).

(3) Directional selection.

Clones of the cDNA library Pb R322-line, recorded on filter paper Whatman S41, and by hybridization of colonies with probe (IWQ) labeled 32, you can select a single clone. A subsequent study by the method of the spots on the Southern [Southern. J. Mol. Biol. 98,503(1975)] showed that this clone also hybridized with probe (A). The nucleotide sequence of this clone was determined by dimethoxymethane [Sanger, Science, 214, 1205 (1981)]

The nucleotide sequence of the obtained CDNA insert is shown in Fig. 2, where one can see that this insert consists of 308 base pairs, including probes (IWQ) and (A), and has an open reading frame encoding a 83 amino acids, the sequence of which pokazanev example 3 (III) pB R 322-derivative containing these 308 base pairs pHCS-1 (example 8).

The DNA fragment containing 308 base pairs obtained from s-I, marked radioactive label by way labeled broadcast (see Molecular Cloning), and using this fragment as a probe, osseotite five clones. Using the same method described above was determined nucleotide sequence of the clone, which was once believed, contains CDNA [Fig. 3-5]

This CDNA insert was a single large open reading frame.

As shown in Fig. 3-5, can be deduced amino acid sequence encoded by this cDNA.

Comparison with the N-terminal amino acid sequence of G-CSF protein described in example 3(1), found that this cDNA contained a nucleotide sequence that corresponded to both the signal peptide encoded by 90 base pairs, starting with the sequence ATG at nucleotide positions 32-34 from the 5'end to the sequence GCC in the provisions 119-121, and G-CSF to the polypeptide encoded 531 base pairs, starting with the ATT sequence in positions 122-124 and ending sequence ZZZ in the provisions 650-652. Therefore, the polypeptide with the amino acid sequence l, shown in Fig. 6-7, consisted of 207 amino acids and a calculated molecular weight 22292, 67D. The polypeptide with the amino acid sequence II consisted of 177 amino acids, its molecular weight was calculated in the amount of 18986, 74 D (example 9).

Should Otmetim H Escherichia coli, containing pB R 322, which had this CDNA (+VSE) in the website oRI was placed for safekeeping in the Institute for the study of fermentation Bureau of industrial science and technology (Fermentation Research Institute, the Agency of Industrial Science and Technology (FERM BP-954).

In Fig.18 shows the restriction sites of this DNA.

This CDNA ligated with Pb R327 Soberon with TCS. [Soberon et al. Gene, 9,287(1980)] the website RI and the resulting plasmid is called here below pBRG4.

Thus obtained pBRG4 was treated with restriction enzyme RI to obtain the DNA fragment containing the CDNA of approximately 1500 base pairs. This fragment was labeled radioactive label method labeled broadcast (see Molecular Cloning) and, using as a probe the DNA fragment labeled with a radioactive label, again held directional selection library gt 10 CDNA clones, using hybridization spots (see Benton and Davis).

In this method, hybridization spots prepare two sheets of nitrocellulose filter paper with a related DNA phage, one of these sheets are used in the above hybridization spots, and the other is subjected to hybridization spots with the already described probe (LC). Selected phages that were positive for both probes. Selected clone, which had "polname. -10.

This cDNA had a single large open reading frame was determined amino acid sequence that can be encoded by this cDNA is shown in Fig. 8-10.

Comparison with the N-terminal amino acid sequence of G-CSF protein shown in example 3(1), revealed that this CDNA contained a nucleotide sequence that corresponded to both the signal peptide encoded with 90 base pairs, starting with the sequence ATG at positions of nucleotides 31-33 from the 5'end to the sequence GCC in positions 118-120, and finished G-CSF to the polypeptide encoded using 522 base pairs, starting with the ATT sequence in positions 121-123 and ending sequence ZZZ in the provisions 640-642. Therefore, the polypeptide with the amino acid sequence l, shown in Fig. 11 and 12, consisted of 204 amino acids, and it has been calculated that its molecular weight was 21977,35 daltons. The polypeptide with the amino acid sequence'll consisted of 174 amino acids, it was estimated that its molecular weight was 18671,42 daltons (example 10).

It should be noted that the ATG at positions 58-60 or in positions 67-69 also be considered was placed for safekeeping in the Institute for the study of fermentation Bureau of industrial science and technology (Fermentation Research Institute, the Agency of Idustrial Science and Technology (FERM BP-955).

In Fig.18 shows the restriction sites of this DNA.

This CDNA ligated with pBR327 in the website oRI to obtain plasmid, which here below called a plasmid pBRV 2.

(4) Directional selection library of the human chromosomal gene.

Library of the human chromosomal gene, obtained in accordance with the methods described by Manation and variety. (Maniatis et al. Molecular Cloning, ibid), were subjected to directed selection with the above S-I. Probes that can be used for directional selection included: pHCS-I DNA fragment length 308 base pairs, pBRG4 DNA fragment of approximately 1500 base pairs pBRV 2 DNA fragment of approximately 1500 base pairs, the DNA fragment of an appropriate length containing a part of one or more of these DNA fragments, as well as the above-mentioned oligonucleotide probes [i.e., (IWQ), (a) and (IC). Here below is described a case of using a DNA fragment pHCS-I.

This DNA fragment was labeled radioactive label32P in accordance with a method labeled broadcast [see Roop et al. ll, 15, 431 (1978)] Using as a probe received 32P-labeled fragment library of the human chromosomal gene was subjected napravlennom">

After extraction of DNA from clones with known methods received the card restriction [Fritsch et al. ll, 19, 959 (1980)]

Using the same probe DNA was carried out by hybridization to Southern (see ibid Southern), it was found that the DNA fragment length of about 4 thousand bases, cut through oRI and hoI, may potentially contain plot, encoding a human polypeptide G-CSF. Therefore, the DNA fragment of about 4 thousands of bases inserted in BR327 and website coRI using the linker coRI, in order to get pBRCE3 . Using this plasmid as DNA with a specific sequence of bases was determined by dideoxy-way nucleotide sequence of the site approximately 3 thousand bases of the DNA fragment length of about 4 thousand bases. In the result, it was found that the DNA fragment is the gene coding for the polypeptide of the human G-CSF (Fig.5).

Strain H E. coli carrying pBRC3 (i.e., plasmid pBR327 that has the specified DNA fragment of about 4 thousands of bases inserted in the website oRI) was placed for safekeeping in the Institute for the study of Fermentation Bureau of industrial science and technology (Fermentation Research Institute, the Agency of Industrial Science and hnolohgy (FE RM BP-956).

Comparison of the insertion pBRG4 cDNA, dormancy is ü exon parts, and it encodes the amino acid sequence deduced from pB RG4 and pB RV 2.

In Fig.19 shows the restriction sites of the obtained gene.

This DNA fragment contained a chromosomal gene of human G-CSF, or the preceding section, transcribed in mRNA of human G-CSF, together with the nucleotide sequence involved in the control of transcription [Benoist and Chambon, Nature, 290, 304 (1981), and Breathnack and Chambon, Ann. Rev. Biochem. 50, 349 (1981)]

(5) Construction of recombinant vector for expression in E. coli.

And/ Recombinant vector line +ANY.

From the plasmid pB RG4 obtained in (3) (example 9), cut the cDNA fragment of the polypeptide G-CSF using restrictase, and built a recombinant vector using one of the following methods: (I) using denaturirovannyj synthetic linker fragment associated with the fragment obtained from the tac-promoter-containing RCC-3 (Pharmacia Fine Chemicals) (example 12 and Fig.8);

(II) three fragments obtained from PL-promoter-containing PL-lambda (Pharmacia Fine Chemicals) associated with denaturirovannym synthetic linker, and product bundling and the cDNA fragment was subjected to the stages of re-fetch to build a recombinant vector (example 13, Fig. 21-22) or

(III) ispor-containing plasmids pOYI (example 14 and Fig.23),

(B) a Recombinant vector line-ANY.

In the same way described above, constructed three recombinant vector using plasmid pBRV2 (example 10), shown in example 19 and Fig. 24, 25, 26, 27.

(6) Obtaining transformed E. coli and their cultivation and expression of

Using three recombinant vector of each line +VSE and ANY strain of E. coli D1, N 4830 or JM 105 transformed when handling calcium chloride or rubidium chloride, described in Molecular Cloning ibid (examples 12,13,14 and 19). Each of the obtained transformants were cultured in the medium of Luria (Luria) containing ampicillin, and then spent the induction, as it is necessary to carry out the expression (examples 15 and 20).

(7) Isolation and purification of the polypeptide G-CSF from E. coli and its amino acid analysis.

The culture solution of the transformants was subjected to centrifugation to obtain a cell sediment after centrifugation. The collected cells were treated with Lisitsina and after lysis by cyclic freezing and thawing received a superficial solution. Alternative cells were treated with chloride guanine, centrifuged, and received a superficial solution.

Surface the solution was subjected to as a device.

Then an aqueous solution triperoxonane acid containing n-propanol was added to the concentrate, and after incubation in ice, the mixture was ofcentrifugal and absorbed on a column of reversed phase C18. After elution checked faction on their activity. Active fractions were collected and subjected to the same cleaning methods, which are described above. Purified fractions were dried by freezing, and the powder was dissolved and subjected to high-resolution liquid chromatography separation by size. The resulting polypeptides were subjected to SDS-polyacrylamide to gelelectrophoresis, and the only band confirmed the presence of the desired polypeptide (G-CSF) (examples 16 and 20). Thus obtained polypeptide was active human polypeptide G-CSF (examples 17 and 20). Polypeptide G-CSF were analyzed by the method of amino acid analysis using an automatic amino acid analyzer Hitachi 835" (Hitachi, Ltd). For analysis of N-terminal amino acids used gas-phase determiner sequence (for decomposition by Admino), high performance liquid chromatograph chromium - adopted and column Ultrasphere-ODS (examples 18 and 21).

(8) Construction of recombinant vectors for animal cells.

each CDNA line +VSE and ANY and chromosomal gene. Recombinant vectors (dhar) for use with cells SNO were also constructed for each CDNA line +VSE, and-for ANY chromosomal gene. Were also built of recombinant vectors for use in cells oS. Described are illustrative examples, and for a more detailed description reference should be made to the relevant working examples.

(A) Construction of recombinant vectors line +ANY

The cDNA fragment (+ANY) obtained in (3), inserted into a vector pdKCR to obtain the plasmid pGA 410, (example 22, Fig.28), which was partially digested with Eco RI, and then was treated with DNA polymerase I (fragment maple) to obtain blunt ends. To the DNA attached to the linker Hind III, then it was treated with Hind III, and T4 DNA ligase-treated DNA was used for transformation of strain DN E. coli using rubidium chloride (see Molecular Cloning). The obtained plasmid was named pH 410 GA (N) (Fig.29). raffia and column Ultrasphere-ODS (examples 18 and 21).

(8) Construction of recombinant vectors for animal cells.

Recombinant vectors (derived BPV) for use in host cells With 127 and N NTS were constructed for each CDNA line +VSE and ANY and chromosomal gene. Recombinant vectors (dhfr) for use with combinatie vectors for use in cells oS. Described are illustrative examples, and for a more detailed description reference should be made to the relevant working examples.

(A) Construction of recombinant vectors line +ANY

The cDNA fragment (+ANY) obtained in (3), inserted into a vector pdKCR to obtain the plasmid pGA 410, (example 22, Fig.28), which was partially digested with Eco RI, and then was treated with DNA polymerase I (fragment maple) to obtain blunt ends. To the DNA attached to the linker Hind III, then it was treated with Hind III, and T4 DNA ligase-treated DNA was used for transformation of strain DN E. coli using rubidium chloride (see Molecular Cloning). The obtained plasmid was named pH 410 GA (N) (Fig.29).

pH GA 410(H) was treated with SalI and after receiving the blunt ends of her processed again Hind III and separated fragment Hind III Sal I. the Plasmid pdBPV-1, which has transformed the fragment of the virus bovine papilloma, processed Hind III and Pvu II, and highlighted the large fragment of DNA and attached to a separately prepared fragment Hind III Sal I. the United fragments were used to transform strain DHI E. Li, to obtain plasmid pTN-G4, which had a pH of GA 410 fed SF cDNA (Fig.29, example 23) (CSF-CSF, Approx. recently.

Any of the plasmids, pH 410 GA, or GA pH 410 (N), Soter (+ANY for use with CHO cells (Fig. 30, 31, example 26).

The DNA fragment length 2 thousand bases containing the dhfr gene was isolated from pAdD26SVpA by treatment with oRI and Wamn selected fragment was inserted into the GA 410(H) in the site Hind III c, in order to build pG4DR1 and pG4DR2 (Fig.32, example 25).

(C) Construction of recombinant vectors line-ANY.

The CDNA fragment (-ANY) obtained in (3), inserted into a vector pdKCR to obtain plasmid pH GV2 (example 28), which was partially digested with Eco RI followed by treatment with DNA polymerase I (fragment of Klenow) to obtain blunt ends.

The linker Hind III was added to the DNA, which has consistently treated Hind III and T4 DNA ligase. The treated DNA was used for transformation of strain DH1 E. coli using rubidium chloride (see ibid Melecular Cloning). The obtained plasmid was named pH GV2(N) (Fig.34).

Plasmid pH GV2 (N) processed Sal after I obtain blunt ends, it has processed its Hind III and separated fragment Hind III Sal I. the Plasmid pdBPV-1, which has transformed the fragment of the virus bovine papilloma, processed Hind III and Pvu II and separated the larger DNA fragment and joined to a separately prepared fragment Hind III Sal I.

Attached fragments were used to transform strain D1 E. Li, which is the means or plasmid pH GV2, either plasmid pH GV2(N), in combination with the plasmid dD 26SV pA used to build the pH GV2-dhfr, which was a recombinant vector (-ANY) for use with cells Cho (Fig.35, 36, example 31).

The DNA fragment of about 2 thousand bases containing the dhfr gene, was isolated from the pAdD 26 SV pA processing using oRI and Wamn 1, and the selected fragment was inserted into the LV GV2(N), site Hind III c, in order to build pV2DR1 and pV2DR2 (Fig.37, example 31).

(C) Construction of recombinant vectors containing chromosomal gene.

Plasmid pBRCE3 received in (4) and which contained a chromosomal gene, shown in Fig. 13-17, processed RI. Plasmid pSV+K+described Baner J1 et al. in the Cell, 27, 299(1981), processed Kpn I to remove glubinoy gene. Then the plasmids were subjected to partial digestion with Hind III c in order to remove part of the late gene SV40. The fragments are re-combined to obtain the expression vector pML-E+.

This vector was treated with restriction enzyme, Eco RI and was subjected to dephosphorylation alkaline phosphatase (Takara Shuzo Co. Ltd.), to obtain the vector DNA, which was associated with the above fragment of chromosomal DNA using T4 DNA ligase (Takava Shuzo Co. Ltd.), to get pMLC , which was presented in SV 40, begin replication of SV40, early replication of pBR322 and-lactamase gene (Ampr) pBR322 and human G-CSF chromosomal gene, located behind the enhancer SV 40.

The expression vector for cells Is built in the following ways. A DNA fragment containing a chromosomal gene CSF, cut out using a suitable restrictase of pML cèze which represented the expression vector for cells OS. This fragment was joined with T4 DNA ligase to the fragment joined using T4 DNA ligase to a DNA fragment containing GDP virus bovine papillomavirus (BPV) and the DNA fragment that contains the early promoter of SV40. Received RT NC3 represented the expression vector, which had a chromosomal gene CSF, located behind early promoter, SV 40, and which contained 65% of the BPV.

The expression vector for cells SNO had two DNA fragments, United with T4 DNA ligase, one fragment contained a chromosomal gene CSF and early promoter, SV 40, as in the case of the expression vector for cells C, and the other fragment contained a gene pAdD 26 SV RA-dhfr. Received pD26SVCE3 represented the expression vector, which had a chromosomal gene CSF, located at the promoter, SV 40 and dhfr gene for the major late promoter of adenovirus.

(9) EC Is I. The treated plasmid used for transformation of cells S (pre-grown by cultivation) by the method using calcium phosphate. Grew transformed cells, and selected clones with a high rate of production of CSF. Allocated glycoproteins containing Expressway CSF and purified from the culture solution of the transformed cells. Found that they have activity of human G-CSF. The presence of the desired glycoprotein was also confirmed by amino acid analysis and analysis of sugar content in the sample.

To analyze the sugar content of the CSF sample used in amino acid analysis, were subjected to the determination of the amino sugar method Elson-Margana (lson-Margan), determination of neutral sugars by the method using itinysoft or determination of sialic acid by the method using thiobarbiturate. Methods each determination described in "ohshitsu no Called "hemistry of Saccharides" (including 2 of two parts), including 13, T. 4 course in Biochemical experiments (Biochemical periments, published by Tokyo Called Dojin). Transfer of measured values in weight percent showed that the sugar content in the received G-CSF was distributed in the range of 1-20 m is major depression in cells OS.

Cells OS, which were derived from cells of the monkey CV-I and which have been transformed with a mutant devoid of early SV40, for the expression of the large T antigen of SV 40 [see Gluzman et al. ell. 32, 175(1981)] were transformed vector pML cèze , which was obtained in (5) (C) and which contained the human chromosomal gene for G-CSF. The supernatant of the cell culture OS was active human G-CSF (example 35).

Cells OS were isolated from the analyzed mRNA, which showed the presence of two mRNA corresponding to the amino acid sequences shown in Fig.3 and Fig.4, respectively. The following example illustrates the determination of the activity of CSF.

Sample activity, stimulating the formation of colonies (a) With cells of human bone marrow:

Cultivation in the monolayer in soft agar was performed in accordance with the methodology Brandley, T. R. and Metcalf, D. (ust. J. Of Exp. Biol. Med. Sci. 44, 287-300, 1966). Mixed with 0.2 ml of serum bovine embryo, 0.1 ml samples, 0.1 ml of a suspension of non-consolidation of bone marrow cells (1-2 x 105nuclear cells), 0.2 ml of the modified culture solution MC-Coy 5A (Mc Coy) and 0.4 ml of the modified culture solution MC-Coy 5A, containing 0.75 percent ageaboutWith 5% CO2/95% air at 100% humidity. Ten days has calculated the number of obtained colonies (one colony consisted of at least 50 cells) and determined colonystimulating activity, and believed that one unit is the activity required to form one colony.

(C) bone marrow cells of the mouse:

Mixed horse serum (0.4 ml) 0.1 ml sample of 0.1 ml of a suspension of bone marrow cells (females) mice SN/No (0.5 to 1x x105nuclear cells) and 0.4 ml of the modified culture solution MC-Coy 5A, containing about 0.75% agar, poured the mixture into a plastic Cup for tissue culture (diameter 35 mm) was subjected to coagulation and were cultured for 5 days at 37aboutWith 5% CO295% air and 100% humidity. Estimate the number of formed colonies (one colony consisted of at least 50 cells) and determined colonystimulating activity, assuming that one unit corresponds to the activity, necessary for the formation of one colony.

P R I m e R 1. The establishment of the NDA-2.

The tumor of the patient with cancer of the oral cavity, in which the observed marked increase in the number of neutrophils, transplanted in mice nu/nu. After about 10 is adki the tumor was removed aseptically, cut into cubes 1-2 mm3and were grown as follows.

From ten to fifteen cubes tumor was placed in 50 ml plastic centrifuge tube. After adding 5 ml of trypsin solution (containing 0.25% trypsin and 0.02% EDTA) tube was shaken for 10 minutes in a warm bath at 37aboutC and the supernatant liquid was decanted. Added another 5 ml portion of the same solution of trypsin, and trypsinogen digestion was performed under stirring for 15 min at 37aboutC. Allocated supernatant cell suspension and kept it in ice after trypsin were decontaminated by adding 1 ml serum bovine embryo.

After another repetition of these methods, the cell suspension was singled out, mixed with the previously obtained suspension and subjected to centrifugation at 15000 rpm for 1 min for 10 min to obtain the cell sediment after centrifugation. The precipitate after centrifugation washed twice F-10 containing 10% serum bovine embryo, and then placed in a plastic flask culture (25 cm2to obtain a cell concentration of 5 x 106cells/flask. After incubation over night in an incubator with CO2(5% CO2and 100% RH is together with dead cells, and the cultivation was continued with fresh infusion of culture solution. Six days after the beginning of cultivation, the flask was filled with cells, and the culture solution was replaced with fresh. The next day the culture solution was poured, and the flask was loaded with 2 ml of anti-mouse erythrocyte antibodies (appel), diluted 5 times with RPMI-1640 and 2 ml of chromosomal Guinea pigs (Kyokuto Seiyaku Co. Ltd.), diluted 2.5 times with RPMI-1640. After incubation for 20 min at 37aboutWith culture, washed twice F-10 containing 10% serum bovine embryo and deleted murine fibroblasts. Then added F-1 culture solution containing 10% serum bovine embryo and culturing was performed for 2 days. After this part of the cells was isolated and subjected to cloning by the method of limiting dilution.

The resulting II clones tested their CSF activity, it was found that one clone (SN U-2) was active about 10 times higher than other clones.

P R I m m e R 2. The allocation of CSF.

Cells obtained in example 1 were grown in a completely dense populations in the two culture flasks (150 cm3). Cells were isolated, suspended in 500 ml culture of the m3(Belco) and subjected to vortex cultivation in rotation with a speed of 0.5 rpm 1 min When it was found that the cells grew in a completely dense populations on the inner wall of the rotating bottle, the culture solution was replaced by R PMI 1640 containing no serum. After culturing for 4 days allocated supernatant of culture and kultivirovanie continued with the addition of F-10 solution containing 10% serum bovine embryo. After three days of cultivation, the culture solution was again replaced with RPMI 1640 containing no serum, and 4 days later allocated the supernatant of the culture. By repeating these methods each allotment received 500 ml containing no serum supernatant per bottle. In addition, this method allows to extract the supernatant of the culture while maintaining cells within a considerably long period.

Portion containing 5000 ml of the supernatant liquid obtained culture was mixed with 0.01% tween-20 and concentrated approximately 1000-fold by ultrafiltration through a filter Hollow Fiber DC-4 and Amicon PM-10 (Amicon).

The concentrate was purified in the following ways:

(I) a Portion (5 ml) concentrated skorosti flow of approximately 50 ml/h with 0.01 mol/l Tris-HCl buffer (pH 7.4), containing 0.15 mol/l NaCl and 0.01% tween-20 (Nakai Called Co. Ltd. ). Column have calibrated using bovine serum albumin (mol.m. 67000), ovalbumin (mol. m 45000) and cytochrome C (mol.m. 12400). After gel filtration of 0.1 ml of each fraction was diluted 10 times, and spent directional selection active fractions using the above-described method of determining the activity of stimulating the colony (KSA) (b). It is found that the fraction with Ve 400-700 ml are macrophage-dominant colonystimulating activity, while the fraction with Ve 800-1200 ml are granulocyte-dominant activity, stimulating the colony. Therefore, the latter fraction was collected and concentrated to a volume of approximately 5 ml per ultrafiltration device with PM-10 (Amso).

(II) To the concentrated fractions were added an aqueous solution of 0.1% triperoxonane acid containing 30% n-propanol (to determine the amino acid sequence supplied by the company "Tokyo Kasei K. K."). After keeping the mixture in ice for about 15 min the precipitate was removed by centrifugation for 10 min at 15000 rpm. The supernatant was adsorbing on a column of C18-Bondapak (8 mm x 30 cm for prepreparation use, Waters), balanced water concrete is luxusni acid, containing n-propyl alcohol, which had a linear concentration gradient of 30-60% Used liquid chromatograph high pressure firm Hitachi, model 685-50(Hitahi. Ltd.) and detector "Hitachi model 638-41 (Hitachi, Ltd.).

To determine the values of absorption at the same time at 220 nm and 280 nm. After elution, 10 μl of each fraction was diluted 100 times, and spent directional selection active fractions using the above-described method of determining colonystimulating activity (). It is found that the peaks of the elution of 40% n-propanol, have activity stimulating colony, so they were collected, subjected to repeated chromatographicaliy in the same conditions and in the same manner determined their activity stimulating colony. Again watched the activity at the peaks of 40% n-propanol. Therefore, these peaks were collected (4 fraction 4 ml) and dried by freezing.

(III) the Obtained freeze-dried powder was dissolved in 200 μl of an aqueous solution of 0.1% triperoxonane acid containing 40% n-propanol, and the solution was subjected to liquid chromatography high-pressure column SK-G3000SW (Too Soda Manufacturing Co. Ltd. 7,5 MMX 60 cm). Elution was performed with the same aqueous solution at a flow rate of 0.4 ml/min and fractions with which to determine its colonystimulating activity using the same method, what is described above, and the activity was observed in the fractions with retention times 37-38 min (corresponding to molecular mass of about 2 x 104). Active fractions were isolated and purified on an analytical column-Bondapak C18 (4.6 mm x 30 cm). The main peaks were isolated and dried by freezing. The obtained sample was analyzed using the method of determining colonystimulating activity (a). Found that he possessed the activity of the human G-CSF.

P R I m e R 3. Determination of amino acid sequences

(I) Determination of N-terminal amino acid sequence.

The sample was subjected to decomposition of Edman using gas-phase determinant sequence (Applied Biosystems), and the PTH-amino acids were analyzed by conventional methods using a liquid chromatograph, high pressure (Beckman Instruments) and column Ultrasphere ODS (Beckman Instruments). Column (5 μm, 4.6 mm in diameter and 250 mm in length) was balanced by the source buffer is an aqueous solution containing 15 millimoles/l nitroacetate buffer (pH 4.5) and 40% acetonitrile and injected sample (dissolved in 20 ál buffer). The separation was carried out at the isocratic elution of the source buffer. The flow rate was 1.4 ml/min, and the pace of the deposits in the UV range at wavelengths 269 and 320 nm. Standard samples of PTH-amino acids (Sigma) in 2-nanomolar portions were separated on the same line, to determine their retention times, which were compared with the times of test samples. As a result, found that the sample had the following amino acid sequence of the 40 residues from the N-Terminus. H2N-hr-Pro-LEU-Glu-Pro-Ala-Ser-Ser-

(10) LEU-Pro-GLn-Ser-Ph-LEU-LEU-Lys-Cys-

(20) LEU-Glu-GLn-Val-Arg-Lys-Ile-GLn-Glu-

(30) ASP-Glu - Ala-LEU-GLn-Glu-Lys-LEU-

(40) Cys-Ala-hr-Tight-Liz- (II) Decomposition of the bromine cyan.

The sample was dissolved in 70% formic acid. To the solution was added 200 equivalent quantities of bromine cyan, which was purified by sublimation. The mixture was left overnight at 37aboutFor the reaction. The reaction product was dried by freezing and fractionally using high-performance liquid chromatography and column SKG3000SW (Too Soda Manufacturins CO. Ltd.), having four peak. Peaks called CN-1, CN-2, SP-3, SP-4 in order of decreasing molecular weight. The first two peaks (CN-1 and CN-2) had the best outputs, and their amino acid sequences were analyzed using an automated gas-phase determinant sequence (Applied Biosystems) under the same conditions as used in (I)the taxable amino acid sequence: Pro-Ala-Hairdryer-Ala-Ser-Ala-Hairdryer - GLn-Arg-Arg-Ala-Gli-Gli-Val - LEU-Val-Ala-Ser-GIS-LEU-GLn- (III) Decomposition of trypsin.

The sample was dissolved in 0.1 mol/l Tris-HCl buffer (pH 7.4) containing 8 mol/l urea, and the solution was mixed with 0.1 mol/l Tris-Hcl buffer (pH 7.4) containing 0,1,2-mercaptoethanol, to obtain a final concentration of urea, 2 mol/l was Added TRNC-treated trypsin (Sigma) so that the ratio of sample to the enzyme was 50:1. The mixture was stirred for 4 h at 25aboutFrom and after adding an equal number TRNC-treated trypsin, the mixture was stirred for a further 16 h at 25aboutC. After the reaction product was subjected to high-speed obremeniaet column chromatography on a C8 column (Yamamura Depending K. K.) at the elution of 0.1% triperoxonane acid containing n-propyl alcohol, which had a linear density gradient 5-60% Despite the fact that the observed multiple peaks in the measurement of the absorption at 280 nm, were analyzed main peak to determine its amino acid sequence using automatic gas-phase determinant sequence (Applied Biosystems) under the same conditions as used in (I). As a result, found that the main peak was a peptide having the following sequence, which contained part of CN-2 fragment shown in (II): GLn-LEU-Asia-Ala-Hairdryer-Ala-Ser-

P R I m e R 4. Obtaining DNA probe (I) Synthesis of probe (IWQ)

Thirty consecutive nucleotides (see Fig.1) received on the basis of a sequence of 10 amino acids (Ile-TRP-GLn-GLn-Met-Glu-Glu-LEU-Gli-Met), included in the amino acid sequence obtained in example 3(II). You need to make a remark regarding the designation of nucleotides shown in Fig. 1, for example, the nucleotide at position 9 from the 5'-end is an equimolar mixture of da and dG. Source nucleotides are mostly dimers, but if you also use mononucleotide. In a column equipped with a glass filter, downloaded 20 mg original nucleotide resin Ap-d(G) (Yamasa Snoyu Co. Ltd.). After repeated washing the methylene chloride 4,4'-dimethoxytrityl group was removed by treatment with a solution of methylene chloride, soderzhaschego 3% trichloroacetic acid. Then the column was washed several times with 1 ml of methylene chloride. After the column was washed with anhydrous pyridine, to the Deputy of the solvent there was added 20 mg of nucleotide dimer (DMTr) Arthritis (other3), (Nippon Zeon; other3-triethylammonium; DMr-dimethoxytrityl) and 0.2 ml of pyridine and the interior of the column was dried by vacuum using a vacuum pump. Sardina, and the interior of the column was replaced with gaseous nitrogen. Nucleotide resin concentrate with dimer by reaction for 45 min at room temperature with stirring from time to time. After the reaction column was washed with pyridine and unreacted Oh groups have azetilirovanie pyridine solution containing an excess of acetic anhydride and 4-dimethylaminopyridine. After washing the column with pyridine skondensiroval the following dimers or monomers, written in order, by repeating the above-described methods (DMr)Ip(other3), (DMTr)GpGp(NR3), (DMTr)Ip(other3), an equimolar mixture of (DMTr) Srtr(R3and (DMTr) Trtr(other3), an equimolar mixture of (DMTr) ApAp(other3and (DMTr)ApGp(other3), an equimolar mixture of (DMTr)ApGp(other3and (DMTr)GpGp(other3), (DMTr)GpAp(other3), (DMTr) TpGp(other3), an equimolar mixture of (DMTr)ApAp(other3and (DMTr)GpAp(other3), (DMTr)CPAP(other3), an equimolar mixture of (DMTr)ApAp(other3and (DMTr)ApGp (other3), (DMr)GpCp(other3), (DMTr)pGp(other3), (DMTr)Ip(other3and (DMTr)Arthritis(other3), all of these nucleotides are delivered by the company Nippon Zeon" except (DMr) Ip (other3), which is supplied by the firm "Yamasa Shoyu Co. Ltd.". After the reaction at the final stage the resin is then washed pyridines pyridine (0.5 ml), water (0.2 ml) and dioxane (1 ml) containing 1 mol/l tetramethylguanidine and 1 mol/l-picolylamine. The suspension is left to stand overnight at room temperature and concentrated to a volume of 100-200 μl under vacuum. The concentrate was mixed with a small amount (2-3 drops) of pyridine and 2-3 ml of concentrated aqueous ammonia, the mixture was heated at 55aboutC for 6 hours After extraction with ethyl acetate, the aqueous layer was separated and concentrated under vacuum. The concentrate was dissolved in a solution of 50 mmol/l acetate of triethylamine (pH 7.0) and the solution was subjected to chromatography on a column of C-18, (1,0 15 cm; Waters), and elution was performed with acetonitrile (linear density gradient 10-30%) in a solution of 50 millimoles/l of acetate of triethylamine (pH 7.0). Peak fractions eluruumi in acetonitrile approximately 25% concentrated under vacuum.

To the concentrate was added 80% acetic acid and the mixture is left to stand for 30 min at room temperature. After extraction with ethyl acetate was separated the aqueous layer and concentrated under vacuum. The resulting concentrate was then purified liquid chromatography high pressure on a column of C-18 (supplied by Senshu Depending K. K. SSC-ODS - 272, diameter 6x 200 mm). Suirou is to 7.0). The synthetic DNA was obtained with the yield not lower than 10A260units.

Analysis using the method of sequencing by MAXIMO-Gilbert [Maxam-Gilbert, Meth. nzym. 65,499(1980)] showed that the obtained oligonucleotide had the nucleotide sequence shown in Fig.1.

(II) Synthesis of probe (A).

Fourteen of the nucleotide sequences (see Fig.1) has been based on a sequence of 5 amino acids (Met-Pro-Ala-Hairdryer-Ala), is included in the amino acid sequence obtained in example 3.

Methods of synthesis were similar to those used when receiving probe (IWQ) and the following nucleotides skondensiroval in nucleotide resin Ar-d (T) (Yamasa Shoyu Co. Ltd.) in order of writing: (DMTr)CpAp(other3), (DMTr)GpGp(other3), an equimolar mixture of (DMTr)CpAp(other3), (DMTr)Srtr(other3), (DMTr)CpGp(NR3and (DMTr)CpCp(other3), an equimolar mixture of (DMTr)ApGp(NR3), (DMTr)TpGp(other3), (DMTr)GpGp(other3and (DMTr)CGp(other3), (DMTr)ApAp(other3), an equimolar mixture of (DMTr)CpAp(NR3and (DMTr)CpGp(other3), and (DMr)Gp(NR3), all of the nucleotides are delivered by the company Nippon Zeon. The synthetic DNA was obtained with the yield of about 10A260units. Analysis using the method of determining the sequence GU in Fig.1.

(III) Synthesis of probe (LC).

Automated DNA synthesis was performed using the DNA synthesizer, model A "pplied Biosystems". This technique is based on the principles described ruthers et al. [J. Am. hem. Soc. 103,3185 (1981)] is usually called phosphoramidite method.

Phosphoramidite form (DMTr)-dT, pre-activated by tetrazole, condense to dG-S (Si substrate), which will unlock 5'-dimethoxytrityl group (DMTr). After that, unreacted groups will acetimidoyl and oxidized by iodine in the presence of water to get a group of fostoria. After the release of the DMTr group repeat condensation in the same way, until, until 24 synthesize nucleotides having the sequence shown in Fig.1. These nucleotides otscheplaut from the substrate, this will release and purified using obremeniaet liquid chromatography high pressure on a column of C-18 (Senshu Called Co. Ltd. SSC-ODS-272).

P R I m e R 5. Cultivation of cells CHU-2 and obtaining mRNA

1/the Cultivation and isolation of cells SNI-2.

Adapted cells SNI-2 were grown in a completely dense populations in the two flasks for cultivation (150 cm2provided, suspended in 500 ml of the culture solution RMI 1640 containing 10% sivasothi for 4 days at a rate of 0.5 turnover in 1 minutes When it was found that the cells grew in a completely dense populations on the inner wall of the rotating bottles, the culture solution was removed on the rotary bottle, and it was loaded with 100 ml of preheated (37about(C) physiological saline solution containing 0.02% EDTA. After heating at 37aboutC for 2 min, the cells were separated from the inner wall of the flask by suction pipette. The cell suspension centrifugation at 1500 rpm for 10 min to obtain the cell sediment after centrifugation. Cells re-suspended in 5 ml of physiological saline containing no EDTA. The suspension was centrifuged at 1500 rpm for 1 min for 10 min to obtain the cell precipitate after centrifugation (wet weight approximately 0.8 g). Thus obtained cells were kept frozen at -80aboutWith as long as they are not subjected to the procedure of extraction of RNA.

2) Purification of mRNA.

The allocation of mRNA from cells SNI-2 obtained in 1) was performed using methods that were essentially the same as the methods described in the "Molular Cloning", Maniatis et al. old Spring Harbor, R. 196, 1982. Frozen cells SNI-2 (wet weight 3.8 g) of suspendible-mercaptoethanol and 0.5% sarcosinate sodium] and the suspension is well mixed vortex rotation within 2-3 minutes The mixture was subjected to 10 cycles of absorption and wypracowania syringe (20 ml capacity) equipped with a 18G needle. Approximately 6 ml of viscous guanidinium solution containing the broken cells were layered on a 6-ml cushion of 5.7 mol/l CsCl in 0.1 mol/l EDTA (pH 7.5) in a centrifuge tube, Vismap SW40i polyallomer so that the tube was completely filled. Four centrifuge tubes were prepared in the ways described and centrifuged at a speed of 30,000 rpm for 1 min for 15 h at 20aboutC. the Obtained precipitates after centrifugation was washed three times with a small amount of 70% ethanol.

Precipitation from centrifugation derived from the corresponding test tubes, mixed, dissolved in 550 μl of water and processed to obtain a NaCl concentration of 0.2 mol/l After treatment with a mixture of 1:1 phenol and chloroform, and then one chloroform, was added 2.5 volumes of ethanol to precipitate the whole of RNA (approximately 10.1 mg of total RNA obtained from 3.8 g of wet cells). Poly (A+) RNA was purified from total RNA using the following methods, affinity chromatography, using the advantage of attaching the poly(A)chain to the 3'-end of mRNA. Adsorption on oligo (dt)-cellulose (type 7 P-L Biochemicals) was carried out by passing through oligo (dt)-cellulose columns the RA SDS] after as the solution was heated at 65aboutC for 5 minutes. The column was balanced with the same load buffer. Elution of poly(A+) RNA was performed using the solution [containing 10 millimoles/l Tris-Hcl (pH 7.5) and 1 mm/l EDTA] Neadsorbirovanne output stream again missed through the column again and the eluate obtained by repeating the same very ways, mixed with the first shoulder strap of the eluate. The result was 400 micrograms of poly(A+) RNA.

Thus obtained mRNA was fractionally in size by centrifugation in a density gradient of sucrose in accordance with the techniques described in the Laboratory manual Schleif and Wensink, "Practical Methods in Molecular Biology", Springer-Verlag, New York, idelberg, Berlin (1981).

The density gradient of sucrose 5-25% were created in the centrifuge tube Backman SW40i. Two sucrose solution was prepared by dissolving 5 and 25% sucrose, not containing pH-basics (Schwarz/Mann) solution containing 0.1 mol/l NaCl, 10 mm/l Tris-HCl (pH 7.5), 1 mm/l EDTA and 0.5 SDS. to 7.5) and 1 mm/l EDTA] Neadsorbirovanne output stream again missed through the column again and the eluate obtained by repeating the same very ways, mixed with the first shoulder strap of the eluate. As a result, the Roux by centrifugation in a density gradient of sucrose in accordance with the methods described in the Laboratory manual Schleif and Wensink, "Practical Methods in Molecular Biology", Springer-Verlag, New York, idelberg, Berlin (1981).

The density gradient of sucrose 5-25% were created in the centrifuge tube Backman SW40i. Two sucrose solution was prepared by dissolving 5 and 25% sucrose, not containing pH-basics (Schwarz/Mann) solution containing 0.1 mol/l NaCl, 10 mm/l Tris-HCl (pH 7.5), 1 mm/l EDTA and 0.5 SDS.

Eight hundred micrograms of mRNA [poly (A+)-RNA] received already described, was dissolved in 200-500 Microlitre THE solution. The solution was heated at 65aboutC for 5 min, and after rapid cooling, he was placed on solutions with a density gradient of sucrose, which was subjected to centrifugation at 30,000 rpm for 20 hours Collected fraction by mass of 0.5 ml each, and measured their absorption at 260 nm. Size fractionated RNA was determined on the basis of the provisions of the standard RNA (ribosomal RNA, 28S, 18S, and 5S). At the same time determined the activity of G-CSF in each fraction using oocytes enopus laevis by the following methods. First, mRNA in each fraction was transferred to an aqueous solution with a concentration of 1 μg/1 μl, the oocytes were taken from enopus (at the age of about one year) and the solution mRNA was injectively so about Microtiterwells Cup, oocytes were cultured for 48 h at room temperature in 10 μl environment Bertha (Barth) [88 mmol/l NaCl, 1 mmol/l KCl; 2.4 mmol/l NaHCO3; 0.82 mmol/l MgSO4; 0.33 mmol/l CA(NO3)2; 0.41 mmol/l CaCl2; 7.5 mmol/l Tris-HCl (pH 7,6); penicillin, 10 mg/l and streptomycinresistant 10 mg/l] the supernatant of the culture was isolated, concentrated and purified to a purity sufficient for determining the activity of G-CSF.

Found that the activity of G-CSF was present in fractions 15-17S.

P R I m e R 6. Synthesis of CDNA (construction of CDNA library pBR-line).

On poly (A+) obtained in example 5 was synthesized cDNA by the method of land with TCS. (Land et al. [Nucleic Acids Res. 9, 2251 (1981)] a modified method after Gubler and Hoffman [Gene, 25,263 (1983)]

(I) Synthesis of single-stranded cDNA.

In an Eppendorf tube (capacity 1.5 ml) was placed reagents in the following order: 80 μl of reaction buffer (500 mmol/l KCl, 50 mmol/l MgCl2, 250 mmol/l Tris-Hcl, pH 8.3), 20 ml of 200 mmol/l of dithiothreitol, 32 μl, 12.5 mmol/l deoxynucleotides (containing 12.5 mmol/l of each deoxyadenosines, deoxyguanosine, deoxycytidylate and deoxythymidylate), 10 mg -32P deoxycitidine the RNA level (2.1 μg/ml) and 206 μl of distilled water. A total of 400 μl of the reaction solution was heated at 65aboutC for 5 min, and then heated at 42aboutWith over 5 minutes To the heated solution was added 120 units reversetranscriptase (Takagi Shuzo Co. Ltd.). After the reaction for more than 2 h at 43aboutWith added 2 μl of inhibitor RNA-ASE (Bethesda Research Laboranories), 20 µl of THE solution, 16 μl of 100 mmol/l sodium pyrophosphate and 48 units (4 ál) reversetranscriptase, and the reaction was carried out at 46aboutC for 2 h the Reaction was suppressed by adding 0.5 mol/l EDTA (8 microlitres) and 10% SDS (8 microlitres). By subsequent treatment with phenol/chloroform and precipitation with ethanol (twice) received single-stranded CDNA.

2) Attach the dC circuit to single-stranded CDNA.

Single-stranded cDNA obtained in 1) was dissolved in distilled water. To the solution was added 60 μl of dC-episodebased buffer [400 mmol/l of cacodylate potassium, 50 mmol/l Tris-Hcl (pH 6.9), 4 mmol/l dithiothreitol, 1 mmol/l CoCl2and 1 mmol/l methoxynicotinate (d)] and the mixture was heated at 37aboutWith over 5 minutes To the reaction solution were added 3 μl of terminal transferase (27 units/μl, P-L Biochemicals) and the mixture was heated at 37aboutC for 2.5 min After treatment with phenol/chloroform (once) and the CLASS="ptx2">

3) Synthesis of double-strand CDNA.

To 40 μl of the DNA solution obtained in 2) was added 4 μl of oligo (dG)12-18 (200 μg/ml, P-L Biochemicals) and the mixture was heated at 65aboutC for 5 min, and then at the 43aboutC for 30 minutes while the reaction solution was maintained at 0aboutWith, it added 80 μl of buffer [100 mmol/l Tris-HCl (pH 7.5), 20 mmol/l MgCl2, 50 mmol/l (NH4)2SO4and 500 mmol/l KCl] 4 μl of 4 mmol/l deoxynucleotides (containing 4 mmol/l of each dA, d, d and d), 60 microlitres 1 mmol/l NAD, 210 μl of distilled water, 20 μl of E. coli DNA polymerase I (Takagi Shuzo Co. Ltd. ), 15 μl E. coli DNA ligase (Takagi Shuzo Co. Ltd.) and 15 microliters E. coli PH-basics N (Takagi Shuzo Co. Ltd.) and the mixture was subjected to reaction at the 12aboutC for 1 h After addition of 4 millimoles/l of deoxynucleotides (4 μl), the reaction was carried out at 25aboutC for 1 h By subsequent treatment with phenol/chloroform and precipitation with ethanol(once) got about 8 micrograms double-CDNA. This Dunaeva CDNA was dissolved in THE solution and subjected to 1.2% agarose gel electrophoresis. Fragments corresponding in size to approximately 560 base pairs up to 2 thousand base pairs, adsorbing on Whatman DE 81 and allocated e/P> Dunaeva CDNA obtained in 3) was dissolved in 40 μl of TE solution. Once added 8 μl of carrier dC-tail buffer of the type described in 2), the mixture was heated at 37aboutwithin 2 minutes After adding 1 μl of terminal transferase (27 units/μl) and the mixture was subjected to reaction at 37aboutC for 3 minutes then the reaction solution was immediately cooled to 0aboutC, and the reaction repaid by adding 1 μl of 0.5 mol/l EDTA. After treatment with phenol/chloroform and precipitation with ethanol, the precipitate suspended in 10 μl of TE solution.

5) Construction of CDNA library Pb R-line

Four microliters of commercial oligo (défi)-custom made BR 322 vector (Bethesda Research Laboratories; 10 nano-grams/μl) and 2 μl of dC-double stitched cDNA obtained in 4), senatoriable in THE solution, containing 5 µl of 0.1 mol/l of NaCl. Resaturate consisted of three stages: heating at 65aboutC for 5 min, subsequent heating at 40aboutC for 2 h and then cooled to room temperature.

In accordance with the method described in the laboratory manual Maniatis with TCS. [Maniatis et al. Molecular Cloning, old Spring Harbor, R. 249 and beyond (1982)] (here you can also use other known methods is to obtain transformants.

P R I m e R 7. Synthesis of CDNA (build libraries of phage)

1/Synthesis of single-stranded CDNA.

In accordance with the methods described in example 5, 3.8 g of frozen SNI-2 cells was twice purified on a column of oligo (dt)-cellulose, and then subjected to processing to get 400 micrograms of poly(A+)-RNA.

THE solution (10 μl), which is dissolved 12 μg of poly(A+)-RNA was placed in a reaction tube containing 10 µg actinomycin D (Sigma). After that the tube was loaded reagents in the following order: 20 μl of buffer for reverse transcriptase [250 mmol/l Tris-Hcl (pH 8.3); 40 mmol/l MgCl2; 250 mmol/l KCl] 20 μl of 5 mmol/l deoxynucleotides (containing 5 mmol/l of each of dA, d, d and d), 20 ál of oligo (dt)12-18 (0.2 ág/ml, P-L Biochemicals); 1 μl of 1 mol/l of dithiothreitol, 2 μl of PH-azina (30 units) MCL, Promeda Biotech); 10 µl reversetranscriptase (10 units/μl, Seikagku Kogyo Co. Ltd.); 1 cells / mm32R - d(10 CI; merscham) and 16 μl of water. The reaction solution volume was 100 µl, kept at 42aboutC for 2 h and then the reaction repaid by adding 0.5 mol/l EDTA (5 mm) and 20% SDS (1 ml). Subsequent treatment with phenol/chloroform (100 ml) and precipitation with ethanol (twice) received approximately 4 μg of single-stranded CDNA is a solution prepared by adding the following reagents in a written order: 25 μl of polymerase buffer [400 mm/l Hepes (pH 7,6); 16 mmol/l MgCl2, 63 mmol/l mercaptoethanol and 270 mmol/l KCl] 10 μl of 5 mmol/l deoxynucleotides; 1,0 ál 15 mmol/l NAD; 1,0 cells / mm32-R-dA (10 MCI/ml); 0.2 µl E. coli DNA ligase (60 units/μl, Takagi Shuzo Co. Ltd.); 5,0 ál E. coli DNA polymerase I (New ngland Biolabs; 10 units/ál); ál of 0.1 pH-ASE H (60 units/μl; Takagi Shuzo Co. Ltd.); and 28.7 μl of distilled water.

The reaction solution was incubated at 14aboutC for 1 h, allowed it to cool to room temperature, and then incubated for one hour. Then the reaction repaid by adding 0.5 mol/l EDTA (5 mm) and 20% SDS (1 ml) and treated reaction mixture with phenol/chloroform and held precipitation with ethanol. The obtained DNA was dissolved, and 20 µl, 0.5 mmol/l EDTA and got the reaction solution by adding 3 μl of the buffer, Klenow [500 millimoles/l Tris-Hcl (pH 8.0) and 50 mmol/l MgCl2] 3 μl of 5 mmol/l deoxynucleotides and 4 μl water. After adding 1 μl of DNA polymerase (fragment of Klenow, Takagi Shuzo Co. Ltd.) the reaction solution was incubated at 30aboutC for 15 minutes

Incubated the reaction solution was diluted with 70 μl of THE solution, and the reaction repaid by adding 0.5 mol/l EDTA (5 mm) and 20% SDS (1 ml). By subsequent treatment with phenol/chloroform the th cDNA

An aqueous solution (30 μl) double-cDNA synthesized in 2), was mixed with 40 ál of buffer methylation [500 mmol/l Tris-HCl (pH 8.0), 50 mmol/l EDTA] 20 μl of a solution SAM [800 µmol/l of S-adenosyl-L-methylmethane (SAM), 50 millimoles/l-mercaptoethanol] and 100 Microlitre water. To the mixture was added 15 ml oRI of methylase (New ngland Biolabs; 20 units/μl) to obtain a reaction solution, which volume was 200 ál. After incubation at 37aboutWith in 2 hours spent processing the phenol and ether, and ethanol precipitation to isolate DNA.

4) add a linker oRI.

About to 1.2 μg of methylated DNA double-added 1.5 μl of ligase buffer [250 mmol/l Tris-Hcl (pH 7.5) and 100 mmol/l MgCl2] 0,5 ál of pre-phosphorylated oRI linker (10-Mer; Takagi Shuzo Co. Ltd. ), and 1.5 μl of 10 mmol/l ATP, and 1.5 μl of 100 mmol/l, dithiothreitol and 2 μl of H2About to get the reaction solution, which volume was 15 μl. After adding 0.7 ál of T4DNA ligase (3,4 units/μl, Takagi Shuzo Co. Ltd. ) the reaction was carried out overnight at 4aboutC. After this, the ligase were decontaminated by heating at 65aboutC for 10 minutes the Reaction solution was diluted to volume 50 ál by adding 100 mmol/l eakly was carried out at 37aboutC for 2 h and Then added to 2.5 μl of 0.5 mol/l EDTA, and 0.5 μl of 20% SDS, and then spent processing by phenol/chloroform and precipitation with ethanol in order to isolate DNA. After that, unreacted oRI linker was removed by gelfiltration on Ultrogel34 (LKB) or by agarose gelelectrophoresis in order to allocate about 0.5-0.7 μg double-cDNA-added linker.

5) Attach double-cDNA to vector gt 10

Dunaeva cDNA added linker were mixed with 2.4 µg pre oRI-processed vector gt 10 (cloning vector), with the addition of 1.4 μl ligase buffer (250 mmol/l Tris-HCl and 100 mmol/l MgCl2) and 6.5 μl of distilled water, and the mixture was heated at 42aboutC for 15 minutes then added 1 μl of 10 mmol/l ATP, 1 μl of 0.1 mol/l of dithiothreitol and 0.5 μl of T4DNA ligase to obtain the full volume of 15 μl, and the reaction was carried out over night at the 12aboutC.

6) Packing in vitro

Approximately one third of recombinant DNA obtained in 5), Packed with set for packaging in vitro (Promeda Biotech) to obtain the phage spot.

P R I m e R 8. Directional selection library Pb R-line using a probe (IWQ).

Paper Whatman 541 put rabotal according to the following method tawba and Thompson [aub and Thompson, Anal. Biochem. 126, 222 (1982)]

Colonies are transferred onto the paper 541, grown on agar medium containing chloramphenicol (250 µg/ml), overnight at 37aboutC.

Paper 541 removed and left at room temperature for 3 min on another sheet of filter paper which has been impregnated with 0.5 N. the solution Paon. This procedure was repeated twice. Two similar experiment was carried out for 3 min using a solution of 0.5 mol/l Tris-Hcl (pH 8). At 4aboutWith the spent processing solution of 0.05 mol/l Tris-Hcl (pH 8) for 3 min and a solution of 1.5 mg lysozyme/ml [containing 0.05 mol/l Tris-Hcl (pH 8) and 25% sucrose] for 10 minutes, then at 37aboutWith the spent processing solution 1 SSC (0.15 mol/l NaCl and 0.015 mol/l sodium citrate) for 2 min and a solution of 1 SSC containing 200 μg/ml proteinase K for 30 min, and finally at room temperature was performed processing solution 1 x SSC for 2 min and a solution of 95% ethanol for 2 minutes. The final stage was repeated twice. After this paper 541 dried. The dried paper 541 immersed in a mixture of 25: 24: 1 phenol/chloroform/isoamyl alcohol [balanced 100 millimoles/l Tris-Hcl (pH 8.5), 100 mmol/l NaCl and 10 mmol/l EDTA] for 30 min at room temperature. Then similar prinout. After that dried filter paper.

Probe (IWQ) marked marked atom R in accordance with known methods (see Molecular Cloning), and the hybridization of colonies was carried out according to the method of Wallace et al. [Nucleic Acids Res. 9, 879 (1981)] Prehybridization was carried out at 65oC for 4 h in hybridization buffer containing 6 x NET [0.9 mol/l NaCl; 0.09 mol/l Tris-HCl (pH 7.5) and 6 millimoles/l EDTA] 5 x solution Denhardt (Denhardt), 0,1% SDS and 0.1 mg/ml denatured DNA (calf thymus). After this hybridization was performed over night at 56aboutC in hybridization buffer (see recipe above) containing 1x106bills per minute/ml of the radioactive labeled probe (IWQ). After the reaction paper 541 washed twice with a solution of 6 x SSC (containing 0.1% SDS) for 30 min at room temperature, then washed with 56aboutC for 1.5 min. Washed paper 541 then subjected autoradiography.

The plasmid was separated from the positive clones and subjected to the sunspot on the Southern (Southern) c probe (IWQ). Hybridization and autoradiography were performed under the same conditions as described above.

Similarly spots on the Southern was performed with a probe (A). Using hybridization buffer having the above then hybridization at this temperature for 1 h After the reaction nitrocellulose filter twice washed 6 SSC containing 0.1% SDS for 30 min at room temperature, and then washed with 39aboutC for 3 minutes, Washed paper then subjected autoradiography. mol/l Tris-Hcl (pH 8) for 3 min and a solution of 1.5 mg lysozyme/ml [containing 0.05 mol/l Tris-Hcl (pH 8) and 25% sucrose] for 10 minutes, then at 37aboutWith the spent processing solution 1 x SSC (0.15 mol/l NaCl and 0.015 mol/l sodium citrate) for 2 min and a solution of 1x SSC containing 200 μg/ml proteinase K for 30 min, and finally at room temperature was performed processing solution 1 x SSC for 2 min and a solution of 95% ethanol for 2 minutes. The final stage was repeated twice. After this paper 541 dried. The dried paper 541 immersed in a mixture of 25:24:1 phenol/chloroform/isoamyl alcohol [balanced 100 millimoles/l Tris-Hcl (pH 8.5), 100 mmol/l NaCl and 10 mmol/l EDTA] for 30 min at room temperature. Then the same procedure was repeated three times with a solution of 5 x SSC for 3 min, then twice with 95% ethanol solution for 3 minutes. After that dried filter paper.

Probe (IWQ) marked marked atom32P in accordance with known methods (see Molecular C and 65aboutC for 4 h in hybridization buffer containing 6 x NET [0.9 mol/l NaCl; 0.09 mol/l Tris-HCl (pH 7.5) and 6 millimoles/l EDTA] 5 x solution Denhardt (Denhardt), 0,1% SDS and 0.1 mg/ml denatured DNA (calf thymus). After this hybridization was performed over night at 56aboutC in hybridization buffer (see recipe above) containing 1x106bills per minute/ml of the radioactive labeled probe (IWQ). After the reaction paper 541 washed twice with a solution of 6 x SSC (containing 0.1% SDS) for 30 min at room temperature, then washed with 56aboutC for 1.5 min. Washed paper 541 then subjected autoradiography.

The plasmid was separated from the positive clones and subjected to the sunspot on the Southern (Southern) c probe (IWQ). Hybridization and autoradiography were performed under the same conditions as described above.

Similarly spots on the Southern was performed with a probe (A). Using hybridization buffer having the above composition, hybridization was performed first at the 49aboutC for 1 h, After cooling to 39aboutWith continued then hybridization at this temperature for 1 h After the reaction nitrocellulose filter was twice washed in 6 x SSC containing 0.1% SDS, m subjected autoradiography.

In the result, it was found that a single clone is positive. Determination of a nucleotide sequence using dideoxy-method showed that this clone had a DNA consisting of 308 base pairs, containing part of a probe (IWQ), and probe (A), Pb R 322-derived plasmid containing the insert, called S-1.

P R I m e R 9. Directional selection library line-phage using probe S-1 DNA.

Hybridization spots was carried out according to the method of Benton and Davis (Benton and Davis [Science, 196,180(1977)] S-1 obtained in example 8, was treated with Sau3A and ORI to obtain a DNA fragment of about 600 base pairs. This DNA fragment was labeled with radioactive labeled using labeled broadcast by the usual methods. Nitrocellulose filters (S&S) marked on the phage spot growing on agar medium in order to transfer the phage on the filter. After denaturation of the DNA of the phage using 0.5 mol/l NaOH, filter paper treated in the following ways: treatment of 0.1 mol/l Paon and 1.5 mol/l NaCl for 20 with two processing 0.5 mol/l Tris-Hcl (pH 7.5) and 1.5 mol/l NaCl for 20 s, and finally, processing 120 mmol/l NaCl, 15 mmol/l sodium citrate, 13 mmol/l KH2PO2and 1 mmol/l EDTA (pH of 7.2) for 20 sec.

The filter is then wisoc: 42aboutIn prehybridization buffer containing 5 x SSC, 5 x solution Denhardt, 50 mmol/l phosphate buffer, 50% formamide, 0.25 mg/ml denatured DNA (DNA from salmon sperm) and 0.1% SDS. After this hybridization was carried out at 42aboutC for 20 h in hybridization buffer containing 4 x 105bills per minute/ml of probe pS-1, which was tagged with a radioactive label using labeled broadcast. This hybridization buffer was a mixture of 5 x SSC, 5 x solution Denhardt, 20 mmol/l phosphate buffer (pH 6.0), 50% formamide, 0.1 percent SDS, 10% doctranslate and 0.1 mg/ml denatured DNA (DNA from salmon sperm).

Heriditary nitrocellulose filter was washed for 20 min with 2 x SSC containing 0.1% SDS, at room temperature, then for 30 min with 0.1 x SSC containing 0.1% SDS at 44aboutC, and finally for 10 min with 0.1 x SSC at room temperature. Then spent detection using autoradiography.

The result was five positive clones (G1-G5) Clone containing the "full" cDNA were subjected to testing to determine the nucleotide sequence of its DNA through dideoxy-way, and was identified nucleotide posledovatelnostei to obtain plasmid, you can get on a large scale. This plasmid was named pBRG4.

P R I m e R 10. Directional selection library line-phase with the probe pBRG4 and probe (LC)

Hybridization spots was carried out according to the method of Benton and Davis (see ibid Science) used in example 9. Nitrocellulose filters (S&S) placed on the stain growing phage on Yarovoy environment to put the phages on the filter. After denaturation ragovoy DNA with 0.5 mol/l Paon filter treated in the following ways: treatment with 0.1 mol/l Paon and 1.5 mol/l such as NaCl for 20 s, then the two treatment using 0.5 mol/l Tris-Hcl (pH 7.5) and 1.5 mol/l NaCl for 20 s, nakonec treatment with 120 mmol/l NaCl, 15 mmol/l sodium citrate, 13 mmol/l KN2PO4and 1 mmol/l EDTA (pH of 7.2) for 20 C. the Filter is then dried and heated at 80aboutC for 2 h to mobilitat DNA. Two sheets of the same filter was prepared as described above and subjected them to the directed selection using probe BRG4 derived DNA probe (LC).

Directional selection using probe pBRG4-derived DNA was performed in the following way. BRG4-processed oRI to obtain a DNA fragment of approximately 1500 base pairs. This DNA fragment was labeled the radio is x filters subjected prehybridization during the night when the 42aboutIn prehybridization buffer containing 5 x SSC, 5 x solution Denhardt, 50 mmol/l phosphate buffer, 50% formamide, 0.25 mg/ml denatured DNA (DNA from salmon sperm) of 0.1% SDS. After that, the filter was subjected to hybridization at 42aboutC for 20 h in hybridization buffer containing labeled with radioactive-labeled probe DNA (approximately 1 x 106bills per minute/ml) approximately 1500 base pairs. This hybridization buffer consisted of a mixture of 5 x SSC, 5 x solution Denhardt, 20 mmol/l phosphate buffer (pH 6.0), 50% formamide, 0.1 percent SDS, 10% doctranslate and 0.1 mg/ml denatured DNA (DNA from salmon sperm). Heriditary nitrocellulose filter was washed for 20 min with 2 x SSC containing 0.1% SDS, at room temperature, then for 30 minutes, 0.1 x SSC containing 0.1% SDS, at 44aboutC, and finally for 10 min using a 0.1 SSC at room temperature. Then spent detection using autoradiography.

Directional selection using a probe (LC) was performed as follows. Another filter is pre-processed 3SSC containing 0.1% SDS at 65aboutC for 2 hours Then spent prehybridization at 65aboutC for 2 h in a solution containing 6 x PET, 1 x solution Denhardt and 100 µg/miditation buffer, containing labeled with radioactive-labeled probe (LC) (2 x 106accounts in min/ml). This hybridization buffer also was a mixture of 6 x PET, 1 x solution Denhardt and 100 μg/ml denatured DNA (DNA from salmon sperm). Heriditary nitrocellulose filter was washed three times (each time for 20 min) 6 x SSC containing 0.1% SDS, at room temperature, then washed 6 x security standards containing 0.1% SDS at 63aboutC for 2 minutes

The filter was dried, and de-activated carried out using autoradiography.

In the above-described directional selection was selected clones that were positive for both probes, and a clone containing the "full" cDNA were subjected to testing to determine its nucleotide sequence by using dideoxy-way. It was found that she had the nucleotide sequence shown in Fig. 8-10. This cDNA was cut out from the vector gt 10 and annexed to the pBR 327 in the website RI to obtain plasmid pBU 2.

P R I m e R 11. Directed the selection of the library of human chromosomal gene.

1/build a library of human chromosomal gene

Library of the human chromosomal gene, obtained through the courtesy of Dr. Maniatis of garb of man with phenol or other suitable chemical reagents and subjected to partial digestion with restrictase Hae III and AluI, the resulting DNA fragments were processed by centrifugation in a density gradient of sucrose in order to concentrate fragments having a length of chain approximately 18 to 25 thousand bases, concentrated fragments attached to the shoulder DNA E. coli phage haron 4A, and put short-chained synthetic nucleotides having the sites of cleavage by restriction enzyme oRI in order to obtain infectious phage DNA recombinants, with the aim of obtaining high infectivity, when the package got more refined particle-phage. Theoretically, it is considered that the thus obtained library human gene is a set of recombinants containing human DNA with lengths of chains 18-25 thousand bases, which contain almost all human genes.

2) Directional selection library of the human chromosomal gene using the probe S-1 DNA.

Hybridization spots was carried out according to the method of Benton and Davis [Science, 196,180(1977)] pS-1 obtained in example 8, was treated with Sau3A and oRI to obtain a DNA fragment of about 600 base pairs. This DNA fragment was labeled with radioactive labeled using labeled broadcast by known methods. Nitrocellulose filters (S&S) placed on phage p the l/l NaOH, filter paper treated in the following ways: treatment of 0.1 mol/l Paon and 1.5 mol/l NaCl for 20 with two processing 0.5 mol/l Tris-Hcl (pH 7.5) and 1.5 mol/l NaCl for 20 s, and finally, processing 120 mmol/l NaCl, 15 mmol/l sodium citrate, 13 mmol/l KN2PO4and 1 mmol/l EDTA (pH of 7.2) for 20 sec.

The filter is then dried and heated at 80aboutC for 2 h to immobilized DNA. Prehybridization was performed overnight at 42aboutIn prehybridization buffer containing 5 x SSC, 5 x solution Denhardt, 50 mmol/l phosphate buffer, 50% formamide, 0.25 mg/ml denatured DNA (DNA from salmon sperm) and 0.1% SDS. After this hybridization was carried out at 42aboutC for 20 h in hybridization buffer containing 4 x 105bills per minute/ml of probe S-1, which was radioactively labeled using labeled broadcast. This hybridization buffer is a mixture of 5 x SSC, 5 x solution Denhardt, 20 mmol/l phosphate buffer (pH 6.0), 50% formamide, 0.1 percent SDS, 10% doctranslate and 0.1 mg/ml denatured DNA (DNA from salmon sperm).

Heriditary nitrocellulose filter was washed for 20 min with 2 x SSC containing 0.1% SDS, at room temperature, then for 30 min, 0.1 x SSC, with sterowanie with autoradiography.

The result was ten random positive clones. Recombinant DNA obtained from these clones by the method Maniatis [Maniatic, Cell, 15, 687 (1978)] the resulting DNA was treated with restrictase, such as oRI, BamHI and Bgl II were analysed using agarose gel electrophoresis, and got them restricting map according to the method of Frisch with TCS. [Fritsh et al. ell, ibid)]

Hybridization on Southern was performed using a probe representing labeled with a radioactive label S-1 derived DNA fragment, which was the same as what was used in the above methods of directional selection. The DNA fragment of about 8 thousand base pairs, cut through oRI, selected clones that were hybridisable with the probe. This fragment was subclinically to the site RI pR327. Subcloned DNA was subjected to another processing restrictase, and re-hybridization was performed by Southern. It was found that the DNA fragment of about 4 thousand base pairs, cut through oRI and hoI, contained the gene encoding the polypeptide of the human G-CSF. This fragment was subjected to testing to determine the sequence of part of a length of about 3 thousand base pairs using dideoxy-way was determined nucleotide serial is SS="ptx2">

Directed the selection of the human chromosomal gene was performed using also pBRG4 derived DNA and pBRV-2-derived DNA as probes. In each case, the DNA fragment 1500 base pairs, already processed oRI was directly radioactively labeled using labeled broadcast as described above, or alternatively the DNA fragment of about 700 base pairs, obtained by sequential treatments oRI and DraI, marked radioactive labeled using labeled broadcast. Thus obtained probe used in the method of hybridization spots, which were conducted under the same conditions as described above. The selected clones were analyzed by hybridization to Southern in order to obtain a DNA fragment having the nucleotide sequence shown in Fig.13-17. Thus obtained plasmid was named pBRCE3 .

P R I m e R 12. Construction of E. coli recombinant vector and transformation (using tac-promoteroperator vector)

1) Construction of recombinant vector

1) Receive vector

5 µg tac-promoter-containing vector RK-3 (Pharmacia) was treated with 8 units oRI (Takagi Shuzo Co. Ltd.) for 2 h at 37aboutWith 30 μl of reaction solution (40 IMO the basics (Takara Shuzo Co. Ltd.), and the processing was carried out at 60aboutC for 30 min, the DNA Fragment was identified by three treatments with phenol, one treatment with ether and precipitation with ethanol, and all processing was performed in the usual way.

The selected DNA fragment was dissolved in 50 μl of a mixture consisting of 50 mmol/l Tris-Hcl, 5 mmol/l 10 mmol/l D and 1 mmol/l each d ATP, d, d and d. After addition of 3 μl of E. coli DNA polymerase 1 slice of Klenow (Takara Shuzo Co. Ltd.), the reaction was carried out at the 14aboutC for 2 h to create blunt ends.

(II) Obtaining a synthetic linker.

3 μg of oligonucleotides having sequence synthetic linkers CATHARTICS and AGGGCGCATC, fosforilirovanii by conducting the reaction in 40 μl of reaction solution (consisting of 50 mmol/l Tris-Hcl, 10 mmol/l MgCl2, 10 mmol/l 2-mercaptoethanol and 1 mmol/l ATP) at 37aboutC for 60 min in the presence of 4 units of T4polynucleotide-kinase.

Each phosphorylated oligonucleotides (0.2 ág) was dissolved in 20 μl of 100 mmol/l NaCl containing the solution [10 mmol/l Tris-Hcl (pH 8.0) and 1 mmol/l EDTA] After treatment at 65aboutC for 10 min oligonucleotides were senatoriable by slow ohlau RV RG4, obtained in example 9, which contained the CDNA shown in Fig.3-5, processed 100 units restrictase Era (New ngland Biolabs) and 50 units of Dra I (Takara Shuzo Co. Ltd.) at 37aboutC for 3 h in 200 μl of reaction solution containing 6 mmol/l Tris-Hcl, 6 mmol/l MgCl2and 6 mmol/l 2-mercaptoethanol. About 2 μg of the fragment Are-DraI (approximately 590 base pairs) were identified using a 1.2% agarose gel electrophoresis.

(IV) associating the fragments.

About 0.1 μg of each of the fragments obtained in stages (I)-(III), dissolved in 20 μl of binding solution (66 mmol/l Tris-Hcl, 6.6 mmol/l MgCl2, 10 mmol/l D and 1 mmol/l ATP). After adding 175 units of T4DNA ligase solution was kept overnight at 4aboutIn order to obtain recombinant vector (Fig.20).

2) Transformation.

Using 20 μl of reaction solution containing the recombinant vector obtained in (IV), transformed strain E. coli JM105 by treatment with chloride of rubidium (see T. Maniatis et al. Molecular Cloning, R. 252 (1982)] a Plasmid was isolated from the culture of resistance to ampicillin colony of transformed and processed by restrictase BamHI, Ass and ApaI to confirm receipt of the desired transformants.

P R I m e p 13. On the P CLASS="ptx2">

1) Construction of recombinant vector.

1) Receive vector.

One hundred micrograms PL-promoter-containing vector PL-lambda (Pharmacia) was treated overnight at 37aboutUsing 50 units of restrictase BamHI and 100 μl of reaction solution [10 mmol/l Tris-Hcl (pH 7,6), 7 mmol/l MgCl2, 100 mmol/l NaCl and 10 mmol/l D] Subjecting the reaction solution 1% agarose gel electrophoresis, has allocated approximately 49 mg of fragment length 4 thousand bases and approximately 11 μg of the fragment of about 1.2 thousand bases.

Fragment 4 thousand bases dissolved in 100 µl TE buffer (see above for composition) and subjected her dephosphorylating by reaction with alkaline phosphatase (Takagi Shuzo Co. Ltd.) at 60aboutWith over 60 minutes

Another fragment of approximately 1.2 thousand bases, dissolved in 20 μl of buffer (10 mmol/l Tris-Hcl, 10 mmol/l MgCl2, 6 mmol/l KCl, and 1 mmol/l D) and treated overnight with 20 units of restrictase and Mbo II (New ngland Biolabs) at 37aboutC.

By gel electrophoresis on 4% polyacrylamide gel was allocated about 0.9 μg Bam HI-Mbo II fragment (approximately 200 base pairs) and about 1.9 µg MboII-BamHI fragment (about 310 base pairs).

(II) Obtaining a synthetic linker.

(III) Obtaining expression vector.

One-tenth micrograms fragment length of about 4 thousand reasons, 0.05 µg of each BamHI-MboII fragment with land OLPLand Mb II BamHI fragment with the plot tL1 [three fragments obtained in (1)] and 0.1 µg renaturierung synthetic S/D linker, obtained in (II) was subjected to reaction overnight at the 12aboutWith 40 μl of reaction solution (66 mmol/l Tris-Hcl, 6.6 mmol/l MgCl2, 10 mmol/l DTT, and 1 mmol/l ATP) in the presence of 175 units of T4DNA ligase (Takagi Shuzo Co. Ltd.). Twenty Microlitre reaction solution was used for transformation of strain N99Cl+E. coli (Pharmacia) according to the method using calcium chloride (see Molecular Cloning).

Cultured transformants was isolated plasmid from the culture of their colonies that were resistant to ampicillin. Processing plasmids by restrictase oRI BamHI and SaI showed that it was the target plasmid.

Two micrograms of this plasmid in contact with the restriction enzyme laI (New ngland Biolabs) at 37aboutC for 2 h in 20 μl of buffer (10 mmol/l Tris-Hcl, 6 mmol/l MgCl2/P> One microliter of the reaction solution was contacted during the night when 12aboutWith 175 units of T4DNA ligase [Takagi Shuzo Co. Ltd.) in linking the solution having the above composition. Then the reaction solution was used for transformation of strain N 99 Cl+E. coli (Pharmacia). Plasmid was isolated from the culture resistant to ampicillin colonies transformers and processing RI and Wamn to confirm that this plasmid is desired plasmid.

(IV) preparation of recombinant vector and transformers, expressmusic G-CSF.

Plasmid expression obtained in (III), was treated with restriction enzyme laI. After you create blunt ends of the plasmid and then treated as in example 12, to obtain a recombinant vector with the inserted CDNA fragment of G-CSF. This vector was used for transformation of strain N 4830 E. coli (Pharmacia Fine Chemicals) according to the method using calcium chloride as described in Molecular Cloning (see.above) the Identity of the desired transformants was produced as in example 12 (Fig.21-22).

P R I m e R 14. Construction of E. coli-recombinant vector (+ANY) and transformacija (using trp-promoter-containing vector).

1) Construction of recombinant vector.

1) Obtaining the (approximately 330 base pairs) in pBR322 at the site lAl. Ten micrograms of this plasmid was treated with 7 units restrictase lAl and 8 units of Pvu II at 37aboutC for 3 h in 30 μl of a reaction solution consisting of 10 mmol/l Tris-Hcl, 6 mmol/l MgCl2and 50 mmol/l NaCl.

Then added 2 μl of alkaline phosphatase (Takagi Shuzo Co.0, Ltd.) and the reaction was conducted at 60aboutC for 1 h

The DNA fragment (about 2.5 μg) of about 2.6 thousand bases in length were isolated from the reaction solution by gelelectrophoresis 1% agarose gel.

(II) Obtaining a synthetic linker.

Oligonucleotides having a sequence synthetic linkers CGGAATTACTGGGCGT and AGGGCGCATC, phosphorylation and senatoriable, as in (II) in example 12, to obtain a synthetic linker.

(III) Obtaining a recombinant vector.

Approximately 1 μg of vector fragment obtained in (I), about 1 μg of the synthetic linker, obtained in (II), and about 1 μg of a DNA fragment G-CSF obtained in (III) in example 12, in contact with 175 units of T4DNA ligase overnight at the 12aboutWith 20 ál of binding solution having the composition described in example 12,1) (IV), in order to obtain a recombinant vector (Fig.23).

As in example 12, the plasmid was isolated from resistant to ampicillin colonies of transformants, and processed this plasmid by restrictase Are, Dra I, NruI and Pst I, which showed that the obtained desired transformants.

P R I m e R 15. The cultivation of the transformants.

1) Cultivation of the transformants (tac) obtained in example 12.

Transformants were cultured overnight at 37aboutWith 1 ml of culture was added to 100 ml of medium, Luria (Luria) containing 25 μg/ml or 50 μg/ml ampicillin. Cultivation was carried out for 2-3 h at 37aboutC.

Cultivation was carried out at 37aboutC for 2-4 h after addition of isopropyl- -D-thiogalactoside, to obtain a final concentration of 2 mmol/L.

2) Cultivation of the transformants (with PL) obtained in example 13.

Transformants were cultured overnight at 28aboutWith 1 ml of culture was added to 100 ml of medium, Luria (Luria) containing 25 or 50 μg/ml ampicillin. The cultivation was carried out for approximately 4 h at 28aboutC.

The cultivation was continued for 2-4 hours at 42aboutC.

3) Cultivation of the transformants (trp) obtained in example 14 the M9, containing 0.5% glucose, 0.5% esamination (asamino acids (Difco) and 25 or 50 μg/ml ampicillin. Cultivation was carried out for 4-6 h at 37aboutC. After adding 50 μg/ml 3- -intracrinology acid (IAA), the cultivation was continued for 4-9 hours at 37aboutC.

P R I m e R 16. Isolation and purification of the polypeptide G-CSF from E. coli

1) the Selection.

Three transformants, cultivated in example 15 was subjected to the following methods of allocation.

Culture (100 ml) was subjected to centrifugation to obtain a cell precipitate after centrifugation, suspended in 5 ml of a mixture of 20 millimoles/l Tris-Hcl (pH 7.5) and 30 millimoles/l NaCl.

Then added 0.2 mol/l of phenylmethylsulfonyl 0.2 mol/l EDTA to obtain concentrations of, respectively, 1 mmol/l 10 mmol/l and 0.2 mg/ml, and the suspension was placed for 30 min at 0aboutC.

Cells were literally using three cycles of freezing/thawing, after which it is desirable to process the ultrasound. The lysate was centrifuged to obtain the supernatant. Alternatively, the lysate was treated with 8 mol/l guanidinoacetate so that the final concentration became 6 mol/l of guanidinoacetate then conducted centrifugational liquid, obtained in 1) was subjected to gel filtration on a column of Ultrogel AcA54 (diameter 4.6 mm, length 90 cm, LKB) at a flow rate of about 50 ml/h using 0.01 mol/l Tris-Hcl buffer (pH 7.4) containing 0.15 mol/l NaCl and 0.01% tween-20 (Nakai Called Co. Ltd.).

Selected fractions that showed activity in the analysis method of determining colonystimulating activity (b) (described earlier in this description), and concentrated to a volume of approximately 5 ml using ultrafiltration equipment RM-10 (AMAP).

(II) To the concentrated fractions were added n-propanol (purity suitable for determining amino acid sequences Tokyo Kasei Co. Ltd. and triperoxonane acid, and the mixture is processed to a final concentration of n-propanol and triperoxonane acid was 30 and 0.1%, respectively. The treated mixture was left in ice for about 15 minutes and subjected to centrifugation at 15000 rpm for 10 min to remove the precipitate. The supernatant was adsorbing column-Bondapak C18 (prepreparation varieties; Waters; 8 MMX h cm), which was balanced with an aqueous solution containing n-propanol (see above) and triperoxonane acid. The column was continuously suirable aqueous solution of 0.1% triptone chromatograph high-pressure company "Hitachi"), "Hitachi model 638-41 (detector company "Hitachi"), simultaneously measured values of absorption at wavelengths of 220 and 280 nm. After elution of 10 microlitres aliquot each fraction was diluted 100 times, and the diluted solution was subjected to selection to search active fractions according to the method of determining colonystimulating activity (b). Activity was observed at the peaks, which were suirable at 40% n-propanol. These peaks were mixed and re-chromatographically under the same conditions as used above, and the fractions were subjected to testing to determine their activity according to the method (b). Again, the activity was found in the peaks at 40% n-propanol. These active peaks were collected (four fractions of 4 ml) and dried by freezing.

(III) Dried by freezing the powder was dissolved in 200 μl of an aqueous solution of 0.1% triperoxonane acid containing 40% n-propanol, and the solution was subjected to liquid chromatography high-pressure column SK-G300 SW (7.5 mm x 60 cm, Too Soda Manufacturins Co. Ltd.). Elution was performed at a flow rate of 0.4 ml/min with an aqueous solution of 0.1% triperoxonane acid containing 40% n-propanol, and fractions of 0.4 ml were collected using a collector of fractions FRAC-100 (Pharmacia Fine Chemicals). These fractions have checked the above way is column-Bondapak C18 (4.6 mm x 30 cm), and highlighted the main peak and dried by freezing.

Thus obtained protein was treated with 2-mercaptoethanol and subjected to gel electrophoresis with SDS-polyacrylamide gel (15,0%) (15 mV, 6 h). When staining dye oomassie Blue the desired polypeptide G-CSF can have identificirovat in the form of a single strip.

P R I m e R 17. The determination of the activity of G-CSF (+VSE).

The CSF sample obtained in example 16, were analyzed by the method of analysis of CSF(a), described earlier in this description. The results are shown in table.1.

P R I m e R 18. Amino acid analysis (+ANY)

1) the Analysis of amino acids

The CSF sample, purified in example 16, hydrolyzed in the usual ways, and amino acid composition of the protein part of the hydrolysate was analyzed by the method of amino acid analysis using an automatic amino acid analyzer Hitachi 835" company "Hitachi"). The results are shown in table.2. The hydrolysis was performed under the following conditions: (I) 6 n Hcl solution 110aboutWith 24 hours in vacuum, (II) 4 n solution of methanesulfonate + 0.2% of 3-(2-amino-ethyl) indole, 110aboutC, 24 h, 48 h, 72 h in vacuum.

The sample was dissolved in a solution (1.5 ml) containing 40% n-propanol and 0.1% triperoxonane acid. Ali is s in (I) or (II), containers tightly closed in a vacuum, and then spent the hydrolysis of the content.

Each of the values shown in the table.2 represented an average value of four measurements, values for 24 h to (I) and values for 24,48 and 72 h for (II), except that the content of Tre, Ser, 1/2 Cys, Met, Val, Ile and TRP was calculated using the following methods (see "Tamaki Called (Protein Chemistry) II, A Course in Biochemical periments, Tokuo Depending Dohjin):

For Tre, Ser, 1/2 Cys and Met the curve of the time-dependent values for 24, 48 and 72 h for (I) were extracted to zero hours.

For Val and Ile used value for 72 h for (II).

For the HSP usage: average values for 24,48 and 72 h for (II).

2/ analysis of the N terminal amino acids

The sample was subjected to decomposition by Admino using gas-phase determinant sequence (company "Applied" Biosystems"), and the PTH-amino acids were analyzed by conventional methods using a liquid chromatograph, high pressure (firm Beckman instruments") and column Ultrasphere-ODS (firm "Beckman instruments"). After column (5 µm, diameter 4.6 mm x 250 mm in length) was balanced by the original buffer [aqueous solution containing 15 mmol/l sodium acetate buffer (pH 4, the ri the isocratic elution of the source buffer. During these stages was supported by the flow rate of 1.4 ml/min and the column temperature 40aboutC. Detection of PTH-amino acids was carried out by measuring the magnitude of the absorption in the ultraviolet region of the spectrum at a wavelength of 269 nm and 320 nm. Standard samples (weighing 2 nmol) PTH-amino acids (Sigma) were separated on the same line, to determine their retention times, which were compared with the retention time of the sample to identify the N-terminal amino acids. The result were found PTH-methionine and PTH-threonine.

P R I m e R 19. Construction of E. coli recombinant vector (-ANY) and transformation

1) use the tac-promoter-containing vector.

Repeated the methods of example 12, except that "pBRG 4 obtained in example 9, which contained the CDNA shown in Fig.3-5 [cm.(III) in example 12] was replaced by "pBRV2 obtained in example 10, which contained the CDNA shown in Fig. 8-10". As in example 12, it was confirmed that the obtained transformants are desired transformants Fig.24.

2) Use PL-promoter-containing vector

Repeated the methods of example 13, using CDNA (-ANY), and it was confirmed that the obtained transformants are with is authorily the methods of example 14, using CDNA (-ANY), and it was confirmed that the transformants was a need transformants (Fig.27).

P R I m e R 20. Analysis of the activity of G-CSF (-ANY).

Three types of transformants obtained in example 19, were cultured according to the method described in example 15. From the cultured cells of E. coli were isolated polypeptides G-CSF and purified them by the method described in example 16, resulting in a polypeptide the human G-CSF obtained in the form of a single strip.

Thus obtained sample of CSF was analyzed by the method of analyzing the activity of CSF(a), described earlier in this description.

The results are shown in table.3.

P R I m e R 21. Amino acid analysis (-ANY)

1) Analysis of amino acid composition.

The amino acid composition of the CSF sample, purified in example 20 were analyzed according to the method described in 1) in example 18. The results are shown in table. 4.

2) Analysis of N-terminal amino acid,

The sample was subjected to analysis of N-terminal amino acid by the method described in 2) in example 18. The result were found PTH-methionine and PTH-threonine.

P R I m e R 22. Obtaining vector pH GA410 (for use with animal cells, +ANY line)

Fragment oRI, the value of 2 h, then treated with fragment Klenow DNA polymerase I (Takagi Shuzo Co. Ltd) to create blunt ends. One microgram of the linker (8 mer, Takara Shuzo Co. Ltd. ) was fosforilirovanii with ATF and added separately to the mixture of DNA fragments. Attached fragments were treated with restriction enzyme Bgl II and subjected to gel electrophoresis with agarose gel. Then selected only the large fragment of DNA.

This DNA fragment was equivalent to the coding portion of the polypeptide of the human G-CSF containing approximately 710 base pairs (see Fig.18) the Vector pdKCR [Fukunaga et al. Proc. Natl. Acad. Sci. USA, 81,5086(1984)] was treated with restriction enzyme Wamn, and then dephosphorylated alkaline phosphatase (Takagi Shuzo Co. Ltd. ). The resulting vector DNA was added to the CDNA fragment length 710 base pairs in the presence of T4DNA ligase (Takara Shuzo Co. Ltd.) in order to get G410 (Fig.28). As shown in Fig.28, this plasmid contained early gene, SV40 promoter, origin of replication of SV40, part of the gene rabbit-globin, the site of initiation of replication of pBR322 and pBR322-derived-lactamase gene AMRr), and the gene of the human G-CSF is attached downstream from the promoter of the early gene, SV40.

P R I m e R 23. Construction of recombinant vector (+ANY) used for transfromer 22, dissolved in the reaction solution consisting of 50 mmol/l Tris-Hcl (pH 7.5), 7 mmol/l MgCl2, 100 mmol NaCl, 7 mmol/l 2-mercaptoethanol and 0.01% bovine serum albumin (BSA). Added the restriction enzyme EcoRl (10-15 units; Takara Shuzo Co. Ltd.), and the reaction solution was kept at 37aboutC for about 30 min to induce partial digestion of Eco RI. Then the DNA fragment was subjected to two treatments with 1:1 mixture of phenol/chloroform and then treated with ether and held precipitation with ethanol.

The obtained DNA fragment was dissolved in 50 μl of a solution consisting of 50 mmol/l Tris-Hcl, 5 mmol/l MgCl2, 10 mmol/l D and 1 mmol/l each of d, d, d and d. After adding 5 μl of a fragment of Klenow E. coli DNA polymerase (Takagi Shuzo Co. Ltd.) the solution was incubated at 14aboutC for 2 h to create blunt ends.

By subsequent gel electrophoresis in 0.5% agarose gel gave 6 μg of DNA fragment length of approximately 5.8 thousand base pairs.

Five micrograms of the selected DNA fragment was re-dissolved in 50 µ g of a reaction solution consisting of 50 mmol Tris-Hcl (pH 7,6), 10 mmol/l MgCl2, 10 mmol/l D and 1 mmol/l ATP. After adding 2 μg of linker Hind III (Takara Shuzo Co. Ltd.) and 100 units of T4Gnolam and ether and precipitation with ethanol. The precipitate was dissolved in 30 μl of a solution consisting of 10 mmol/l Tris-Hcl (pH 7.5), 7 mmol/l MgCl2and 60 mmol/l NaCl, and the solution was incubated at 37aboutC for 3 h in the presence of 10 units of Hind III. After re-processing of the T4DNA ligase, the DNA used for transformation of strain DHI E. coli by processing using rubidium chloride (see above Molecular Cloning). From resistant to ampicillin (Ampv) colonies of transformants were selected cells carrying plasmid, which was identical to the GAA 410 except that the Hind III was inserted into the site oRI. Thus obtained plasmid was named pGA410(N) (Fig.29).

2) Construction of recombinant expression vector N-G4

Twenty micrograms thus obtained pH GA410 (H) was dissolved in 50 μl of a reaction solution consisting of 10 mmol/l Tris-Hcl (pH 7.5), 7 mmol/l MgCl2, 175 mmol/l NaCl, 0.2 mmol/l EDTA, 7 mmol/l 2-mercaptoethanol and 0.01% bovine serum albumin. After adding 20 units of SalI (Takagi Shuzo Co. Ltd.), the reaction solution was incubated at 37aboutC for 5 h After treatment with phenol and precipitation with ethanol incubation was performed as in 1), for approximately 2 h at 14aboutWith the presence of a fragment of the Clancy DNA polymerase (That is gel, the reaction solution was immediately subjected to precipitation with ethanol. The obtained DNA fragment was treated with Hind III and allocated 5 g fragment ind III Sal I (about 2.7 thousand base pairs) using gel electrophoresis 1% agarose gel. In a separate stage plasmid pd BPV-I have the virus bichiev papilloma [this plasmid was provided by Dr. Hawley, and it is described in Sarver, N, Sbyrne, J. C. owley, P. M. Proc. Natl. Acad. Sci. USA, 79, 7147-7151 (1982)] was treated with Hind III and Pvu II, as described Nagata with TCS. [Nagata et al.) [Fukunaga, Sokawa and Nagata, Proc. Natl. d. Sci. USA, 81, 5086-5090(1984)] to obtain the DNA fragment length 8.4 thousand bases. This DNA fragment 8.4 thousand reasons and separately obtained Hind III-Sal I DNA fragment (about 2.7 thousand bases) connected with T4DNA ligase. Product link used for transformation of strain E. coli DHI method using rubidium chloride, described in Molecular Cloning, see above. Selected colonies of E. coli carrying a plasmid with the GA 410-derived CDNA G-CSF. This plasmid was named N-G4 (Fig.29).

Similarly handled adenovirus type II [Tanpakushitsu, Kakusan, Koso (proteins, nucleotide acid and enzymes), December 27, 1982, Kyoritsu Shuppan] to obtain plasmid pVA, which contained a SalI-Hind III fragment length of approximately 1700 base pairs carrying VAI and VAII, returns NG4VA and pTNG4VA (Fig.29). Because of the presence of VA gene of adenovirus these plasmids were able to increase the expression product of transcription from the early promoter of SV40.

P R I m e R 24. Transformation of cells S and expression in them G-CSF (+ANY).

Before use for cell transformation mouse S, N-G4, obtained in example 23 was treated with restriction enzyme BamHI. Twenty micrograms of plasmid RT N-G4 was dissolved in 100 μl of reaction solution [10 mmol/l Tris-Hcl (pH 8.0), 7 mmol/l MgCl2, 100 mmol/l NaCl, 2 mmol/l 2-mercaptoethanol, 0.01% of bovine serum albumin] and treated with 20 units of Bam HI (Takagi Shuzo Co. Ltd), and then processed mixture of phenol and ether and held precipitation with ethanol.

Cells of mice S were grown in minimum essential medium of Dulbecco containing 10% serum bovine embryo ("Gibco"). Cells growing on cups (5 cm in diameter), transformed individual DNA, 10 μg, per Cup, according to the method using calcium phosphate/(cm. Haynes, J. Weissman, S. Nuclec ids Res. II, 687-706 (1983)] After treatment with glycerol, the cells were incubated at 37aboutC for 12 h

Incubated cells were transferred to three new cups (5 cm in diameter), and the medium was changed twice a week. On the 16th day of the red Dulbecco, containing 10% serum bovine embryo ("Gibco") in order to select clones with a high rate of production of G-CSF. These clones were produced G-CSF at a concentration of approximately 1 mg/L. Subsequent cloning gave the clones, which could produce G-CSF at concentrations of 10 mg/l or higher. In addition to the cells S, as the host cells can also use cells NI33.

P R I m e R 25. Expression of G-CSF in cells Cho (+ANY)

1) Construction of GG4-dhfr.

Twenty micrograms of plasmid GA410 obtained in example 22 was dissolved in 100 μl of reaction solution containing 10 mmol/l Tris-Hcl (pH 7.5), 7 mmol/l MgCl2, 175 mmol/l NaCl, 0.2 mmol/l EDTA, 0.7 mmol/l 2-mercaptoethanol and 0.01% bovine serum albumin. The reaction was carried out overnight at 37aboutIn the presence of 20 units of restrictase Sal I (Takagi Shuzo Co. Ltd.), then spent the processing of phenol and ether and precipitated with ethanol.

The precipitated DNA was dissolved in 100 μl of a reaction solution consisting of 50 mmol/l Tris-Hcl, 5 mmol/l MgCl2, 10 mmol/l D and 1 mmol/l each of d, d, d and d, and the reaction was carried out at the 14aboutC for 2 h in the presence of fragment Klenow E. coli DNA polymerase (10 microlitres; Takagi Shuzo Co.to DNA in the Deposit using the following methods: DNA was dissolved in 50 μl of reaction solution, consisting of 50 mmol/l Tris-Hcl (pH 7.4), 10 mmol/l D, 0.5 mmol/l spermidine, 2 mmol/l ATP, 2 mmol/l hexamine-Coulthard and 20 μg/ml bovine serum albumin. The reaction was carried out at 4aboutC for 12-16 h in the presence of Eco RI linker (Takagi Shuzo Co. Ltd.) and 200 units of T4DNA ligase (Takagi Shuzo Co. Ltd.). After treatment with phenol, washing with ether and precipitation with ethanol, which was carried out in accordance with conventional methods, the precipitated DNA was partially digested with Eco RI and using gel electrophoresis 1% agarose gel received 3 μg of DNA fragment length of about 2.7 thousand base pairs.

Plasmid pAdD26SVpA [Kaufman, R. G., Sharp, P. A. Mol. ll Biol. 2, 1304-1319 (1982)] was treated with oRI and dephosphorylated by treatment with bacterial alkaline phosphatase. More specifically, 20 µg dD26SVp and 20 units oRI added to the reaction solution [50 mmol/l Tris-Hcl (pH 7.5), 7 mmol/l MgCl2, 100 mmol/l NaCl, 7 mmol/l 2-mercaptoethanol and 0.01% bovine serum albumin and the reaction was conducted at 37aboutC for 10 h and Then 5 units of bacterial alkaline phosphatase was added to the reaction solution, the reaction was carried out at 68aboutC for 30 minutes After treatment with phenol by electrophoresis allocated oRI fragment pAdD26SVpA with access pity. The obtained plasmid was used for transformation of strain E. coli DHI method using rubidium chloride, and selected colonies carrying plasmids pGG4-dhfr. The obtained plasmid was named GG4-dhfr (Fig.30).

An alternative method was as follows: plasmid pH 410 GA treated Sal I and partially digested with Eco RI without attaching any oRI-linker. Highlighted the DNA fragment length of about 2.7 thousand base pairs and treated with fragment Klenow E. coli DNA polymerase to create blunt ends. Fragment oRI having blunt ends, received from pdD26SVp, as described above. This oRI-fragment separately and the resulting fragment (about 2.7 thousand base pairs) was treated with T4 DNA ligase to obtain pGG4-dhfr.

Plasmid pH 410 GA(N) obtained in example 23 was treated with restrictase Hind III and Sal I, as described in 2) in example 23, and the fragment Hind III Sal I annexed obtuse Eco RI fragment pAdD26SVp described above. This method can also be used to obtain GG 4-dhfr (Fig.31)

2) Build pG4DRI and pG4DR2.

Ten micrograms of plasmid pAdD26SVpA specified in (I), dissolved in 50 ml of reaction solution containing 50 mmol/l Tris-Hcl (pH 7.5), 7 mmol/l MgCl2, 100 mmol/l NaCl, 7 mmol/l 2-mercaptoethanol and 0 and at 37 ° C for 10 h, then the mixture was treated with phenol and washed with ether. The DNA fragment of about 2 thousand reasons highlighted by electrophoresis through 1% agarose gel with a low melting point. The selected DNA fragment processed fragment Klenow DNA polymerase according to normal procedures in order to create blunt ends. Blunt DNA fragment was subjected to treatment with phenol, washed with ether and held precipitation with ethanol. if Sal I and partially digested with Eco RI without attaching any oRI-linker. Highlighted the DNA fragment length of about 2.7 thousand base pairs and treated with fragment Klenow E. coli DNA polymerase to create blunt ends. Fragment oRI having blunt ends, received from pdD26SVp, as described above. This oRI-fragment separately and the resulting fragment (about 2.7 thousand base pairs) was treated with T4DNA ligase to obtain pGG4-dhfr.

Plasmid pH 410 GA(N) obtained in example 23 was treated with restrictase Hind III and Sal I, as described in 2) in example 23, and the fragment Hind III Sal I annexed obtuse Eco RI fragment pAdD26SVp described above. This method can also be used to obtain GG 4-dhfr (Fig.31)

2) Build pG4DRI and pG4DR2.

Ten micrograms of plasmid pAdD26SVpA specified in (I), resol/l 2-mercaptoethanol and 0.01% bovine serum albumin. After addition of 10 units each restricts EcoRI and BamHI, the reaction was carried out at 37aboutC for 10 h, after which the mixture was treated with phenol and washed with ether. The DNA fragment of about 2 thousand reasons highlighted by electrophoresis through 1% agarose gel with a low melting point. The selected DNA fragment processed fragment Klenow DNA polymerase according to normal procedures in order to create blunt ends. Blunt DNA fragment was subjected to treatment with phenol, washed with ether and held precipitation with ethanol.

Ten micrograms of the plasmid, RNA 410(H) obtained in 1) of example 23 was dissolved in 50 μl of reaction solution containing 10 mmol/l Tris-Hcl (pH 7.5), 7 mmol/l MgCl2and 60 mmol/l NaCl. The reaction was carried out at 37aboutC for 6 h in the presence of 10 units of Hind III. The DNA fragment was isolated by electrophoresis through 1% agarose gel with a low melting point, which was carried out according to normal procedures. The selected DNA fragment was then treated with bacterial alkaline phosphatase, and blunt ends were obtained by processing the fragment Klenow. After treatment with phenol and washing with ether, the DNA fragment was joined blunt ends to the previously obtained DNA fragment of approximately 2 thousand bases with T2, 5 mmol/l D and 1 mmol/l ATP, and the reaction was carried out in 6aboutC for 12 h in the presence of 50 units of T4DNA ligase. Product link used for transformation of strain DHI E. coli. The result was G4DR1 and pG4DR2 shown in Fig.32.

3) Transformation and expression.

Cells Cho (strain dhfr-courtesy of Dr L. hasin of Columbia University) cultivation for growth in alpha-minimum essential medium containing 10% calf serum ( -MEN with the addition of adenosine, deoxyadenosine and thymidine) in the bowl (diameter 9 cm, Nunc). The cultured cells transformed according to the method using calcium phosphate [Wigler et al. ll, 14,725,(1978)] as follows.

Carrier DNA (DNA calf thymus) was added in a suitable quantity to 1 µg of plasmid GG4-dhfr obtained in 1), and the mixture was dissolved in 375 µg THOSE of the solution then added 125 μl of 1 mol/l CaCl2. After cooling the solution on ice for 3-5 min, to the solution was added 500 ál of 2x xBS (50 mmol/l Hepes, 280 mm NaCl, 1.5 mmol/l phosphate buffer). After re-cooling on ice the solution was mixed with 1 ml of culture Cho cells were transferred into cups and inkubirovali and add 20% glycerol-containing Tris-buffered saline and re-flushing added a non-selective medium (described above-IU N environment except what it added nucleotides). After incubation for 2 days, the culture is diluted 10 times, moved on selective medium (without supplements nucleotides). Continued cultivation, and the medium was replaced with fresh selective medium every 2 days, and the resulting colonies were selected and transferred to fresh Cup, where cells were grown in the presence of 0.02 µmol/l of methotrexate, then produced cloning by growing in the presence of 0.05 mmol/l of methotrexate, the content of which is then increased to 0.01 µmol/L.

The transformation of Cho cells can also be accomplished through co-transformation with GG4 and PAdD26SVpA [cm. Scahill et al. Proc. Natl. d. Sci. USA, 80, 4654-4658 (1983)]

Cells SNO also transformed in the following ways: pG4DRI or pG4DR2 obtained in (2), pre-processed, respectively, by using the SalI and kpni restriction sites, to obtain fragments of DNA, and 10 μg these fragments were used to transform cells of Cho, as described above, the transformed cells were subjected to continuous cultivation in a number of selective media according to the above method, after about 7 days appeared at least 100 individual colonies per Cup, these colonies were transferred entirely to a fresh Cup and subjected to about the wound dozen colonies, the same procedure was repeated with successive increase in the concentration of methotrexate to 0.02 mmol/l 0.05 mmol/l and 0.1 µmol/l, and selected colonies that survived the selection of colonies can be done in a similar way, even if individually to collect received more than 10 colonies and tuck their cultivation with increasing concentrations of methotrexate.

Recombinant vector carrying "a polycistronic gene", also can be used for transformation of Cho cells. An example of this alternative method is as follows: pAdD26SVpA processed Pst I and selected two pieces attached to BRG4-derived CDNA fragment CSF c in order to construct a recombinant vector, in which adenoviruses promoter, CDNA CSF, DHFR and poly(A) site SV40 were inserted in the written order. This recombinant vector was used to transform cells SNO.

P R I m e R 26. Analysis of the activity of G-CSF (+ANY).

The supernatant of cell cultures S and SNO received, respectively, in examples 24 and 25, brought to pH 4 using acetic acid. After adding an equal volume of n-propanol, the precipitate was removed by centrifugation. The supernatant was passed through an open Actulaly using 50% n-propanol. Eluent was diluted by half with water and have it subjected to reversed-phase liquid chromatography high-pressure column (YMC-C8 (Yamamura Kaguka K. K.), and then she suirable n-propanol (30-60% linear density gradient) containing 0.1% triperoxonane acid. Fractions that were suirable at concentrations of n-propanol 40% allocated, dried by freezing and dissolved in 0.1 mol/l glycinol buffer (pH 9). As a result of these procedures, human G-CSF in cells C and SNO concentrated approximately 20-fold.

As control cells transformed with plasmids containing CDNA of human G-CSF, and the supernatant of these cultures were concentrated in accordance with the methods described above. The activity of the human G-CSF samples were analyzed using methods of analysis of activity of the human G-CSF (a), described earlier in this description. If the efficiency of expression is sufficiently high, then the supernatant liquid cultures can be analyzed directly without prior concentration. The results are given in table.5, where data are based on concentrated samples.

P R I m e R 27. Amino acid analyses of sugar (+ANY)

1) Analysis of amino the least 2(III). The purified CSF sample hydrolyzed in the usual ways and protein part of the hydrolyzate was analyzed to determine its amino acid composition using a special method of amino acid analysis using an automatic amino acid analyzer Hitachi 835" company "Hitachi"). The results are shown in table.6. The hydrolysis was performed under the following conditions: (I) 6 n Hcl solution 110aboutWith 24 hours in a vacuum (II) 4 n solution of methanesulfonate + 0.2% of 3-(2-amino-ethyl) indole, 110aboutC, 24 h, 48 h, 72 h, under vacuum.

The sample was dissolved in a solution (1.5 ml) containing 40% n-propanol and 0.1% triperoxonane acid. Aliquots of 0.1 ml each were dried using dry nitrogen gas, and after adding the reagents listed in (I) or (II) containers tightly closed in a vacuum, and then spent the hydrolysis of the content.

Each of the values shown in the table.6, represented the average value of four measurements, values for 24 h to (l) and values for 24 h to (I) and values for 24,48 and 72 h for (II), except that the content of Tre, Ser, 1/2 Cys, Met, Val, Ile and TRP were calculated by using the following methods (see "Tamaki Called (Protein Chemistry) II" And urse in Biochemical periments, Tokuo Depending Dohjin):

For Tre, CE Is>For Val and Ile used value for 72 h For (II).

For TRD used average values for 24,48 and 72 h (II).

2) analysis of the sugar composition.

Internal standard (25 nmol Inositol) was added to 200 ng of purified CSF sample used in the analysis of amino acid composition 1). After adding methanol solution (500 μl) containing 1.5 and a solution of Hcl reaction was performed at 90aboutC for 4 h in nitrogen purged sealed tube. After that, as the tube opened, added silver carbonate (Ag2CO3to neutralize the contents. Then added 50 μl of acetic anhydride, and the tube was shaken for a sufficient period of time. Then the tube was left overnight in the dark at room temperature. The top layer was placed in an exemplary test tube and dried using nitrogen gas. To the precipitate was added methanol, and the mixture is washed and slightly ofcentrifugal. The top layer was placed in the same test tube and dried. After adding 50 ál MS (a mixture of 5:1:1 pyridine, hexamethyldisilazane and trimethylchlorosilane) carried out a reaction at 40aboutC for 20 min, and the reaction product was kept in a low temperature freezer. With The Gal), N-atsetilgalaktozamin (Gal Nac), sialic acid, and any other suitable reagents.

Thus obtained samples were subjected to gas chromatography analysis under the following conditions:

Analysis conditions Column: 2% HP OV, 60-80

17 VIN port mesh, 3M,

glass

Temperature: increasing from

110 to 250aboutWith speeds

4aboutC/min

The pressure of the carrier gas (N2):

initial of 1.2-1.6 kg/cm2< / BR>
end 2-2,5 kg/cm2< / BR>
Sensitivity: range

103M range, 0.1 to 0.4 V

Pressure: H20.8 kg/cm2< / BR>
the air is 0.8 kg/cm2< / BR>
Supply sample: 2,5-3,0 mm.

In the analysis, the CSF sample of the present invention were identified galactose, N-atsetilgalaktozamin and sialic acid.

P R I m e R 28. Obtaining vector pH GV2 (for use with animal cells, the line-VSE).

oRI fragment obtained in example 10, which had the CDNA shown in Fig. 8-10, was treated with restriction enzyme Dra I at 37aboutC for 2 h, after which the processed fragment Klenow DNA polymerase I (akava Shuzo Co. Ltd.), to create blunt ends. One microgram of linker Bgl II (8 mer, Takagi Shuzo Co. Ltd.) was fosforilirovanii using ATP is Riccati Bgl II and subjected to agarose gel electrophoresis. Then selected only the large fragment of DNA.

This DNA fragment was equivalent to about 700 base pairs, contains a section that encodes the polypeptide of human G-CSF (see Fig.18). Vector pdKCR[Fukunag et al. Proc. Natl. d. Sci. USA, 81, 5086 (1984)] was treated with restriction enzyme BamHI, and then dephosphorylated alkaline phosphatase (Takagi Shuzo Co. Ltd.). The resulting vector DNA was added to the CDNA fragment of approximately 700 base pairs in the presence of T4 DNA ligase (Takagi Shuzo Co. Ltd. in order to obtain a pH GV2 (Fig.33). As shown in Fig.33, this plasmid contains the early promoter of the gene SV 40, the site of replication initiation SV 40, part of the gene rabbit-globin, the site of replication initiation R322 and pBR 322 bred-lactamase gene (Ampr), and the gene of the human G-CSF is attached below thread from the early promoter of the gene SV 40.

P R I m e R 29: Construction of recombinant vector (-ANY) for use in transformation of cells S.

1) Construction pH GV 2(H).

Twenty micrograms of plasmid pH GV2 (Fig.17)), obtained in example 28, was treated by the methods described in 1) in example 23, to obtain plasmid, named pH GV2 (N) (Fig.34).

2) Construction of recombinant vectors expressii NV2, tabret E. coli, carrying a plasmid having a pH GV2 CDNA derived G-CSF. This plasmid was named RT N-V2 (Fig.34).

Similarly handled adenovirus type II [ampakushitsu, Kakusan, Koso (Proteins, nucleic acids and enzymes), December 27, 1982, Kyoritsu Shuppan] to obtain plasmid pVA, which contained a SalI-Hind III fragment of approximately 1700 base pairs carrying VAI and VAII and the fragment containing the VAI and VAII, was isolated from this plasmid. This fragment was inserted into pN-V2 site Hind III to obtain NVA and NVA (Fig.18). Because of the presence of VA gene of adenovirus these plasmids could enhance the expression product of transcription from the early promoter of SV40.

P R I m e RNO 30. Transformation of cells S and expression in them G-CSF (-ANY) N-V2 obtained in example 29, was treated with restriction enzyme BamHI before it was used to transform cells of mice S.

Cells mouse Is transformed using the thus obtained DNA to expressivity G-CSF (see example 24), and selected clones with a high rate of production of G-CSF. These clones were produced G-CSF at a concentration of approximately 1 mg/l

By subsequent cloning is possible to select clones that can produce G-CSF at a concentration of 10 mg/L. Similarly, cells C cord is the functioning to produce G-CSF, as for NVA you can get clones capable of producing G-CSF with outputs of 20 mg/l or more, while by transformation with NVA got clones with lower productivity (a few mg/l).

In addition to the cells S, as the host cells can also use cells N1H33.

P R I m e R 31. Expression of G-CSF in cells Cho (-ANY).

1) Construction of GV-2dhfr.

The DNA fragment length of about 2.7 thousand base pairs were prepared from 20 micrograms plasmid GV2 (example 28) using the methods described in 1) in example 25. This fragment (0.5 μg) and RI-fragment pAdD26SVp (0.5 μg) was subjected to renaturation. The obtained plasmid was used for transformation of strain DHI E. cli method using rubidium chloride, and selected colonies carrying plasmids pGV2-dhfr. The obtained plasmid was named GV2-dhfr (Fig.35).

An alternative method was as follows: plasmid GV2 was treated with Sal I and partially digested with Eco RI without attaching any linker Eco RI. Highlighted the DNA fragment length of about 2.7 thousand base pairs, and it was treated with the fragment of Klenow E. Li DNA polymerase to create blunt ends. Blunt RI-fragment prepared from pAD26SVpA as MP4 DNA ligase to obtain GV2-dhfr.

GV2(N) obtained in 1) of example 29, was treated with restrictase Hind III and SalI as described in 2) in example 29, and Hind III Sal I fragment attached to obtuse Eco RI fragment pAdD26SVpA described above. This method can also be used to obtain GG4-dhfr (Fig.36).

2) Build pV2DR1 and pV2DR2

Ten micrograms of plasmid pAdD26SVpA mentioned in 1) was dissolved in 50 ml of reaction solution containing 50 mmol/l Tris-Hcl (pH 7.5), 7 mmol/l MgCl2, 100 mmol/l NaCl, 7 mmol/l 2-mercaptoethanol, and 0.01% bovine serum albumin. The reaction was carried out at 37aboutC for 10 h in the presence of 10 units each restricts oRI and BamHI. Therefore, the processing of phenol and washing the ether was performed in the usual way. The DNA fragment length of about 2 thousand reasons highlighted by electrophoresis through 1% agarose gel with a low melting point. The selected DNA fragment was treated with the fragment of Klenow DNA polymerase in the usual way in order to create blunt ends. Blunt DNA fragment was treated with phenol, washed with ether and held precipitation with ethanol.

Ten micrograms of plasmid GV2(N) obtained in 1) of example 29 was dissolved in 50 μl of the reaction PP>C for 6 h in the presence of 10 units of Hind III. The DNA fragment was isolated by electrophoresis through 1% agarose gel with a low melting point, which is conducted in the usual way. The selected DNA fragment was then treated with bacterial alkaline phosphatase, and blunt ends were created by processing the fragment maple. After treatment with phenol and washing with ether, the DNA fragment was joined blunt ends to the previously obtained DNA fragment of approximately 2 thousand bases with T4DNA ligase according to the following procedure: 1 micrograms of each DNA fragment was dissolved in 50 micrograms reaction solution containing 66 mm/l Tris-Hcl (pH 7.5), 6.6 millimoles/l MgCl2, 5 mmol/l D and 1 mmol/l ATP, and the reaction was carried out in 6aboutC for 12 h in the presence of 50 units of T4DNA ligase. Product link used for transformation of strain DHI E. coli. The result was plasmids pV2DR1 and pV2DR2 shown in Fig. 37.

3) Transformation and expression.

Cells SNO transformed with the plasmid pH GV2-dhfr for the expression of G-CSF in accordance with the methods described in 3) in example 25.

Transformation of cells SNO can also be implemented through joint transformation predvaritelno treated with Sal I and Kpn I, respectively, to obtain DNA fragments, and 10 µg in these fragments were used to transform cells of Cho, as described above, the transformed cells were subjected to continuous cultivation in a number of selective media thus, as described above, approximately 7 days was at least 100 individual colonies per Cup, these speakers are transferred entirely to a fresh Cup and subjected to continuous cultivation in a number of selective media in the presence of 0.01 µmol/l of methotrexate, then appeared dozen colonies, the same methods were repeated at a concentration of methotrexate, consistently increasing to 0.02 mmol/l 0.05 mmol/l and 0.1 µmol/l, and selected colonies that survived the selection of colonies can be performed in a similar manner, the dal if you receive 10 + colonies individually selected and subjected to cultivation with increasing concentrations of methotrexate.

Recombinant vector carrying "a polycistronic gene" can also be used to transform cells of SSC. An example of this alternative method consists in the following: pAdD26SV processed > PST and selected two pieces attached to pBRV2 derived CDNA fragment CSF in order to construct a recombinant vector, in to the sector used for cell transformation SNO.

P R I m e R 32. Analysis of the activity of G-CSF (-ANY)

Using the methods described in example 26, the human G-CSF was obtained from the supernatant fluids of cell cultures S cells and SNO, which did in example 30 and 31, respectively. The activity of the human G-CSF each of the selected samples was analyzed as in example 26. The results are shown in table.7.

P R I m e R 33. Amino acid analysis and sugar analysis (-VSE)

1) Analysis of amino acid composition.

The crude CSF sample obtained in example 32, was purified in accordance with the techniques described in example 2(III). The purified CSF sample was subjected to analysis of its amino acid composition by the methods described in 1) in example 27. The results are shown in table.8.

2) analysis of the sugar composition.

The purified CSF sample used for the analysis of amino acid composition in 1), were analyzed to determine the sugar composition using the same methods and under the same conditions as described in 2) in example 27. As a result of this analysis was confirmed by the presence of galactose, N-atsetilgalaktozamin and sialic acid in the CSF sample of the present invention.

P R I m e R 34. Construction of recombinant vector in example 11 and which contained a chromosomal gene, it is shown in Fig. 13-17, processed RI. Plasmid SVH+K+described Banerji et al. ll, 27, 299 (1981), processed Kpn I to remove glubinoy gene. Then the plasmids were subjected to partial digestion with Hind III and to remove part of the late gene SV40. Fragments connected again to obtain the expression vector pML-E+.

This vector was treated with restriction enzyme RI and dephosphorylated alkaline phosphatase (Takagi Shuzo Co. Ltd) to obtain the vector DNA, which is connected with the above chromosomal DNA with T4DNA ligase (Takagi Shuzo Co. Ltd.), to get L cèze . As shown in Fig. 20, this plasmid contained the amp gene of SV40, early replication of SV40, early replication pB R322 and pBR322 derived-lactamase gene (AMRr), and had a chromosomal gene of human G-CSF, connected downstream from the amp gene SV40.

P R I m e R 35. The expression of the chromosomal gene for human CSF in COS cells.

Cells COS-I (obtained by courtesy of Dr. Gluzman from laboratory Cold spring Harbor, USA), which were grown to a density of about 70% in Petri dishes (diameter 9 cm, Nunc) using DMEM (modified Dulbecco environment Needles supplied by the company Nissui Seiyaku K. K. under LCIA [Wigler et al. ell, 14, 725, (1978)] or a method using DE-dextran, chloroquine, see for example, Gordon al. Sciencå, 228, 810 (1985)]

Transformation method using calcium phosphate was carried out as follows: 160 µg plasmid pMLC obtained in example 34 was dissolved in 320 μl of THE solution and after adding distilled water (3.2 ml) was added 504 μl of 2 mol/l CaCl2.

To the resulting solution was added 4 ml of 2 BS (50 mmol/l Hepes, 280 mmol/l NaCl, 1.5 mmol/l phosphate buffer, pH 7,12) and the mixture was cooled on ice for 20-30 minutes the Cooled mixture was added dropwise to the environment in quantities of 1 ml per Petri dish growing cells OS-1. After culturing for 4 h at 37aboutWith the incubator with CO2the cells were washed in serum-free DMEM medium, and then left to stand for approximately 3 minutes at room temperature in 5 ml of DMEM medium containing 20% glycerol, and then washed repeatedly serum-free medium D. After removal of the serum-free medium D, added 10 ml of medium D containing 10% calf serum, and the cultivation was continued over night in an incubator with CO2. After the medium was replaced with fresh medium of the same type, cultivation was continued for another 3 days.

aboutC for 12 h, and then cells were twice washed in serum-free medium D and subjected to further cultivation at 37aboutC for 2 h in DMEM containing 10% calf serum and 1 mmol/l chloroquine, after that, cells were washed twice in serum-free medium D and cultivated at 37aboutC for an additional 3 days in the environment D containing 10% calf serum.

The supernatant thus obtained cell culture OS-1 brought to pH 4 with 1 n solution of acetic acid. After adding an equal volume of n-propanol, the precipitate was removed by centrifugation. Surface solution was passed through a short column (diameter 1 and length 2 cm) filled with 68 reversed-phase media (Yamamura Depending K. K.), and elution was performed using 50% n-propanol. The eluate was diluted two times with water and subjected to reversed-phase liquid chromatography high pressure is), containing 0.1% triperoxonane acid. Allocated fractions, which were suirable at concentrations of n-propanol is about 40% and then dried by freezing and dissolved in 0.1 mol/l goldikova buffer (pH 9). As a result of these procedures, human G-CSF in the supernatant of cell culture OS-1 was concentrated approximately 20-fold.

As a control, cells OS-1 transformed pML-E+not containing chromosomal gene G-CSF, with the help of the method described above, and concentrated supernatant obtained culture.

The activity of the human G-CSF in the obtained samples were analyzed in "Method of analyzing the activity of the human G-CSF (a), described earlier in this description. The results are shown in table.9.

P R I m e R 36. RNA analysis of G-CSF (chromosomal gene).

Of COS cells grown to a cell concentration of 8 x 106cells/Cup (diameter 9 cm), transformed with 80 µg plasmid ML cèze After 48 h received all RNA according to the method hirgwin [Biochemistry, 18, 5294-5299 (1979)]

Plasmid BRG4 obtained in example 9 was digested with restrictase Aha III, and received pBRG4-derived DNA fragment was labeled with radioactive labeled with [32P] ATP, use the Fragment was isolated and used as probe DNA. Once was denaturiruet probe DNA (1,5 x105bills per minute, the 2.8 x106accounts in 1 min/µg DNA), it was mixed with 20 μg of total RNA obtained from cells OS. Hybridization was carried out at 45aboutC for 15 h, the Mixture was digested with 200 units/ml or 400 units/ml nuclease S1 (P. L. Biochemicals) according to the methods Weaver and Weissmann [Nucleic Acids Res. 7, 1175-1193(1979)] then conducted gel electrophoresis through a 4% polyacrylamide gel in the presence of 8.3 mol/l urea. Then spent detection using autoradiography.

In the observed band corresponding 722 base pairs, in the form of highly radioactively labeled bands in cells OS, which also marked the band corresponding 487 base pairs.

Therefore, it was found that RNA cells OS contained the endogenous G-CSF both lines +and ANY-ANY.

P R I m e R 37. Amino acid analysis and sugar assay (chromosomal gene).

1) Analysis of amino acid composition.

The crude CSF sample obtained in example 35, was purified in accordance with the techniques described in example 2 (III). The purified CSF sample was subjected to analysis of its amino acid composition by the methods described in 1) in example 27. The results are shown in table.10.

2) Analysis of sah who were subjected to analysis of its sugar composition using the same methods and under the same conditions, what is described in 2) in example 27. As a result of this analysis was confirmed by the presence of galactose, N-atsetilgalaktozamin and sialic acid in the CSF sample of the present invention.

P R I m e R 38. The expression of the chromosomal gene for human G-CSF in cells C.

Plasmid pML cèze obtained in example 34 was treated RI, and have identified a fragment of approximately 4 thousand bases using the methods described in Molecular Cloning, ibid. The selection used as the source of chromosomal gene G-CSF.

The fragment was treated fragment Klenow DNA polymerase I to create blunt ends (A).

The SV40 promoter (Eco RI-RI fragment of about 0.4 thousand bases) was cut out from plasmid GA410 (obtained as in example 22) using the methods described in Molecular Cloning, ibid, and then treated with fragment Klenow DNA polymerase (B).

In a separate stage of plasmid pdBPV-I have the virus bovine papillomavirus (BPV) [this plasmid was obtained by courtesy of Dr. Hawley, and it is described in Sarver, N. Sbyrne, J. C. owle, P. M. Proc. Natl. d. Sei. USA, 79, 7147-7151 (1982)] was treated with Hind III and Pvu II to obtain a DNA fragment of about 8.4 thousand bases. This fragment was treated with the fragment of Klenow DNA polymerase I and dephosphorylated the th, dissolved in 20 μl of reaction solution [50 mmol/l Tris-Hcl (pH 7,6), 10 mmol/l MgCl2, 10 mmol/l D, 1 mmol/l ATP] and the reaction was carried out overnight at 4aboutIn the presence of 180 units T4DNA ligase.

The reaction solution is then processed according to the method of using the rubidium chloride, described in Molecular Cloning, ibid, in order to obtain plasmid pNCE3 (Fig.39).

The DNA fragment(A), used as a source of chromosomal gene G-CSF, can be replaced by a DNA fragment of about 1,78 thousand reasons, which received as follows: 20 μg pML cèze dissolved in 100 μl of a mixture of 10 mmol/l Tris-Hcl (pH 8.0), 7 mmol/l MgCl2, 100 mmol/l NaCl, 7 mmol/l 2-mercaptoethanol and 0.01% bovine serum albumin solution were incubated at 37aboutC for 5 h in the presence of 20 units of StuI, and then was subjected to electrophoresis through a 1.2% agarose gel.

Thus obtained plasmid the NCE used to transform cells of mice S 1, as in example 24, and selected clones were expressively chromosomal gene of human G-CSF and who had a high ability to produce G-CSF.

P R I m e R 39. The expression of the chromosomal gene for human G-CSF in cells SNO.

As in the case of expression in cells C, plasmid L cèze processed Stu I identified a DNA fragment of about 1,78 thousand reasons, alternative, the same plasmid was treated RI and allocated oRI fragment of approximately 4 thousand bases. Any piece can be used as the source of chromosomal gene G-CSF.

The fragment used as a source, treated with fragment Klenow DNA polymerase I (a).

As in example 38, the SV40 promoter (RI oRI fragment) was cut out from GA410) to obtain a fragment of about 0.4 thousand reasons, which similarly treated with fragment Klenow DNA polymerase (b).

In a separate stage plasmid pAdD26SV pA [Kaufman, R. G., Sharp, P. A. Mol. ll. Biol. 2, 1304-1319 (1982)] was treated with RI, and then fragment Klenow DNA polymerase, and finally have dephosphorylated by treatment with bacterial alkaline phosphatase ().

Fragments (a), (b) and (C), weight 0.1 μg each, were dissolved in 20 μl of reaction solution [50 mm/l Tris-Hcl (pH 7,6), 10 millimoles/l MgCl4DNA ligase.

The reaction solution is then processed according to the method of using the rubidium chloride, described in Molecular Cloning, ibid, in order to transform a strain of E. coli DHI. From the obtained Facercolonies were selected those that contained plasmid pD26SVCE

As shown in Fig.40, plasmid pD26SVCE has CSF gene attached to early gene SV40 gene and the dhfr connected downstream from the main late promoter of adenovirus.

Plasmid dD26SVp treated with RI and BamHI, as in 2) of example 25 to obtain a DNA fragment (about 2 thousand bases) containing the dhfr gene. This fragment was tied to the fragment (a) and RI-Sal I fragment GA410(N) in order to construct the expression vector AmprpDR cèze (Fig.40).

Cells SNO transformed with the thus obtained plasmids pD26SVCE and pDR cèze as in example 25. Through re-selection when grown in the presence of methotrexate have been clones of the strain producing G-CSF.

P R I m e R 40. Analysis of the activity of G-CSF in transformed (expressmusic chromosomal gene of a person).

The supernatant of cell cultures S cells and SNO, which were obtained in examples 38, 39, respectively, were processed as in examples is="ptx2">

P R I m er 41. Molecular weight and isoelectric point of transformants.

Purified CSF samples used for the analysis of amino acid composition in examples 16, 20, 27, 33 and 37, used for measurement of their molecular weights and isoelectric points using the following methods.

1) Molecular weight.

Molecular weight of CSF were determined using gel electrophoresis sodium dodecyl sulphate-polyacrylamide gene (SDS-PAGE). Used electrophoretic installation PRONTN(16 cm supplied by the company Bio-Rad Corporation, using a gel made from polyacrylamide slab gel (T 15% From 2.6%), size 140 x 160 x 1.5 mm, and the concentration of the gel (T 3% To 20% ). The denatured sample CSF prepared in the following way: CSF boiled for 3 min in a solution containing 2% sodium dodecyl sulfate at 0.46 mol/l 2-mercaptoethanol. After electrophoresis with 4 µg of the sample at a constant current of 30 mA for 4 h, the gel was removed and held staining with 0.25% of the dye brilliant blue (oomassie Brilliant Blue R 250, produced by the company Sigma chemical company") for the detection of bands. The following substances were used as labels molecular weight after a similar loungers (mol. m 31000), soy tripeny inhibitor (mol.m. 21500) and litozin (mol.m. 14400).

The result found a single band corresponding to a molecular weight of 185,000 1000, for each of the CSF samples obtained example 16 [E. coli (CDNA (+VSE)] in example 20 [E. coli/CDNA (-VSE)] and a single band corresponding to a molecular weight of 19000 1000, for each of the CSF samples obtained in example 27 [C127, Cho/CDNA (+VSE)] in example 33 [S, SNO/CDNA (-VSE)] in example 37 (OS/g DNA).

2) Isoelectric point.

Isoelectric point CSF of the present invention was determined using the device for isoelectric electrophoresis in a flat layer F-3000 (izgotovlena company "Pharmacia fine chemicls"). After 2 hours of electrophoresis at a constant power of 30 W (Vmax. 2000) on polyacrylamide gel (T 5% 3% 115x hmm 230 mm) containing formalit (pH 4 to 6.5, the company "Pharmacia fine chemicls and 4 mol/l urea, CSF recorded using 30% methanol/10% trichloroacetic acid (35% sulfosalicylic acid and dyed using the dye brilliant blue R-250 (oomassie). As labels isoelectric points used set with low pI (pH: 2,5-6,5 supplied by the company "Pharmacia Fine Chemicals).

Analysis of the division pimero 16 and 20, and gave three separate bands corresponding to PI of 5.5, and 5.8 and 6.1 for each of the CSF samples obtained in examples 27, 33 and 37.

P R I m e R 42. Protective effect of human G-CSF against microbial infection.

Test method 1. Protection against infection with Pseudomonas aeruginosa Endoxan (ndoan, trade name of product of the company Shionogi Co. Ltd.) was injected intravenously in ICR mice at the age of 8-9 weeks (males, body weight 35,3 to 1.38 g) at a dose of 200 mg/kg Then mice were divided into three groups, two groups were injected at intervals of 24 h for four subcutaneous injections (dose each 0.1 ml) solution [1% propanol in 0.5 (weight/volume) of mouse serum albumin and physiological saline] containing human G-CSF (25000 or 50000 units/mol), while the third group was injected only solvent in the same way. Three hours after the last injection mice in each group were infected Pseudomanas aeruginosa GNB-139 using subcutaneous injections (3.9 x 105craniopathy units/mouse). Twenty-one hours after infection in the first two groups have introduced one more subcutaneous injection of a solution containing human G-CSF (25000 or 50000 units/mouse), and the third group was injected only solvent.

Protective effect of human CSF on emochnoy suspension.

Pseudomonad aeruginosa GNB-139 were cultured overnight with shaking at 37aboutWith in a liquid medium for heart infusion (trade name "Difco"). Culture suspended in physiological saline.

2. Protection against intrusion ndida Endoxan (ndoxan trading name of the company Shionogi Co. Ltd."), was administered intraperitoneally to ICR mice aged 8 weeks (males, body weight 40,51,60 g) at a dose of 200 mg/kg Then mice were divided into two groups, one group was injected with 24-hour intervals for four subcutaneous injections (dose each 0.1 ml) of the solvent [1% propanol and 10% (weight/volume) ICR mouse serum in physiological saline] containing human G-CSF (50,000 units/mouse), while the other group was injected only solvent in the same way. Four hours after the last injection, mice in each group were infected ndida albicans U-50-1 (strain isolated from urine lakeline patients, courtesy of the Bacteriological laboratory, Tohoku University, medical school) via intravenous injection (5,6 x 105kolonialismus units/mouse). Protective effect of human G-CSF was determined by counting the number of mice that survived ten days after infection.

PoWith in containing yeast extract Sabouraud's liquid medium (2% dextrose from the firm "Junsei Pure Chemicals Co. Ltd."; 10% trypticase peptone, trade name "BBL", 5% yeast extract from "Difc"; pH 5, 6). The culture was twice washed with physiological saline and suspended in physiological saline.

3. Protection against infection with the intracellular parasite Listeria Endotoxin (ndotoxan, trade name of product of the company Shionogi Co. Ltd.) was administered intraperitoneally to ICR mice aged 7 weeks (males, body weight 34,7 1.24 g at a dose of 200 mg/kg Then mice were divided into two groups; one group with 24 hour intervals was introduced four subcutaneous injections (dose each 0.1 ml) of the solvent [1% n-propanol and 10% (weight/volume) ICR mouse serum in physiological saline] containing human G-CSF (50,000 units/mouse), while the other group was injected only solvent in the same way. Four hours after the last injection, mice in each group were infected by Listeria monocytogenes 46 (courtesy of the microbiological laboratory of the medical school of Tohoku University) by intravenous injection of 1.0 x 107kolonialismus units/mouse. Protective effect of human G-CSF was determined by calculating the Chi

Listeria monocytogenes 46 were cultured overnight with shaking at 37aboutWith in a liquid medium brain heart infusion (trade name of product "Difco"). Culture suspended in physiological saline.

Results

l) Test 1, 2 and 3 were performed with the polypeptide. G-CSF E. Li (+VSE) obtained in example 16. The results are shown in table.12, 13 and 14.

ll) l Test was performed with the polypeptide G-CSF E. coli (-VSE) obtained in example 20. The results are shown in table. 15.

lll) l Test was performed with purified specimen of human G-CSF derived cells CHO (+VSE), which was the same as the sample used in the analysis of amino acid composition in example 27. The results are in table.16.

Essentially the same results were obtained when test 1 was performed with purified specimen of human G-CSF derived T cell, which was the same as the sample used in the analysis of amino acid composition in example 27.

IV) the Test I conducted with purified specimen of human G-CSF derived Cho cell (-ANY), which was the same as the sample used in the analysis of amino acid composition in example 33. The results are shown in TM human G-CSF, the extracted cell S, which was the same as the sample used in the analysis of amino acid composition in example 33.

A method of OBTAINING a FACTOR THAT STIMULATES the FORMATION of COLONIES of GRANULOCYTES, providing for the construction of recombinant plasmid DNA containing the DNA fragment encoding the factor stimulate the formation of colonies of granulocytes, the transformation strains of Escherichia coli or cultured animal cells recombinant plasmid DNA, culturing the transformants and the selection of the target product, wherein the construct recombinant plasmid DNA with a DNA fragment coding for the factor stimulate the formation of colonies of granulocytes with the following amino acid sequence listed in the description.

 

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