RU2057809C1 - Method of preparing factor stimulating granulocyte colony formation - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves the preparing polypeptide or glycoproteide showing activity of human factor that stimulates the formation of granulocyte colonies. Method involves the preparing double-stranded cDNA which is complementary to messenger RNA (mRNA) encoding polypeptide indicated above and human chromosomal gene encoding polypeptide that shows factor activity stimulating granulocyte colony formation, gene insertion into vector, transformation of prepared recombinant DNA of recipient cells, isolation and purification of the end product. EFFECT: improved method of stimulating factor preparing. 40 dwg, 17 tbl

Description

 The invention relates to a method for producing a polypeptide or glycoprotein having G-CSF granulocyte colony stimulating factor activity using recombinant DNA molecule technology.

 The biological effect of G-CSF is their ability to differentiate leukemic bone marrow cells and enhance the function of mature granulocytes, which is why their use in the treatment and prevention of leukemia is promising. Attempts have been made to isolate and purify G-CSF from the cell culture supernatant, but homogeneous G-CSF was not obtained in large quantities because it is produced in a low concentration and after the purification procedure it is obtained in "trace" amounts from a large volume of the culture fluid.

Figure 1 presents the sequence of various probes, IWQ, A and LC; 2, the nucleotide sequence of the pHCS-1 insert; in FIG. 3-5 nucleotide sequence of cDNA insertion into pBRG4; Fig.6 amino acid sequence of the precursor of human G-CSF, displayed on pBRG4 cDNA; 7, the amino acid sequence of human G-CSF derived from pBRG4 cDNA; in FIG. 8-10 nucleotide sequence of the insertion of cDNA into pBRV2; in Fig.11 the amino acid sequence of the precursor of human G-CSF derived from pBRV2 cDNA, Fig.12 amino acid sequence of the human G-CSF derived from pBRV2 cDNA; 13-17, the nucleotide sequence of a human chromosomal gene encoding G-CSF; on Fig sites of cleavage by rastrictase pBRG4 or PBRV2 cDNA of human G-CSF; in FIG. 19 restriction enzyme cleavage sites of the human chromosomal gene encoding G-CSF; on Fig partially a method for producing a tac-promoter-containing vector (+ VSE line); on Fig.21-22 a method of obtaining a P _L -promoter-containing vector (+ VSE line); on Fig the method of obtaining t _rp promoter-containing vector (+ VSE line); on Fig partially a method for producing a tac-promoter-containing vector (VSE line); in FIG. 25-26 a method for producing a P _L promoter-containing vector (-VSE line), FIG. 27 a method for producing a t _rp promoter-containing vector (-VSE line); on Fig schematically the structure of the pHGA410; on Fig methods of constructing recombinant expression vectors pTN-G4, pTN-G4VAα and pTN-G4VA β in Fig.30-31 two methods of constructing pH GG4-dhfr; on Fig methods for constructing pG4DR1 and pGDR2; on Fig schematically the pH structure of GV2; on Fig methods of constructing recombinant expression vectors pTN-V2, pTN-VAα and pTN-VAβ in Fig. 35-36 two methods for constructing a recombinant pHGV2-dhfr expression vector; in FIG. 37 methods of constructing pV2DR1 and pV2DR 2; on Fig schematically the structure of the pML CEZ α; on Fig schematically the structure of pTN CEZ α; on Fig schematically in the structure of pD26SVCEЗα and pDRCEЗ α.

 A gene encoding a polypeptide having human G-CSF activity (G-CSF) is DNA (cDNA), which is complementary and messenger RNA (mRNA), which is obtained as fractions 15-17S by centrifugation in a sucrose density gradient and which encode a polypeptide having human G-CSF activity.

 Got two lines of cDNA.

 A single line cDNA contains the complete sequence or part of a gene encoding a polypeptide I or II (FIGS. 6 and 7). More specifically, this cDNA has a nucleotide sequence extending from ATG at nucleotide positions 32-34, measured from the 5'-end (Figs. 3-5), to CCC at positions of nucleotides 650-652, or from ACC at positions 122-124 to the CCC in the provisions of 650-652. Alternatively, the cDNA has a nucleotide sequence or part thereof, the cDNA of this line is hereinafter called cDNA (+ VSE).

 Another line cDNA has all or part of the gene encoding the polypeptide I or II shown in FIG. 6-7. More specifically, this cDNA has a nucleotide sequence extending from ATG at nucleotide positions 31-33, counted from the 5 'end [see FIG. 8-10] to the CCC and the nucleotide positions 640-642, or from the ACC at positions 121-123 to the CCC at positions 640-642. Alternatively, the cDNA may have the nucleotide sequence shown in FIG. 8-10, or part of the cDNA of this line, hereinafter referred to as cDNA (-VSE).

 The gene described above can be obtained in the following ways: first, mRNA encoding G-CSF is obtained from mammalian animal cells or from other host cells capable of producing a polypeptide having G-CSF activity, then the mRNA is converted into a double-stranded cDNA by any known method; obtained by the cDNA library is then subjected to directional selection by known methods.

 The gene of the present invention also includes a human chromosomal gene encoding a polypeptide having G-CSF activity. 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 from the kidneys, or from malignant cells. A library of the human chromosomal gene can be obtained from human cells by any of the known methods [see Maniatis et all, Cell, 15, 687 (1978); and Manidtis et al. Molecular Cloning, Cold Spring Harbor Laboratory, p. 269, ff. (1982)] which is illustrated below:
human chromosomal DNA is extracted from sources such as the liver of a human embryo, phenol or other suitable reagents, the extracted DNA is partially or completely digested with a suitable restriction enzyme to obtain a DNA fragment of suitable length, the DNA fragment is inserted into the vector fragment of the λ-phage DNA using T4 DNA- ligase or other suitable ligase, optionally connecting a linker containing a restriction site for a suitable enzyme, such as EcoRI, then receive particles of λ-phage by laying in vitro and transform host cells, such as E. coli, using the resulting particles of λ-phage.

 Examples of λ phage that can be used as a vector in these methods include Charon 4A and EMBL-3 and EMBL-4.

A mammalian cell that can be used as a source of mRNA is a strain of cells, for example, derived from human oral cancer cells, CHU-2 deposited in the National Collection of Microorganism Cultures or CN.M. under inventory number I-483). Obtaining and RNA can be carried out in one of the ways that have already been proposed for cloning the genes of a number of other physiologically active proteins: for example, all RNA is obtained first by treatment with a surfactant and phenol in the presence of a ribonucleoside inhibitor such as a vanadyl ribonucleoside complex [see Berger and Birkenmeier, Biochemistry, 18-5143 (1979)] or by centrifugation in a density gradient of CsCl followed by treatment with guanidine thiocyanate [see Chirgwin et al. Biochemistry, 18.5294 (1979)] then produce poly (A ⁺ ) RNA (IRNA) by subjecting all RNA to periodic adsorption or affinity chromatography on an oligo (dT) cellulose column or on poly-U-sepharose using Sepharose 2B as carrier. Poly (A ⁺ ) RNA can be further fractionated by a suitable method, such as centrifugation in a sucrose density gradient. The ability of the mRNA thus obtained to encode a polypeptide having G-CSF activity can be confirmed by a number of methods: for example, the mRNA is translated into a protein and its physiological activity is checked, alternatively, the authenticity of this protein is determined using an anti-G-CSF antibody. More specifically, mRNA is introduced into xenopus Laevis oocytes for translation [see Gurdon et al. Nature, 233, 177 (1972)] or it is possible to carry out translational reactions with rabbit reticulocytes or with wheat germ [Schleif and Wensink, "Practical Methods in Molecular Biology". Springer-Verlag, NY (1981)] G-CSF activity can be tried using a soft-agar culture method using bone marrow cells, and the methodology has already been described [Metcalf, "Hemopoietic Colonies", Springer-Verlag, Berlin , Neidelberg, NY 1977)]
Single-stranded cDNA is synthesized using the mRNA thus obtained, which is used as a template, then double-stranded cDNA is synthesized from this single-stranded cDNA and double-stranded cDNA is inserted into a suitable DNA vector to obtain a recombinant plasmid. This recombinant plasmid can be used to transform a suitable host, say Escherichia coli, in order to obtain a cDNA library.

 A double-stranded cDNA can be obtained from mRNA in one of the following two ways: mRNA is processed by reverse transcriptase with oligo (dT), which is complementary to the poly (A) chain at the 3 'end, used as a seed, or an oligonucleotide that corresponds to part the amino acid sequence of the protein G-CSF, and cDNA is synthesized, which is complementary to mRNA, by reverse scripting, and the synthesized oligonucleotide is used as seed. Double-stranded cDNA can also be obtained in the following ways: mRNA is decomposed and removed by treatment with alkali, and the obtained single-stranded cDNA is treated first with reverstranscriptase or DNA polymerase I (for example, fragment Klenow), then S1 nuclease, alternatively, mRNA can be directly treated with RNase H and DNA polymerase (e.g., E. coli polymerase I). For more information see Maniatis et al. "Molecular Clonig", Spring Harbor Laboramory (1982); and Gubler and Hoffman, Gene, 25, 263 (1983) .

 The double-stranded cDNA thus obtained is inserted into a suitable vector, such as, for example, one of the EC-type plasmid vectors, typical representatives of which are pS CIOI, pDF 41, Sol E1, pMB9, pBR322, pBR327 and pACYC1, or one of the phage vectors typical examples of which are λgt, λc, λgt 10 and λgtWES, and then a recombinant vector is used to transform the E. coli strain (e.g., X1776, HB101, DN1 or C600) in order to obtain a cDNA library (see, for example, " Molecular Cloning ", above).

 A double-stranded cDNA can be attached to a vector in the following ways: the end of the cDNA is provided with a attachable end by attaching a suitable chemically synthesized DNA fragment, and the DNA vector that has been cleaved with restriction enzyme is attached to the cDNA by treatment of T4 phase DNA ligase in the presence of ATP. Alternatively, the dC, dG units (or dT, dA units) of the double-stranded cDNA, respectively, are attached to the DNA vector that is digested with restrictase and the solution containing both types of DNA is renatured (see above "Molecular Cloning").

The host cell can be transformed with the recombinant DNA thus obtained by any of the known methods. If the host cell is E. coli, then the method developed by Hanahan [J. Mol. Biol. 166, 557 (1983)] where recombinant DNA is added to a competent cell obtained in the presence of CaCl ₂ , MgCl ₂ or RbCl.

 Directed selection of cells carrying the desired gene can be carried out using a number of methods, which include: plus or minus the method used for cloning interferon cDNA [Taniguchi et al. Proc. Jpn. Acad. 55, ser. B. 464 (1979)] a method for translational hybridization analysis [Nagata et al. Nature, 284, 316 (1980)] and a method for hybridizing a colony or spot using an oligonucleotide probe chemically synthesized based on the amino acid sequence of a protein having human G-CSF activity [Wallace et al. Nucleic Acids Res. 9, 879 (1981); and Benton Davis, Science, 196, 180 (1977).

 A fragment comprising a gene thus cloned encoding a polypeptide having human G-CSF activity was inserted into a suitable DNA vector to transform other prokaryotic or eukaryotic host cells (Proc. Natl. Acad. Sei, USA82, July 1985, pp 4360-4364 ) (prototype).

 E. coli can be transformed with pBR 322, which is a vector capable of replication in E. coli [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 needed for genetic expression in prokaryotic hosts include the promoter of the β-lactamase gene [Chang et al. Nature, 275, 615 (1978)] lactose promoter [see Goeddel et al. Nature 281, p. 544 (1979)] and tryptophan promoter [see Goeddel et al. Nucleic Acid Res. 8, 4057 (1980)], etc.

 Any of these promoters can be used in the preparation of a polypeptide having human G-CSF activity of the present invention.

A eukaryotic microorganism, such as Saccharomyces cerevisiae, can be transformed with a vector such as the plasmid YRp7 [see Stinchcomb et al. Nature, 282, 39, (1979)] This plasmid has the TKP1 gene as a selection marker for yeast strains lacking the ability to produce tryptophan, so transformants can be selected by growing in the absence of tryptophan. Examples of a promoter that can be used to express a gene include an acidic phosphatase gene promoter [Miyanohara et al. Proc. Natl. Acad Sci. USA, 80.1 (1983)] and alkkholdehydrogenase gene promoter [Valenzuela et al. Nature, 298, 347 (1982)]
For transformation of mammalian cells, such as COS cells, Chinese hamster ovary cells (CHO), C-127 cells and Hela cells, for example, pSV2-gpt can be used [see Milligan and Berg; Proc. Natl. Acad. Sci. USA, 78, 2072 (1981)] The vectors used to transform these cells contain an initiation site, a selectable marker, a promoter whose position is preceded by the position of the expressed gene, a polyadenylation signal, etc. For gene expression in mammalian cells, promoters of retrovirus, polyoma virus, adenovirus, monkey virus 40 (SV 40), etc. can be used. If the SV 40 promoter is used, then the desired gene expression can be easily carried out according to the method of Mulligan et al. (Mulligan et al. Described in Nature, 277.108 (1979).

 The initiation sites that can be used are derived from SV 40, polio virus, adenovirus, bovine papilloma virus (BPV), etc. Selective markers used include the phosphorotransferase (ARN (3 ') II or I gene, thymidine kinase gene, E. coli xanthine guanine phosphoribosyltransferase gene (Ecogpt), dihydrofolate reductase gene (DHFR), etc.

 Eukaristic genes usually exhibit polymorphism, as is known for the human interferon gene [see Nishi et al. J. Biochem. 97,153 (1985)] wherein either a change in the nucleotide sequence does not lead to any change in the 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 have 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.

 The following describes a method for producing a factor in accordance with the invention.

 (I) Obtaining a probe.

A homogeneous protein of human CSF was obtained by purification from the surface layer of a culture cell of tumor cells of the CHU-2 line and its amino acid sequence was determined from the N-type. Fragments were obtained by decomposition with bromocyanine and trypsin treatment, and the amino acid sequences of these fragments were also determined [example 3 (I), (II) and (III)]
Based on the determined amino acid sequences, three nucleotide probes (A), (LC) and (IWQ) were synthesized having the sequences shown in FIG. 1 (Example 4). The probe (A) was a mixed type and consisted of 14 consecutive nucleotides. The probe (IWQ) consisted of 30 consecutive nucleotides with deoxyinosine and was of the type used for cloning the human cholecystokinin gene [Takahashi et al. Proc. Natl. Acad. Sci. USA 82, 1931 (1985)] The probe (LC) was a 24-nullotide probe that was synthesized from nucleotides at 32-39 positions from the N-terminus of the amino acid sequence shown in example 3 (I), based on the nucleotide sequence shown figure 3-5.

The chemical synthesis of nucleotides can be carried out by applying the improved phosphotriether method to the solid phase method, and this was reviewed by Hapang (Narang) [Tetrahedron, 39.3-22 (1983)]
Amino acid sequence based probes may also be used at positions that are different from those used in the aforementioned probes.

 (2) Construction of a cDNA library.

 CHU-2 cells were homogenized after addition of guanidine thiocyanate solution, and all RNA was obtained by density gradient centrifugation, CsCl.

Poly (A ⁺ ) RNA was isolated from all RNA by column chromatography on oligo (dT) cellulose. After that, single-stranded cDNA was synthesized using reverstranscriptase, and ribonuclease H and E. coli DNA polymerase I were added to obtain double-stranded cDNA. The dC chain was attached to the resulting double-stranded cDNA, which was attached to the pB vector R 322, to which the dG chain was attached at the Pst-I cleavage site; the resulting recombinant DNA was used to transform the E. coli X1776 stamp, and a library of rB R 322 line cDNA was constructed (examples 5 and 6).

 In a similar way, a double-stranded cDNA was attached to the λgt 10 vector using the Eco RI linker and a cDNA library of the λ-phage line was constructed (Example 7).

 (3) Directional selection.

Clones of the rB R322 line cDNA library were fixed on Whatman S41 filter paper, and by hybridizing the colonies with a probe (IWQ) labeled 32, a single clone could be selected. Subsequent Southern Spot Method [Southern. J. Mol. Biol. 98,503 (1975)] showed that this clone also hybridizes with probe (A). The nucleotide sequence of this clone was determined by a dideoxymethod [Sanger, Science, 214, 1205 (1981)]
The nucleotide sequence of the resulting cDNA insert is shown in FIG. 2, where it can be seen that this insert consists of 308 base pairs, including probes (IWQ) and (A), and has an open reading frame encoding 83 amino acids, the sequence of which is shown in Example 3 (III) pB R 322-derivative containing these 308 base pairs pHCS-1 (example 8).

A DNA fragment containing 308 base pairs obtained from rNCs-I was labeled with a radioactive label according to the method of labeled translation (see ibid. Molecular Cloning), and using this fragment as a probe, directional selection of the λgt 10 cDNA library was performed using spot hybridization [ Benton and Davis, Science, 196, 180 (1977)] to obtain five clones. Using the same method as described above, the nucleotide sequence of the clone, which was believed to contain cDNA, was determined [Fig. 3-5]
This cDNA insert had a single large open reading frame.

 As shown in FIG. 3-5, the amino acid sequence encoded by this cDNA can be derived.

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

 It should be noted that ATG at positions 32-34 or at positions 68-70 can also be considered a protein initiation site.

 Strain X1776 of Escherichia coli containing pB R 322 that had this cDNA (+ VSE) at the EcoRI site was deposited with the Fermentation Research Institute, the Agenc of Industrial Science and Technology (FERM BP- 954).

 On Fig shows the restriction sites of this DNA.

 This cDNA was ligated with pB R327 Soberon et al. [Soberon et al. Gene, 9.287 (1980)] at the EcoRI site and the resulting plasmid is referred to here below as pBRG4.

 Thus obtained pBRG4 was digested with restriction enzyme EcoRI to obtain a DNA fragment containing cDNA with a length of about 1500 base pairs. This fragment was radiolabeled using the labeled translation method (see ibid. Molecular Cloning) and, using this radiolabeled DNA fragment as a probe, we again targeted the λgt library of 10 cDNA clones using spot hybridization (see Benton and Davis )

 In this spot hybridization method, two sheets of nitrocellulose filter paper with coupled λ-phage DNA are prepared, one of these sheets is used for the above spot hybridization, and the other is subjected to spot hybridization with the probe already described (LC). Phages that were positive for both probes were selected. A clone that had a "full length" cDNA was selected and the nucleotide sequence of the cDNA insert determined by the dideoxy method is shown in FIG. 8-10.

 This cDNA had a single large open reading frame, the amino acid sequence that this cDNA shown in FIG. 8-10.

 Comparison with the N-terminal amino acid sequence of the G-CSF protein shown in Example 3 (1) revealed that this cDNA contained a nucleotide sequence that corresponded as a signal peptide encoded using 90 base pairs, starting from the ATG sequence at nucleotide positions 31 -33 from the 5'-end and ending with the MCC sequence at positions 118-120, and the completed G-CSF polypeptide encoded using 522 base pairs, starting from the ACC sequence at positions 121-123 and ending with the MCC sequence in dix 640-642. Therefore, a polypeptide with the amino acid sequence l shown in FIG. 11 and 12, consisted of 204 amino acids, and it was calculated that its molecular weight was 21977.35 daltons. A polypeptide with the amino acid sequence ll consisted of 174 amino acids, it was calculated that its molecular weight was 18671.42 daltons (example 10).

 It should be noted that ATG at positions 58-60 or at positions 67-69 can also be considered as a protein initiation site.

 Strain X1776 of Esherichia coli carrying pBR322, which had this cDNA (-VSE) at the EcoRI site, was deposited with the Institute of Fermentation Research of the Bureau of Industrial Sciences and Technology (Fermentation Research Institute, the Agency of Industrial Science and Technology (FERM BP-955) .

 On Fig shows the restriction sites of this DNA.

 This cDNA was ligated with pBR327 at the EcoRI site to obtain a plasmid, which is hereinafter referred to as pBRV 2 plasmid.

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

 The library of the human chromosomal gene, obtained in accordance with the methods described by Manyatis and grade. (Maniatis et al. Molecular Cloning, ibid.) Were subjected to directional selection with the above pCS-I. Probes that can be used in targeted sampling included: a pHCS-I DNA fragment of 308 base pairs long, pBRG4 DNA fragment of approximately 1,500 base pairs pBRV 2 DNA fragment of approximately 1,500 base pairs, a suitable length DNA fragment containing a portion of one or more of of these DNA fragments, as well as the above oligonucleotide probes [i.e. (IWQ), (A) and (IC). The following describes the use of a pHCS-I DNA fragment.

This DNA fragment was labeled with a radioactive label ³² P according to the method of labeled translation [see Roop et al. Sell, 15, 431 (1978)] Using the obtained ³² P labeled fragment as a probe, the library of the human chromosome gene was targeted by spot hybridization (see Benton and Davis) to obtain ten-odd clones.

After isolation of the DNA from the clones using known methods, a restriction map was obtained [Fritsch et al. Sell, 19, 959 (1980)]
Using the same DNA probe, Southern hybridization was performed (see ibid. Southern), it was found that a DNA fragment with a length of about 4 thousand bases cut with EcoRI and XhoI could potentially contain a region encoding the human G-CSF polypeptide. Therefore, a DNA fragment of approximately 4 thousand bases was inserted into pBR327 and the EcoRI site using the EcoRI linker in order to obtain pBRCE3 β. Using this plasmid as DNA with a specific base sequence, the nucleotide sequence of a portion of approximately 3 thousand bases of this DNA fragment of approximately 4 thousand bases in length was determined by the dideoxy method. As a result, it was found that this DNA fragment is a gene encoding the polypeptide of human G-CSF (Fig. 5).

 The E. coli strain X1776 carrying pBRCЕ3β (i.e., the pBR327 plasmid having the indicated DNA fragment of about 4 thousand bases inserted into the EcoRI site) was deposited with the Fermentation Research Institute, the Emergency of Industrial Science and Thehnolohgy (FE RM BP-956).

 A comparison of the pBRG4 cDNA insert shown in FIGS. 3-5 and the pB RV2 cDNA insert shown in FIGS. 8-12 shows that the DNA fragment under discussion contained five exon parts and it encoded the amino acid sequences derived from pB RG4 and pB RV 2.

 On Fig shows the restriction sites of the obtained gene.

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

 A / Recombinant line vector + VSE.

From the plasmid pB RG4 obtained in (3) (Example 9), a cDNA fragment of the G-CSF polypeptide was cut using a restriction enzyme and a recombinant vector was constructed using one of the following methods: (I) using a renatured synthetic linker, the fragment was linked to the fragment, obtained from tac-promoter-containing pKK223-3 (Pharmacia Fine Chemicals) (example 12 and Fig. 8);
(II) three fragments obtained from a PL-promoter-containing PL-lambda (Pharmacia Fine Chemicals) were coupled to a renatured synthetic linker, and the binding product and cDNA fragment were subjected to re-preparation steps to construct a recombinant vector (Example 13, FIG. 21 -22) or
(III) using a renatured synthetic linker, the fragment was linked to the fragment obtained from the t _rp promoter-containing plasmid pOYI (example 14 and FIG. 23),
(B) Recombinant line vector-VSE.

 In the same manner as described above, three recombinant vectors were constructed using the plasmid pBRV2 (Example 10) shown in Example 19 and in FIG. 24, 25, 26, 27.

(6) Obtaining transformants of E. coli and their cultivation and expression
Using three recombinant vectors of each of the + VSE and -VSE lines, the E. coli strain DH1, N 4830 or JM 105 was transformed by treatment with calcium chloride or rubidium chloride described in Molecular Cloning in the same place (examples 12,13,14 and 19). Each of the obtained transformants was cultured in Luria medium containing ampicillin, and then induction was carried out as necessary to effect expression (examples 15 and 20).

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

 The transformants culture solution was centrifuged to obtain a cell pellet after centrifugation. The harvested cells were treated with lysozine and, after lysis, a surface solution was obtained by cyclic freezing and thawing. Alternatively, the cells were treated with guanidium chloride, centrifuged, and a surface solution was obtained.

 The surface solution was subjected to gel filtration on an Ultrogel ACA54 (LKB) column, and the active fractions were concentrated using an ultrafiltration device.

 Then, an aqueous trifluoroacetic acid solution containing n-propanol was added to the concentrate, and after standing in ice, the mixture was centrifuged and absorbed on a C18 reverse phase column. After elution, the fractions were checked for their activity. Active fractions were collected and subjected to the same purification methods as described above. The purified fractions were freeze-dried, and the powder was dissolved and subjected to size resolution liquid chromatography. The resulting polypeptides were subjected to SDS-polyacrylamide gel electrophoresis, and a single lane confirmed the presence of the desired polypeptide (G-CSF) (examples 16 and 20). Thus obtained polypeptide showed the activity of the human G-CSF polypeptide (examples 17 and 20). The G-CSF polypeptide was analyzed by amino acid analysis using a Hitachi 835 automatic amino acid analyzer (Hitachi, Ltd). For analysis of N-terminal amino acids, a gas phase sequence determinant (for Edman decomposition), a high performance liquid chromatography chromatograph, and an Ultrasphere-ODS column were used (Examples 18 and 21).

 (8) Construction of recombinant vectors for animal cells.

 Recombinant vectors (BPV derivatives) for use in host cells With 127 and N IN3T3 were constructed for each cDNA line + VSE and -VSE and for the chromosomal gene. Recombinant vectors (with dhar) for use with CHO cells were also constructed for each cDNA line + VSE and -VSE for the chromosomal gene. Recombinant vectors have also been constructed for use in CoS cells. Illustrative examples are described, and for a more detailed description, reference should be made to the corresponding working examples.

(A) Construction of recombinant line vectors + VSЕ
The cDNA fragment (+ VSE) obtained in (3) was inserted into the pdKCR vector to obtain the plasmid pHGA 410, (Example 22, FIG. 28), which was partially digested with Eco RI and then treated with DNA polymerase I (fragment Klenova) to get blunt ends. The Hind III linker was attached to the DNA, then it was treated with Hind III, and T4 DNA with a ligase, the treated DNA was used to transform E. coli DNI strain using rubidium chloride (see also Molecular Cloning). The resulting plasmid was called pH GA 410 (H) (FIG. 29). raffia and column Ultrasphere-ODS (examples 18 and 21).

 (8) Construction of recombinant vectors for animal cells.

 Recombinant vectors (BPV derivatives) for use in host cells With 127 and N IN3T3 were constructed for each cDNA line + VSE and -VSE and for the chromosomal gene. Recombinant vectors (with dhfr) for use with CHO cells were also constructed for each cDNA line + VSE and -VSE for the chromosomal gene. Recombinant vectors have also been constructed for use in CoS cells. Illustrative examples are described, and for a more detailed description, reference should be made to the corresponding working examples.

(A) Construction of recombinant line vectors + VSЕ
The cDNA fragment (+ VSE) obtained in (3) was inserted into the pdKCR vector to obtain the plasmid pHGA 410, (Example 22, FIG. 28), which was partially digested with Eco RI and then treated with DNA polymerase I (fragment Klenova) to get blunt ends. The Hind III linker was attached to the DNA, then it was treated with Hind III, and T4 DNA with a ligase, the treated DNA was used to transform E. coli DNI strain using rubidium chloride (see also Molecular Cloning). The resulting plasmid was called pH GA 410 (H) (FIG. 29).

 pН GA 410 (H) was treated with SalI and after blunt ends it was treated again with Hind III and a Hind III Sal I fragment was isolated. The plasmid pdBPV-1 having a transformed fragment of bovine papilloma virus was treated with Hind III and Pvu II and a large fragment was isolated DNA and attached to a separately prepared Hind III Sal I. fragment. The combined fragments were used to transform E. coli DHI strain to obtain the plasmid pTN-G4, which had pH GA 410 derived CSF cDNA (Fig. 29, Example 23) (CSF-CSF , Note transl.).

 Any of the plasmids, pH GA 410, or pH GA 410 (H), in combination with plasmid pAdD26SVpA, was used to construct the pH of GG4-dhfr, which was a recombinant vector (+ VSЕ for use with CHO cells (Fig. 30, 31, example 26).

 A 2,000-base DNA fragment containing the dhfr gene was isolated from pAdD26SVpA by treatment with EcoRI and BamHI1, the selected fragment was inserted into pHGA 410 (H) at the Hind III site in order to construct pG4DR1 and pG4DR2 (Fig. 32, example 25) .

 (B) Construction of recombinant line vectors-VSE.

 The cDNA fragment (-VSE) obtained in (3) was inserted into the pdKCR vector to obtain the plasmid pH GV2 (Example 28), which was partially digested with Eco RI followed by DNA polymerase I (Klenow fragment) to obtain blunt ends .

 The Hind III linker was attached to the DNA, which was sequentially treated with Hind III and T4 DNA ligase. The treated DNA was used to transform E. coli DH1 strain using rubidium chloride (see Melecular Cloning). The resulting plasmid was called pH GV2 (H) (Fig. 34).

 Plasmid pH GV2 (H) was treated with Sal I after blunt ends were obtained, it was treated with Hind III times and a Hind III Sal I fragment was isolated. Plasmid pdBPV-1 having a transformed bovine papilloma virus fragment was treated with Hind III and Pvu II and a larger fragment was separated DNA and attached to a separately prepared fragment of Hind III Sal I.

 The attached fragments were used to transform E. coli strain DH1 to obtain plasmid pTN V2, which had a pH of GV-2 deduced by CSF cDNA (Fig. 34, Example 29).

 By similar methods, either plasmid pH GV2 or plasmid pH GV2 (H), in combination with plasmid pADD 26SV pA, was used to construct pH GV2-dhfr, which was a recombinant vector (-VSE) for use with CHO cells (Figs. 35, 36 , example 31).

 A DNA fragment of about 2 thousand bases containing the dhfr gene was isolated from pAdD 26 SV pA by treatment with EcoRI and BamH 1, and the isolated fragment was inserted into pH GV2 (H) at the Hind III site in order to construct pV2DR1 and pV2DR2 ( Fig. 37, example 31).

 (C) Construction of recombinant vectors containing the chromosomal gene.

The plasmid pBRCE3 β, which was obtained in (4) and which contained the chromosomal gene shown in FIG. 13-17, treated with EcoRI. Plasmid pSVH ⁺ K ⁺ described by Baner J1 et al. in Cell, 27, 299 (1981), were treated with Kpn I to remove the globin gene. The plasmid was then partially digested with Hind III so as to remove part of the late SV40 gene. Fragments were re-combined to obtain the expression vector pML-E ⁺ .

This vector was digested with restrictase, Eco RI and dephosphorylated with alkaline phosphatase (Takara Shuzo Co. Ltd.) to obtain a DNA vector that was linked to the above chromosomal DNA fragment using T4 DNA ligase (Takava Shuzo Co. Ltd.) to get pMLCEZ α, which was a recombinant vector for COS cells (example 34). As shown in FIG. 31, this plasmid contained the enhancer of the SV 40 gene, the start of SV40 replication, the start of pBR322 replication, and the β-lactamase gene (Amp ^r ) pBR322 and the human G-CSF chromosome gene located behind the SV 40 enhancer.

 The expression vector for C127 cells was constructed in the following ways. A DNA fragment containing the CSF chromosomal gene was excised using a suitable restriction enzyme from pML CEZα which was an expression vector for COS cells. This fragment was attached using a T4 DNA ligase to the fragment was attached using a T4 DNA ligase to a DNA fragment containing bovine papillomavirus origin (BPV) and a DNA fragment containing the SV40 early promoter. The resulting pT NCE3 α was an expression vector that had the CSF chromosomal gene located behind the early promoter SV 40, and which contained 65% of BPV.

 The expression vector for CHO cells had two DNA fragments connected using T4 DNA ligase, one fragment contained the CSF chromosome gene and early promoter SV 40, as in the case of the expression vector for C127 cells, and the other fragment contained the pAdD 26 SV pA-dhfr gene . The resulting pD26SVCE3α was an expression vector that had the CSF chromosomal gene located behind the SV 40 promoter and the dhfr gene behind the main late adenovirus promoter.

(9) Expression in animal cells
(A) Expression in C127 Mouse Cells
Plasmid pTN-G4 or pTN-V2 was treated with BamHI. The treated plasmid was used to transform C127 cells (previously grown by cultivation) according to the method using calcium phosphate. Transformed cells were grown, and clones having a high CSF production rate were selected. Glycoproteins containing expressed CSF were isolated and purified from the transformed cell culture solution. It was found that they possess human G-CSF activity. The presence of the desired glycoprotein was also confirmed by amino acid analysis and analysis of the sugar content in the sample.

 To analyze the sugar content, the CSF sample used in the amino acid analysis was subjected to the determination of the amino sugar according to the Elson-Margan method, the determination of neutral sugar by the method using orcinol sulfate, or the determination of sialic acid by the method using thiobarbiturate. The methods for each determination are described in the Tohitsitsu no Kagaku Chemistry of Saccharides (part 2 of two parts), part 13, volume 4 of course A in Biochemical Experiments (published by Tokyo Kagaku Dojin). weight percent showed that the sugar content in the obtained G-CSF was distributed in the range of 1-20 wt. depending on the type of host cell, expression vectors and cultivation conditions.

 (B) Expression in COS cells.

 COS cells that were removed from the cells of monkeys CV-I and which were transformed using a mutant devoid of the beginning of SV40, to express the large-sized T-antigen SV 40 [see Gluzman et al. Cell. 32, 175 (1981)] were transformed with the pML CEZ α vector, which was obtained in (5) (C) and which contained the human G-CSF chromosomal gene. The supernatant of the cell culture COS showed activity of human G-CSF (example 35).

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

A sample of activity that stimulates the formation of colonies (a) With human bone marrow cells:
Cultivation in a monolayer of soft agar was carried out in accordance with the method of Brandley, T.R. and Metcalf, D. (Aust. J. Exp. Biol. Med. Sci. 44, 287-300, 1966). 0.2 ml of bovine embryo serum were mixed, 0.1 ml of samples, 0.1 ml of a suspension of non-assembled human bone marrow cells (1-2 x 10 ⁵ nuclear cells), 0.2 ml of a modified McCoy 5A culture solution (Mc Coy) and 0.4 ml of the culture solution of the modified McCoy 5A containing 0.75% agar, poured into a plastic dish for tissue culture (35 ml in diameter) was subjected to coagulation, and cultured at ³⁷ ° C in 5% CO _2/95% air at 100% humidity. Ten days later, the number of obtained colonies was calculated (one colony consisted of at least 50 cells) and the colony-stimulating activity was determined, and one unit was considered to be the activity necessary for the formation of one colony.

(c) With mouse bone marrow cells:
Horse serum (0.4 ml) was mixed 0.1 ml of a sample 0.1 ml of a suspension of bone marrow cells (females) of C3H / He mice (0.5-1x x10 ⁵ nuclear cells) and 0.4 ml of a modified culture solution Coy 5A containing 0.75% of agar, the mixture was poured into a plastic tissue culture dish (35 mm diameter), subjected to coagulation and cultured for 5 days at ³⁷ ° C in 5% CO ₂ and 95% air at 100% humidity. The number of colonies formed was calculated (one colony consisted of at least 50 cells) and determined colony-stimulating activity, assuming that one unit corresponds to the activity necessary for the formation of one colony.

 PRI me R 1. The establishment of CHU-2.

A tumor of a patient having oral cancer, in which a marked increase in the number of neutrophils was observed, was transplanted into a nu / nu mouse. Approximately 10 days after transplantation, an increase in tumor weight and the number of neutrophils was noted. Twelve days after transplantation, the tumor was removed aseptically, diced into 1-2 mm ³ cubes and grown as follows.

Ten to fifteen tumor cubes were placed in a 50 ml plastic centrifuge tube. After adding 5 ml trypsin solution (containing 0.25% trypsin and 0.02% EDTA) tube was shaken for 10 minutes in a warm bath at 37 ^{° C} and the supernatant was decanted. Was added an additional 5 ml portion of the same solution of trypsin and trypsin digestion was performed under agitation for 15 min at 37 ^C. The Have allocated supernatant cell suspension and it was stored on ice after trypsin was deactivated by the addition of 1 ml of fetal bovine serum.

After repeating these methods again, the cell suspension was isolated, mixed with the previously prepared suspension and centrifuged at 15,000 rpm for 1 min for 10 min to obtain a cell pellet after centrifugation. After centrifugation, the pellet was washed twice with F-10 containing 10% bovine embryo serum, and then placed in a plastic culture flask (25 cm ² ) to obtain a cell concentration of 5x10 ⁶ cells / flask. After overnight incubation in a CO ₂ incubator (5% CO ₂ and 100% humidity) with an F-10 culture solution containing 10% bovine embryo serum, the supernatant was removed along with non-fused cells, and cultivation was continued with fresh infusion of the culture solution . Six days after the start 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 2 ml of anti-mouse erythrocyte antibody (Cappel) diluted 5 times with RPMI-1640 and 2 ml of a guinea pig chromosome kit (Kyokuto Seiyaku Co. Ltd.) diluted in 2 , 5 times using RPMI-1640. After incubation for 20 min at ³⁷ ° C the culture was washed twice with F-10 containing 10% fetal bovine serum was removed and mouse fibroblasts. Then an F-1 culture solution containing 10% bovine embryo serum was added, and cultivation was carried out for another 2 days. After that, part of the cells was isolated and subjected to cloning by the method of limiting dilution.

 The obtained II clones were checked for their CSF activity, it was found that one clone (CH U-2) showed activity about 10 times higher than other clones.

 PRI me R 2. The allocation of CSF.

The cells obtained in example 1 were grown in a completely dense population in two culture flasks (150 cm ³ ). Cells were isolated, suspended in 500 ml of an F-10 culture solution containing 10% bovine embryo serum, transferred to a 1580 cm ³ glass rotating bottle (Belco), and subjected to vortex culture at 0.5 rpm rotation for 1 minute. When it was found that the cells had grown in a completely dense population on the inner wall of the rotating bottle, the culture solution was replaced with serum-free PMI 1640. After culturing for 4 days, the culture supernatant was isolated and cultivation was continued by adding with F-10 a solution containing 10% bovine embryo serum. After a three-day cultivation, the culture solution was again replaced with serum-free RPMI 1640, and after 4 days the culture supernatant was isolated. By repeating these methods, each plot received 500 ml of serum-free supernatant per bottle. In addition, this method makes it possible to isolate the supernatant of the culture while maintaining the cells for a substantially extended period.

 A portion containing 5000 ml of the supernatant of the obtained culture was mixed with 0.01% Tween 20 and concentrated approximately 1000 times by ultrafiltration through Hollow Fiber DC-4 and Amicon PM-10 (Amicon) filters.

 The concentrate was purified by the following methods.

 (I) A portion (5 ml) of the concentrated surface layer of the culture was subjected to gel filtration on an Ultrogel ACA54 column (diameter 4.6 cm, length 90 cm, LKB) at a flow rate of about 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 Kagaku Co. Ltd.). The column was calibrated using bovine albumin serum (mol. M. 67000), ovoalbumin (mol. M. 45000) and cytochrome C (mol. M. 12400). After gel filtration, 0.1 ml of each of the fractions was diluted 10 times, and a directed selection of active fractions was carried out using the above-described method for determining the activity of a stimulating colony (CSA) (c). It was found that fractions with Ve 400-700 ml exhibit macrophage-dominant colony stimulating activity, while fractions with Ve 800-1200 ml exhibit granulocyte-dominant colony stimulating activity. Therefore, the latter fractions were collected and concentrated to a volume of about 5 ml on an ultrafiltration device with PM-10 (Amico).

 (II) To the concentrated fractions was added an aqueous solution of 0.1% trifluoroacetic acid containing 30% n-propanol (to determine the amino acid sequence supplied by Tokyo Kasei K.K.). After keeping the mixture in ice for about 15 minutes, the precipitate was removed by centrifugation for 10 minutes at 15,000 rpm. The supernatant was adsorbed on a C18 μ-Bondapak column (8 mm x 30 cm for semi-preparative use, Waters) balanced with an aqueous solution containing n-propanol and trifluoroacetic acid; the column was continuously eluted with an aqueous solution of 0.1% trifluoroacetic acid containing n-propanol which had a linear concentration gradient of 30-60% A Hitachi high pressure liquid chromatograph, model 685-50 (Hitahi. Ltd.) and a Hitachi detector model 638-41 (Hitachi, Ltd.) were used.

 To determine the absorption values simultaneously at 220 nm and 280 nm. After elution, 10 μl of each of the fractions was diluted 100 times, and a directed selection of the active fractions was carried out using the above method for determining colony stimulating activity (c). It was found that the peaks, eluting with 40% n-propanol, have the activity of a stimulating colony, therefore they were collected, re-chromatographed under the same conditions, and their activity of the stimulating colony was determined in the same way. Again activity was observed at peaks in 40% n-propanol. Therefore, these peaks were collected (4 fractions 4 ml) and dried by freezing.

(III) The freeze-dried powder was dissolved in 200 μl of an aqueous solution, 0.1% trifluoroacetic acid containing 40% n-propanol, and the solution was subjected to high pressure liquid chromatography on a TSK-G3000SW column (Touo 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 were collected in 0.4 ml portions using a fraction collector FRAC-100 (Pharmacia Fine Chemical). Each of the fractions taken was checked to determine its colony-stimulating activity using the same method as described above, and activity was observed in fractions with retention times of 37-38 min (corresponding to a molecular weight of about 2 x ^{10 4} ). Active fractions were isolated and purified on a μ-Bondapak C18 analytical column (4.6 mm x 30 cm). The main peaks were isolated and freeze dried. The resulting sample was analyzed using the method for determining colony-stimulating activity (a). It was found that he possessed the activity of human G-CSF.

PRI me R 3. Determination of amino acid sequence
(I) Determination of the N-terminal amino acid sequence.

The sample was digested with Edman using a gas phase sequencer (Applied Biosystems), and the resulting PTH amino acid was analyzed by conventional methods using a Beckman Instruments HPLC and an Ultrasphere ODS column (Beckman Instruments). A column (5 μm, 4.6 mm diameter and 250 mm long) was equilibrated with an initial buffer in an aqueous solution containing 15 mmol / L sodium acetate buffer (pH 4.5) and 40% acetonitrile, and a sample was introduced (dissolved in 20 μl of the original buffer) . Separation was carried out with isocratic elution with the original buffer. The flow rate was 1.4 ml / min and the column temperature was maintained at 40 ^C. Detection of PTH-amino acids was performed using the absorption in the UV range at wavelengths between 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 the tested samples. As a result, it was found that the sample had the following amino acid sequence of 40 residues from the N-terminus. H ₂ N-Thr-Pro-Lei-Glu-Pro-Ala-Ser-Ser-
(10) Lei-Pro-Gln-Ser -Phe-Lei-Lei-Liz-Cis-
(20) Ley-Glu-Gln-Val-Arg-Liz-Ile-Gln-Glu-
(30) Asp-Glu-Ala-Ley-Gln-Glu-Liz-Ley-
(40) Cis-Ala-Thr-Tur-Liz- (II) Decomposition by bromine cyan.

The sample was dissolved in 70% formic acid. 200 equivalent amounts of cyanogen bromide were added to the solution, which was purified by sublimation. The mixture was left overnight at ³⁷ ° C for reaction. The reaction product was freeze dried and fractionated by high performance liquid chromatography and on a TSKG3000SW column (Toyo Soda Manufacturins CO. Ltd.) to obtain four peaks. The peaks were named CN-1, CN-2, CN-3, CN-4 in decreasing molecular weight. The first two peaks (CN-1 and CN-2) had better yields, and their amino acid sequences were analyzed using an automatic gas phase sequence determiner (Applied Biosystems) under the same conditions as those used in (I).

 As a result, it was found that CN-1 is a peptide from the N-terminus of the G-CSF protein, and CN-2 had the following amino acid sequence: Pro-Ala-Fen-Ala-Ser-Ala-Fen-Gln-Arg-Arg-Ala -Gly-Gli-Val- Lei-Val-Ala-Ser-Gis-Lei-Gln- (III) Trypsin decomposition.

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 urea concentration of 2 mol / L. TRPC-treated trypsin (Sigma) was added so that the ratio of sample to enzyme was 50: 1. The mixture was kept for 4 hours at ²⁵ ° C and after adding an equal amount of TPCK-treated trypsin, and the mixture was heated for another 16 hours at 25 ^C. Thereafter, the reaction product was subjected to reversed phase high column chromatography on C8 column (Yamamura Kagaku KK) at eluting with 0.1% trifluoroacetic acid containing n-propanol, which had a linear density gradient of 5-60% Although several peaks were observed when measuring absorbance at 280 nm, the main peak was analyzed to determine its amino acid last consistency using an automatic gas phase sequencer (Applied Biosystems) under the same conditions as used in (I). As a result, it was found that the main peak was a peptide having the following sequence, which contained part of the CN-2 fragment shown in (II): Gln-Lei-Asp-Val-Ala-Asp-Fen-Ala-Tre-Tre-Ile -Trp-Gln-Gln-Met-Glu-Glu-Lei-Gli-Met-Ala-Pro-Ala-Lei-Gln-Pro-Tre-Gln-Gli-Ala-Met-Pro-Ala-Fen-Ala-Ser -
PRI me R 4. Obtaining DNA probe (I) Synthesis of the probe (IWQ)
Thirty consecutive nucleotides (see FIG. 1) were obtained on the basis of the sequence of 10 amino acids (Ile-Trp-Gln-Gln-Met-Glu-Glu-Lei-Gli-Met) included in the amino acid sequence obtained in example 3 (II) . One point to be made regarding the designation of the nucleotides shown in FIG. 1, for example, the nucleotide at position 9, from the 5 ′ end, is an equimolar mixture of dA and dG. The starting nucleotides are for the most part dimers, but if necessary, monoculeotides are also used. A column equipped with a glass filter was charged with 20 mg of the starting nucleotide resin Ap-d (G) (Yamasa Snoyu Co. Ltd.). After repeated washing with methylene chloride, the 4,4'-dimethoxytrityl group was excluded by treatment with a methylene chloride solution containing 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 replace the solvent, 20 mg of the nucleotide dimer (DMTr) ApTp (NHR ₃ ), (Nippon Zeon; NHR ₃ -triethylammonium; DMT-dimethoxytrityl) and 0.2 ml of pyridine and the inside were added the columns were vacuum dried using a vacuum pump. Then 20 mg of 2,4,6-trimethylbenzenesulfonyl-3-nitrotriazolid (MSNT of Wako Pure Chemical Inductries, Ltd.) and 0.2 ml of anhydrous pyridine were added, and the inside of the column was displaced with nitrogen gas. The nucleotide resin was concentrated with the dimer by reaction for 45 minutes at room temperature with stirring from time to time. After completion of the reaction, the column was washed with pyridine, and unreacted OH groups were acetylated with a pyridine solution containing an excess of acetic anhydride and 4-dimethylaminopyridine. After washing the column with pyridine, the following dimers or monomers, written in order, were condensed by repeating the above methods (DMTr) Ip (NHR ₃ ), (DMTr) GpGp (NHR ₃ ), (DMTr) Ip (NHR ₃ ), equimolar mixture (DMTr) СрТр (ТНR ₃ ) and (DMTr) ТТр (NHR ₃ ), equimolar mixture (DMTr) ApAp (NHR ₃ ) and (DMTr) ApGp (NHR ₃ ), equimolar mixture (DMTr) ApGp (NHR ₃ ) and (DMTr) GpGp (NHR ₃ ), (DMTr) GpAp (NHR ₃ ), (DMTr) TpGp (NHR ₃ ), an equimolar mixture (DMTr) ApAp (NHR ₃ ) and (DMTr) GpAp (NHR ₃ ), (DMTr) СрАр (NHR ₃ ), an equimolar mixture of (DMTr) ApAp (NHR ₃ ) and (DMTr) ApGp (NHR ₃ ), (DMTr) GpCp (NHR ₃ ), (DMTr) TPp (NHR ₃ ), (DMTr) Ip (NHR ₃ ) and ( DMTr) ApTp (NHR ₃ ), all of these nucleotides being supplied by Nippon Zeon, except (DMTr) Ip (NHR ₃ ), which is supplied by Yamasa Shoyu Co. Ltd. After the reaction at the final stage, the resin was washed successively with pyridine, methylene chloride and ether without acylation and then dried. The dried resin was suspended in 1.7 ml of a mixture of pyridine (0.5 ml), water (0.2 ml) and dioxane (1 ml) containing 1 mol / L tetramethylguanidine and 1 mol / L α-picoline aldoxime. The suspension was 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 3.2 ml of concentrated aqueous ammonia, the mixture was heated at 55 ^{° C} for 6 hours. After extraction with ethyl acetate, the aqueous layer was separated and concentrated in vacuo. The concentrate was dissolved in a solution of 50 mmol / L triethylammonium acetate (pH 7.0) and the solution was chromatographed on a C-18 column, (1.0-15 cm; Waters), eluting with acetonitrile (linear density gradient 10-30%) in a solution of 50 mmol / L triethylammonium acetate (pH 7.0). The peak of the fraction eluted with an acetonitrile concentration of approximately 25% was concentrated in vacuo.

80% acetic acid was added to the concentrate, and the mixture was allowed to stand for 30 minutes at room temperature. After extraction with ethyl acetate, the aqueous layer was separated and concentrated in vacuo. The resulting concentrate was then purified by high pressure liquid chromatography on a C-18 column (supplied by Senshu Kagaku KK SSC-ODS-272, diameter 6x200 mm). Elution was performed with acetonitrile (linear gradient of density 10-20%) in a solution of 50 mmol / L triethylammonium acetate (pH 7.0). Synthetic DNA was obtained with a yield of no lower than 10A ₂₆₀ units.

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

 (II) Synthesis of the probe (A).

 Fourteen nucleotide sequences (see FIG. 1) were obtained based on the sequence of 5 amino acids (Met-Pro-Ala-Fen-Ala) included in the amino acid sequence obtained in example 3.

The synthesis methods were similar to those used in the preparation of the probe (IWQ) and the following nucleotides were condensed into the nucleotide resin Ap-d (T) (Yamasa Shoyu Co. Ltd.) in the order of writing: (DMTr) CpAp (NHR ₃ ), (DMTr ) GpGp (NHR ₃ ), equimolar mixture (DMTr) CpAp (NHR ₃ ), (DMTr) CpTp (NHR ₃ ), (DMTr) CpGp (NHR ₃ ) and (DMTr) CpCp (NHR ₃ ), equimolar mixture (DMTr) ApGp (NHR ₃ ), (DMTr) TpGp (NHR ₃ ), (DMTr) GpGp (NHR ₃ ) and (DMTr) CpGp (NHR ₃ ), (DMTr) ApAp (NHR ₃ ), equimolar mixture (DMTr) CpAp (NHR ₃ ) and (DMTr) CpGp (NHR ₃ ), and (DMTr) Gp (NHR ₃ ), all nucleotides being supplied by Nippon Zeon. Synthetic DNA was obtained with a yield of approximately 10A ₂₆₀ units. Analysis using the Maxam-Gilbert sequence determination method showed that the obtained oligonucleotide had the nucleotide sequence shown in FIG.

 (III) Synthesis of the probe (LC).

 Automatic DNA synthesis was performed using a DNA synthesizer, model 380A "Applied Biosystems". This technique is based on the principles described by Suthers et al. [J. Am. Chem. Soc. 103.3185 (1981)] is commonly referred to as the phosphoramidite technique.

 The phosphoramidite form (DMTr) -dT preactivated with tetrazole is condensed to dG-S (Si substrate), where the 5'-dimethoxytrityl group (DMTr) is released. After this, the unreacted groups are acetylated and oxidized with iodine in the presence of water to obtain phosphoryl groups. After the release of the DMTr group, the condensation is repeated in the same way until 24 nucleotides having the sequence shown in FIG. 1 are synthesized. These nucleotides are cleaved from the support, released and purified by high pressure reverse phase liquid chromatography on a C-18 column (Senshu Kagaku Co. Ltd. SSC-ODS-272).

PRI me R 5. Cultivation of CHU-2 cells and obtaining mRNA
1 / Cultivation and isolation of CHU-2 cells.

Adapted CHU-2 cells were grown in a completely dense population in two culture flasks (150 cm ² ), isolated, suspended in 500 ml of a culture solution of RPMI 1640 containing 10% bovine embryo serum, transferred to a 1580 cm ² glass rotating bottle (Belco) and cultured under rotation for 4 days at a speed of 0.5 revolution in 1 min. When it was found that the cells grew in a completely dense population on the inner wall of the rotating bottle, the culture solution was removed on the rotating bottle and 100 ml of pre-heated (37 ^° C) physiological saline solution containing 0.02% EDTA was loaded into it. After heating at ³⁷ ° C for 2 minutes, the cells were separated from the inside wall of the flask by suction pipette. The resulting cell suspension was centrifuged at 1,500 rpm for 10 minutes to obtain a cell pellet after centrifugation. Cells were resuspended in 5 ml of physiological saline without EDTA. The suspension was centrifuged at 1500 rpm for 1 min for 10 min to obtain a cell pellet after centrifugation (wet weight of about 0.8 g). The thus-obtained cells were stored frozen at ^-80 ° C until the moment they are subjected to RNA extraction procedure.

 2) Purification of mRNA.

Isolation of mRNA from CHU-2 cells obtained in 1) was carried out using methods that were essentially the same as the methods described in "Molesular Cloning", Maniatis et al. Сold Spring Harbor, r. 196, 1982. Frozen CHU-2 cells (wet weight 3.8 g) were suspended in 20 ml of a solution of 6 mol / L guanidine [6 mol / L guanidinium isothiodianate, 5 mmol / L sodium citrate (pH 7.0) 0.1 mol / L β-mercaptoethanol and 0.5% sodium sarcosyl sulfate] and the suspension were mixed well by vortex rotation for 2-3 minutes. The mixture was subjected to 10 suction and injection cycles with a syringe (20 ml capacity) equipped with an 18G needle. About 6 ml of viscous guanidinium solution containing torn cells was layered on a 6 ml pad of 5.7 mol / L CsCl in 0.1 mol / L EDTA (pH 7.5) in a Beckman SW40Ti polyallomer centrifuge tube so that the tube was completely filled. Four centrifuge tubes were prepared by methods described above and centrifuged at 30,000 rpm for 1 minute for 15 hours at 20 ^C. The resulting precipitates after centrifugation, washed three times with a small amount of 70% ethanol.

The centrifugation pellets obtained from the respective tubes were mixed, dissolved in 550 μl of water and treated to obtain a NaCl concentration of 0.2 mol / L. After treatment with a 1: 1 mixture of phenol and chloroform, and then with one chloroform, 2.5 volumes of ethanol were added to precipitate all RNA (approximately 10.1 mg of all RNA was obtained from 3.8 g of wet cells). Poly (A ⁺ ) RNA was purified from all RNA using the following affinity chromatography methods, taking advantage of the attachment of a poly (A) chain to the 3 ′ end of the mRNA. Adsorption on oligo (dT) -cellulose (type 7 Р-L Biochemicals) was carried out by passing through the oligo (dT) -cellulose column all RNA in loading buffer [containing 10 mmol / L Tris-Hcl (pH 7.5) 0.5 mol / l NaCl, 1 mmol / l EDTA and 0,1% SDS solution] after the solution was heated at ⁶⁵ ° C for 5 minutes. The column was balanced with the same load buffer. Poly (A ⁺ ) RNA was eluted with a TE solution [containing 10 millimol / L Tris-Hcl (pH 7.5) and 1 millimol / L EDTA]. The non-adsorbed effluent was re-passed through the column again and the eluate obtained by repeating those the methods themselves, mixed with the first epaulet shoulder strap. The result was 400 micrograms of poly (A +) RNA.

 The mRNA thus obtained was size fractionated by sucrose density gradient centrifugation according to the procedures described in the Schleif and Wensink Laboratory Manual, Practical Methods in Molecular Biology, Springer-Verlag, New York, Heidelberg, Berlin (1981).

A sucrose density gradient of 5-25% was created in a Backman SW40Ti centrifuge tube. Two sucrose solutions were prepared by dissolving 5 and 25% sucrose, not containing pHase (Schwarz / Mann), in a solution containing 0.1 mol / L NaCl, 10 mmol / L Tris-HCl (pH 7.5), 1 millimol / l EDTA and 0.5 SDS. 7.5) and 1 millimol / L EDTA] The non-adsorbed effluent was re-passed through the column again and the eluate obtained by repeating the same methods was mixed with the first eluate stream. The result was 400 micrograms of poly (A ⁺ ) RNA.

 The mRNA thus obtained was size fractionated by sucrose density gradient centrifugation according to the procedures described in the Schleif and Wensink Laboratory Manual, Practical Methods in Molecular Biology, Springer-Verlag, New York, Heidelberg, Berlin (1981).

 A sucrose density gradient of 5-25% was created in a Backman SW40Ti centrifuge tube. Two sucrose solutions were prepared by dissolving 5 and 25% pH-free sucrose (Schwarz / Mann) in a solution containing 0.1 mol / L NaCl, 10 mmol / L Tris-HCl (pH 7.5), 1 millimol / l EDTA and 0.5 SDS.

Eight hundred micrograms of mRNA [poly (A ⁺ ) -RNA] obtained by the method already described, was dissolved in 200-500 microliters of a TE solution. The solution was heated at ⁶⁵ ° C for 5 min, and after quenching, it was placed in solution with sucrose density gradient which was centrifuged at 30,000 rpm for 20 hours. The mass of collected fractions of 0.5 ml each, and measured their absorption at 260 nm. The sizes of fractionated RNAs were determined based on the positions of standard RNAs (ribosomal RNAs, 28S, 18S, and 5S). At the same time, the activity of G-CSF of each fraction was determined using oocytes of Henopus laevis according to the following methods. First, the mRNA of each fraction was transferred to an aqueous solution with a concentration of 1 μg / 1 μl, oocytes were taken from Henopus (about one year old) and the mRNA solution was injected so that 50 nanograms of mRNA were introduced into one oocyte, ten of these oocytes were placed in each of 96 recesses in a microtiter dish, oocytes were cultured for 48 hours at room temperature in 10 μl of Barth medium [88 mmol / L NaCl; 1 mmol / L KCl; 2.4 mmol / L NaHCO ₃ ; 0.82 mmol / L MgSO ₄ ; 0.33 mmol / L Ca (NO ₃ ) ₂ ; 0.41 mmol / L CaCl ₂ ; 7.5 mmol / L Tris-HCl (pH 7.6); penicillin 10 mg / l and streptomycin sulfate 10 mg / l] culture supernatant was isolated, concentrated and purified to a purity sufficient to determine G-CSF activity.

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

 PRI me R 6. Synthesis of cDNA (building a library of cDNA pBR-line).

On poly (A ⁺ ) obtained in example 5, cDNA was synthesized according to the Land method with al. (Land et al. [Nucleic Acids Res. 9, 2251 (1981)] modified by the method of Gubler and Hoffman [Gene, 25,263 (1983)]
(I) Synthesis of single-stranded cDNA.

The reagents were placed in an Eppendorf tube (with a capacity of 1.5 ml) in the following order: 80 μl of reaction buffer (500 mmol / L KCl, 50 mmol / L MgCl ₂ , 250 mmol / L Tris-Hcl, pH 8.3), 20 μl 200 mmol / L of dithiotreptol, 32 μl, 12.5 mmol / L of deoxynucleotide triphosphate (containing 12.5 mmol / L of each of deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytosine triphosphate and deoxytimidentrifosphate), 10 μg of α- ³² P-5-D-5-R-5-R-5-D-5-R-5-R-5-R-5 ), 32 microliters of oligo (dT) 12-18 (from the firm PL Biochemicals, 500 ug / ml), 20 micrograms of poly (A) ⁺ RNA (2.1 ug / ml) and 206 .mu.l dis illirovannoy water. Total 400 l of the reaction solution was heated at ⁶⁵ ° C for 5 min and then heated at 42 ^{° C} for 5 min. 120 units of reverstranscriptase (Takara Shuzo Co. Ltd.) were added to the heated solution. After reacting for 2 hours at 43 ^{° C} was added 2 .mu.l of an inhibitor of RNA-ase (Bethesda Research Laboranories), 20 l of TE solution, 16 l of 100 mmol / l of sodium pyrophosphate and 48 units (4 l) reverstranskriptazy and reacted at 46 ^{° C} for 2 hours. The reaction was quenched by adding 0.5 mol / l EDTA (8 microliters) and 10% SDS (8 microliters). Subsequent treatment with phenol / chloroform and ethanol precipitation (twice) produced single-stranded cDNA.

 2) Attachment of the dC chain to single-stranded cDNA.

Single-stranded cDNA obtained in 1) was dissolved in distilled water. To the solution was added 60 μl of dC-chain-containing buffer [400 mmol / L potassium cacodylate, 50 mmol / L Tris-Hcl (pH 6.9), 4 mmol / L dithiothreitol, 1 mmol / L CoCl ₂ and 1 mmol / L deoxycytosine triphosphate ( dTsTF)] and the mixture was heated at ³⁷ ° C for 5 min. To the reaction solution was added 3 l of terminal transferase (27 units / ml, P-L Biochemicals) and the mixture was heated at 37 ^C for 2.5 min. After treatment with phenol / chloroform (once) and precipitation with ethanol (twice), the dC-bound cDNA was dissolved in 40 μl of a TE solution containing 100 mmol / L NaCl.

 3) Synthesis of double-stranded cDNA.

To 40 microliters of DNA solution obtained in 2) was added 4 ul of oligo (dG) 12-18 (200 micrograms / ml, P-L Biochemicals) and the mixture was heated first at ⁶⁵ ° C for 5 min and then at 43 ^{° C} within 30 minutes While the reaction solution was maintained at ⁰ ° C, to it was added 80 .mu.l of buffer [100 mmol / l Tris-HCl (pH 7.5), 20 mmol / l MgCl _2, 50 mmol / l (NH _{4) 2} SO ₄ , and 500 mmol / L KCl] 4 μl 4 mmol / L deoxynucleotide triphosphate (containing 4 mmol / L each dATP, dCTP, dGTP and dTTP), 60 microliters 1 mmol / L, β-NAD, 210 μl distilled water, 20 μl of E. coli DNA polymerase I (Takara Shuzo Co. Ltd.), 15 μl of E. coli DNA polymerase I (Takara Shuzo Co. Ltd.) and 15 microliters of E. coli PHase AS (Takara Shuzo Co. Ltd. .) and the mixture was subjected to reaction at 12 ^{° C} for 1 hour. After adding 4 mmol / l deoxynucleotide triphosphate (4 l), p action was carried out at ²⁵ ° C for 1 hour. By subsequent treatment with phenol / chloroform and precipitation with ethanol (once) received approximately 8 micrograms of double-stranded cDNA. This double-stranded cDNA was dissolved in a TE solution and subjected to 1.2% agarose gel electrophoresis. Fragments corresponding to a size of from about 560 base pairs to 2 thousand base pairs were adsorbed onto Whatman DE 81, and about 0.2 μg double-stranded cDNA was isolated by elution.

 4) Connection of the dC chain with double-stranded cDNA.

The double-stranded cDNA obtained in 3) was dissolved in 40 μl of TE solution. Once added 8 l of dC-tail rotor of this type of buffer as that described in 2) The mixture was heated at ³⁷ for 2 min. After adding 1 ul terminal transferase (27 units / microliter), the mixture was reacted at ³⁷ ° C for 3 min. Thereafter, the reaction solution was immediately cooled ^to 0 ° C, and the reaction was quenched by adding 1 .mu.l of 0.5 mol / l EDTA. After treatment with phenol / chloroform and ethanol precipitation, the resulting precipitate was suspended in 10 μl of a TE solution.

5) Construction of a cDNA library of pB R-line
Four microliters of a commercial oligo (dG) cross-linked pBR 322 vector (Bethesda Research Laboratories; 10 nanograms / microliter) and 2 μl of the dC cross-linked double-stranded cDNA obtained in 4) were renatured in a TE solution containing 5 μl of 0.1 mol / l NaCl solution. Renaturation consisted of three stages: heating at ⁶⁵ ° C for 5 min, then heating ^at 40 ° C for 2 h followed by cooling to room temperature.

 In accordance with the method described in the laboratory manual Manyatis et al. [Maniatis et al. Molecular Cloning, Spring Spring Harbor, b. 249 et seq. (1982)] (other known methods can also be used here), competent cells were obtained from E. coli strain X 1776 and transformed using a renatured plasmid to obtain transformants.

PRI me R 7. Synthesis of cDNA (construction of a library of λ-phage)
1 / Synthesis of single-stranded cDNA.

In accordance with the methods described in Example 5, 3.8 g of frozen CHU-2 cells were purified twice on an oligo (dT) -cellulose column and then processed to obtain 400 μg of poly (A ⁺ ) -RNA.

A TE solution (10 μl) in which 12 μg of poly (A ⁺ ) RNA was dissolved was placed in a reaction tube containing 10 μg of antinomycin D (Sigma). After that, the reagents were loaded into the tube in the following order: 20 μl reverse transcriptase buffer [250 mmol / L Tris-Hcl (pH 8.3); 40 mmol / L MgCl ₂ ; 250 mmol / L KCl] 20 μl 5 mmol / L deoxynucleotide triphosphate (containing 5 mmol / L each of dATP, dGTP, dTCP and dTTP), 20 μl oligo (dT) 12-18 (0.2 μg / ml, P- L Biochemicals); 1 μl 1 mol / L dithiothreitol, 2 μl RN-azine (30 units) μl, Promeda Biotech); 10 μl of reverstranscriptase (10 units / μl, Seikagaku Kogyo Co. Ltd.); 1 μl of α- ³² P-dATP (10 μCi; Amerscham) and 16 μl of water. The reaction solution whose volume was 100 ul, heated at 42 ^{° C} for 2 hours and then the reaction was quenched by adding 0.5 mol / L EDTA (5 L) and 20% SDS (1 l). Subsequent treatment with phenol / chloroform (100 μl) and ethanol precipitation (twice) gave approximately 4 μg single-stranded cDNA.

2) Synthesis of double-stranded cDNA
The cDNA obtained in 1) was dissolved in 29 μl of TE solution, and the reaction solution was prepared by adding the following reagents in the written order: 25 μl of polymerase buffer [400 mmol / L Hepes (pH 7.6); 16 mmol / L MgCl ₂ , 63 mmol / L β-mercaptoethanol and 270 mmol / L KCl] 10 μl 5 mmol / L deoxynucleotide triphosphate; 1.0 μl 15 mmol / L β-NAD; 1.0 μl α - ³² -P-dATP (10 μi / μl); 0.2 μl of E. coli DNA ligase (60 units / μl, Takara Shuzo Co. Ltd.); 5.0 μl of E. coli DNA polymerase I (New England Biolabs; 10 units / μl); 0.1 μl pH-H (60 units / μl; Takara Shuzo Co. Ltd.); and 28.7 μl of distilled water.

The reaction solution was incubated at 14 ^{° C} for 1 h, allowed to cool to room temperature, and then it was incubated for an additional hour. The reaction was then quenched by adding 0.5 mol / L EDTA (5 μl) and 20% SDS (1 μl) and the reaction mixture was treated with phenol / chloroform and ethanol precipitated. The resulting DNA was dissolved and 20 μl, 0.5 mmol / L EDTA and the reaction solution was obtained by adding 3 μl Klenow buffer [500 mmol / L Tris-Hcl (pH 8.0) and 50 mmol / L MgCl ₂ ] 3 μl 5 mmol / l deoxynucleotide triphosphate and 4 μl of water. After addition of 1 l of DNA polymerase (Klenow fragment Takara Shuzo Co. Ltd.), the reaction solution was incubated at ³⁰ ° C for 15 min.

 The incubated reaction solution was diluted with 70 μl of TE solution, and the reaction was quenched by adding 0.5 mol / L EDTA (5 μl) and 20% SDS (1 μl). Subsequent treatment with phenol / chloroform and ethanol precipitation gave approximately 8 micrograms of double-stranded cDNA.

3) Methylation of double-stranded cDNA
An aqueous solution (30 μl) of the double-stranded cDNA synthesized in 2) was mixed with 40 μl of methylation buffer [500 mmol / L Tris-HCl (pH 8.0), 50 mmol / L EDTA] 20 μl SAM solution [800 μmol / L S-adenosyl-L-methylmethionine (SAM), 50 mmol / L β-mercaptoethanol] and 100 microliters of water. 15 ml of EcoRI methylase (New England Biolabs; 20 units / μl) was added to the mixture to obtain a reaction solution with a volume of 200 μl. After incubation at ³⁷ ° C for 2 hours and had a treatment with phenol and ether, and ethanol precipitation to isolate DNA.

 4) Adding an EcoRI linker.

1.5 μl of ligase buffer [250 mmol / L Tris-Hcl (pH 7.5) and 100 mmol / L MgCl ₂ ] 0.5 μl of a pre-phosphorylated EcoRI linker (10 mer) were added to approximately 1.2 μg of methylated double-stranded DNA. ; Takara Shuzo Co. Ltd.), 1.5 μl of 10 mmol / L ATP, 1.5 μl of 100 mmol / L, dithiothreitol and 2 μl of H ₂ O to obtain a reaction solution of 15 μl. After addition of 0.7 l of T reaction was performed overnight at ₄ April DNA ligase (3.4 units / microliter, Takara Shuzo Co. Ltd.) ^C. Thereafter ligase inactivated by heating at ⁶⁵ ° C for 10 min . The reaction solution was diluted to a total volume of 50 μl by adding 100 mmol / L Tris-HCl (pH 7.5), 5 mmol / L MgCl ₂ , 50 mmol / L NaCl and 100 μg / ml gelatin. After the addition of EcoRI (3.5 L, 10 units / ml), the reaction was carried out at ³⁷ ° C for 2 hours. Then 2.5 L of 0.5 mol / l EDTA, and 0.5 .mu.l 20% SDS, and then conducted phenol / chloroform treatment and ethanol precipitation in order to isolate DNA. After that, the unreacted EcoRRI linker was removed by gel filtration on UltrogelAcA34 (LKB) or agarose gel electrophoresis so as to isolate approximately 0.5-0.7 μg double stranded cDNA with an added linker.

5) Attachment of double-stranded cDNA to the λ gt 10 vector
Double-stranded cDNA with an added linker was mixed with 2.4 μg of a pre-Eco-treated λgt 10 vector (vector cloning system), with the addition of 1.4 μl of ligase buffer (250 mmol / L Tris-HCl and 100 mmol / L MgCl ₂ ) and 6 , 5 l of distilled water, and the mixture was heated at 42 ^{° C} for 15 min. Then 1 l of 10 mmol / l ATF, 1 l 0.1 mol / l DTT and 0.5 l of _T4 DNA ligase to obtain a total volume of 15 uL, and the reaction was carried out overnight at 12 ° ^C.

6) In vitro packaging
About a third of the recombinant DNA obtained in 5) was packaged using an in vitro packaging kit (Promeda Biotech) to obtain phage spots.

 PRI me R 8. Directional selection of library pB R-line using a probe (IWQ).

Whatman 541 paper placed on agar medium with growing colonies and left at ³⁷ ° C for 2 h. The filter paper was then treated by the following method Taub and Thompson [Taub and Thompson, Anal. Biochem. 126, 222 (1982)]
Colonies were transferred onto paper 541, and then grown on agar medium containing hloranfenikol (250 ug / ml) overnight at 37 ° ^C.

Paper 541 was removed and left at room temperature for 3 minutes on another sheet of filter paper, which was impregnated with 0.5 N. NaOH solution. This procedure was repeated twice. Two similar experiments were carried out for 3 min using a solution of 0.5 mol / L Tris-Hcl (pH 8). At 4 ^° C, the treatment was carried out with a solution of 0.05 mol / L Tris-Hcl (pH 8) for 3 min and a solution of lysozyme 1.5 mg / ml [containing 0.05 mol / L Tris-Hcl (pH 8) and 25 % sucrose] for 10 minutes, then at ³⁷ ° C was performed with 1 processing SSC (0,15 mol / l NaCl and 0.015 mol / l sodium citrate) for 2 minutes and a solution of 1 SSC, coderzhaschim 200 ug / ml proteinase K , for 30 minutes, finally, at room temperature, treatments with a 1 x SSC solution for 2 minutes and a solution of 95% ethanol for 2 minutes were performed. The final stage was repeated twice. After that, paper 541 was dried. The dried paper 541 was immersed in a mixture of 25: 24: 1 phenol / chloroform / isoamyl alcohol [balanced 100 mmol / L Tris-Hcl (pH 8.5), 100 mmol / L NaCl and 10 mmol / L EDTA] for 30 min at room temperature. Then, similar procedures were repeated three times with a solution of 5 x SSC for 3 minutes, then twice with 95% ethanol solution for 3 minutes. After that, the filter paper was dried.

The probe (IWQ) was labeled with a 32P-labeled atom according to known methods (see Molecular Cloning), and colony hybridization was carried out according to the method of Wallace et al. [Nucleic Acids Res. 9, 879 (1981)] Prehybridization was carried out at 65 ° C for 4 hours in a hybridization buffer containing 6 x NET [0.9 mol / L NaCl; 0.09 mol / L Tris-HCl (pH 7.5) and 6 mmol / L EDTA] 5 x Dengardt solution (Denhardt), 0.1% SDS and 0.1 mg / ml denatured DNA (calf thymus). Thereafter, hybridization was carried out overnight at ⁵⁶ ° C in hybridization buffer (see recipe it. Above), containing 1x10 ⁶ counts per minute / ml radiolabelled probe (IWQ). After completion of the reaction 541 paper washed twice with 6 x SSC solution (containing 0,1% SDS) for 30 min at room temperature, then washed at ⁵⁶ ° C for 1.5 min. The washed paper 541 was then subjected to autoradiography.

 The plasmid was separated from the positive clones and spotted by Southern (Southern) probe (IWQ). Hybridization and autoradiography were performed under the same conditions as described above.

Similarly, Southern spotting was performed with a probe (A). Using a hybridization buffer having the aforementioned composition, hybridization was performed first at 49 ^{° C} for 1 hour. After cooling to 39 ^{° C} was continued and then hybridizing at this temperature for 1 hour. After the reaction, the nitrocellulose filter was washed twice with 6 SSC, containing 0 , 1% SDS, for 30 min at room temperature and then washed at 39 ^{° C} for 3 min. The washed paper was then subjected to autoradiography. mol / L Tris-Hcl (pH 8) for 3 min and a solution of lysozyme 1.5 mg / ml [containing 0.05 mol / L Tris-Hcl (pH 8) and 25% sucrose] for 10 minutes, then at 37 ^° C was treated with a solution of 1 x SSC (0.15 mol / L NaCl and 0.015 mol / L sodium citrate) for 2 min and a solution 1x SSC containing 200 μg / ml proteinase K, for 30 min, finally, at room temperature was treated with a solution of 1 x SSC for 2 minutes and a solution of 95% ethanol for 2 minutes. The final stage was repeated twice. After that, paper 541 was dried. The dried paper 541 was immersed in a mixture of 25: 24: 1 phenol / chloroform / isoamyl alcohol [balanced 100 mmol / L Tris-Hcl (pH 8.5), 100 mmol / L NaCl and 10 mmol / L EDTA] for 30 min at room temperature. Then, similar procedures were repeated three times with a solution of 5 x SSC for 3 minutes, then twice with 95% ethanol solution for 3 minutes. After that, the filter paper was dried.

A probe (IWQ) was labeled with a ³² P labeled atom according to known methods (see Molecular Cloning), and colony hybridization was carried out according to the method of Wallace et al. [Nucleic Acids Res. 9, 879 (1981)] Prehybridization was performed at 65 ° ^C for 4 hours in a hybridization buffer containing 6 x NET [0,9 mol / l NaCl; 0.09 mol / L Tris-HCl (pH 7.5) and 6 mmol / L EDTA] 5 x Dengardt solution (Denhardt), 0.1% SDS and 0.1 mg / ml denatured DNA (calf thymus). Thereafter, hybridization was carried out overnight at ⁵⁶ ° C in hybridization buffer (see recipe it. Above), containing 1x10 ⁶ counts per minute / ml radiolabelled probe (IWQ). After completion of the reaction 541 paper washed twice with 6 x SSC solution (containing 0,1% SDS) for 30 min at room temperature, then washed at ⁵⁶ ° C for 1.5 min. The washed paper 541 was then subjected to autoradiography.

 The plasmid was separated from the positive clones and spotted by Southern (Southern) probe (IWQ). Hybridization and autoradiography were performed under the same conditions as described above.

Similarly, Southern spotting was performed with a probe (A). Using a hybridization buffer having the aforementioned composition, hybridization was performed first at 49 ^{° C} for 1 hour. After cooling to 39 ^{° C} was continued and then hybridizing at this temperature for 1 hour. After the reaction, the nitrocellulose filter was washed twice with 6 x SSC, containing 0,1% SDS, for 30 min at room temperature and then washed at 39 ^{° C} for 3 min. The washed paper was then subjected to autoradiography.

 As a result, it was found that a single clone is positive. Sequencing of the nucleotides using the dideoxy method showed that this clone had 308 base pairs DNA containing parts of both the probe (IWQ) and probe (A), the pB R 322-derived plasmid containing this insert was called pCS -1.

 PRI me R 9. Directional selection of the library of the λ-phage line using the probe rNS-1 DNA.

Hybridization of the spots was carried out according to the method of Benton and Davis (Benton and Davis [Science, 196,180 (1977)] pNS-1 obtained in example 8, was treated with Sau3A and ECORI to obtain a DNA fragment of approximately 600 base pairs. This DNA fragment was labeled with a radioactive label with using labeled translation according to conventional methods. A nitrocellulose filter (S&S) was marked on a phage spot growing on an agar medium to transfer the phages to the filter. After denaturing phage DNA using 0.5 mol / L NaOH, the filter paper was processed in the following ways: processing 0 , 1 mol / L NaOH and 1.5 mol / L NaCl in t 20 s, two treatments with 0.5 mol / L Tris-Hcl (pH 7.5) and 1.5 mol / L NaCl for 20 s, finally, treatment with 120 mmol / L NaCl, 15 mmol / L sodium citrate, 13 mmol / L KH ₂ PO ₂ and 1 mmol / L EDTA (pH 7.2) for 20 s.

The filter was then dried and heated at ⁸⁰ ° C for 2 hours to immobilize DNA. Prehybridization was carried out overnight at 42 ^{° C} in prehybridization buffer containing 5 x SSC, 5 x Denhardt's solution, 50 mmol / l phosphate buffer, 50% formamide, 0.25 mg / ml denatured DNA (salmon sperm DNA) and 0 , 1% SDS. Thereafter, hybridization was carried out at 42 ^{° C} for 20 hours in hybridization buffer containing 4 x 10 ⁵ CPMs / ml pNSS-1 probe which was labeled using radiolabelled labeled translation. This hybridization buffer was a mixture of 5 x SSC, 5 x Dengardt solution, 20 mmol / L phosphate buffer (pH 6.0), 50% formamide, 0.1% SDS, 10% dextransulfate and 0.1 mg / ml denatured DNA (DNA from salmon sperm).

The hybridized nitrocellulose filter was washed for 20 min in 2 x SSC, containing 0,1% SDS, at room temperature, then for 30 minutes using 0.1 x SSC, containing 0,1% SDS at 44 ^{° C,} and finally for 10 min using 0.1 x SSC at room temperature. Then, detection was performed using autoradiography.

 Five positive clones (G1-G5) were obtained. A clone containing the "full" cDNA was checked to determine the nucleotide sequence of its DNA using the dideoxy method, and the nucleotide sequence shown in FIG. 3-5. This cDNA was excised from the λ gt vector and attached to pBR 327 (Soberon et al. Gene, 9, 287 (1980)] at the EcoRI site to obtain a plasmid that can be obtained on a large scale. This plasmid was named pBRG4.

PRI me R 10. Directional selection of the library of the λ-phase line using the probe pBRG4 and the probe (LC)
Hybridization of the spot was carried out according to the method of Benton and Davis (see Science there), used in Example 9. A nitrocellulose filter (S&S) was placed on the spot of the growing phage on an anaric medium to transfer the phages to the filter. After denaturing phage DNA using 0.5 mol / L NaOH, the filter was treated in the following ways: processing with 0.1 mol / L NaOH and 1.5 mol / L NaCl for 20 s, then two treatments with 0.5 mol / l Tris-Hcl (pH 7.5) and 1.5 mol / L NaCl for 20 s, but treatment with 120 mmol / L NaCl, 15 mmol / L sodium citrate, 13 mmol / L KH ₂ PO ₄ and 1 mmol / L EDTA (pH 7.2) for 20 s. The filter was then dried and heated at ⁸⁰ ° C for 2 hours to immobilize DNA. Two sheets of the same filter were prepared as described above and subjected to directional selection using the pBRG4 probe of the extracted DNA and probe (LC).

Directed selection using the probe pBRG4-derived DNA was carried out as follows. pBRG4-treated with EcoRI to obtain a DNA fragment of approximately 1,500 base pairs. This DNA fragment was radiolabeled using labeled translation in accordance with conventional methods. One of the two nitrocellulose filters were subjected to prehybridization overnight at 42 ^{° C} in prehybridization buffer containing 5 x SSC, 5 x Denhardt's solution, 50 mmol / l phosphate buffer, 50% formamide, 0.25 mg / ml denatured DNA (DNA from salmon sperm) 0.1% SDS. Thereafter, the filter was subjected to hybridization at 42 ^{° C} for 20 hours in hybridization buffer containing radiolabeled DNA probe (about 1 × 10 ⁶ counts per minute / ml) was approximately 1,500 base pairs. This hybridization buffer was a mixture of 5 x SSC, 5 x Dengardt solution, 20 mmol / L phosphate buffer (pH 6.0), 50% formamide, 0.1% SDS, 10% dextransulfate and 0.1 mg / ml denatured DNA (DNA from salmon sperm). The hybridized nitrocellulose filter was washed for 20 minutes in 2 x SSC coderzhaschim 0,1% SDS, at room temperature, then for 30 minutes, 0.1 x SSC, containing 0,1% SDS, at 44 ^{° C,} and finally, for 10 min using 0.1 SSC at room temperature. Then, detection was performed using autoradiography.

Directional sampling using a probe (LC) was carried out as follows. Another filter was pretreated 3hSSC that records the 0,1% SDS, at ⁶⁵ ° C for 2 hours. Then, prehybridization was performed at 65 ° ^C for 2 hours in a solution containing 6 x NET, 1 x Denhardt's solution and 100 ug / ml denatured DNA (salmon sperm DNA). Then, hybridization was performed overnight ^at 63 ° C in hybridization buffer containing radiolabeled probe (LC) (2 x 10 ⁶ cpm / ml). This hybridization buffer was also a mixture of 6 x NET, 1 x Dengardt solution and 100 μg / ml denatured DNA (salmon sperm DNA). The hybridized nitrocellulose filter was washed three times (each time for 20 minutes) 6 x SSC, containing 0,1% SDS, at room temperature, then washed with 6 x SSC containing 0,1% SDS, ^at 63 ° C for 2 min.

 The filter was dried and deactivation was performed using autoradiography.

 In the directed selection described above, clones that were positive for both probes were selected, and the clone containing the “full” cDNA was checked to determine its nucleotide sequence using the dideoxy method. It was found that it had the nucleotide sequence shown in FIG. 8-10. This cDNA was excised from the λgt 10 vector and attached to pBR 327 at the EcoRI site to obtain the plasmid pBKU 2.

 PRI me R 11. The directed selection of the library of the human chromosomal gene.

1 / Building a library of the human chromosomal gene
The library of the human chromosomal gene, obtained thanks to the courtesy of Dr. Maniatis from Harvard University, was prepared as follows: all chromosomal DNA was extracted from the liver of a human embryo using phenol or other suitable chemicals and partially digested with Hae III and AluI restrictase, the obtained DNA fragments were centrifuged in sucrose density gradient to concentrate fragments having chain lengths of about 18-25 thousand bases, concentrated fra cops attached E. coli phage λ Charon 4A phage to the shoulder, and inserted short-chain synthetic nucleotides having EcoRI restriction enzyme cleavage sites in order to obtain infectious phage DNA recombinants, in order to obtain increased infectivity, when packaging received more purified particles of λ-phage . It is theoretically believed that the human gene library thus obtained is a set of recombinants containing human DNA with chain lengths of 18-25 thousand bases, which contain almost all human genes.

 2) Directional selection of a human chromosomal gene library using a pNS-1 DNA probe.

Hybridization of the spots was carried out according to the method of Benton and Davis [Science, 196,180 (1977)] pHS-1 obtained in Example 8 was treated with Sau3A and EcoRI to obtain a DNA fragment of approximately 600 base pairs. This DNA fragment was tagged with radioactive labeling using known techniques. A nitrocellulose filter (S&S) was placed on a phage spot growing on an agar medium to transfer phages to the filter. After denaturing phage DNA using 0.5 mol / L NaOH, the filter paper was treated in the following ways: processing 0.1 mol / L NaOH and 1.5 mol / L NaCl for 20 s, two treatments 0.5 mol / L Tris -HCl (pH 7.5) and 1.5 mol / L NaCl for 20 s; finally, treatment with 120 mmol / L NaCl, 15 mmol / L sodium citrate, 13 mmol / L KH ₂ PO ₄ and 1 mmol / L EDTA (pH 7.2) for 20 s.

The filter was then dried and heated at ⁸⁰ ° C for 2 hours to immobilize DNA. Prehybridization was carried out overnight at 42 ^{° C} in prehybridization buffer containing 5 x SSC, 5 x Denhardt's solution, 50 mmol / l phosphate buffer, 50% formamide, 0.25 mg / ml denatured DNA (salmon sperm DNA) and 0 , 1% SDS. Thereafter, hybridization was carried out at 42 ^{° C} for 20 hours in hybridization buffer containing 4 × 10 ⁵ scores cpm / ml probe rNSS-1, which was radioactively labeled using the labeled translation. This hybridization buffer is a mixture of 5 x SSC, 5 x Dengardt solution, 20 mmol / L phosphate buffer (pH 6.0), 50% formamide, 0.1% SDS, 10% dextransulfate and 0.1 mg / ml denatured DNA (DNA from salmon sperm).

The hybridized nitrocellulose filter was washed for 20 min in 2 x SSC, containing 0,1% SDS, at room temperature, then for 30 min, 0.1 x SSC, coderzhaschim 0,1% SDS at 44 ^{° C,} and finally for 10 min 0.1 x SSC at room temperature. Then, detection was performed using autoradiography.

As a result, ten random positive clones were obtained. Recombinant DNAs were obtained from these clones by the Maniatis method [Maniatic, Cell, 15, 687 (1978)]. The obtained DNAs were treated with restriction enzymes such as EcoRI, BamHI and Bgl II, analyzed by agarose gel electrophoresis, and their restriction enzyme map obtained by the method Frisha et al. [Fritsh et al. Cell, in the same place)]
Southern hybridization was carried out using a probe, which was a radiolabeled DNA fragment labeled with a radioactive label pns-1, which was the same as that used in the above methods of directional selection. A DNA fragment of approximately 8 thousand base pairs cut using EcoRI was selected from clones that hybridized with the probe. This fragment was subcloned to the EcoRI pHR327 site. The subcloned DNA was subjected to another restriction enzyme treatment, and Southern hybridization was repeated. It was found that a DNA fragment of approximately 4 thousand base pairs, cut with EcoRI and XhoI, contained a gene encoding a human G-CSF polypeptide. This fragment was checked to determine the sequence of its part with a length of approximately 3 thousand base pairs using the dideoxy method; the nucleotide sequence shown in FIG. 13-17. This DNA fragment had restriction enzyme cleavage sites shown in FIG.

 Directed selection of human chromosomal genes was carried out using pBRG4-derived DNA and pBRV-2-derived DNA as probes. In each case, a 1500 bp DNA fragment already processed by EcoRRI was directly radioactively labeled using the labeled translation as described above, or alternatively a DNA fragment of approximately 700 base pairs obtained by sequential ECORI and DraI treatments was radiolabeled using labeled translation. The probe thus obtained was used in a spot hybridization method which was carried out under the same conditions as described above. Selected clones were analyzed by Southern hybridization so as to obtain a DNA fragment having the nucleotide sequence shown in Fig.13-17. The plasmid thus obtained was called pBRCE3 β.

PRI me R 12. Construction of E. coli recombinant vector and transformation (using tac-promoter vector)
1) Construction of a recombinant vector
1) Getting the vector
5 ug tac-promoter-containing vector pKK223-3 (Pharmacia) was treated with 8 units of EcoRI (Takara Shuzo Co. Ltd.) for 2 hours at ³⁷ ° C in a 30 ul reaction solution (40 mmol / l Tris-HCl, 7 mM / l MgCl ₂ , 100 mmol / l NaCl and 7 mmol / l 2-mercaptoethanol). Then add 3 ul of alkaline phosphatase (Takara Shuzo Co. Ltd.), and treatment was carried out at ⁶⁰ ° C for 30 min. The DNA fragment was isolated by three treatments with phenol, one treatment with ether and ethanol precipitation, all treatments being carried out in the usual manner.

The isolated DNA fragment was dissolved in 50 μl of a mixture consisting of 50 mmol / L Tris-Hcl, 5 mmol / L 10 mmol / L DTT and 1 mmol / L each of d ATP, d-PTF, d-GTF and dTTP. After addition of 3 .mu.l E. coli DNA polymerase 1 Klenow fragment (Takara Shuzo Co. Ltd.), reaction was conducted at 14 ^{° C} for 2 hours to create blunt ends.

 (II) Obtaining a synthetic linker.

3 μg of oligonucleotides having sequences of synthetic linkers TSGAATGAZTSTSTSTSTSTTGHHTSTs and TSAGHYYYYTTATTSTsG were phosphorylated by reaction in 40 µl of a reaction solution (consisting of 50 mmol / L Tris-Hcl, 10 mmol / L MgCl ₂ , 10 mmol / L 2-mer l ATF) at 37 ^{° C} for 60 min in the presence of 4 units of _T4 polynucleotide kinase.

Each phosphorylated oligonucleotide (0.2 ug) were dissolved in 20 l 100 mmol / l NaCl containing TE solution [10 mmol / l Tris-HCl (pH 8.0), 1 mmol / l EDTA] After treatment at ⁶⁵ ° C for 10 min, the oligonucleotides were renatured by slow cooling to room temperature.

(III) Obtaining cDNA fragment G-CSF
Sixty micrograms Pb RG4, obtained in Example 9 which contained the cDNA, illustrated in Figures 3-5, treated with 100 units of restriction enzyme ApaI (New England Biolabs) and 50 units of Dra I (Takara Shuzo Co. Ltd.) at ³⁷ ° C. for 3 hours in 200 μl of a reaction solution consisting of 6 mmol / L Tris-Hcl, 6 mmol / L MgCl ₂ and 6 mmol / L 2-mercaptoethanol. Approximately 2 μg of the AraI-DraI fragment (approximately 590 base pairs) was isolated using 1.2% agarose gel electrophoresis.

 (IV) Binding of fragments.

About 0.1 μg of each of the fragments obtained in stages (I) to (III) was dissolved in 20 μl of binding solution (66 mmol / L Tris-Hcl, 6.6 mmol / L MgCl ₂ , 10 mmol / L DTT and 1 mmol / L ATP). After addition of 175 units of _T4 DNA ligase solution was incubated overnight at ⁴ ° C to obtain a recombinant vector (Figure 20).

 2) Transformation.

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

 PRI me R 13. Construction of a recombinant vector E. Coli (+ VSE) and transformation (using PL-promoter-containing vector).

 1) Construction of a recombinant vector.

 1) Getting the vector.

One-hundred micrograms PL-promoter-containing vector PL-lambda (Pharmacia) was treated overnight at ³⁷ ° C with 50 units of restriction enzyme BamHI, and 100 .mu.l of a reaction solution [10 mmol / l Tris-HCl (pH 7.6), 7 mM / l MgCl ₂ , 100 mmol / l NaCl and 10 mmol / l DTT] By subjecting the reaction solution to 1% agarose gel electrophoresis, approximately 49 μg of a fragment of 4 thousand bases and approximately 11 μg of a fragment of approximately 1.2 thousand bases were isolated.

Fragment 4 tys.osnovany dissolved in 100 l of TE buffer (see. The above composition) and subjected to dephosphorylation by its reaction with an alkaline phosphatase (Takara Shuzo Co. Ltd.) at ⁶⁰ ° C for 60 min.

Another fragment with a length of approximately 1.2 thousand bases was dissolved in 20 μl of buffer (10 mmol / L Tris-Hcl, 10 mmol / L MgCl ₂ , 6 mmol / L KCl and 1 mmol / L DTT) and treated overnight with 20 units of restrictase and Mbo II (New England Biolabs) at 37 ^about C.

 Approximately 0.9 μg Bam HI-Mbo II fragment (approximately 200 base pairs) and approximately 1.9 μg MboII-BamHI fragment (approximately 310 base pairs) were isolated by gel electrophoresis of a 4% polyacrylamide gel.

 (II) Obtaining a synthetic linker.

 Oligonucleotides having sequences of synthetic linkers TAAGGAGATTZATTSGAT and TTsGATGAATTTSTSTSTTAG were subjected to phosphorylation and renaturation, as in (II) in example 12, in order to prepare a synthetic S / D linker.

 (III) Obtaining an expression vector.

One tenth microgram of a fragment with a length of about 4 thousand bases, 0.05 μg of each BamHI-MboII fragment having an O _L P _L and Mbо II BamHI fragment having a tL1 region [three fragments were obtained in (1)] and 0.1 μg reneturirovannogo synthetic S / D linker prepared in (II), was reacted overnight at 12 ^{° C} in 40 ul reaction solution (66 mmol / l tris-HCl, 6.6 mmol / l MgCl _2, 10 mmol / l DTT and 1 mmol / L ATP) in the presence of 175 units of T ₄ DNA ligase (Takara Shuzo Co. Ltd.). Twenty microliters of the reaction solution was used to transform strain N99Cl ⁺ E. coli (Pharmacia) according to the method using calcium chloride (see also Molecular Cloning).

 The transformants were cultured and a plasmid was isolated from the culture of their colonies, which were resistant to ampicillin. Processing of the plasmid with restriction enzymes EsoRI BamHI and SmaI showed that it was the target plasmid.

Two micrograms of this plasmid is contacted with the restriction enzyme SlaI (New England Biolabs) at ³⁷ ° C for 2 h in 20 l of buffer (10 mmol / l Tris-HCl 6 mmol / l MgCl ₂ and 50 mmol / liter (NaCl). After this enzyme was inactivated by heating at 65 ^about C for 10 minutes

One microliter of the reaction solution were contacted overnight at 12 ^{° C} with 175 units _T4 DNA ligase [Takara Shuzo Co. Ltd.) in a binder solution having the above composition. Then the reaction solution was used to transform strain N 99 Cl ⁺ E. coli (Pharmacia). The plasmid was isolated from the culture of ampicillin-resistant transformer colonies and the treatment with EcoRI and BamHI to confirm that the plasmid was the desired plasmid.

 (IV) Obtaining a recombinant vector and transformers expressing G-CSF.

 The expression plasmid obtained in (III) was digested with restriction enzyme ClaI. After creating blunt ends, the plasmid was then processed as in example 12 to obtain a recombinant vector with the insertion of a cDNA fragment of G-CSF. This vector was used to transform E. coli strain N 4830 (Pharmacia Fine Chemicals) according to the method using calcium chloride described in Molecular Cloning (see above). Identification of the desired transformants was carried out as in example 12 (Fig.21-22).

 PRI me R 14. Construction of E. coli-recombinant vector (+ VSE) and throne formation (using trp-promoter-containing vector).

 1) Construction of a recombinant vector.

 1) Getting the vector.

Plasmid pOY1 was obtained by inserting a tryptophan promoter containing the HpaII-TagI fragment (approximately 330 base pairs) in pBR322 into the ClAl site. Ten micrograms of this plasmid was treated with 7 units of the restriction enzyme SlAl and 8 units of Pvu II at ³⁷ ° C for 3 h in 30 l of a reaction solution consisting of 10 mmol / l Tris-HCl 6 mmol / l MgCl ₂ and 50 mmol / l NaCl .

Then we added 2 .mu.l of alkaline phosphatase (Takara Shuzo Co.0, Ltd.) and reacted at ⁶⁰ ° C for 1 hour.

 A DNA fragment (approximately 2.5 μg) of approximately 2.6 thousand base lengths was isolated from the reaction solution by gel electrophoresis of a 1% agarose gel.

 (II) Obtaining a synthetic linker.

 Oligonucleotides having sequences of synthetic linkers TSHGCAATGATSTSTSTSTSTSTGHGTSTS and TSAGGYYYYTTSTATTsG, phosphorylation and renatured, as in (II) in example 12, in order to obtain a synthetic linker.

 (III) Preparation of a recombinant vector.

About 1 μg of the vector fragment obtained in (I), about 1 μg of the synthetic linker obtained in (II), and about 1 μg of the G-CSF DNA fragment obtained in (III) in Example 12 were contacted with 175 units of T ₄ DNA -ligazy overnight at 12 ^C. 20 l of the binding solution having the composition described in example 12,1) (IV), in order to obtain a recombinant vector (23).

 2) Transformation.

 Twenty microliters of the reaction solution obtained in (III) were used to transform E. coli DHI according to the method using rubidium chloride described above in Molecular Cloning.

 As in example 12, the plasmid was isolated from ampicillin-resistant colonies of transformants, and the plasmid was treated with restriction enzymes ApaI, Dra I, NruI and Pst I, which showed that the desired transformants were obtained.

 PRI me R 15. The cultivation of transformants.

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

Transformants were grown overnight at ³⁷ ° C and 1 ml of culture was added to 100 ml of LB (Luria), containing 25 ug / ml or 50 .mu.g / ml ampicillin. Cultivation was carried out for 2-3 hours at 37 ^about C.

Cultivation was performed at ³⁷ ° C for 2-4 hours after addition of isopropyl -D-thiogalactoside to a final concentration of 2 mmol / l.

2) Cultivation of transformants (with P _L ) obtained in example 13.

Transformants were grown overnight at ²⁸ ° C and 1 ml of culture was added to 100 ml of LB (Luria), containing 25 or 50 micrograms / ml ampicillin. Cultivation was carried out for approximately 4 hours at 28 ^about C.

Cultivation was continued for 2-4 hours at 42 ^about C.

 3) Cultivation of transformants (with trp) obtained in example 14.

Transformants were grown overnight at ³⁷ ° C, and 1 ml of culture was added to 100 mL of M9 medium containing 0.5% glucose, 0.5% casamino acids (Sasamino acids (Difco)) and the 25 or 50 .mu.g / ml ampicillin. Cultivation was carried out for 4-6 hours at 37 ^C. After addition of 50 ug / ml 3-β -indolakrilovoy acid (IAA), cultivation was continued for 4-9 hours at 37 ° ^C.

PRI me R 16. Isolation and purification of the G-CSF polypeptide from E. coli
1) Isolation.

 Three types of transformants cultured in Example 15 were subjected to the following isolation methods.

 The culture (100 ml) was centrifuged to obtain a cell pellet after centrifugation, which was suspended in 5 ml of a mixture of 20 mmol / L Tris-Hcl (pH 7.5) and 30 mmol / L NaCl.

Then was added 0.2 mol / L phenylmethylsulfonyl fluoride 0.2 mol / l EDTA, to obtain a concentration, respectively, 1 mmol / l, 10 mmol / l and 0.2 mg / ml, and the suspension was set at 30 minutes at 0 ° ^C.

 Cells were lysed using three freeze / thaw cycles, after which it was desirable to sonicate. The lysate was centrifuged to obtain a supernatant. Alternatively, the lysate was treated with 8 mol / L guanidine hydrochloride so that its final concentration became 6 mol / L guanidine hydrochloride, after which it was centrifuged at 30,000 rpm for 5 hours and the supernatant was isolated.

2) Cleaning
(I) The supernatant obtained in 1) was subjected to gel filtration on an Ultrogel AcA54 column (4.6 mm in diameter, 90 cm long, 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 Kagaku Co. Ltd.).

 The fractions that were active in the analysis by the method for determining colony-stimulating activity (b) (described earlier in this description) were selected and concentrated to a volume of about 5 ml using a pM-10 ultrafiltration device (Amicon).

 (II) n-propanol (purity suitable for determining the amino acid sequence, Tokyo Kasei Co. Ltd.) and trifluoroacetic acid were added to the concentrated fractions, and the mixture was treated so that the final concentrations of n-propanol and trifluoroacetic acid were 30 and 0.1 % respectively. The treated mixture was left on ice for about 15 minutes and centrifuged at 15,000 rpm for 10 minutes to remove the precipitate. The supernatant was adsorbed on a μ-Bondapak C18 column (semi-preparative grade; Waters; 8 mmx x30 cm), which was equilibrated with an aqueous solution containing n-propanol (see above) and trifluoroacetic acid. The column was continuously eluted with an aqueous solution of 0.1% trifluoroacetic acid containing n-propanol with a linear density gradient of 30-60% Using a Hitachi model 685-50 (Hitachi high-pressure liquid chromatograph), a Hitachi model 638-41 (Hitachi detector), the absorption values at wavelengths of 220 and 280 nm were simultaneously measured. After elution, a 10-liter oliquot of each fraction was diluted 100 times, and the diluted solutions were selected to search for active fractions by the method for determining colony-stimulating activity (b). Activity was observed at peaks that were eluted at 40% n-propanol. These peaks were mixed and rechromatographed under the same conditions as used above, and the fractions were checked to determine their activity by method (b). Again activity was found at peaks at 40% n-propanol. These active peaks were collected (four fractions of 4 ml) and freeze dried.

 (III) Freeze-dried powder was dissolved in 200 μl of an aqueous solution of 0.1% trifluoroacetic acid containing 40% n-propanol, and the solution was subjected to high pressure liquid chromatography on a TSK-G300 SW column (7.5 mm x 60 cm, Toyo Soda Manufacturins Co. Ltd.). Elution was carried out at a flow rate of 0.4 ml / min with an aqueous solution of 0.1% trifluoroacetic acid containing 40% n-propanol, and 0.4 ml fractions were collected using a FRAC-100 fraction collector (Pharmacia Fine Chemicals). These fractions were checked as described above for the analysis of colony stimulating activity, and the active fractions were isolated. They were then purified on a μ-Bondapak C18 analytical column (4.6 mm x 30 cm), and the main peak was isolated and freeze dried.

 The protein thus obtained was treated with 2-mercaptoethanol and subjected to gel electrophoresis with SDS-polyacrylamide gel (15.0%) (15 mV, 6 h). When staining with Сomassie Blue dye, the desired G-CSF polypeptide could be identified as a single band.

 PRI me R 17. Determination of the activity of G-CSF (+ VSE).

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

PRI me R 18. Amino acid analysis (+ VSE)
1) Analysis of amino acid composition
A CSF sample purified in Example 16 was hydrolyzed by conventional methods, and the amino acid composition of the protein portion of the hydrolyzate was analyzed by amino acid analysis using a Hitachi 835 automatic amino acid analyzer (Hitachi). The results are shown in table.2. Hydrolysis was carried out under the following conditions: (I) 6 N. a solution of Hcl 110 ^about With, 24 hours in vacuum, (II) 4 N. a solution of methanesulfonic acid + 0.2% 3- (2-aminoethyl) indole, 110 ^° C, 24 hours, 48 hours, 72 hours in vacuum.

 The sample was dissolved in a solution (1.5 ml) containing 40% n-propanol and 0.1% trifluoroacetic acid. Aliquots of 0.1 ml each were dried with dry gaseous nitrogen and, after the addition of the reagents listed in (I) or (II), the containers were sealed in vacuum, after which the contents were hydrolyzed.

Each of the values given in Table 2 was an average of four measurements, values for 24 h for (I) and values for 24.48 and 72 h for (II), except that the contents of Tpe, Ce, 1/2 Cis, Met, Val, Ile and Trp were calculated using the following methods (see "Tampaku Kagaku (Protein Chemistry) II", A Course in Biochemical Expert, Tokyo Kagaku Dohjin):
For Tre, Ser, 1/2 Cis and Met, the time-dependent curve for 24, 48, and 72 h for (I) was extracted to zero hours.

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

 For Trp use: average values for 24.48 and 72 hours for (II).

2 / Analysis of N terminal amino acids
The sample was digested according to Edman using a gas phase sequence determiner (Apple Biosystems), and the resulting PTH amino acid was analyzed by conventional methods using a high pressure liquid chromatograph (Beckman instruments) and an Ultrasphere-ODS column (Beckman instruments) ) After the column (5 microns, diameter 4.6 mm x length 250 mm) was equilibrated with the original buffer [aqueous solution containing 15 mmol / L sodium acetate buffer (pH 4.5) and 40% acetonitrile], a sample was injected (dissolved in 20 μl of the original buffer) and separation was carried out by isocratic elution with the original buffer. During these stages was maintained flow rate 1.4 ml / min and the column temperature was 40 ^C. The detection of PTH-amino acids was carried out by measuring the absorption values in the ultraviolet region at wavelengths of 269 nm and 320 nm. Standard samples (weighing 2 nmol) of 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 N-terminal amino acids. As a result, PTH methionine and PTH threonine were detected.

PRI me R 19. Construction of E. coli recombinant vector (-VSE) and transformation
1) Use of a 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 [see (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 the desired transformants of Fig.24.

2) Use of a PL-promoter-containing vector
Repeated the methods of example 13 using cDNA (-VSE), and it was confirmed that the obtained transformants are the desired transformants (Fig.25-26).

 3) Use of trp-promoter-containing vector.

 The methods of Example 14 were repeated using cDNA (-VSE), and it was confirmed that the transformants were the desired transformants (FIG. 27).

 PRI me R 20. Analysis of the activity of G-CSF (-VSE).

 Three types of transformants obtained in example 19 were cultured according to the method described in example 15. G-CSF polypeptides were isolated from cultured E. coli cells and purified by the method described in example 16, whereby the human G-CSF polypeptide was obtained as single strip.

 The CSF sample thus obtained was analyzed by the method for analyzing CSF activity (a) described earlier in this description.

 The results are shown in table.3.

PRI me R 21. Amino acid analysis (-VSE)
1) Analysis of amino acid composition.

 The amino acid composition of the CSF sample purified in Example 20 was analyzed by the method described in 1) in Example 18. The results are shown in table. 4.

2) Analysis of N-terminal amino acids,
The sample was subjected to N-terminal amino acid analysis according to the method described in 2) in Example 18. As a result, PTH methionine and PTH threonine were detected.

PRI me R 22. Obtaining a pH vector GA410 (for use with animal cells, + any line)
The EcoRI fragment obtained in Example 9, which had the cDNA shown in FIG. 3-5 was treated with restriction enzyme Dra I at ³⁷ ° C for 2 hours, then treated with the Klenow fragment of DNA polymerase I (Takara Shuzo Co. Ltd), to create blunt ends. One microgram of the linker (8 mer, Takara Shuzo Co. Ltd.) was phosphorylated with ATP and attached to a separately prepared mixture of DNA fragments. The attached fragments were digested with Bgl II restriction enzyme and subjected to agarose gel electrophoresis. Then only the largest DNA fragment was isolated.

This DNA fragment was equivalent to the coding portion of the human G-CSF polypeptide containing approximately 710 base pairs (see FIG. 18) pdKCR vector [Fukunaga et al. Proc. Natl. Acad Sci. USA, 81.5086 (1984)] was treated with BamHI restrictase and then dephosphorylated with alkaline phosphatase (Takara Shuzo Co. Ltd.). The resulting DNA vector was attached to a 710 bp cDNA fragment in the presence of T ₄ DNA ligase (Takara Shuzo Co. Ltd.) in order to obtain pHGA410 (FIG. 28). As shown in FIG. 28, this plasmid contained the early SV40 promoter gene, the start of SV40 replication, part of the rabbit β-globin gene, the replication initiation site of pBR322 and pBR322-derived β-lactamase gene Amp ^r ), the human G-CSF gene being attached below upstream of the promoter of the early gene SV40.

PRI me R 23. The construction of the recombinant vector (+ VSE) used for the transformation of C127 cells
1) Construction of pHGA410 (N)
Twenty micrograms of the plasmid pHGA410 (FIG. 28) obtained in Example 22 was dissolved in a reaction solution consisting of 50 mmol / L Tris-HCl (pH 7.5), 7 mmol / L MgCl ₂ , 100 mmol NaCl, 7 mmol / l of 2-mercaptoethanol and 0.01% bovine albumin serum (BSA). Was added the restriction enzyme EcoRl (10-15 units; Takara Shuzo Co. Ltd.), and the reaction solution was heated at ³⁷ ° C for about 30 minutes to cause partial digestion with Eco RI. The DNA fragment was then subjected to two treatments with a 1: 1 phenol / chloroform mixture, then treated with ether and ethanol precipitated.

The obtained DNA fragment was dissolved in 50 μl of a solution consisting of 50 mmol / L Tris-Hcl, 5 mmol / L MgCl ₂ , 10 mmol / L DTT and 1 mmol / L each of dATP, d-PTF, d-HTP and dTTP. After addition of 5 .mu.l of Klenow fragment E.soli DNA polymerase (Takara Shuzo Co. Ltd.) solution was incubated at 14 ^{° C} for 2 hours to create blunt ends.

 Subsequent gel electrophoresis of 0.5% agarose gel yielded 6 μg of the DNA fragment with a length of approximately 5.8 thousand base pairs.

Five micrograms of the isolated DNA fragment was redissolved in 50 μg of a reaction solution consisting of 50 mmol Tris-Hcl (pH 7.6), 10 mmol / L MgCl ₂ , 10 mmol / L DTT and 1 mmol / L ATP. After addition of 2 ug linker Hind III (Takara Shuzo Co. Ltd.) and 100 units of _T4 DNA ligase (Takaro Shuzo Co. Ltd.) reaction was performed overnight at 4 ° ^C.

Then, phenol and ether treatments and ethanol precipitation were carried out. The precipitate was dissolved in 30 l of a solution consisting of 10 mmol / l Tris-HCl (pH 7.5), 7 mmol / l MgCl ₂ and 60 mmol / l NaCl and the solution was incubated at ³⁷ ° C for 3 hours in the presence of 10 units Hind iii. After re-treatment of T _{4 with} DNA ligase, the obtained DNA was used to transform E. coli DHI strain by treatment using rubidium chloride (see Molecular Cloning above). Cells bearing a plasmid that was identical to pHGAA 410 were selected from ampicillin (Amp ^v ) resistant transformant colonies, except that Hind III was inserted into the EcoRI site. The plasmid thus obtained was called pHGA410 (H) (Fig. 29).

2) Construction of a recombinant expression vector pTN-G4
Twenty micrograms of the 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 MgCl ₂ , 175 mmol / L NaCl, 0.2 mmol / l EDTA, 7 mmol / l 2-mercaptoethanol and 0.01% bovine albumin serum. After adding 20 units of SalI (Takara Shuzo Co. Ltd.), the reaction solution was incubated at ³⁷ ° C for 5 hours. After phenol treatment and ethanol precipitation were carried out incubating as in 1), for about 2 hours at ^about 14 C. the presence of a Klensu fragment of DNA polymerase (Takara Shuzo Co. Ltd.) in order to create blunt ends. Without DNA isolation by agarose gel electrophoresis, the reaction solution was immediately precipitated with ethanol. The resulting DNA fragment was treated with Hind III and 5 g of the Hind III Sal I fragment (approximately 2.7 thousand base pairs) was isolated by gel electrophoresis of 1% agarose gel. In a separate step, plasmid pd BPV-I having a bovine papilloma virus [this plasmid was obtained by Dr. Hawley and is described in Sarver, N, Sbyrne, J.C. Howley, PM Proc. Natl. Acad Sci. USA, 79, 7147-7151 (1982)] processed with Hind III and Pvu II as described by Nagata et al. [Nagata et al.) [Fukunaga, Sokawa and Nagata, Proc. Natl. Acad. Sci. USA, 81, 5086-5090 (1984)] to obtain a DNA fragment with a length of 8.4 thousand bases. This DNA fragment of 8.4 thousand bases and the separately obtained Hind III-Sal I DNA fragment (approximately 2.7 thousand bases) were linked using T ₄ DNA ligase. The binding product was used to transform E. coli DHI strain according to the method using rubidium chloride described in Molecular Cloning, see above. E. coli colonies carrying the plasmid having a pHGA 410-derived G-CSF cDNA were selected. This plasmid was named pTN-G4 (FIG. 29).

 Similarly, type II adenovirus was processed [Tanpakushitsu, Kakusan, Koso (proteins, nucleotide acids and enzymes), December 27, 1982, Kyoritsu Shuppan] to obtain the ΔpVA plasmid, which contained a SalI-Hind III fragment of approximately 1,700 base pairs carrying VAI and VAII and a fragment containing VAI and VAII was isolated from this plasmid. This fragment was inserted into pTNGA at the Hind III site in order to obtain pTNG4VAα and pTNG4VAβ (Fig. 29). Due to the presence of the adenovirus VA gene, these plasmids were able to increase the expression of the transcription product from the early SV40 promoter.

 PRI me R 24. Transformation of C127 cells and expression of G-CSF (+ VSE) in them.

Before using C127 for transformation of mouse cells, the pTN-G4 obtained in Example 23 was treated with BamHI restrictase. Twenty micrograms of plasmid pT N-G4 was dissolved in 100 μl of the reaction solution [10 mmol / L Tris-Hcl (pH 8.0), 7 mmol / L MgCl ₂ , 100 mmol / L NaCl, 2 mmol / L 2-mercaptoethanol, 0 , 01% bovine serum albumin] and treated with 20 Bam HI units (Takara Shuzo Co. Ltd), after which the mixture was treated with phenol and ether and ethanol precipitated.

C127 mouse cells were grown in Dulbecco's minimal essential medium containing 10% bovine embryo serum (Djibco). Cells growing on plates (5 cm in diameter) were transformed with the individual DNA obtained, in an amount of 10 μg, per plate, by the method using calcium phosphate / (see Haynes, J. Weissman, C. Nuclec Acids Res. II, 687- 706 (1983)] After treatment with glycerol, the cells were incubated at 37 ^about C for 12 hours

 The incubated cells were transferred to three new plates (5 cm in diameter), and the medium was changed twice a week. On day 16, the foci were transferred to fresh plates and cultured sequentially on Dulbecco's minimal essential medium containing 10% bovine embryo serum (Djibco) in order to select clones with a high rate of G-CSF production. These clones produced G-CSF at a concentration of about 1 mg / L. Subsequent cloning yielded clones that could produce G-CSF at concentrations of 10 mg / L or higher. In addition to C1271 cells, NIH3T3 cells can also be used as host cells.

PRI me R 25. Expression of G-CSF in CHO cells (+ VSE)
1) Construction of pHGG4-dhfr.

Twenty micrograms of the plasmid pHGA410 obtained in Example 22 was dissolved in 100 μl of a reaction solution containing 10 mmol / L Tris-HCl (pH 7.5), 7 mmol / L MgCl ₂ , 175 mmol / L NaCl, 0.2 mmol / l and EDTA, 0.7 mmol / l 2-mercaptoethanol and 0.01% bovine serum albumin. The reaction was conducted overnight at ³⁷ ° C in the presence of 20 units of restriction enzyme Sal I (Takara Shuzo Co. Ltd.), which was followed by phenol and ether treatment and ethanol precipitated.

The DNA pellet was dissolved in 100 μl of a reaction solution consisting of 50 mmol / L Tris-Hcl, 5 mmol / L MgCl ₂ , 10 mmol / L DTT and 1 mmol / L each of dATP, d-PTF, d-HTP and dTTP, and the reaction was carried out at 14 ^{° C} for 2 hours in the presence of Klenow fragmanta E. coli DNA polymerase (10 microliters; Takara Shuzo Co. Ltd.), which was followed by treatment with phenol and ether and precipitated with ethanol.

The Eco RI linker was attached to the DNA in the precipitate using the following methods: DNA was dissolved in 50 μl of a reaction solution consisting of 50 mmol / L Tris-Hcl (pH 7.4), 10 mmol / L DTT, 0.5 mmol / L spermidine, 2 mmol / L ATP, 2 mmol / L hexamine-cobalt chloride and 20 μg / ml bovine serum albumin. The reaction was performed at ⁴ ° C for 12-16 h in the presence of an Eco RI-linker (Takara Shuzo Co. Ltd.) and 200 units of _T4 DNA ligase (Takara Shuzo Co. Ltd.). After phenol treatment, washing with ether, and ethanol precipitation, which were carried out in accordance with conventional methods, the DNA pellet was partially digested with Eco RI and 3 μg of DNA fragment with a length of approximately 2.7 thousand base pairs was obtained by gel electrophoresis of 1% agarose gel.

Plasmid pAdD26SVpA [Kaufman, RG Sharp, R.A. Mol. Sell Biol. 2, 1304-1319 (1982)] was treated with EcoRI and dephosphorylated by treatment with bacterial alkaline phosphatase. More specifically, 20 μg pADd26SVpA and 20 EcoRI units were added to the reaction solution [50 mmol / L Tris-HCl (pH 7.5), 7 mmol / L MgCl ₂ , 100 mmol / L NaCl, 7 mmol / L 2-mercaptoethanol and 0.01% bovine serum albumin and reacted at ³⁷ ° C for 10 hours. Then, 5 units of bacterial alkaline phosphatase was added to the reaction solution, the reaction was conducted at 68 ^{° C} for 30 min. After phenol treatment by electrophoresis, an EcoRI pAdD26SVpA fragment was isolated in approximately 5 μg yield. A fragment of approximately 2.7 thousand base pairs and pAdD26SVpA, which weighed 0.5 μg each, was renatured. The resulting plasmid was used to transform E. coli DHI strain according to the method using rubidium chloride, and colonies carrying the plasmid pHGG4-dhfr were selected. The resulting plasmid was called pHGG4-dhfr (FIG. 30).

 An alternative method was as follows: plasmid pH GA 410 was treated with Sal I and partially digested with Eco RI without the addition of any Eco-linker. A DNA fragment of approximately 2.7 thousand base pairs was isolated and processed with a Klenow fragment of E. coli DNA polymerase to create blunt ends. The EcoRI fragment having blunt ends was obtained from pADD26SVpA as described above. This EcoRI fragment and a separately obtained fragment (approximately 2.7 thousand base pairs) were treated with T4 DNA ligase to obtain pHGG4-dhfr.

The plasmid pH GA 410 (H) obtained in Example 23 was digested with the restriction enzymes Hind III and Sal I as described in 2) in Example 23, and the Hind III Sal I fragment was attached to the blunt-ended Eco RI fragment pAdD26SVpA described above. This method can also be used to obtain pHGG 4-dhfr (Fig.31)
2) Construction of pG4DRI and pG4DR2.

Ten micrograms of the plasmid pAdD26SVpA indicated in I) was dissolved in 50 ml of a 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. After adding 10 units of each of the restriction enzymes EcoRI and BamHI, the reaction was carried out at 37 ° C for 10 hours, after which the mixture was treated with phenol and washed with ether. A DNA fragment of approximately 2 thousand bases was isolated by electrophoresis through 1% low melting point agarose gel. The isolated DNA fragment was treated with a Klenow fragment of DNA polymerase according to the usual procedure in order to create blunt ends. The blunt DNA fragment was treated with phenol, washed with ether and ethanol precipitated. whether Sal I was partially digested with Eco RI without the addition of any Ecoo linker. A DNA fragment of approximately 2.7 thousand base pairs was isolated and processed with a Klenow fragment of E. coli DNA polymerase to create blunt ends. The EcoRI fragment having blunt ends was obtained from pADD26SVpA as described above. This EcoRI fragment and a separately obtained fragment (approximately 2.7 thousand base pairs) were treated with T ₄ DNA ligase to obtain pHGG4-dhfr.

The plasmid pH GA 410 (H) obtained in Example 23 was digested with the restriction enzymes Hind III and Sal I as described in 2) in Example 23, and the Hind III Sal I fragment was attached to the blunt-ended Eco RI fragment pAdD26SVpA described above. This method can also be used to obtain pHGG 4-dhfr (Fig.31)
2) Construction of pG4DRI and pG4DR2.

Ten micrograms of the plasmid pAdD26SVpA indicated in I) was dissolved in 50 ml of a reaction solution containing 50 mmol / L Tris-Hcl (pH 7.5), 7 mmol / L MgCl ₂ , 100 mmol / L NaCl, 7 mmol / L 2 -mercaptoethanol and 0.01% bovine serum albumin. After addition of 10 units each of the restriction enzymes EcoRI and BamHI, the reaction was conducted at ³⁷ ° C for 10 h, whereupon the mixture was treated with phenol and washed with ether. A DNA fragment of approximately 2 thousand bases was isolated by electrophoresis through 1% low melting point agarose gel. The isolated DNA fragment was treated with a Klenow fragment of DNA polymerase according to the usual procedure in order to create blunt ends. The blunt DNA fragment was treated with phenol, washed with ether and ethanol precipitated.

Ten micrograms of the plasmid pHRA 410 (H) obtained in 1) of Example 23 was dissolved in 50 μl of a reaction solution containing 10 mmol / L Tris-Hcl (pH 7.5), 7 mmol / L MgCl ₂ and 60 mmol / L NaCl . The reaction was conducted at ³⁷ ° C for 6 hours in the presence of 10 units of Hind III. A DNA fragment was isolated by electrophoresis through 1% low melting point agarose gel, which was carried out according to the usual method. The isolated DNA fragment was then treated with bacterial alkaline phosphatase, and the blunt ends were obtained by treatment with the Klenow fragment. After phenol treatment and washing with ether, the DNA fragment was attached with blunt ends to a previously obtained DNA fragment of approximately 2 thousand bases using T ₄ DNA ligase according to the following procedure: 1 μg of each DNA enzyme was dissolved in 30 μl of a reaction solution containing 66 mmol / L tris-HCl, (pH 7.5), 6.6 mmol / l MgCl _2, 5 mmol / l DTT, 1 mmol / l of ATF, and the reaction was carried out at 6 ^{° C} for 12 hours in the presence of 50 units T ₄ DNA ligases. The binding product was used to transform E. coli DHI strain. As a result, pG4DR1 and pG4DR2 shown in FIG. 32 were obtained.

 3) Transformation and expression.

 CHO cells (dhfr strain, kindly provided by Dr. L. Chasin from Columbia University) cultivation for growth in alpha-minimal essential medium containing 10% calf serum (α-MEN with the addition of adenosine, deoxenosine and thymidine) in cups (9 cm in diameter, Nunc). Cultured cells were transformed according to the method using calcium phosphate [Wigler et al. Sell, 14,725, (1978)] as follows.

The DNA carrier (calf thymus DNA) was added in an appropriate amount to 1 μg of the plasmid pHGG4-dhfr obtained in 1), and the mixture was dissolved in 375 μg of TE solution, after which 125 μl of 1 mol / L CaCl _{2 was} added. After cooling the solution on ice for 3-5 minutes, 500 μl of 2x xHBS (50 mmol / L Hepes, 280 mmol NaCl, 1.5 mmol / L phosphate buffer) was added to the solution. After repeated cooling on ice, the solution was mixed with 1 ml of CHO cell culture, transferred to plates and incubated for 9 hours in a CO ₂ incubator. The medium was removed from the dish after washing with Tris-buffered saline and adding 20% glycerol-containing Tris-buffered saline and re-washing added a non-selective medium (the α-ME N medium described above except that nucleotides were added). After incubation for 2 days, the culture, diluted 10 times, was transferred to a selective medium (without the addition of nucleotides). Cultivation was continued, the medium being replaced with fresh selective medium every 2 days, and the obtained colonies were selected and transferred to fresh plates where cells were grown in the presence of 0.02 μmol / L methotrexate, after which cloning was performed by growing in the presence of 0.05 μmol / l methotrexate, the content of which was then increased to 0.01 μmol / l.

The transformation of CHO cells can also be accomplished by co-transformation with PHGG4 and PAdD26SVpA [see Scahill et al. Proc. Natl. Acad. Sci. USA, 80, 4654-4658 (1983)]
CHO cells were also transformed in the following ways: pG4DRI or pG4DR2 obtained in (2) were pretreated with SalI and KpnI, respectively, to obtain DNA fragments, and 10 μg of these fragments were used to transform CHO cells, as described above, transformed cells subjected to continuous cultivation in a number of selective media according to the method described above, after about 7 days at least 100 separate colonies per dish appeared, these colonies were transferred whole to a fresh cup and subjected to continuous cultivation in a number of selective media in the presence of 0.01 μmol / L metatrexate, after which ten or more colonies appeared, the same procedures were repeated with a successive increase in the concentration of methotrexate to 0.02 μmol / L 0.05 μmol / L and 0.1 μmol / l, and the colonies that survived were selected, the selection of colonies can be carried out in a similar way, even if individually collect the obtained 10-odd colonies and tuck them into cultivation with increasing concentrations of methotrexate.

 A recombinant vector carrying the "polycistronic gene" can also be used to transform CHO cells. An example of this alternative method is as follows: pAdD26SVpA was treated with Pst I and the selected two fragments were coupled to a pBRG4-derived CSF cDNA fragment to construct a recombinant vector in which the ananovirus promoter, CSF cDNA, DHFR and poly (A) SV40 site were inserted in written order. This recombinant vector was used to transform CHO cells.

 PRI me R 26. Analysis of the activity of G-CSF (+ VSE).

 The supernatants of the C127 and CHO cell cultures obtained in Examples 24 and 25, respectively, were adjusted to pH 4 with acetic acid. After adding an equal volume of n-propanol, the resulting precipitate was removed by centrifugation. The supernatant was passed through an open column (1 cm in diameter and 2 cm long) filled with C8 reverse phase support (Yamamura Kagaku K. K.) and elution was performed using 50% n-propanol. The eluent was diluted twice with water and subjected to high pressure reverse phase liquid chromatography on a YMC-C8 column (Yamamura Kaguka K.K.), followed by elution with n-propanol (30-60% linear density gradient) containing 0.1% trifluoroacetic acid. Fractions that were suirable at n-propanol concentrations of about 40% were isolated, freeze dried, and dissolved in 0.1 mol / L glycine buffer (pH 9). As a result of these procedures, human G-CSF in C127 and CHO cells was concentrated approximately 20 times.

 As a control, cells were transformed with plasmids not containing human G-CSF cDNA, and the supernatants of these cultures were concentrated in accordance with the methods described above. The activity of human G-CSF samples was analyzed using methods for analyzing the activity of human G-CSF (a) described earlier in this description. If the expression efficiency is sufficiently high, then the supernatant of the cultures can be analyzed directly without prior concentration. The results are summarized in table 5, where the data are based on concentrated samples.

PRI me R 27. Amino acid analysis and analysis of sugar (+ VSE)
1) Analysis of amino acid composition
The crude CSF sample obtained in Example 26 was purified by the methods described in Example 2 (III). The purified CSF sample was hydrolyzed by conventional methods and the protein part of the hydrolyzate was analyzed to determine its amino acid composition using a special amino acid analysis method using a Hitachi 835 automatic amino acid analyzer (Hitachi company). The results are shown in table.6. Hydrolysis was carried out under the following conditions: (I) 6 N. a solution of Hcl 110 ^about C, 24 hours, in vacuum (II) 4 N. solution of methanesulfonic acid + 0.2% 3- (2-amino-ethyl) indole, 110 ^{° C,} 24 hours, 48 hours, 72 hours, in vacuo.

 The sample was dissolved in a solution (1.5 ml) containing 40% n-propanol and 0.1% trifluoroacetic acid. Aliquots of 0.1 ml each were dried with dry gaseous nitrogen, and after adding the reagents listed in (I) or (II), the containers were sealed in vacuum, after which the contents were hydrolyzed.

Each of the values given in Table 6 was an average of four measurements, values for 24 hours for (l) and values for 24 hours for (I) and values for 24.48 and 72 hours for (II), for except that the contents of Tre, Ser, 1/2 Cis, Met, Val, Ile and Trp were calculated by the following methods (see "Tampaku Kagaku (Protein Chemistry) II", And Course in Biochemical Expert, Tokyo Kagaku Dohjin):
- For Tre, Ser, 1/2 Cis and Met, the time dependence of the values for 24.48 and 72 hours for (II) was extrapolated to zero hours.

 - For Val and Ile, a value of 72 h was used For (II).

 - For Trp, average values were used for 24.48 and 72 hours (II).

 2) Analysis of sugar composition.

An internal standard (25 nmol of inositol) was added to 200 ng of the purified CSF sample used in the analysis of amino acid composition 1). After adding a methanol solution (500 .mu.l) containing 1.5 N HCl and the solution was reacted at 90 ^{° C} for 4 hours in a nitrogen purged sealed tube. After that, as the tube was opened, silver carbonate (Ag ₂ CO ₃ ) was added to neutralize the contents. After that, 50 μl of acetic anhydride was added and the tube was shaken for a sufficient period of time. The tube was then left overnight in the dark at room temperature. The top layer was placed in a sample tube and dried with nitrogen gas. Methanol was added to the precipitate, and the mixture was washed and slightly centrifuged. The top layer was placed in the same tube and dried. After addition of 50 .mu.l TMS reagent (a mixture of 5: 1: 1 pyridine, hexamethyldisilazane and trimetilhlorosilana) were reacted ^at 40 ° C for 20 min, and the reaction product was stored in a deep freezer. A standard sample was prepared by mixing 25 nmol of inositol with 50 nmol of each of the reagents, galactose (Gal), N-acetylgalactosamine (Gal Nac), sialic acid and any other suitable reagents.

The samples thus obtained were subjected to gas chromatographic analysis under the following conditions:
Analysis conditions Column: 2% OV HP, 60-80
17 VIN port mesh, 3M,
glass
Temperature: rising from
110 to 250 ^about With a speed
4 ^about C / min.

Carrier gas pressure (N ₂ ):
initial 1.2-1.6 kg / cm ²
final 2-2.5 kg / cm ²
Sensitivity: Range
10 ³ M Ω range, 0.1-0.4 V
Pressure: H ₂ 0.8 kg / cm ²
air 0.8 kg / cm ²
Sample flow: 2.5-3.0 μl.

 As a result of the analysis, galactose, N-acetylgalactosamine and sialic acid were identified in the CSF sample of the present invention.

 PRI me R 28. Obtaining a pH vector GV2 (for use with animal cells, line -VSE).

The EcoRI fragment obtained in Example 10, which had the cDNA shown in FIG. 8-10 was treated with restriction enzyme Dra I at ³⁷ ° C for 2 hours, then treated with Klenow fragment of DNA polymerase I (Takava Shuzo Co. Ltd.), to create blunt ends. One microgram of the Bgl II linker (8 mer, Takara Shuzo Co. Ltd.) was phosphorylated with ATP and attached to approximately 1 μg of the individual fragment mixture obtained. The combined fragments were digested with Bgl II restriction enzyme and subjected to agarose gel electrophoresis. Then only the largest DNA fragment was isolated.

This DNA fragment was equivalent to approximately 700 base pairs containing a region encoding a human G-CSF polypeptide (see FIG. 18). The pdKCR vector [Fukunaga et al. Proc. Natl. Acad. Sci. USA, 81, 5086 (1984)] was treated with BamHI restrictase and then dephosphorylated with alkaline phosphatase (Takara Shuzo Co. Ltd.). The resulting DNA vector was attached to a cDNA fragment of approximately 700 base pairs in the presence of T4 DNA ligase (Takara Shuzo Co. Ltd.) in order to obtain a pH of GV2 (FIG. 33). As shown in FIG. 33, this plasmid contained the SV 40 early gene promoter, the SV 40 replication initiation site, part of the rabbit β-globin gene, the pHR322 and pBR 322 replication initiation site deduced β-lactamase gene (Amp ^r ), the human G gene Α-CSF is attached downstream of the promoter of the early gene SV 40.

 PRI me R 29: Construction of a recombinant vector (-VSE) for use in the transformation of C127 cells.

 1) Construction of pH GV 2 (N).

 Twenty micrograms of the plasmid pH GV2 (Fig. 17)) obtained in Example 28 was processed by the methods described in 1) in Example 23 so as to obtain a plasmid called pH GV2 (H) (Fig. 34).

 2) Construction of recombinant expression vectors pTNV2, pTNVAα and pTNVA β.

 Using 20 micrograms of pH GV2 (H), the methods described in 2) were repeated in Example 23 to select E. coli carrying a plasmid having pH GV2 derived G-CSF cDNA. This plasmid was named pT N-V2 (FIG. 34).

 Similarly, type II adenovirus was treated [Tampakushitsu, Kakusan, Koso (Proteins, Nucleic Acids and Enzymes), December 27, 1982, Kyoritsu Shuppan] to obtain the ΔpVA plasmid, which contained SalI-Hind III, a fragment of approximately 1700 base pairs, carrying VAI and VAII and a fragment containing VAI and VAII was isolated from this plasmid. This fragment was inserted into pTN-V2 at the Hind III site in order to obtain pTNVAα and pTNVA β (Fig. 18). Due to the presence of the adenovirus VA gene, these plasmids could enhance expression of the transcription product from the early SV40 promoter.

 Example 30. Transformation of C127 cells and expression of G-CSF (-VSE) pTN-V2 obtained in Example 29 was treated with BamHI restrictase enzyme before it was used to transform C127 mouse cells.

 C1271 mouse cells were transformed with the DNA thus obtained to express G-CSF (see Example 24), and clones having a high G-CSF production rate were selected. These clones produced G-CSF at a concentration of about 1 mg / L.

 By subsequent cloning, clones that can produce G-CSF at a concentration of 10 mg / L can be selected. Similarly, C127 cells were transformed with pTNVAα and pTNVAβ obtained in Example 29, and transformants were selected for clones with high ability to produce G-CSF, as for pTNVAα, clones capable of producing G-CSF with yields of 20 mg / L or more, while while, by transformation with pTNVA β, clones having lower productivity (several mg / L) were obtained.

 In addition to C1271 cells, N1H3T3 cells can also be used as host cells.

 PRI me R 31. Expression of G-CSF in CHO cells (-VSE).

 1) Construction of pHGV-2dhfr.

 A DNA fragment of approximately 2.7 thousand base pairs in length was prepared from 20 micrograms of the plasmid pHGV2 (Example 28) using the methods described in 1) in Example 25. This fragment (0.5 μg) and the EcoRI fragment pAdD26SVpA (0.5 mcg) subjected to renaturation. The obtained plasmid was used to transform E. coli DHI strain by the method using rubidium chloride, and colonies carrying the plasmid pHGV2-dhfr were selected. The resulting plasmid was called pHGV2-dhfr (Fig. 35).

An alternative method was as follows: the plasmid pHGV2 was treated with Sal I and partially digested with Eco RI without the addition of any Eco Eco RI linker. A DNA fragment of approximately 2.7 thousand base pairs was isolated and processed with the Klenow fragment of E. coli DNA polymerase to create blunt ends. The blunt-ended EcoRI fragment was prepared from pAD26SVpA as described above. This EcoRI fragment and a separately obtained fragment (approximately 2.7 thousand base pairs) were treated with T ₄ DNA ligase to obtain pHGV2-dhfr.

 The pH GV2 (H) obtained in 1) of Example 29 was treated with restriction enzymes Hind III and SalI as described in 2) in Example 29, and the Hind III Sal I fragment was attached to the blunt-ended Eco RI fragment of pAdD26SVpA described above. This method can also be used to obtain pHGG4-dhfr (Fig. 36).

2) Build pV2DR1 and pV2DR2
Ten micrograms of the plasmid pAdD26SVpA mentioned in 1) was dissolved in 50 ml of a reaction solution containing 50 mmol / L Tris-Hcl (pH 7.5), 7 mmol / L MgCl ₂ , 100 mmol / L NaCl, 7 mmol / L 2 -mercaptoethanol, and 0.01% bovine serum albumin. The reaction was conducted at ³⁷ ° C for 10 hours in the presence of 10 units each of restriction endonucleases EcoRI and BamHI. Therefore, phenol treatment and washing with ether were carried out in the usual way. A DNA fragment of approximately 2 thousand bases in length was isolated by electrophoresis through 1% low melting point agarose gel. The isolated DNA fragment was treated with the Klenow fragment of the DNA polymerase in the usual manner in order to create blunt ends. The blunt-ended DNA fragment was treated with phenol, washed with ether, and ethanol precipitated.

Ten micrograms of the plasmid pHGV2 (H) obtained in 1) of Example 29 was dissolved in 50 μl of a reaction solution containing 10 mmol / L, Tris-HCl (pH 7.5), 7 mmol / L MgCl ₂ and 60 mmol / L NaCl . The reaction was conducted at ³⁷ ° C for 6 hours in the presence of 10 units of Hind III. The DNA fragment was isolated by electrophoresis through 1% low melting point agarose gel, which was carried out in the usual way. The isolated DNA fragment was then treated with bacterial alkaline phosphatase, and blunt ends were created by treatment with a Klenov fragment. After treatment with phenol and washing with ether, the DNA fragment was attached with blunt ends to a previously obtained DNA fragment with a length of approximately 2 thousand bases using T ₄ DNA ligase according to the following procedure: 1 microgram of each DNA fragment was dissolved in 50 micrograms of a reaction solution containing 66 mmol / tris-HCl (pH 7.5), 6.6 mmol / l MgCl _2, 5 mmol / l DTT, 1 mmol / l of ATF, and the reaction was carried out at 6 ^{° C} for 12 hours in the presence of 50 units T ₄ DNA ligases. The binding product was used to transform E. coli DHI strain. As a result, plasmids pV2DR1 and pV2DR2 shown in FIG. 37.

 3) Transformation and expression.

 CHO cells were transformed from plasmids pH GV2-dhfr for the expression of G-CSF in accordance with the methods described in 3) in example 25.

 CHO cell transformation can also be accomplished by co-transformation with pHGV2 and pAdD26SVpA.

 CHO cells were also transformed in the following ways: pV2DR1 or pV2DR2 obtained in 2) was pretreated with Sal I and Kpn I, respectively, to obtain DNA fragments, and 10 μg of these fragments were used to transform CHO cells, as described above, the transformed cells were subjected continuous cultivation in a number of selective media in such a way as described above, after about 7 days there were at least 100 separate colonies per cup, these columns were transferred entirely to a fresh cup and subjected to continuous cultivation in a number of villages of objective media in the presence of 0.01 μmol / L methotrexate, after which ten or more colonies appeared, the same methods were repeated with a concentration of methotrexate successively increasing to 0.02 μmol / L 0.05 μmol / L and 0.1 μmol / l, and the colonies that survived were selected, the selection of colonies can be carried out in a similar way, further if the obtained 10-odd colonies are individually taken and cultured with increasing concentrations of methotrexate.

 A recombinant polycistronic gene vector can also be used to transform CHO cells. An example of this alternative method is as follows: pAdD26SVpA was treated with PstI and the selected two fragments were attached to pBRV2 derived CSF cDNA fragment in order to construct a recombinant vector in which the adenovirus promoter, CSF DFF cDNA and the poly (A) SV 40 site are inserted in written order . This recombinant vector was used to transform CHO cells.

PRI me R 32. Analysis of the activity of G-CSF (-VSE)
Using the methods described in example 26, human G-CSF was obtained from supernatants of cultures of C127 cells and CHO cells, which were obtained in example 30 and 31, respectively. The activity of human G-CSF of each of the selected samples was analyzed as in example 26. The results are shown in table.7.

PRI me 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 procedures described in Example 2 (III). The purified CSF sample was subjected to analysis of its amino acid composition according to the methods described in 1) in example 27. The results are shown in table.8.

 2) Analysis of sugar composition.

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

 PRI me R 34. Construction of a recombinant vector containing the chromosomal gene for expression in COS cells.

Plasmid pBRCEC β, which was obtained in Example 11 and which contained the chromosomal gene shown in FIG. 13-17, treated with EcoRI. Plasmid pSVH ⁺ K ⁺ described by Banerji et al. Sell, 27, 299 (1981), treated with Kpn I to remove the globin gene. The plasmid was then partially digested with Hind III and to remove part of the late SV40 gene. Fragments were re-combined to obtain the expression vector pML -E ⁺ .

This vector was digested with EcoRI restriction enzyme and dephosphorylated with alkaline phosphatase (Takara Shuzo Co. Ltd) to obtain a DNA vector that was coupled to the above chromosomal DNA using T ₄ DNA ligase (Takara Shuzo Co. Ltd.) to obtain pML CEZα. As shown in fig. 20, this plasmid contained the SV40 gene enhancer, the start of SV40 replication, the start of pB R322 replication and pBR322 deduced β-lactamase gene (Amp ^r ), and had the human G-CSF chromosome gene attached downstream of the SV40 gene enhancer.

 PRI me R 35. Expression of the chromosomal gene of human CSF in the cells of the COS.

COS-I cells (obtained thanks to the courtesy of Dr. Gluzman of the Could Spring Harbor Laboratory, USA), which were grown to a density of about 70% in Petri dishes (9 cm diameter, Nunc) using DMEM medium (Dulbecco modified Eagle medium supplied by Nissui Seiyaku KK under the trade name "Nissui") containing 10% calf serum was transformed either by treatment with calcium phosphate [Wigler et al. Cell, 14, 725, (1978)] or by a method using DEAE-dextran, chloroquine, see, for example, Gordon et al. Science, 228, 810 (1985)]
The transformation according to the method using calcium phosphate was carried out as follows: 160 μg of the plasmid pMLCЕЗα obtained in Example 34 was dissolved in 320 μl of TE solution and after adding distilled water (3.2 ml), 504 μl of 2 mol / L CaCl _{2 was} added.

To the resulting solution was added 4 ml of 2 HBS (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 medium in an amount of 1 ml per Petri dish, where COS-1 cells were grown. After culturing for 4 hours at 37 ^{° C} in an incubator with CO _2, the cells were washed with serum-free DMEM medium, then left to stand for about 3 minutes at room temperature in 5 ml of DMEM medium containing 20% glycerol, and then washed repeatedly serum free medium DMEM. After removal of serum-free DMEM medium, 10 ml of DMEM medium containing 10% calf serum was added, and cultivation was continued overnight in a CO ₂ incubator. After the medium was replaced with fresh medium of the same type, cultivation was continued for another 3 days.

Transformation according to the method using DEAE-dextran, chloroquine was carried out as follows: as in the method using calcium phosphate, COS-1 cells were cultured to grow to a density of 70% and washed twice with serum-free DMEM medium, serum-free DMEM medium was added to the washed cells containing 250 micrograms / ml DEAE-dextran in 2 ug / ml plasmid pML CEZ α obtained in example 34, and the culturing was continued at ³⁷ ° C for 12 hours, then the cells were washed twice with serum-free DMEM medium and subjected to further kultivi tion at ³⁷ ° C for 2 hours in DMEM medium containing 10% calf serum and 1 mmol / L chloroquine, washed twice with serum-free medium DMEM and cultured thereafter the cells at ³⁷ ° C for an additional 3 days in medium DMEM containing 10 % calf serum.

 The supernatant of the thus obtained COS-1 cell culture was adjusted to pH 4 with 1 N. acetic acid solution. After adding an equal volume of n-propanol, the resulting precipitate was removed by centrifugation. The surface solution was passed through an open column (1 cm in diameter and 2 cm long) filled with 68 reverse phase support (Yamamura Kagaku K.K.), and elution was performed using 50% n-propanol. The eluate was diluted twice with water and subjected to high pressure reverse phase liquid chromatography on a YMC-C8 column (Yamamura Kagaku KK), followed by elution with n-propanol (30-60% linear density gradient) containing 0.1% trifluoroacetic acid . Fractions were isolated which were eluted at n-propanol concentrations of about 40%, then they were freeze dried and dissolved in 0.1 mol / L glycidine buffer (pH 9). As a result of these procedures, human G-CSF in the supernatant of the COS-1 cell culture was concentrated approximately 20 times.

As a control, COS-1 cells were transformed with pML-E ⁺ lacking the G-CSF chromosomal gene using the method described above, and the supernatant of the resulting culture was concentrated.

 Human G-CSF activity in the obtained samples was analyzed by the "Method for the analysis of human G-CSF activity (a)" described earlier in this description. The results are shown in table.9.

 PRI me R 36. RNA analysis of G-CSF (chromosomal gene).

COS cells grown to a cell concentration of 8 x 10 ⁶ cells / dish (9 cm diameter) were transformed with 80 μg of plasmid pML CEZ α. After 48 h, all RNA was obtained by the method of Chirgwin [Biochemistry, 18, 5294-5299 (1979)]
The plasmid pBRG4 obtained in Example 9 was digested with Aha III restriction enzyme, and the resulting pBRG4-derived DNA fragment was radiolabeled with [γ- ³² P] ATP using T ₄ polynucleotide kinase to obtain a 2.8 thousand DNA fragment .bases containing G-CSF cDNA. The fragment was isolated and used as a DNA probe. After the DNA probe was denatured (1.5 x10 ⁵ counts per minute, 2.8 x10 ⁶ counts per 1 min / μg of DNA), it was mixed with 20 μg of all RNA obtained from COS cells. Hybridization was carried out at ⁴⁵ ° C for 15 hours. The mixture was digested with 200 units / ml or 400 units / ml of nuclease S1 (PL Biochemicals) according to the procedures Weaver and Weissmann [Nucleic Acid Res. 7, 1175-1193 (1979)] after which gel electrophoresis was performed through 4% polyacrylamide gel in the presence of 8.3 mol / L urea. Then, detection was performed using autoradiography.

 As a result, a band corresponding to 722 base pairs was observed as a strongly radioactively labeled band in COS cells, where a band corresponding to 487 base pairs was also noted.

 Therefore, it was found that the RNA of COS cells contained G-CSF mRNA of both lines + VSЕ and -VSE.

 PRI me R 37. Amino acid analysis and sugar analysis (chromosomal gene).

 1) Analysis of amino acid composition.

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

 2) Analysis of sugar composition.

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

 PRI me R 38. Expression of the chromosomal gene of human G-CSF in C127 cells.

 The plasmid pML CEZ α obtained in Example 34 was digested with EcoRI and a fragment of approximately 4 thousand bases was isolated using the methods described in Molecular Cloning, ibid. The isolated fragment was used as a source of the G-CSF chromosomal gene.

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

 The SV40 promoter (Eco RI-EcoRI fragment of approximately 0.4 thousand bases) was excised from the plasmid pHGA410 (obtained as in Example 22) using the methods described in Molecular Cloning, ibid., And then processed with the Klenow fragment of DNA polymerase ( IN).

 In a separate step, the pdBPV-I plasmid having bovine papilloma virus (BPV) [this plasmid was obtained thanks to the courtesy of Dr. Hawley, and it is described in Sarver, N. Sbyrne, J. C. Howle, P.M. Proc. Natl. Acad. Sei. USA, 79, 7147-7151 (1982)] treated Hind III and Pvu II to obtain a DNA fragment of approximately 8.4 thousand bases. This fragment was treated with a Klenow fragment of DNA polymerase I and dephosphorylated using bacterial alkaline phosphatase (C).

DNA fragments (A), (B) and (C), each weighing 0.1 μg, were dissolved in 20 μl of the reaction solution [50 mmol / L Tris-Hcl (pH 7.6), 10 mmol / L MgCl ₂ , 10 mmol / l DTT, 1 mmol / l ATF] and reaction was performed overnight at ⁴ ° C in the presence of 180 units of _T4 DNA ligase.

 The reaction solution was then processed according to the method using rubidium chloride described in Molecular Cloning, in the same place, in order to obtain the plasmid pTNCE3α (Fig. 39).

The DNA fragment (A) used as the source of the G-CSF chromosomal gene can be replaced by a DNA fragment of approximately 1.78 thousand bases, which was obtained as follows: 20 μg pML CEZ α was dissolved in 100 μl of a mixture of 10 mmol / L Tris- HCl (pH 8.0), 7 mmol / l MgCl _2, 100 mmol / l NaCl, 7 mmol / l 2-mercaptoethanol and 0.01% bovine serum albumin, the solution was incubated at ³⁷ ° C for 5 hours in the presence of 20 units of StuI, and then subjected to electrophoresis through 1.2% agarose gel.

 The plasmid pTNCE3 α thus obtained was used to transform C127 1 mouse cells, as in Example 24, and clones were selected that expressed the human G-CSF chromosome gene and which had high ability to produce G-CSF.

 PRI me R 39. Expression of the chromosomal gene of human G-CSF in CHO cells.

 As with expression in C127 cells, the pML plasmid CEZ was treated with Stu I, a DNA fragment of approximately 1.78 thousand bases was isolated, alternatively, the same plasmid was treated with EcoRI and an EcoRI fragment of approximately 4 thousand bases was isolated. Any fragment can be used as a source of the G-CSF chromosomal gene.

 As with expression in C127 cells, the pML CEZα plasmid was treated with Stu I, a DNA fragment of approximately 1.78 thousand bases was isolated, alternatively, the same plasmid was treated with EcoRI and an EcoRI fragment of approximately 4 thousand bases was isolated. Any fragment can be used as a source of the G-CSF chromosomal gene.

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

 As in Example 38, the SV40 promoter (EcoRI EcoRI fragment) was excised from pHGA410) to obtain a fragment of approximately 0.4 thousand bases, which was similarly treated with the Klenow fragment of DNA polymerase (b).

 In a separate step, plasmid pAdD26SV pA [Kaufman, R.G. Sharp, R.A. Mol. Sell. Biol. 2, 1304-1319 (1982)] was treated with EcoRI and then with the Klenow fragment of DNA polymerase, and finally dephosphorylated by treatment with bacterial alkaline phosphatase (c).

Fragments (a), (b) and (c), each weighing 0.1 μg, were dissolved in 20 μl of the reaction solution [50 mmol / L Tris-Hcl (pH 7.6), 10 mmol / L MgCl ₂ , 10 mmol / l DTT, 1 mmol / l ATP] and reaction was performed overnight at ⁴ ° C in the presence of 180 units of _T4 DNA ligase.

The reaction solution was then processed according to the method using rubidium chloride described in Molecular Cloning, in the same place, in order to transform the E. coli DHI strain. From the obtained Tet ^r colonies, those containing the plasmid pD26SVCE3 α were selected
As shown in FIG. 40, the plasmid pD26SVCE3α has a CSF gene attached to the early SV40 gene and a dhfr gene attached downstream of the main late adenovirus promoter.

Plasmid pADd26SVpA was processed with EcoRI and BamHI as in 2) of Example 25 in order to obtain a DNA fragment (approximately 2 thousand bases) containing the dhfr gene. This fragment was bound to fragment (a) and to the EcoRI-Sal I fragment of pHGA410 (H) in order to construct an expression vector of Amp ^r pDR CEZα (Fig. 40).

 CHO cells were transformed with the plasmids pD26SVCEЗα and pDR CEЗα thus obtained, as in Example 25. Clones of the strain producing G-CSF were obtained by repeated selection when grown in the presence of methotrexate.

 PRI me R 40. Analysis of the activity of G-CSF in transformants (expressing the human chromosomal gene).

 The supernatants of the cultures of C127 cells and CHO cells, which were obtained in Examples 38, 39, respectively, were processed as in Example 26 to obtain human G-CSF and their activity was analyzed. The results are shown in table.11.

 PRI me R 41. Molecular weight and isoelectric point of transformants.

 The purified CSF samples used to analyze the amino acid composition in examples 16, 20, 27, 33 and 37 were used to measure their molecular weights and isoelectric points using the following methods.

 1) Molecular weight.

The molecular weight of CSF was determined by gel electrophoresis of the sodium dodecyl sulfate polyacrylamide gene (SDS-PAGE). The PROTEAN ^TN electrophoretic apparatus (16 cm, supplied by Bio-Rad Corporation) was used using a gel made of polyacrylamide tile gel (T 15% C 2.6%), size 140 x 160 x 1.5 mm, at a gel concentration (T 3% C 20%). A denatured CSF sample was prepared as follows: CSF was boiled for 3 min in a solution containing 2% sodium dodecyl sulfate in 0.46 mol / L 2-mercaptoethanol. After electrophoresis with 4 μg of the sample at constant 30 milliamps for 4 hours, the gel was removed and stained with 0.25% br Illuminant blue (Сoomassie Brilliant Blue R 250, manufactured by Sigma Chemical Company) to detect bands. The following substances were used as molecular weight marks after similar treatments: phosphorylase B (mol.m. 92500), bovine serum albumin (BSA, mol m. 67000), ovalbumin (OVA, mol.m. 45000), carbonic anhydrase (mol. m. 31000), soybean trypsin inhibitor (mol. m. 21500) and lysozine (mol. 14400).

 As a result, a single band corresponding to a molecular weight of 185,000 ± 1000 was found for each of the CSF samples obtained in Example 16 [E. coli (KDNA (+ VSE)] and in Example 20 [E. coli / KDNA (-VSE)] and the only the band corresponding to a molecular weight of 19,000 ± 1000 for each of the CSF samples obtained in Example 27 [C127, CHO / CDNA (+ VSE)] in Example 33 [C127, CHO / KDNA (-VSE)] and in Example 37 (COS / g DNA).

 2) Isoelectric point.

The CSF isoelectric point of the present invention was determined using an FEE-3000 flat layer isoelectric electrophoresis device (manufactured by Pharmacy Fine Chemicals). After 2-hour electrophoresis at a constant power of 30 W (V _max . 2000 V) on a polyacrylamide gel (T 5% C 3% 115x x 230 mm) containing formalite (pH 4 6.5, Pharmacy Fine Chemicals and 4 mol / L urea, CSF was fixed with 30% methanol / 10% trichloroacetic acid (35% sulfosalicylic acid and stained with diamond blue dye R-250 (Сoomassie). A set with low pI (pН: 2 was used as isoelectric point marks) 5-6.5 is supplied by Pharmacia Fine Chemicals).

 Analysis of the separation of the bands at pH from 4 to 6.5 gave a single band corresponding to pI 6.1 for each of the CSF samples obtained in examples 16 and 20, and gave three separate bands corresponding to pI 5.5, 5.8 and 6 , 1 for each of the CSF samples obtained in examples 27, 33 and 37.

 PRI me R 42. The protective effect of human G-CSF against microbial infection.

Test method 1. Protection against infection with Pseudomonas aeruginosa Endoxan (Endohan, trade name of the product of Shionogi Co. Ltd. ") was administered intravenously in ICR mice aged 8-9 weeks (males, body weight 35.3 ± 1.38 g) when a dose of 200 mg / kg, then the mice were divided into three groups, the two groups were injected at intervals of 24 hours with four subcutaneous injections (each 0.1 ml dose) of a solution of [1% propanol in 0.5 (w / v) mouse serum albumin and physiological saline solution] containing human G-CSF (25,000 or 50,000 units / mol), while the third group was administered only erator in the same way. Three hours after the last injection, the mice in each group were infected Pseudomanas aeruginosa GNB-139 via a subcutaneous injection (3,9 x ¹⁰ May koloniiobrazuyuschih units / mouse). Twenty-one hours after infection in the first two groups introduced another subcutaneous injection of a solution containing human G-CSF (25,000 or 50,000 units / mouse), and only the solvent was introduced into the third group.

 The protective effect of human CSF was determined by counting the number of mice that survived 10 days after infection.

 Obtaining cell suspension.

Pseudomonad aeruginosa GNB-139 was cultured overnight with shaking at ³⁷ ° C in a liquid heart infusion medium (trade name "Difco"). The culture was suspended in physiological saline.

2. Protection against infection Sandida Endoxan (Endoxan, trade name of Shionogi Co. Ltd. "), was administered intraperitoneally in ICR mice at 8 weeks of age (males, body weight 40.5 ± 1.60 g) at a dose of 200 mg / kg. Then the mice were divided into two groups, one subcutaneous injection (dose of 0.1 ml each) of solvent [1% propanol and 10% (weight / volume) ICR mouse serum in physiological saline] containing human G- was administered at 24-hour intervals CSF (50,000 units / mouse), while the other group was administered only the solvent according to the same scheme. After the last injection, mice in each group were infected with Candida albicans U-50-1 (a strain isolated from the urine of leukemia patients, kindly provided by the Bacteriological Laboratory, Tohoku University Medical School) by intravenous injection (5.6 x 10 ⁵ colony forming units / mouse) The protective effect of human G-CSF was determined by counting the number of mice that survived ten days after infection.

 Learning cell suspension.

Sandida albicand U-50-1 was cultured overnight with shaking VARIATIONS ³⁷ ° C containing yeast extract liquid medium, Sabouraud (2% dextrose, from the firm "Junsei Pure Chemicals Co. Ltd."; 10% triptokaznogo peptone, trade name "BBL" , 5% yeast extract from Difco; pH 5, 6). The culture was washed twice with physiological saline and suspended in physiological saline.

3. Protection against infection with the intracellular parasitic Listeria Endotoxan (Endotoxan, trade name of the product from Shionogi Co. Ltd.) was injected intraperitoneally into ICR mice at 7 weeks of age (males, body weight 34.7 ± 1.24 g at a dose of 200 mg / kg The mice were then divided into two groups: in one group, four subcutaneous injections (each 0.1 ml dose) of a solvent [1% n-propanol and 10% (w / v) ICR mouse serum in physiological saline] containing human G-CSF (50,000 units / mouse), while the other group was administered only solvent of the same pattern. Four hours after the last injection, the mice in each group are infected with Listeria monocytogenes 46 (kindly provided by microbiological laboratory Tohoku University Medical School) by intravenous injection of 1,0 x ¹⁰ July koloniyobrazuyuschih units / mouse. The protective effect of human G-CSF was determined by counting the number of mice that survived 12 days after infection.

 Obtaining cell suspension.

46 Listeria monocytogenes was cultured overnight with shaking at ³⁷ ° C in a liquid medium for brain heart infusion (product name "Difco"). The culture was suspended in physiological saline.

results
l) Tests 1,2 and 3 were performed with a polypeptide. G-CSF E. coli (+ VSE) obtained in example 16. The results are shown in tables 12, 13 and 14.

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

 lll) Test l was performed with a purified sample of human G-CSF excreted by CHO (+ VSE) cells, which was the same as the sample used in the amino acid composition analysis in Example 27. The results are summarized in Table 16.

 Essentially, the same results were obtained when Test 1 was carried out with a purified sample of human G-CSF excreted by a C127 cell, which was the same as the sample used in the analysis of the amino acid composition in Example 27.

 IV) Test I was carried out with a purified sample of human G-CSF excreted by a CHO cell (-VSE), which was the same as the sample used in the analysis of the amino acid composition in Example 33. The results are shown in Table 17.

 Essentially, the same results were obtained when test l was performed with a purified sample of human G-CSF excreted by C127 cell, which was the same as the sample used in the amino acid composition analysis in Example 33.

Claims (1)

 A METHOD FOR PRODUCING A FACTOR STIMULATING THE FORMATION OF GRANULOCYTIC COLONIES, which involves the construction of a recombinant plasmid DNA containing a DNA fragment encoding a factor for stimulating the formation of granulocyte colonies, the transformation of Escherichia coli strains or cultured animal cells, recombinant plasmid production, and transformed plasmid recombinant plasmid DNA with a DNA fragment encoding a colony stimulating factor ulotsitov of the amino acid sequence shown in the description.

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