JPH0657156B2 - New glycoprotein manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a specific stimulating factor, namely a colony stimulating factor (hereinafter, referred to as “CSF”), which is necessary for colonization of a novel human granulocyte lineage cell. (Abbreviated as)) and a method for producing the same.

[Conventional technology]

In the two-layer soft agar culture method, when bone marrow cells as target cells in the upper layer and renal cells or fetal cells in the lower layer are put into culture, a part of the cells in the upper layer proliferate and differentiate, resulting in neutrophil granulocytes (hereinafter referred to as " It is known that there is a factor that promotes colony formation in the living body since colonies composed of granulocytes) and monocyte macrophages are formed (Pluznik and Sach; J. Cell. Comp. Physiol, Volume 66 , 319
Page (1965), Bradley and Metcalf: Aust. J. Exp. Biol. M
ed. Sci. 44 , 287 (1966)).

It is known that this factor, which is collectively referred to as CSF, is produced by cells that are normally widely distributed in the body, such as T cells, monocyte macrophages, fibroblasts, and endothelial cells. CSF-granulocyte-monocyte that acts on stem cells of granulocyte / monocyte macrophage, stimulates its proliferation and induces differentiation to form colonies composed of granulocytes and monocyte macrophages in soft agar Sphere macrophage CSF
(GM-CSF), pluripotent CSF that acts on undifferentiated pluripotent stem cells mainly from monocyte macrophage CSF (abbreviated as M-CSF) that has an action of forming colonies of monocyte macrophages. -CSF
Or a granulocyte CSF (G-) having an action of mainly forming a granulocyte colony as in the present invention.
There are subclasses such as CSF), and it has been considered that the differentiation stage of the target cell is different depending on each subclass [Asano: Metabolism-Metabol
ism and Disease, 22 : 249 (1985), Yunis et al .; “Growt
h and Maturation Factors ”edited by Guroff, John W
iley & Sons, NY, Vol. 1 , p. 209 (1983)]. Therefore, it is extremely important to purify individual subclasses and examine their chemical and biological properties in more detail for analysis of hematopoietic mechanism and pathological conditions of various hematological diseases.

Among them, as the biological action of G-CSF, the induction of differentiation of myeloid leukemia cells and the enhancement of the function of mature granulocytes have attracted attention, and the clinical usefulness of G-CSF for the treatment and prevention of leukemia is particularly expected. It has been.

[Problems to be solved by the invention]

A conventional attempt for isolation and purification of G-CSF is a method of isolating G-CSF from its culture supernatant using a cell culture method, but G-CSF is produced only at a low concentration. However, it has not yet been possible to obtain a large amount of uniform G-CSF due to the difficulty that a complicated purification process is required to obtain a small amount of G-CSF from a large amount of culture solution. Therefore, there has been a long-felt desire to produce G-CSF in large quantities using recombinant DNA technology.

[Means for solving problems]

In such a situation, the present inventors succeeded in isolating a gene encoding a polypeptide having human G-CSF activity and expressing the gene in a host cell.

The present invention provides a method for producing a glycoprotein according to claim 1, that is, a human chromosome-derived gene containing a nucleotide sequence encoding a specific amino acid sequence having human granulocyte colony stimulating factor activity is obtained, and then this gene is incorporated. Characterized in that a recombinant vector is prepared, a host mammalian cell is transformed with the vector, the resulting transformant is cultured, and the target glycoprotein is collected from the culture. The present invention provides a method for producing a glycoprotein having human granulocyte colony stimulating factor activity.

Hereinafter, the present invention will be described in detail.

The gene encoding the polypeptide having human G-CSF activity used in the present invention is human G-CS obtained as a 15 to 17S fraction by sucrose density gradient centrifugation.
There is DNA (cDNA) complementary to messenger RNA (mRNA) that encodes a polypeptide with F activity.

Applicants have obtained two series of such cDNAs. One of the series of cDNAs is polypeptide I of FIG. 3 (B).
Or having a gene encoding II or a part thereof, and more specifically, from the 5'-end of the base sequence of FIG. 3 (A) to ATG at 32-34 nucleotide positions to CCC at 650-652 nucleotide positions. Sequence from ACC at positions 122-124
It has a sequence from CCC at positions 650 to 652, the sequence shown in FIG. 3 (A), or a part thereof.

This series of cDNA is called cDNA (+ VSE).

Another series of cDNAs is the polypeptide I or II of FIG. 4 (B).
4A has a gene or a part thereof, and more specifically, the sequence from the 5'-end of the base sequence of FIG. 4 (A) to ATG at 31-33 nucleotide positions to CCC at 640-642 nucleotide positions. 64 from ACC 121-123
It has the sequence from 0 to 642st CCC, the sequence shown in FIG. 4 (A), or a part thereof.

This series of cDNA is called cDNA (-VSE).

The above-mentioned gene is, for example, from a mammalian cell or the like having the ability to produce a polypeptide having G-CSF activity.
It is obtained by preparing mRNA encoding CSF, converting it into a double-stranded cDNA by a known method, and screening from a set of recombinants containing this DNA (hereinafter referred to as a cDNA library) by a known method.

Moreover, the gene used in the present invention may be a gene derived from a human chromosome that encodes a polypeptide having human G-CSF activity. This gene contains a nucleotide sequence involved in transcription regulation, and specifically has the nucleotide sequence shown in FIG. 5 or a part thereof.

The gene derived from the chromosome can be obtained, for example, by preparing a set of recombinants containing a human chromosomal gene (hereinafter referred to as a human chromosomal gene library) from human cells and then screening by a known method.

In this case, as the source of the human chromosomal gene, all human cells can be used, and isolated cells such as liver and kidney and cultured cells such as tumor cells can be used.

In addition, a known method for preparing a human chromosome gene library from human cells [Maniatis et al .; Cell 15: 687].
(1978) and Maniatis et al .; Molecular cloning, Cold Spr.
ing Harbor Laboratory p. 269 (1982) etc.], for example, from human fetal liver to human chromosome DN.
A is extracted with phenol or the like, and the obtained DNA is partially or completely digested with a restriction enzyme to obtain a fragmented DNA having an appropriate length. The fragmented DNA is converted into T4 DNA ligase. Etc., and further, if necessary, by adding a linker containing a cleavage site such as EcoRI,
Insert into the phage vector DNA fragment. It can then be prepared by obtaining lambda phage particles by the in vitro packaging method and thereby transforming host cells such as E. coli.

The λ phage used as the above vector is, for example, Cha.
ron4A, EMBL-3, 4 and the like.

On the other hand, in the present invention, the mammalian cell that is the source of the mRNA is a human oral floor cancer-derived cell line CHU-2.
(Collection Nationale De Cultures De Microorganis
mes (CNM) deposit number I-483), but not limited to tumor cell lines, cells that can be isolated from mammals or other established cell lines may be used.

Also, the preparation of mRNA is the method already used when cloning the genes of several other physiologically active proteins,
For example, a vanadium complex or the like is used to perform a surfactant treatment and a phenol treatment in the presence of a ribonuclease inhibitor (Berger and Birkenmeier; Biochemistry 18 : 5143).
Page (1979)) or CsCl density gradient centrifugation after guanidine thiocyanate treatment (Chirgwin et al .; Bioche
mistry 18 (p. 5294 (1979))
After obtaining A, poly (A) was prepared by an affinity column method or a batch method using poly (U-sepharose) having oligo (dT) -cellulose or sepharose 2B as a carrier.
⁺ ) RNA (mRNA) can be obtained. Further, the poly (A ⁺ ) RNA can be further fractionated by a sucrose density gradient centrifugation method or the like.

In order to confirm that the mRNA obtained as described above encodes a polypeptide having C-CSF activity, the mRNA is translated into a protein and the physiological activity is examined or anti-G-CSF is used. A method such as identifying the protein using an antibody may be performed. For example, mRNA is injected into oocytes of Xenopus laevis to be translated (Gurdon et al .; Nature, 233 : 177 (1).
972)), or rabbit reticulocytes (Reticulocy
te) and wheat germ (Wheat germ) systems are used for translation reaction (Schleif and Wensink; “Practical Meth
ods in Molecular Biology ”, Springer-Verlag, NY,
(1981)).

The assay of G-CSF activity can be carried out by applying the soft agar culture method using bone marrow cells. There is a review of these methods (Metcalf; “Hemopoietic Colonies”, Springer
-Verlag, Berlin, Heideiberg, NY (1977)).

Single-stranded cD using the mRNA obtained by the above-mentioned method as a template
After synthesizing NA, double-stranded cDNA was converted from this single-stranded cDNA.
NA is synthesized to prepare a recombinant plasmid with an appropriate vector DNA. Escherichia coli or the like is transformed with this to obtain a DNA group (cDNA library) of the transformed strain.

To obtain a double-stranded cDNA from mRNA, for example, mRN
The oligo (d) complementary to the poly A-strand at the 3'-end of A (d
T) is treated with reverse transcriptase as a primer, or an oligonucleotide corresponding to a part of the amino acid sequence of G-CSF protein is synthesized, and this is treated with reverse transcriptase as a primer to prepare a cDNA complementary to mRNA. To synthesize. The double-stranded cDNA may be obtained by decomposing / removing mRNA by alkaline treatment, treating the obtained single-stranded cDNA with reverse transcriptase or DNA polymerase I (eg Klenow fragment etc.) and then treating with Sl nuclease or the like, Alternatively,
It can also be obtained by directly treating with RNase H and DNA polymerase (eg, DNA polymerase I of Escherichia coli etc.) and the like (eg, Maniatis et al .;
Molecular cloning, Cold Spring Harbor Laboratory (198
2) and Gubler and Hoffman; Gene 25 : 263 (1983)).

The double-stranded cDNA thus obtained is used as an appropriate vector, for example, pSC101, pDF41, ColE1, pM.
EK type plasmid vectors represented by B9, pBR322, pBR327, pACYC1, etc., λgt, λc, λgt
10, E. coli (X1776; HB 101; DH) after being incorporated into a phage vector typified by λgtWES
1, a C600 strain, etc. can be transformed to obtain a cDNA library (for example, “Molecular cl” described above).
oning ”).

In order to ligate the double-stranded cDNA with the vector, a suitable chemically synthesized DN is added so as to attach a ligable end to the DNA end.
It can be carried out by adding the A fragment and treating with T4 phage DNA ligase in the presence of ATP and the vector DNA previously cleaved with a restriction enzyme. Alternatively,
The dG and dc-chain (or d
After addition of (A, dT-chain), it can also be carried out, for example, by gradually cooling the solution containing both DNAs (Mo above).
See lecular cloning).

Transformation of a host cell with the thus obtained recombinant DNA is carried out by a method described in detail by Hanahan when the host cell is Escherichia coli (J. Mol. Biol; 166 , 577).
(1983)), that is, CaCl ₂ , MgCl ₂ or Rb
It can be carried out by adding the recombinant DNA strain to competent cells prepared in the presence of Cl.

To search for cells carrying the gene of interest, the plus-minus method used in cloning interferon cDNA (Taniguchi et al .; Proc. Jpn. Acad, Vol. 55 Se.
r. B. 464 (1979) and hybridization-translation assay (Nagata et al .; Nature 284 , 3).
16 (1980)) or a colony or plaque hybridization method using an oligonucleotide probe chemically synthesized based on the amino acid sequence of the protein (Wallace et al .; Nucleic Acids Res. 9 : 879 (198).
1) and Benton and Davide: Science 196, p. 180 (1977)).
Etc. may be used.

The fragment containing the gene encoding the polypeptide having human G-CSF activity thus cloned is transformed into another prokaryotic or eukaryotic host cell by reintegration into a suitable vector DNA. be able to. Furthermore, it is possible to express the gene in each host cell by introducing an appropriate promoter and a sequence for expression in these vectors.

Examples of mammalian-derived host cells include COS cells, Chinese hamster ovary (CHO) cells, C-127 cells, and HeLa cells, and examples of vectors for transforming these cells include pSV2-gpt (Mull.
igan and Berg; Proc. Natl. Acad. Sci. USA; 78 , p. 2072 (see (1981)). These vectors contain origins of replication, selectable markers, promoters located in front of the gene to be expressed, RNA splice sites, polyadenylation signals and the like.

As a promoter for gene expression in mammalian cells, promoters such as retrovirus, polyoma virus, adenovirus, simian virus 40 (SV40) may be used. For example, when using the promoter of SV40, a method such as Mulligan (Nature 277, Volume 108
Page (1979)).

As the origin of replication, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV), etc. can be used.
Phosphotransferase APH (3 ') II or I
(Neo) gene, thymidine kinase (TK) gene,
E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (DHFR) gene and the like can be used.

To obtain a polypeptide having human G-CSF activity using the host-vector system as described above, a host cell is transformed with a recombinant DNA body in which the gene is incorporated into an appropriate site of the above vector, The obtained transformant may be cultured. Further, in order to separate and purify the polypeptide from the inside of the cell or from the culture medium, known means can be used.

Generally, as the gene of eukaryotic organisms are known in the human interferon gene and the like, thought to represent a polymorphism (polymorphysm) (e.g. Nishi like;. J. Biochem 97, Volume 15
(See p. 3 (1985)), this polymorphism may result in the substitution of one or more amino acids, or in some cases, even if there is a change in the nucleotide sequence, the amino acids do not change at all.

Also, for example, a polypeptide lacking or adding one or more amino acids in the amino acid sequence of FIG. 3 (B) or FIG. 4 (B), or one or more amino acids having one or more amino acids. The polypeptide substituted with the amino acid may also have G-CSF activity. For example, a polypeptide obtained by converting a base sequence corresponding to cysteine of human interleukin 2 (IL-2) gene into a base sequence corresponding to serine is interleukin 2
It is already known to retain activity (Wang
Et al., Science, 244 , 1431 (1984)). Therefore, animal cells (transformed by a recombinant vector containing a gene encoding such naturally-occurring or artificially-synthesized polypeptide have human G-CSF activity). All glycoproteins obtained by culturing the body are included in the present invention.

Genes required for obtaining the glycoprotein having human G-CSF activity of the present invention, recombinant vector having the gene, transformants containing the same, and target sugars obtained by culturing the transformants The outline of the method for producing each of the proteins is as follows.

(1) Preparation of probe The amino acid sequence of the homogeneous human CSF protein obtained by purifying from the culture supernatant of the tumor cell line CHU-2 was determined from the N-terminal, and further fragmented by bromocyan degradation, trypsin treatment, etc. The amino acid sequence of the fragment was also determined [Example 3 (i), (ii), (iii)].

From the amino acid sequence, three types of nucleotide probes (A) and probes (LC) corresponding to the sequences shown in FIG.
And a probe (IWQ) were synthesized (Example 4).

The probe (A) is a mixed probe consisting of 14 consecutive nucleotides. The probe (IWQ) was used as in the cloning of the human cholecystokinin gene (Taka
hashi et al .; Proc. Natl. Acad. Sci. , USA, vol. 82 , p. 1931
(1985)) 30 consecutive nucleotides using deoxyinosine. The probe (LC) is a probe consisting of 24 nucleotides synthesized based on the nucleotide sequence shown in FIG. 3 at the portion corresponding to Nos. 32 to 39 from the N-terminal of the amino acid sequence shown in Example 3 (i). Is.

The chemical synthesis of nucleotides can be performed by applying the improved phosphotriester method to the solid phase method and is described in the review by Narang (Tetrahedron 39 : 3-22 (1983)).

The probe used may be based on the amino acid sequence at a position other than the probe used in the present invention.

(2) Construction of cDNA library The guanidine thiocyanate solution was added to CHU-2 cells and homogenized, and total R was measured by CsCl density gradient centrifugation.
Get NA.

After selecting poly (A ⁺ ) RNA from this total RNA with an oligo (dT) cellulose column, single-stranded cDNA was synthesized with reverse transcriptase, and RNase H and E. c
Double-stranded cDNA using oliDNA polymerase I
Got DC chain was added to the obtained double-stranded cDNA,
Escherichia coli X1776 strain was transformed with pBR322 vector in which dG chain was added at the PstI cleavage site, and p
BR322 cDNA library was constructed (Examples 5 and 6).

Similarly, a double-stranded cDNA was ligated to a λgt10 vector using an EcoRI linker to construct a λ phage cDNA library (Example 7).

(3) Screening Recombinant derived from pBR322 cDNA library was immobilized on Whatman 541 filter paper, and colony hybridization was performed using a probe (IWQ) radiolabeled with ³² P. As a result, one clone was selected. did it. This clone was cloned by Southern blotting (Southern; J.
Mol. Biol, vol. 98 , p. 503 (1975)), it was hybridized with the probe (A).

The nucleotide sequence of this clone was determined by the dideoxy method (Sanger; Scie
nce 214 , p. 1205 (1981)).

The nucleotide sequence of the obtained cDNA insert is shown in FIG.
As shown in FIG. 2, this cDNA insert consists of 308 base pairs containing the probe (IWQ) and the probe (A), and an open reading that encodes 83 amino acids including the amino acid sequence shown in Example 3 (iii). It turned out to have a frame.

This pBR322-derived plasmid containing 308 base pairs is abbreviated as pHCS-1 (Example 8).

A DNA fragment containing 308 base pairs obtained from pHCS-1 was nick-translated (Molecular Clon, supra).
ing)) and use this as a probe for λ
Plaque hybridization (Benton and Davis; Science 196 pages 180 (1)
977) to obtain 5 clones, c
The nucleotide sequence of a clone suspected of containing DNA was determined by the same method as described above (FIG. 3 (A)).

As shown in FIG. 3 (A), this cDNA insert has one large open reading frame.

The amino acid sequence encoded by this cDNA is shown in FIG.
It can be performed as shown in (A).

N of G-CSF protein shown in Example 3 (i)
By comparison with the terminal amino acid sequence, this cDNA was 5'-
A signal peptide encoded by 90 base pairs starting from the ATG sequence 32 to 34 nucleotides from the end and ending with a GCC sequence 119 to 121 and an AC at 122 to 124
531 starting with the C sequence and ending with the CCC sequence at positions 650 to 652
It was found to contain a base sequence corresponding to the mature G-CSF polypeptide encoded by the base pair. Therefore, the polypeptide of amino acid sequence I shown in FIG. 3 (B) consists of 207 amino acids and has a molecular weight of 22292.67.
Calculated as Dalton. Similarly, the polypeptide of amino acid sequence II consists of 177 amino acids and has a molecular weight of 1898.
It was 6.74 Daltons (Example 9).

However, regarding the initiation site of the protein, ATGs at positions 32 to 34 or 68 to 70 may be considered in the same manner. PBR322 with this cDNA (+ VSE) inserted at the EcoR1 cleavage site
Escherichia coli strain X1776, which retains Escherichia coli, has been deposited at the Institute of Microbial Technology, Institute of Industrial Science and Technology (FERM BP-954).

FIG. 6 shows the restriction enzyme cleavage site of the obtained gene.

In addition, this cDNA was cloned into pBR327 [Soberon et al .; Gene 9 vol.
P287 (1980)] and the plasmid bound at the EcoRI site are called pBRG4.

The pBRG4 thus obtained was digested with the restriction enzyme Eco.
A DNA fragment containing a cDNA of about 1500 base pairs obtained by treating with RI was subjected to the nick translation method (Mole described above).
radiolabeling (see cular cloning), and using this as a probe, the cDNA liplary derived from λgt10 was screened again by plaque hybridization (see Benton and Davis, supra). At the same time, 2 pieces of nitrocellulose filter paper on which λ phage DNA was immobilized was simultaneously prepared.
A single plate was prepared, and the same plaque hybridization was performed using the above-mentioned probe (LC) to select positive phages with both probes. Select clones that appear to be full-length and use the dideoxy method to
When the base sequence of the insert was determined, it was as shown in FIG. 4 (A).

This cDNA has one large open reading frame and the encoded amino acid sequence can be deduced as shown in Figure 4 (A).

By comparison with the N-terminal amino acid sequence of the G-CSF protein shown in Example 3 (i), this cDNA begins with the ATG sequence at the 31-33 nucleotide position from the 5'-end,
Mature G-CSF polypeptide encoded by a signal peptide encoded by 90 base pairs ending in the GCC sequence at positions 118-120 and 522 base pairs starting at the ACC sequence at positions 121-123 and ending at the CCC sequence at positions 640-642. It was found to contain a nucleotide sequence corresponding to. Therefore, the polypeptide of amino acid sequence I shown in FIG. 4 (B) consisted of 204 amino acids, and its molecular weight was calculated to be 21977.35 daltons. Similarly, the polypeptide of amino acid sequence II is 17
It was composed of 4 amino acids and had a molecular weight of 18671.42 daltons (Example 10).

However, regarding the start site of the protein,
ATGs at positions 58-60 or 67-69 are also conceivable.

Escherichia coli harboring pBR327 having the cDNA (-VSE) inserted at the EcoRI cleavage site (E.co.
li) X1776 strain has been deposited at the Institute of Microbial Technology of the Agency of Industrial Science and Technology (FERMBP-955).

FIG. 6 shows the restriction enzyme cleavage site of the obtained gene.

A plasmid obtained by ligating this cDNA to pBR327 at the EcoRI site is called pBRV2.

(4) Screening of human chromosomal gene library A human chromosomal gene library prepared according to the method described by Maniatis et al. (See above Molecular Cloning) was screened using pHCS-1 described above.

As a probe used for screening, pHCS-
In addition to the 308 base pair DNA fragment derived from No. 1, about 1500 base pair DNA fragment derived from pBRG4 and about 1500 derived from pBRV2
A DNA fragment having a base pair or a DNA fragment having an appropriate length containing a part of these DNA fragments, and further the above-mentioned oligonucleotide probes [(IWQ), (A), (L
C)] can also be used.

A DNA fragment derived from pHCS-1 was radiolabeled with [ ³² P] according to the nick translation method [Roop et al .; Cell 15: 431 (1978)], and the human chromosome-derived gene library was plaque-highed using this as a probe. Screening by hybridization (see Benton and Davis, supra) yielded dozens of clones.

After recovering the DNA from the obtained clone, a restriction enzyme map was prepared according to a known method [Fritsch et al .; Cell 19: 959 (1980)].

Then, Southern blotting (see the above-mentioned Southern literature) was performed using the above DNA probe, and a region encoding human G-CSF polypeptide was present in an approximately 4 kilobase pair DNA fragment excised with EcoRI and XhoI. It turns out that there is a possibility. Therefore, a DNA fragment of about 4 kilobase pairs was inserted into the EcoRI site of pBR327 using an EcoRI linker to obtain pBRCE3β.
Got D for determining the nucleotide sequence of this plasmid
When NA was used and the nucleotide sequence of about 3 kilobase pairs was determined by the dideoxy method, it was found to be a gene encoding a human G-CSF polypeptide (FIG. 5).

An E. coli strain harboring the plasmid pBR327 (pBRCE3β) in which this DNA fragment of about 4 kilobase pairs was inserted into the EcoRI site. E. coli strain X1776 has been deposited at the Institute of Microbial Technology, Institute of Industrial Science and Technology. (FERM BP-
956).

By comparison with the pBRRG4 and pBRV2 cDNA inserts shown in Figures 3 and 4, this DNA fragment contains 5 exon parts and encodes the amino acid sequence deduced from pBRG4 and pBRV2. I understood it.

In addition, this DNA fragment contains a preregion to be transcribed into human G-CSF mRNA, that is, a gene in human G-CSF chromosome, and further has a nucleotide sequence involved in transcriptional regulation [Benoist And Chambon; Nature 29
0 304 (1981) and Breathnack and Chambon; Ann. Re
v. Biochem. 50 , p. 349 (1981)].

(5) Construction of Recombinant Vector for Animal Cells Recombinant vector (derived from BPV) when C127 cells and NIH3T3 cells are used as host cells and recombinant vector (including DHFR) when CHO cells are used
Was constructed for + VSE system cDNA, -VSE system cDNA and chromosome-derived gene, respectively. Also, the same recombinant vector was constructed for COS cells. A typical example will be described here, but for details, refer to Examples.

Construction of (A) + VSE Recombinant Vector The cDNA (+ VSE) fragment obtained in (3) above was incorporated into vector pdKCR to form pHGA410 plasmid (Example 12) (FIG. 8), which was partially digested with EcoRI, Make the end blunt end. A HindIII linker was added to this DNA, followed by Hin
After treatment with dIII and T4 DNA ligase, the rubidium chloride method (see Molecular Cloning, supra) was used to transform E. coli. The E. coli strain DHI was transformed. The resulting plasmid is named pHGA410 (H) (Fig. 9).

pHGA410 (H) was treated with SalI, then the ends were blunt-ended and then again treated with HindIII to obtain Hi.
The ndIII-SalI fragment is recovered.

On the other hand, pd containing a transforming fragment of bovine papilloma virus
The BPV-1 plasmid was treated with HindIII and PvuII to separate the larger DNA fragment.
The dIII-SalI fragment is ligated. Using this, E. col
pHGF4-derived CSF-c obtained by transforming the iDHI strain
A plasmid having DNA, pTN-G4, is obtained (FIG. 9) (Example 13).

On the other hand, pHGA410 plasmid or pHGA410
A recombinant vector for CHO cells (+ VSE), pHGG4-dhfr, was constructed using the (H) plasmid and the pAdD26SVpA plasmid (Fig. 10a and b) (Example 15).

Furthermore, a DNA fragment of about 2 Kb containing the DHFR gene from pAdD26SVpA was recovered by EcoRI and BamHI treatment, and inserted into the HindIII site of pHGA410 (H) to construct pG4DR1 and pG4DR2 (Fig. 10c) (Example 15).

(B) -Construction of VSE-based recombinant vector The cDNA (-VSE) fragment obtained in (3) above was incorporated into the vector pdKCR to form a pHGV2 plasmid (Example 18), which was partially digested with EcoRI to terminate the ends. Use blunt end. Hi to this DNA
ndIII linkers were added, followed by HindIII treatment and T4 DNA ligase treatment, and then using the rubidium chloride method (see Molecular Cloning, supra). coli
The DHI strain was transformed. The obtained plasmid was pHG
It is named V2 (H) (Fig. 12).

pHGV2 (H) was treated with SalI, the ends were blunt-ended, and then treated with HindIII again to be HindI.
While recovering the II-SalI fragment, the pdBPV-1 plasmid containing the transforming fragment of bovine papilloma virus was transformed into H
The larger DNA fragment is separated by treatment with indIII and PvuII, and this is ligated to the above HindIII-SalI fragment. Using this, E. The E. coli strain DHI is transformed to obtain pTN-V2, a plasmid having CSF-cDNA derived from pHGV2 (Fig. 12) (Example 19).

PHGV2 plasmid or pHGV2 as well as + VSE
A recombinant vector (-VSE) for pH cells, pHGV2-dhfr, was constructed using the (H) plasmid and the pAdD26SVpA plasmid (Fig. 13a and 13b) (Example 21).

Furthermore, a DNA fragment of about 2 Kb containing the DHFR gene from pAdD26SVpA was recovered by EcoRI and BamHI treatment and inserted into the HindIII site of pHGV2 (H) to construct pV2DR1 and pV2DR2 (FIG. 13).
c) (Example 21).

(c) Construction of recombinant vector containing chromosome-derived gene The plasmid pBRCE3β containing the chromosomal gene shown in FIG. 5 obtained in (4) above was treated with EcoRI.

On the other hand, the pSVH ⁺ K ⁺ plasmid described in Banerji et al. (Cell 27, p. 299 (1981)) was treated with KpnI to remove the globinin gene, and further partially digested with HindIII to obtain one of the late genes of SV40. After removing the parts,
The expression vector pML-E ⁺ was prepared by religation.

This vector was treated with a restriction enzyme EcoRI and then dephosphorylated with alkaline phosphatase (Takara Shuzo), and the resulting vector DNA was added to T4 DNA ligase (Takara Shuzo) to ligate with the above chromosomal DNA fragment to form COS.
A recombinant vector pMLCE3α for cells was obtained (Example 2
Four). As shown in Figure 14, this plasmid
Gene enhancer, replication initiation region of SV40, pB
Replication origin region of R322 and β-derived from pBR322
A plasmid containing the lactamase gene (Amp ^r ),
The human G-CSF chromosomal gene is connected downstream of the enhancer of the SV40 gene. The expression vector for C127 cells was constructed as follows.

That is, a DNA fragment containing a CSF gene derived from a chromosome was excised from a expression vector pMLCE3α in COS cells with a restriction enzyme, and a DNA fragment containing a replication origin of bovine papilloma virus (BPV) and a DNA fragment having an SV40 early promoter were added to T4 DNA ligase. Was ligated using. The obtained pTNCE3α has the CSF gene derived from the chromosome linked to the downstream of the SV40 early promoter,
An expression vector containing a 65% portion of V.

On the other hand, the expression vector for CHO cells is a DNA fragment containing the same CSF gene derived from the chromosome and the SV40 early promoter portion as described above, and pAdD26SVpA-derived DHF
A DNA fragment containing the R gene is ligated with T4 DNA ligase. The obtained pT26SVCE3α is a chromosome-derived CSF gene downstream of the SV40 promoter,
An expression vector having a DHFR gene downstream of the adenovirus major late promoter.

(6) Expression by Animal Cell Here, a representative example will be described, but in other cases, see each example.

(A) Expression by mouse C127 cells The pTN-G4 plasmid or pTN-V2 plasmid is treated with BamHI, and the C127 cells that have been cultured and grown using this are transformed by the phosphate-calcium method. The obtained transformed cells are cultured and a clone having a high CSF-producing ability is selected.

The expressed G-CSF was recovered and purified from the culture medium of these transformed cells, and it was confirmed that it exhibits human-G-CSF activity. The target glycoprotein was also confirmed by amino acid analysis and sugar content analysis of the sample.

Regarding sugar content analysis, CS used for amino acid analysis
The F sample was subjected to quantification of amino sugars by the Elson-Morgan method, quantification of neutral sugars by the orcinol sulfate method, and quantification of sialic acid by the thiobarbitur method.

The quantification method is described in Chapter 13 of Biochemistry Experiment Course Vol. 4, "Sugar Chemistry (2nd Volume)" (Tokyo Kagaku Dojin). As a result of converting weight% from each quantitative value, the sugar content of the obtained G-CSF was 1 due to the difference in the expression vector and the culture conditions.
It was distributed in the range of up to 20 (% by weight).

(B) Expression by COS cells G-CSF derived from human chromosome obtained in (5)-(C) above
The vector containing the gene pMLCE3α is CV-
As a host cell, a COS cell (see Gluzman et al .; Cell 32, 175 (1981)) expressing a large T antigen of SV40, which is derived from one cell and is transformed with a replication defective mutant of SV40, is used as a host cell. The expression of the phenotype was found to show human G-CSF activity (Examples).
twenty five).

When COS cells were further collected and analyzed for mRNA, mRNA corresponding to each of the amino acid sequences shown in FIGS. 3 (A) and 4 (A) was present.

〔Example〕

Hereinafter, the present invention will be described in detail with reference to Examples, but before that, a method for measuring CSF activity will be described in Reference Examples.

<Reference Example> Method for measuring CSF activity The method for measuring CSF activity (hereinafter abbreviated as CSA) used in the present invention is as follows.

"Method for measuring CSA" (a) When using human bone marrow cells: Bradley T. R. , Metcalf D.D. Etc. (Aust.
J. Exp. Biol. Med. Sci, 44 , 287-300, 1966)
The monolayer soft agar culture method was performed according to That is, fetal bovine serum 0.2 ml, test sample 0.1 ml, human bone marrow non-adherent cell suspension 0.1 ml (1-2 × 10 ⁵ nucleated cells), modified McCoy's
Modified McCoy's 5A containing 0.2 ml of 5A culture and 0.75% of agar
After mixing 0.4 ml of the culture solution and placing it in a tissue culture plastic dish with a diameter of 35 mm to solidify, it is cultured at 37 ° C., 5% carbon dioxide / 95% air, 100% humidity, and then 10
The number of colonies formed after one day (a colony consisting of 50 or more cells is defined as one colony) was counted, and the activity for forming one colony was defined as 1 unit (Unit) to determine CSA.

(b) When using mouse bone marrow cells: Horse serum 0.4 ml, test sample 0.1 ml, C3H / He (female) mouse bone marrow cell suspension 0.1 ml (0.5 to 1 × 10 ⁵ nucleated cells), agar 0.75 % Modified McCoy's 5A Culture Solution 0.4 ml
After mixing and placing in a plastic dish for tissue culture with a diameter of 35 mm to solidify, 37 ° C, 5% carbon dioxide gas / 95%
After culturing for 5 days in air and 100% humidity, the number of colonies formed (a colony consisting of 50 or more cells is defined as 1 colony) is counted, and the activity of forming 1 colony is 1
CSA was calculated as a unit.

The “modified McCoy ′” used in the above methods (a) and (b) was used.
The s5A culture medium and the human bone marrow non-adherent cell suspension used in (a) were prepared as follows.

"Modified McCoy's 5A culture solution (double concentration)"McCoy's 5A culture solution (manufactured by GIBCO) 12 g, MEM
2.55 g of an amino acid vitamin medium (manufactured by Nissui Pharmaceutical Co., Ltd.), 2.18 g of sodium bicarbonate and 50,000 units of penicillin G potassium were dissolved twice in 500 ml of distilled water, and then sterilized by filtration with a 0.22 μm Millipore filter.

"Human bone marrow non-adherent cell suspension" A bone marrow fluid obtained by sternal puncture of a healthy person was diluted 5 times with RPMI1640 culture solution and overlaid with Ficol-Paque solution (Pharmacia) to prepare 400xg, 30 Centrifuge at 25 ℃ for min.
The interface cell layer (specific gravity <1.077) is collected. After washing the cells, the concentration was adjusted to 5 × 10 ⁶ cells / ml with RPMI1640 culture solution containing 20% fetal bovine serum, and the cells were placed in a 25 cm ² tissue culture plastic flask and kept in a carbon dioxide incubator for 30 minutes. After incubation, collect the non-adherent cells in the supernatant and place them again in a 25 cm ² plastic flask for 2 hours 30 min.
After incubation for minutes, the supernatant non-adherent cells were collected and used.

Example 1 Establishment of "CHU-2" Tumors of a cancer of the floor of the mouth with a marked increase in neutrophils were transplanted into nu / nu mice. About 10 days after transplantation, this tumor showed a marked increase in tumor and an increase in neutrophils. This tumor was aseptically removed 12 days after the transplantation and cut into 1-2 mm ³ squares, which were cultured as follows.

10-15 pieces of the above-mentioned minced tumor mass were placed in a 50 ml plastic centrifuge tube, and 5 ml of trypsin solution (trypsin 0.25%,
(Containing 0.02% EDTA), shaken in a 37 ° C warm bath for 10 minutes, discard the supernatant, and again add 5 ml of the same trypsin solution.
Was added, and trypsin digestion was performed while stirring at 37 ° C for 15 minutes.

The supernatant cell suspension was collected, 1 ml of fetal bovine serum was added to stop the action of trypsin, and the cells were stored in ice.

The above operation was performed again to collect the cell suspension, and the cell pellet was obtained by centrifugation at 1,500 rpm for 10 minutes together with the previous time.

This cell pellet was washed twice with F-10 containing 10% fetal bovine serum and then seeded in a 25 cm ² plastic culture flask at a cell concentration of 5 × 10 ⁶ cells / flask. After incubating overnight in a carbon dioxide gas incubator (carbon dioxide gas concentration 5%, humidity 100%) using F-10 culture solution containing 10% fetal bovine serum, the supernatant was removed together with non-adherent cells, and a fresh The culture solution was added to continue the culture. On the 6th day after the start of the culture, the cells were fully grown, so the culture medium was replaced with a new one at this point. The next day, this culture solution was discarded, and R was the same as 2 ml of anti-mouse erythrocyte antibody (manufactured by Cappel) diluted 5 times with RPMI1640.
2 ml of guinea pig complement (manufactured by Kyokuto Pharmaceutical Co., Ltd.) diluted 2.5 times with PMI1640 was added, and the mixture was incubated at 37 ° C. for 20 minutes. After the incubation, F-10 containing 10% fetal calf serum
After washing twice with NU / nu to remove fibroblasts derived from nu / nu mice, F-10 culture solution containing 10% fetal bovine serum was added, and the cells were further cultured for 2 days. Cloning was performed by the method. When the CSF activity of the 11 clones obtained was examined, a clone showing about 10 times higher activity than other clones (C
HU-2) was obtained.

Example 2 Isolation of CSF The cells established as described above grew completely densely 15
The cells were collected from two 0 cm ² culture flasks, suspended in 500 ml of F-10 culture solution containing 10% fetal bovine serum, and then placed on a glass roller bolt (Belc) of 1580 cm ^2.
(manufactured by O Co.) and subjected to rotary culture at a speed of 0.5 rpm. When the cells were completely and densely grown on the inner wall of the roller bottle, the culture solution was replaced with serum-free RPMI1640,
After culturing for 4 days, the culture supernatant was collected and fetal bovine serum was added.
F-10 containing 10% is added to continue the culture. After culturing for 3 days, the serum-free RPMI1640 was changed again, and after 4 days, the culture supernatant was collected. By repeating the same procedure below, 500 ml of serum-free culture supernatant can be obtained from 1 volt each week, and this method allows the cells to be maintained and recovered for a considerably long period of time. Met. The obtained culture supernatant 5 was made into 1 batch, and 0.01% Tween 20 was added to this, and Hollow Fiber was added.
After being concentrated about 1000 times by an ultrafiltration method using DC-4 and Amicon PM-10 (manufactured by Amicon), it was purified in the following order.

(i) Ultrogel AcA54 column with a diameter of 4.6 cm and a length of 90 cm (LK
B), 0.15M NaCl and 0.01% Tween
0.01M Tris-HCl buffer (pH 20) (manufactured by Hanai Chemical Co., Ltd.)
7.4) using 5 ml of the above-mentioned concentrated culture supernatant at a flow rate of about 50 ml
/ H gel filtration. The column is preliminarily bovine serum albumin (molecular weight 67,000), ovalbumin (molecular weight
45,000) and cytochrome C (molecular weight 12,400) were calibrated. After completion of gel filtration, 0.1 ml was collected from each fraction and diluted 10-fold, and then the active fraction was examined by the above-mentioned "CSA measurement method (b)". As a result, first, it was found that the fraction of Ve = 400 to 700 ml showed CSA in which macrophages were dominant, and the fraction of Ve = 800 to 1200 ml showed CSA in which granulocytes were dominant. Therefore, the latter fraction was collected and PM- Concentrated to about 5 ml with an ultrafilter using 10 (Amicon).

(ii) n-propanol (manufactured by Tokyo Kasei Co., Ltd.,
0.1% trifluoroacetic acid aqueous solution containing 30% (for amino acid sequencing) is added and left on ice for about 15 minutes.
The precipitate was removed by centrifugation at 00 rpm for 10 minutes. Then a μ Bondapak C18 column (Waters) equilibrated with an aqueous solution containing the above n-propanol and trifluoroacetic acid.
Adsorbed on a semi-preparative, 8 mm × 30 cm) (manufactured by the same company), and then sequentially eluted with a 0.1% trifluoroacetic acid aqueous solution containing n-propanol having a linear concentration gradient of 30 to 60%. Hitachi 685-50 type was used as the high performance liquid chromatograph, and Hitachi 638-41 type detector (both manufactured by Hitachi Ltd.) was used for detection, and absorption at 220 nm and 280 nm was measured at the same time. After elution, 10 μ of each fraction was fractionated and diluted 100-fold, and then the active fraction was examined by the above-mentioned “CSA assay method (b)”. As a result, activity was observed in the peak eluted with 40% of n-propanol. Therefore, when this peak was collected and re-chromatographed again under the same conditions and CSA was examined in the same manner as described above, it was found that n-propanol 40 was also obtained. %
Since activity was observed in the peak at the position, the peaks were collected (4 fractions = 4 ml) and lyophilized.

(iii) The freeze-dried powder contains 40% of n-propanol.
Dissolved in 200μ of 0.1% trifluoroacetic acid aqueous solution, TSK
It was subjected to high performance liquid chromatography (HPLC) using a G3000SW column (Toyo Soda Co., Ltd., 7.5 mm × 60 cm). Elution was performed with the same aqueous solution at a flow rate of 0.4 ml / min, and 0.4 ml each was collected with a fraction collector FRAC-100 (manufactured by Pharmacia). The CSA of each of the fractions collected was examined in the same manner as above, and the retention time was 37
Since activity was observed in a fraction of ~ 38 minutes (equivalent to a molecular weight of about 20,000), this fraction was collected and further analyzed by μ Bondapak.
After purification by C18 column (4.6 mm x 30 cm),
The main peak was collected and freeze-dried. The obtained preparation was assayed by the above-mentioned “method for measuring CSA (a)” and found to have human G-CSF activity.

Example 3 Determination of amino acid sequence (i) Determination of N-terminal amino acid sequence A sample was subjected to Edman degradation using a gas phase sequencer (manufactured by Applied Biosystems), and the obtained PTH amino acid was analyzed by a high performance liquid chromatography device. (Manufactured by Beckman Instruments) and Ultrasph
An ere-ODS column (manufactured by Beckman Instruments) was used for analysis by a conventional method. Start column (5 μm, diameter 4.6 mm, length 250 mm) buffer (15 mM sodium acetate buffer pH 4.5, aqueous solution containing 40% acetonitrile)
After equilibration with, the sample (dissolved in 20 μl of starting buffer) was injected, and separation was performed by isocratic elution with the starting buffer. Flow rate is 1.4 ml / min, column temperature is 4
Hold at 0 ° C. Detection of PTH amino acids is 269 nm and 320 nm
The ultraviolet absorption of was used. 2 nmol of each standard PTH amino acid (manufactured by Sigma) was separated in the same system to determine the retention time, and the retention time of the test sample was identified.

As a result, the amino acid sequence from the N-terminal to the 40th residue was determined as follows.

_{H 2 N-Thr-Pro-} Leu-Gly-Pro-A
la-Ser-Ser-Leu-Pro-Gln-Se
r-Phe-Leu-Leu-Lys-Cys-Leu
-Glu-Gln-Val-Arg-Lys-Ile-
Gln-Gly-Asp-Gly-Ala-Ala-L
eu-Gln-Glu-Lys-Leu-Cys-Al
a-Thr-Tyr-Lys- (ii) Bromcian decomposition A sample of Bromcyan was dissolved in 70% formic acid and purified by sublimation.
0 equivalent was added, and the mixture was reacted overnight at 37 ° C. Next, the reaction product was freeze-dried and then fractionated by HPLC using a TSK G3000SW column (manufactured by Toyo Soda Co., Ltd.) to obtain four peaks. The peaks are arranged in descending order of molecular weight, CN-1, CN-2, CN-
3, CN-4, which is a good yield of CN-1, CN-2
The amino acid sequence of was analyzed using an automatic gas phase sequencer (manufactured by Applied Biosystems) under the same conditions as in (i).

As a result, it was found that CN-1 is a peptide from the N terminus of G-CSF protein. Furthermore, CN-2 had the following amino acid sequence.

Pro-Ala-Phe-Ala-Ser-Ala-P
he-Gln-Arg-Arg-Ala-Gly-Gl
y-Val-Leu-Val-Ala-Ser-His
-Leu-Gln- (iii) trypsin degradation The sample was 0.1M Tris-HCl buffer (pH7.4) containing 8M urea.
0.1M containing 0.1% 2-mercaptoethanol
Tris-hydrochloric acid buffer (pH 7.4) was added to adjust the final concentration to 2M urea. Next, TPCK-treated trypsin (trade name, manufactured by Sigma) was added so that the sample and the enzyme became 50: 1, and after reacting at 25 ° C for 4 hours, the same amount of TPCK-treated trypsin was further added and again at 25 ° C. The reaction was carried out for 16 hours. After the reaction, the reaction product was subjected to high-speed reverse phase column chromatography using a C8 column (manufactured by Yamamura Chemical Co., Ltd.). Elution was performed by using n-propanol containing 0.1% TFA and increasing the n-propanol concentration linearly from 5% to 60%. Among the peaks obtained by measuring the ultraviolet absorption at 280 nm, the amino acid sequence of the main peak was analyzed using an automatic gas phase sequencer (manufactured by Applied Biosystems) under the same conditions as in (i). As a result, the main peak was found to be a peptide having the following sequence containing a part of the CN-2 fragment of (ii).

Gln-Leu-Asp-Val-Ala-Asp-P
he-Ala-Thr-Thr-Ile-Trp-Gl
n-Gln-Met-Glu-Glu-Leu-Gly
-Met-Ala-Pro-Ala-Leu-Gln-
Pro-Thr-Gln-Gly-Ala-Met-P
ro-Ala-Phe-Ala-Ser- Example 4 Preparation of DNA probe (i) Synthesis of probe (IWQ) Example 3. Il from the amino acid sequence obtained in (iii)
e-Trp-Gln-Gln-Met-Glu-Glu
Based on the sequence of 10 amino acids shown by -Leu-Gly-Met, 30 consecutive nucleotides were obtained (Fig. 1). In the sequence of FIG. 1, for example, the nucleotide at position 9 from the 5'-end is shown to be a mixture containing equal amounts of dA and dG. The starting material nucleotides were mainly dimers, and if necessary, mononucleotides were also used. 20 mg of nucleotide resin Ap-d (G) (manufactured by Yamasa Shoyu Co., Ltd.) as a starting material was placed in a column with a glass filter, and washing with methylene chloride was repeated, and then 4,4 with a methylene chloride solution containing 3% trichloroacetic acid The'-dimethoxytrityl group was eliminated and then the column was washed several times with 1 ml of methylene chloride. After washing with anhydrous pyridine to replace the solvent, nucleotide dimer (DMTr) Ap
20 mg of Tp (NHR ₃ ) (manufactured by Zeon Corporation; NHR ₃ is triethylammonium, DMTr is dimethoxytrityl) and 0.2 ml of pyridine were added, and the inside of the column was vacuum dried with a vacuum pump. Then, 2,4,6-trimethylbenzenesulfonyl-3-nitrotriazolide (MS
(NT, Wako Pure Chemical Industries, Ltd.) 20 mg and anhydrous pyridine 0.2 ml were added, the column was replaced with nitrogen gas, and the nucleotide resin and the dimer were condensed by occasional shaking at room temperature for 45 minutes. After completion of the reaction, the column was washed with pyridine, and then the unreacted OH group was treated with excess 4-acetic anhydride.
After acetylation with a pyridine solution containing dimethylaminopyridine, the column was washed again with pyridine. Similarly, (DMTr) Ip (NHR ₃ ), (DMTr) GpGp (NHR ₃ ), (DMTr) Ip (N
HR _3), (DMTr) equal mixture of CpTp (NHR ₃₎ and _{(DMTr) TpTp (NHR 3)} , an equal mixture of (DMTr) ApAp (NHR ₃₎ and _{(DMTr) ApGp (NHR 3)} , (DMTr ) ApGp (NHR ₃ ) and (DMTr) GpGp (NHR ₃ ) equal mixture, (DMTr) GpAp (NHR ₃ ), (DMTr) TpGp (NHR ₃ ), (DMTr) ApAp
Equivalent mixture of (NHR ₃ ) and (DMTr) GpAp (NHR ₃ ), (DMTr) CpAp (N
HR _3), (mixture of equal amounts DMTr) APAP and (NHR ₃₎ and _{(DMTr) ApGp (NHR 3)} , (DMTr) GpCp (NHR 3), (DMTr) TpGp (NHR 3), (DMTr) Ip
(NHR ₃ , (DMTr) ApTp (NHR ₃ ) [(DMTr) Ip (NHR ₃ ) is manufactured by Yamasa Shoyu Co., Ltd., and all others are manufactured by Nippon Zeon Co., Ltd.] and condensed by repeating the above-described procedure. After completion of the reaction in the final step, the resin was washed with pyridine, methylene chloride, and ether in this order without acetylation, and then dried.
Dioxane containing tetramethylguanidine and 1M α-picoline aldoxime 1 ml, pyridine 0.5 ml, water 0.2 ml
The mixture was suspended in 1.7 ml of the mixed solution of (1), left overnight at room temperature, and then concentrated under reduced pressure to 100 to 200 µ. After adding a small amount (2 to 3 drops) of pyridine to this concentrated liquid, concentrated ammonia water 2
~ 3 ml was added and heated at 55 ° C for 6 hours. Then, ethyl acetate was added for extraction and separation, and the obtained aqueous layer was concentrated under reduced pressure,
It was dissolved in 50 mM triethylammonium acetic acid solution (pH 7.0) and subjected to column chromatography using a C-18 column (1.0 x 15 cm, manufactured by Waters). Elution is with 50 mM triethylammonium acetic acid solution (pH 7.0)
Perform with a linear gradient of 10% to 30% acetonitrile in
The peak fraction eluted at a position where the acetonitrile concentration was around 25% was concentrated under reduced pressure.

80% acetic acid was added to this concentrated liquid, and the mixture was allowed to stand at room temperature for 30 minutes, and then ethyl acetate was added to extract and separate the water layer, which was concentrated under reduced pressure. The concentrate obtained was a C18 column (SSC-ODS-272, 6φ × 200 mm, manufactured by Senshu Scientific Co., Ltd.).
Further purification was performed by high performance liquid chromatography using. Elution was performed using acetonitrile with a linear concentration gradient of 10% to 20% in a 50 mM triethylammonium acetic acid solution (pH 7.0), and synthetic DNA was obtained in a yield of 10A ₂₆₀ units or more.

(ii) Synthesis of probe (A) Example 3. From the amino acid sequences obtained in (iii), Me
14 contiguous nucleotides were obtained based on the sequence of 5 amino acids shown in t-Pro-Ala-Phe-Ala (Fig. 1).

The synthesis was carried out in the same manner as the probe (IWQ), and the nucleotide resin AP-d (T) (manufactured by Yamasa Shoyu Co., Ltd.) (DMTr) was used.
CpAp (NHR ₃ ); (DMTr) GpGp (NHR ₃ ); (DMTr) CpAp (NHR ₃ ), (D
MTr) CpTp (NHR ₃ ), (DMTr) CpGp (NHR ₃ ), and (DMTr) CpCp (N
HR ₃ ) equal mixture; (DMTr) ApGp (NHR ₃ ), (DMTr) TpGp (NHR
₃ ), (DMTr) GpGp (NHR ₃ ), and (DMTr) CpGp (NHR ₃ ), an equal mixture; (DMTr) ApAp (NHR ₃ ); (DMTr) CpAp (NHR ₃ ) and (DMTr) Cp
An equal amount mixture of Gp (NHR ₃ ); (DMTr) Gp (NHR ₃ ) (all manufactured by Nippon Zeon Co., Ltd.) was condensed in this order to obtain about 10A ₂₆₀ units of synthetic DNA.

The nucleotide sequence of the obtained oligonucleotide was analyzed by Maxam-Gilbe
When examined by the rt method, it was confirmed to have the base sequence shown in FIG.

(iii) Synthesis of probe (LC) Automatic synthesis was performed using a DNA synthesizer 380A manufactured by Applied Biosystems. This method is based on the principle described by Caruthers et al. (J. Am. Chem. Soc, 103 , 3185 (1981)).
And is called the phosphoramidite method.

After the 5'-dimethoxytrityl group (DMTr) was deprotected, dG-S (S is a support) was condensed with the (DMTr) -dT phosphoamidite body previously activated with tetrazole, and then the unreacted hydroxyl group was replaced with acetyl. Then, it was oxidized with iodine in the presence of water to give a phosphate. The DMTr group was deprotected, and then the condensation was repeated in the same manner to synthesize 24 nucleotides having the sequence shown in FIG. The resulting nucleotide is cleaved from the support and deprotected, then C18
It was purified by reverse phase high performance liquid chromatography using a column (SSC-ODS-272 manufactured by Senshu Scientific Co., Ltd.).

Example 5 CHU-2 cell culture and mRNA purification 1) CHU-2 cell culture and cell recovery After the established CHU-2 cells were completely and densely grown in two 150 cm ² culture flasks, 10% of this with fetal bovine serum
After suspending in 500 ml of RPMI1640 culture solution,
It was transferred to a 580 cm ² glass roller bottle (manufactured by Belco) and subjected to rotary culture at a speed of 0.5 rpm for 4 days. When the cells have completely grown on the inner wall of the roller bottle, remove the culture medium from the roller bottle and pre-heat to 37 ° C.
100 ml of physiological saline containing 0.02% of warmed EDTA was added to the solution, and after heating at 37 ° C. for 2 minutes, the cells were separated by pipetting. The obtained cell suspension is centrifuged at 1500 rpm for 10 minutes to obtain a cell pellet. The cells were resuspended in 5 ml of physiological saline containing no EDTA and centrifuged at 1500 rpm for 10 minutes to obtain a cell pellet (wet weight: about 0.8 g). The cells thus obtained were subjected to RNA extraction operation. -80 ° C
Store frozen at.

2) Purification of mRNA mRNA from CHU-2 cells obtained as described above
Isolation is essentially “Molecular cloning” [Maniatis
Et al., Cold Spring Harbor, p. 196 (1982)]. CHU that was frozen and preserved
-2 cells (wet weight 3.8 g) in 20 ml of a 6 M guanidine solution (6 M guanidine thiocyanate, 5 mM sodium citrate (pH 7.0), 0.1 M β-mercaptoethanol, 0.
After suspending in 5% sodium sarcosyl sulfate) and mixed well with a Vortex mixer for 2 to 3 minutes, inhalation and discharge were repeated 10 times using a 20 ml syringe equipped with an 18G injection needle. 6ml 5.7M CsCl-0.1M in a polyallomer centrifuge tube that fits the Beckman SW40Ti rotor.
EDTA, (pH 7.5) was added in advance, and about 6 ml of a guanidine solution in which the above-mentioned cells had broken and became viscous so as to fill the tube was overlaid. Four centrifuge tubes thus prepared were centrifuged at 30,000 rpm at 20 ° C. for 15 hours, and the resulting pellet was washed 3 times with a small amount of 70% ethanol.

Combine the pellets from each tube to 550 μ
Dissolved in water and adjusted to a NaCl concentration of 0.2 M, then treated with phenol-chloroform (1: 1),
After the chloroform treatment, 2.5 times volume of ethanol was added and ethanol precipitation was performed to obtain total RNA (about 10.1 mg of total RNA was obtained from 3.8 g of wet cells).

Purification of poly (A ⁺ ) -RNA from total RNA was performed as follows. This method is affinity chromatography which utilizes the fact that mRNA has a poly A chain added to the 3'end. Oligo (dT) -cellulose (PL
Using Biochemicals Type 7), for adsorption, total RNA was adsorbed with an adsorption buffer (10 mM Tris-HCl (pH 7.5), 0.5 M Na).
Cl, 1 mM EDTA, 0.1% SDS solution is included), heated at 65 ° C. for 5 minutes, passed through an oligo (dT) -cellulose column filled with the same solution, and eluted with TE solution ( 10 mM Tris-hydrochloric acid (pH 7.5),
1 mM EDTA was included). The unadsorbed flow-through solution was passed through the same column again to carry out the same elution operation, and mixed with the first eluate. Using such an operation, 400 μg of poly (A ⁺ ) -RNA was obtained.

The mRNA prepared in this manner was used in Schleif and Wensink's Practical Methods in Molecular Biolog.
y, Springer-Verlag, New York, Heiderberg, Berli
n, (1981)), with the same operation as described in
Size fractionation was performed by sucrose density gradient centrifugation.

That is, a sucrose density gradient of 5% to 25% is created in a SW40Ti rotor (Beckman) tube. The sucrose solution is 0.1 M NaCl, 10 mM Tris-HCl (pH 7.5), 1
5% and 2% in solutions of mM EDTA and 0.5% SDS, respectively.
5% RNase-free sucrose (Schwarz / Ma
nn).

800 μg of mRNA (poly (A ⁺ ) -RNA) prepared by the method as described above was added to 200 μ-500 μ of TE.
It is dissolved in a solution, heated at 65 ° C. for 5 minutes and then rapidly cooled, and then placed on a sucrose density gradient solution. After centrifuging at 30,000 rpm for 20 hours, 0.5 ml fractions were collected, the absorbance at 260 nm was measured, and the fractionated RNA was determined based on the position of the standard RNA (28S, 18S, 5S ribosomal RNA) which was similarly measured. , And the G-CSF activity of each fraction was examined at the same time by using an oocyte system of Xenopus laevis. That is, mRNA of each fraction was prepared in an aqueous solution at a concentration of 1 μg / μ, and 50 ng of mRNA was injected into one oocyte taken out from Xenopus laevis (about 1 year old), followed by 96-well microtiter plate. Put 10 oocytes in each well and add 100μ each of Barth's medium (88mM
NaCl, 1 mM KCl, 2.4 mM NaHCO ₃ , 0.82 mM
MgSO ₄ , 0.33 mM Ca (NO ₃ ) ₂ , 0.41 mM CaCl
₂ , 7.5 mM Tris-HCl (pH 7.6), penicillin 10 mg /
, Streptomycin sulfate (10 mg /) for 48 hours at room temperature, and then collect the supernatant, concentrate and purify it for G-CSF.
Measure activity.

As a result, G-CSF activity was observed in the 15-17S fraction.

Example 6 Synthesis of cDNA (Construction of pBR cDNA Library) Lan from poly (A ⁺ ) -RNA obtained by the above-mentioned method.
d etc. [Nucleic Acids Res. , Vol. 9 , pp. 2251 (198
1)] based on Gubler and Hoffman's method [Gene, 25
Volume 263 (1983)] was added to synthesize the cDNA.

1) Synthesis of single-stranded cDNA Put reagents into a 1.5 ml tube manufactured by Eppendorf in the following order. 80μ reaction buffer (500mM K
Cl, 50 mM MgCl ₂ , 250 mM Tris-HCl, pH
8.3), 20μ of 200mM dithiothreitol, 32μ of 1
2.5 mM dNTP (dATP, dGTP, dCTP, d
Each containing 12.5 mM of TTP), 10 μ of α- ³² P-d
CTP (Amersham, PB10205), 32μ oligo (dT) _12-18 (PL Biochemicals, 500)
μg / ml), 20μ of poly (A ⁺ ) -RNA (2.1 μg
/ Μ), distilled water 206μ, total 400μ of reaction solution is heated at 65 ° C for 5 minutes and then heated at 42 ° C for 5 minutes. To this reaction solution, add 120 units of reverse transcriptase (Takara Shuzo), and further at 42 ° C for 2
After reacting for a time, RNase inhibitor (Bethesda
(Manufactured by Research Laboratories) 2 μ, 20 μ TE solution, 16 μ of 100 mM sodium pyrophosphate, 48 units (4 μ) of reverse transcriptase were added, and reacted at 46 ° C. for 2 hours this time. 0.5M EDTA 8μ, 10% SDS
After the reaction was stopped by adding 8 μm, phenol-chloroform treatment and ethanol precipitation (twice) were performed to obtain single-stranded cDNA.

2) Addition of dC-chain to single-stranded cDNA After dissolving the single-stranded cDNA obtained above in 60 µ of distilled water, 60 µ of dC-chain addition buffer (400 mM potassium cacodylate; 50 mM Tris-hydrochloric acid (pH 6 .9); 4 mM dithiothreitol; 1 mM CoCl ₂ ; 1 mM dCTP)
In addition, the mixture was heated at 37 ° C for 5 minutes. Add terminal transferase (27 unit / μ, PL Biochemica
3 μl (manufactured by ls) and reacted at 37 ° C. for 2.5 minutes, followed by phenol-chloroform treatment (once) and ethanol precipitation (twice), and TE containing 100 mM NaCl.
It was dissolved in 40 μl of the solution.

3) Synthesis of double-stranded cDNA 4 μ of oligo (dG) was added to the above 40 μ DNA solution.
_12-18 (200 μg / ml, manufactured by PL Biochemicals) was added and heated at 65 ° C. for 5 minutes and then at 42 ° C. for 30 minutes, and then the reaction solution was kept at 0 ° C. Buffer solution 80μ (100m
M Tris-HCl, pH 7.5, ₂₀ mM MgCl ₂ , 50 mM
(NH ₄ ) ₂ SO ₄ , 500 mM KCl), 4μ 4
mM dNTP (dATP, dCTP, dGtp, dT
TP containing 4 mM each), 60 μm of 1 mM β-NAD
And 210μ distilled water, 20μ E. coli DNA
Polymerase I (Takara Shuzo), 15 μE. coli
DNA ligase (Takara Shuzo), 15 μE. col
After adding iRNase H (Takara Shuzo) and reacting at 12 ° C for 1 hour, 4 µm of 4 mM dNTP was further added, and reacted at 25 ° C for 1 hour, followed by phenol-chloroform treatment and ethanol precipitation (once). Go, about 8 μg of 2
A single-stranded cDNA was obtained. This double-stranded cDNA was dissolved in TE solution and subjected to 1.2% agarose gel electrophoresis.
The portion corresponding to the size of 60 base pairs (bp) to 2 kilo base pairs (Kbp) is Whatman DE81 (manufactured by Whatman).
When it was adsorbed on and collected by elution, about 0.2 μg was recovered.

4) Addition of dC-chain to double-stranded cDNA The double-stranded cDNA obtained as described above was dissolved in 40 μl of TE solution, and 8 μd of dC-chain addition buffer described in 2) was added.
Was added and heated at 37 ° C. for 2 minutes, 1 μl of terminal transferase (27 unit / μ) was added and reacted at 37 ° C. for 3 minutes. The reaction solution was immediately cooled to 0 ° C and 0.5M
After the reaction was stopped by adding 1 μ of EDTA, phenol-chloroform treatment and ethanol precipitation were performed, and the obtained precipitate was suspended in 10 μ of a TE solution.

5) Construction of pBR cDNA library Commercially available oligo (dG) chain-added pBR322 vector (manufactured by Bethesda Research Laboratories, 10 ng / μ) 4 μ
And the above-mentioned dC-strand-added double-stranded cDNA 2 μm of 75 μm.
Annealed in TE solution containing 1 M NaCl.
Anneal at 65 ℃ for 5 minutes, then at 40 ℃ for 2 hours,
Then, it was left to stand until it reached room temperature.

On the other hand, Maniatis et al.'S experiment book [Molecular cloning, Cold
Spring Harbor, p. 249 (1982)] and the like to prepare competent cells from Escherichia coli X1776 strain, and transform with the above annealed plasmid to obtain a transformant. Was given.

Example 7 cDNA Synthesis (Construction of λ Phage-Based Library) 1) Synthesis of Single-Stranded cDNA According to the method described in Example 5, 3.8 g of cryopreserved CHU was stored.
-2 cells were purified twice with an oligo (dT) cellulose column to obtain 400 μg of poly (A ⁺ ) -RNA.

TE solution 10 in which 12 μg of this poly (A ⁺ ) RNA was dissolved
μ was put in a reaction tube containing 10 μg of actinomycin D (manufactured by Sigma), and then reagents were added in the following order: 20 μ of reverse transcription buffer (250 mM Tris-HCl (pH
8.3), 40 mM MgCl ₂ , 250 mM KCl), 20μ
5 mM dNTP (dATP, dGTP, dCT
P and dTTP each contained 5 mM), 20 μl oligo (d
T) _12-18 (0.2 μg / ml PL Biochemicals), 1 μM 1M dithiothreitol, 2 μ30un
it / μ RNasin (Promega Biotech), 10
μ reverse transcriptase (10unit / μ Seikagaku Corporation), 1
μ- [ ³² p] dATP (manufactured by 10 μCi Amersham Co.) and 16 μ of water make a total reaction volume of 100 μ.
After keeping the reaction solution at 42 ℃ for 2 hours, 5μ 0.5M ED
The reaction was stopped by adding TA and 1 μ of 20% SDS.
Phenol-chloroform (100 µ) treatment and ethanol precipitation (twice) were performed to obtain about 4 µg of single-stranded cDNA.

2) Synthesis of double-stranded cDNA The cDNA obtained as described above was dissolved in 29 μl TE solution and reagents were added in the following order to prepare a reaction solution; 25 μl polymerase buffer (400 mM Hepes (pH 7.6) ;
16 mM MgCl ₂ ; 63 mM β-mercaptoethanol; 270 mM KCl); 10 μm 5 mM dNTPs;
1.0 μ of 15 mM β-NAD; 1.0 μ of α-
[ ³² p] dATP (10 μCi / μ); 0.2 μE.
coli DNA ligase (60 unit / μ Takara Shuzo);
5.0 μE. coli DNA polymerase I (New E
ngland Biolabs, 10 unit / μ); 0.1μ RNa
se H (60unit / μ Takara Shuzo); 28.7μ distilled water.

The reaction solution was incubated at 14 ° C. for 1 hour, then returned to room temperature and further incubated for 1 hour. Then 5
The reaction was stopped by adding μ 0.5 M EDTA and 1 μ 20% SDS, followed by phenol-chloroform treatment and ethanol precipitation. The obtained DNA is treated with 0.5 mM EDT
Dissolve in 20 μA of A, and add 3 μ of Klenow buffer (500 mM Tris-HCl (pH8.0), 50 mM MgC).
1 ₂ ), 3 μm of 5 mM dNTP, and 4 μm of water were added to prepare a reaction solution, and then 1 μm of DNA polymerase (Klenow fragment) (Takara Shuzo) was added and incubated at 30 ° C. for 15 minutes.

The reaction solution was diluted with 70 μl of TE solution, and the reaction was stopped by adding 5 μm of 0.5 M EDTA and 1 μm of 20% SDS. The reaction solution was treated with phenol-chloroform and precipitated with ethanol to give about 8 μg of double-stranded cD.
I got NA.

3) Methylation of double-stranded cDNA 30 µL of the aqueous solution of double-stranded cDNA synthesized in 2), methylation buffer (500 mM Tris-HCl (pH8.0), 50 mM)
EDTA) 40 μ, SAM solution (800 μM S-adenosyl-L-methylmethionine (SAM), 50 mM β
-Mercaptoethanol) (20μ) and water (100μ) were added to the mixture, EcoRI methylase (New England Biolabs
Company, 20unit / μ) Add 15μ to make the total reaction liquid 200μ
And incubated at 37 ° C. for 2 hours. After phenol treatment and ether treatment, ethanol precipitation was performed to collect DNA.

4) Addition of EcoRI linker About 1.2 μg of the above methylated double-stranded DNA was added to a ligase buffer (250 mM Tris-HCl (pH 7.5), 100 mM).
MgCl ₂ ) 1.5μ, Ec pre-phosphorylated
oRI linker 0.5μ (10mer, Takara Shuzo), 1.5μ
10 mM ATP, 100 mM dithiothreitol 1.5μ
, 2μ H ₂ O was added to make the reaction solution 15μ, and then T4
After adding 0.7 μl of DNA ligase (3.4 u / μ, manufactured by Takara Shuzo) and reacting at 4 ° C. overnight, the ligase was inactivated by heating at 65 ° C. for 10 minutes. This reaction solution was further added with 100 mM Tris-hydrochloric acid (pH 7.5), 5 mM MgCl ₂ , 50 mM.
NaCl, 100 μg / ml gelatin concentration, the total liquid volume is 50
After preparing it to give μ, EcoRI (10 unit / μ
) 3.5 μm was added, and the mixture was reacted at 37 ° C. for 2 hours. Then 0.5
M EDTA (2.5 μm) and 20% SDS (0.5 μm) were added, followed by phenol-chloroform treatment and ethanol precipitation to recover DNA. After that, unreacted EcoRI linker was removed by gel filtration method of Ultrogel AcA34 (manufactured by LKB) or agarose gel electrophoresis method, and about 0.5 to 0.7 μg of linker-added double-stranded cDNA was recovered.

5) Ligation of double-stranded cDNA and λgt10 vector 2.4 μg of the above-mentioned linker-added double-stranded cDNA was EcoRI-treated with λgt10 vector (Vector Cloning System), ligase buffer (250 mM Tris-HCl, 100 mM MgCl _2). ) 1.4μ, distilled water 6.5μ
Was added and treated at 42 ° C for 15 minutes, then 10 mM ATP
1μ, 0.1M dithiothreitol 1μ, T ₄ DNA
Ligase (0.5 μm) was added to adjust the total amount to 15 μm, and the mixture was reacted at 12 ° C. overnight.

6) In vitro packaging About 1/3 of the recombinant DNA obtained in 5) above was packaged using an in vitro packaging kit (Promega Biotech) to obtain phage plaques.

Example 8 Screening of pBR system library by probe (IWQ) Whatman 541 filter paper was put on an agar medium on which colonies were grown, and left at 37 ° C. for 2 hours. The method of Taub and Thompson [Anal, Biochem. 126 , 222 (1982)].

That is, the colonies were transferred to 541 filter paper, then transferred to an agar medium containing chloramphenicol (250 μg / μ), and further left overnight at 37 ° C.

After the filter paper was taken out, it was left at room temperature for 3 minutes on the filter paper soaked with 0.5N NaOH solution, and this was repeated twice. The same procedure is repeated twice for 3 minutes using a 0.5 M Tris-hydrochloric acid (pH 8) solution, and further for 3 minutes with a 0.05 M Tris-hydrochloric acid (pH 8) solution at 4 ° C. for 1.5 mg / ml of lysozyme solution ( 0.05M Tris-hydrochloric acid (pH8), including 25% sucrose)
10 minutes at 37 ° C, then 1 x SSC (0.15M NaC
1 and 0.015M sodium citrate) solution for 2 minutes, 200
A 1 × SSC solution containing μg / ml protease K was used for 30 minutes, a 1 × SSC solution was used for 2 minutes, and a 95% ethanol solution was used for 2 minutes twice at room temperature, and then the 541 filter paper was dried. The dried 541 filter paper obtained was allowed to stand at room temperature with phenol: chloroform: isoamyl alcohol (25: 24: 1,100 m).
M Tris-hydrochloric acid (pH8.5), 100 mM NaCl, 10 mM
Soaked in EDTA solution) for 30 minutes. The same operation is repeated with a 5 × SSC solution for 3 minutes 3 times, and then
After performing the treatment twice with a 95% ethanol solution for 3 minutes, the filter paper was dried.

The probe (IWQ) was radiolabeled with ³² P according to the standard method (see Molecular cloning) and then Wallace
Colony hybridization was carried out according to the method described above (Nucleic Acids Res, vol. 9 , p. 879 (1981)). 6x
NET [0.9M NaCl, 0.09M Tris-HCl (pH 7.
5), 6 mM EDTA], 5 x Denhardt's solution, 0.1% S
Pre-hybridization was performed at 65 ° C. for 4 hours in a hybridization buffer containing DS, 0.1 mg / ml denatured DNA (calf thymus DNA), and then radiolabeled probe (IWQ) 1 × 10 ⁶ cpm / ml Hybridization was carried out at 56 ° C. overnight using the above-mentioned hybridization buffer containing After completion of the reaction, use 541 filter paper at room temperature for 0.1%
30 minutes twice with 6 × SSC solution containing SDS and 56 ° C.
After washing for 1.5 minutes, autoradiography was performed.

After separating the plasmid from the clone that gave a signal,
Southern blotting was performed using a probe (IWQ). Hybridization and autoradiography were performed under the same conditions as described above.

Similarly, Southern blotting was performed using the probe (A). Hybridization was performed at 49 ° C. for 1 hour using the above-mentioned hybridization buffer, gradually cooled to 39 ° C., and further performed at 39 ° C. for 1 hour. After the reaction was completed, use a nitrocellulose filter with 6xSS containing 0.1% SDS.
After being washed twice at room temperature for 30 minutes at C and then at 39 ° C. for 3 minutes, autoradiography was performed.

As a result, one clone was obtained as a positive clone, and the nucleotide sequence was determined by the dideoxy method. As shown in FIG. 2, a DNA consisting of 308 base pairs containing the probe (IWQ) and probe (A) portions was obtained. The pBR322-derived plasmid containing this insert was named pHCS-1.

Example 9 Screening of λ Phage-based Library with pHCS-1 Derived DNA Probe Method of Benton and Davis [Science 196 , 180, (197)
7)] and plaque hybridization was performed. The pHCS-1 obtained in Example 8 was treated with Sau3A and EcoRI to obtain a DNA fragment of about 600 base pairs, which was radiolabeled by nick translation according to a conventional method. Nitrocellulose filter paper (S & S) was placed on the agar medium in which the phage plaque was generated, the phage was transferred, the DNA was denatured with 0.5 M NaOH, and the filter paper was treated in the following order. 0.1M NaOH,
20 seconds with 1.5M NaCl followed by 0.5M Tris-HCl (pH 7.
5), twice with 1.5 M NaCl for 20 seconds, and finally with 120 mM N
aCl, 15 mM sodium citrate, 13 mM KH ₂ PO
₄ , treated with 1 mM EDTA, pH 7.2 for 20 seconds.

The filter paper was then dried and heated at 80 ° C. for 2 hours to fix the DNA. 5 x SSC, 5 x Denhardt's solution, 50 mM phosphate buffer, 50% formamide, 0.25 mg / ml denatured DNA
(Salmon testis DNA) and 0.1% SDS in a hybridization buffer at 42 ° C. overnight for prehybridization, and radiolabeled pHCS-1 probe 4 × 10 ⁵ cpm / ml by nick translation. Hybridization buffer containing (5 x SSC, 5 x De
nhardt solution, 20 mM phosphate buffer (pH 6.0), 50% formamide, 0.1% SDS, 10% dextran sulfate, 0.1 mg
/ Ml of denatured DNA (salmon testis DNA) mixture) was hybridized at 42 ° C for 20 hours.

Nitrocellulose filter paper containing 0.1% SDS at room temperature 2
Rinse with SSC for 20 minutes, then 0.1% SDS at 44 ° C.
In 0.1 × SSC for 30 minutes, then 0.1 × SS at room temperature
After washing at C for 10 minutes, the cells were detected by autoradiography.

As a result, 5 positive clones (G1-5) were obtained. Therefore, of the obtained clones, full-length cDNA
When the DNA base sequence of the clone suspected of containing NA was examined by the dideoxy method, the base sequence shown in FIG. 3 (A) was obtained. Therefore, this cDNA was cut out from the λg10 vector, and pBR327 [Soberon et al .; Gene 9 , p. 287].
(1980)] at the EcoRI site to prepare a large amount of plasmid. This plasmid is called pBRG4.

Example 10. Screening of λ phage system library with pBRG4-derived DNA probe and probe (LC) Plaque hybridization was performed according to the method of Benton and Davis used in Example 9 (see the above-mentioned document). Place the nitrocellulose filter paper (S & S) on the agar medium in which the phage plaque was generated, transfer the phage, and
The DNA was denatured with 5M NaOH and the filter paper was treated in the following order. 20 with 0.1M NaOH and 1.5M NaCl
Second, followed by 0.5M Tris-HCl (pH 7.5), 1.5M Na
20 times twice with Cl, and finally 120 mM NaCl, 15 mM sodium citrate, 13 mM KH ₂ PO ₄ , 1 mM EDT
A, (pH 7.2) was treated for 20 seconds. The filter paper is then dried and
DNA was fixed by heating at 0 ° C. for 2 hours. In this way, two identical filter papers were prepared and subjected to screening with the pBRG4-derived DNA probe and the probe (LC). In the case of using the pBRG4-derived DNA probe, pBRG4 was treated with EcoRI to obtain a DNA fragment of about 1500 base pairs, and this DNA fragment was radiolabeled by nick translation according to a conventional method. 5 pieces of the above filter paper
X SSC, 5 x Denhardt's solution, 50 mM phosphate buffer, 50%
Formamide, 0.25 mg / ml denatured DNA (salmon testis DN
A) and a hybridization buffer containing 0.1% SDS at 42 ° C. overnight for prehybridization, and the above-mentioned radiolabeled DNA probe of about 1500 base pairs (about 1 × 10 ⁶ cpm / ml). Prehybridization buffer containing [5 x SSC, 5 x Denhardt's solution, 20 mM
Phosphate buffer (pH 6.0), 50% formamide, 0.1% SD
S, 10% dextran sulfate, 0.1 mg / ml denatured DNA
Hybridization was performed at 42 ° C. for 20 hours with a mixed solution of (salmon testis DNA). Rinse the nitrocellulose filter paper at room temperature with 2 × SSC containing 0.1% SDS for 20 minutes,
Then, at 44 ° C., after washing with 0.1 × SSC containing 0.1% SDS for 30 minutes and then with 0.1% SSC at room temperature for 10 minutes,
It was detected by autoradiography.

In the case of probe (LC), filter paper was pretreated with 3 × SSC containing 0.1% SDS at 65 ° C. for 2 hours, and then 6 × NE.
Prehybidization was carried out at 65 ° C. for 2 hours in a solution containing T, 1 × Denhardt's solution, and 100 μg / ml denatured DNA (salmon testis DNA).

Hybridization buffer containing radiolabeled probe (LC) (2 × 10 ⁶ cpm / ml) [6 × NET, 1 × De
nhardt solution, 100μg / ml denatured DNA (salmon testis DN
A)] at 63 ° C. overnight, and then the nitrocellulose filter paper is washed at room temperature with 6 × SSC containing 0.1% SDS for 20 minutes, and then washed 3 times, and then 0.1%. It was washed with 6 × SSC containing SDS at 63 ° C. for 2 minutes.

The filter paper was dried and then detected by autoradiography.

In the screening carried out in this manner, positive clones were selected for both of the two probes, and the nucleotide sequence of the clones that were considered to contain the full-length cDNA was examined by the dideoxy method. A base sequence as shown in A) was obtained. Therefore, this cDNA was cut out from the λgt10 vector, and pBR327 and EcoR were cut out.
Ligation at the I site gave plasmid pBRV2.

Example 11 Screening of human chromosomal gene library 1) Construction of human chromosomal gene library The human chromosomal gene library is Maniatis (Harvard Un
iversity), which was created as follows.

Chromosomal total DNA is extracted from human fetal liver with phenol or the like and partially digested with restriction enzymes HaeIII and AluI.
Among the DNA fragments thus obtained, the chain length is 18 to 25 Kb
Concentrated fragments are concentrated by sucrose density gradient centrifugation, and then Escherichia coli phage λCharon4 is mediated by a short-chain synthetic nucleotide having a restriction enzyme EcoRI fragment site.
Infectious phage DN that is connected to the arm DNA of A
Make A recombinant. Next, for the purpose of further increasing the infectivity, complete phage λ particles are formed by a packaging method. In principle, the human gene library created in this way has a chain length containing almost all human genes.
It is considered to be a set of recombinants containing human DNA of 18 to 25 Kb.

2) Screening of human chromosomal gene library with pHCS-1-derived DNA probe Method of Benton and Davis [Science 196, 180 (1977)]
The plaque hybridization was carried out according to. The pHCS-1 obtained in Example 8 was treated with Sau3A and Ec.
A DNA fragment of about 600 base pairs was obtained by treatment with oRI, and this DNA fragment was radiolabeled by nick translation according to a conventional method. Nitrocellulose filter paper (S & S) was placed on the agar medium in which phage plaque was generated, the phage was transferred, the DNA was denatured with 0.5 M NaOH, and the filter paper was treated in the following order. 0.1M NaOH, 1.5M N
20 seconds with aCl, followed by 0.5 M Tris-HCl (pH 7.5), 1.
5M NaCl twice for 20 seconds and finally 120 mM NaCl,
15 mM sodium citrate, 13 mM KH ₂ PO ₄ , 1 mM
It was treated with EDTA, (pH 7.2) for 20 seconds.

Then, the filter paper was dried and heated at 80 ° C. for 2 hours to fix the DNA. 5 x SSC, 5 x Denhardt's solution, 50m
Hybridization buffer containing M phosphate buffer, 50% formamide, 0.25 mg / ml denatured DNA (salmon testis DNA) and 0.1% SDS hybridized at 42 ° C overnight and radiated by nick translation Labeled pHCS-1 probe 4 × 10 ⁵ cpm / ml
Hybridization buffer containing [5 × SSC, 5
× Denhardt's solution, 20 mM phosphate buffer (pH 6.
0), 50% formamide, 0.1% SDS, 10% dextran sulfate, and a mixed solution of 0.1 mg / ml denatured DNA (salmon testis DNA)] at 42 ° C. for 20 hours.

Nitrocellulose filter paper containing 0.1% SDS at room temperature 2
Wash with × SSC for 20 minutes, then 0.1 × SSC containing 0.1% SDS at 44 ° C for 30 minutes, then 0.1 × SSC at room temperature.
After 10 minutes of washing, the cells were detected by autoradiography.

As a result, dozens of positive clones were obtained.

Recombinant DNA was prepared from these clones by the method of Maniatis (Cell 15: 687 (1978)).

The obtained DNA was treated with each restriction enzyme such as EcoRI, BamHI, BglII, analyzed by agarose gel electrophoresis, and a restriction enzyme map was prepared according to the method of Fritsch (see the above-mentioned literature).

Radiolabeled pHCS- used in the above screening
Southern hybridization was carried out using a probe of the DNA fragment derived from 1. As a result, a DNA fragment of about 8 kilobase pairs cleaved by EcoRI was selected from among the hybridized with the probe, and EcoRI of pBR327 was selected.
Subcloned into the site.

Furthermore, the subcloned DNA is treated with the restriction enzymes as described above, and Southern hybridization is repeated to encode a human G-CSF polypeptide into a DNA fragment of about 4 kilobase pairs cut out with EcoRI and XhoI. It turned out that there is a gene that does.

Then, when the sequence of about 3 kilobase pairs of this DNA fragment was examined by the deoxy method, the base sequence shown in FIG. 5 was obtained.

The restriction enzyme cleavage site of this DNA fragment was as shown in FIG.

In addition to the above-mentioned probes used for screening human chromosomal genes, pBRG4-derived DNA and pBRV2
It was carried out with the DNA of the origin.

Both DNAs are 1500 bp DN treated with EcoRI
The A fragment was directly radiolabeled by the nick translation method as described above, or treated with EcoRI and then with DraI, and the resulting DNA fragment of about 700 base pairs was also radiolabeled under the same conditions as described above. After selecting clones by performing plaque hybridization, a Southern fragment analysis was performed to obtain a DNA fragment having the nucleotide sequence shown in FIG. The resulting plasmid was named pBRCE3β.

Example 12. Preparation of pHGA-410 vector (for animal cells, + VSE system) E of cDNA obtained in Example 9 and shown in FIG. 3 (A)
The coRI fragment was treated with the restriction enzyme DraI at 37 ° C. for 2 hours and then treated with the Klenow fragment of DNA polymerase I (Takara Shuzo) to make the ends blunt. 1μ
AT of ggl BglII linker (8mer; manufactured by Takara Shuzo)
After phosphorylation with P, it was ligated with about 1 μg of the DNA fragment mixture obtained above. Then the restriction enzyme Bgl
After treatment with II, agarose gel electrophoresis was performed, and only the largest DNA fragment was recovered.

As shown in FIG. 6, this DNA fragment corresponded to about 710 base pairs containing the portion encoding the human G-CSF polypeptide. Vector pdKCR (Fukunaga et al .; Proc.Natl.
Acad.Sci.USA; 81: 5086 (1984)) with the restriction enzyme BamH
After treatment with I, the vector DNA obtained by dephosphorylating with alkaline phosphatase (Takara Shuzo) was added to T4DN.
A ligase (Takara Shuzo) was added and ligated with the cDNA fragment to obtain pHGA410 (FIG. 8). As shown in FIG. 8, this plasmid contains the promoter of the SV40 early gene, the replication initiation region of SV40, a part of the rabbit β-globin gene, the replication initiation region of pBR322 and pBR322.
322-derived β-lactamase gene (Amp ^r ) is included, and human G is located downstream of the SV40 early gene promoter.
-The CSF gene is connected.

Example 13. Construction of recombinant vector for C127 cells (+ V
SE) 1). Construction of pHGA410 (H) The pHGA410 plasmid obtained in Example 12 (Fig. 8).
20 μg to 50 mM Tris-HCl (pH 7.5), 7 mM MgC
l2, 100 mM NaCl, 7 mM _2- mercaptoethanol, 0.01% bovine serum albumin (BSA) dissolved in the reaction solution, EcoRI (Takara Shuzo, 10-15 units)
Was added and reacted at 37 ° C. for about 30 minutes to perform partial digestion with EcoRI. Then, phenol-chloroform (1: 1) treatment was performed twice, ether treatment and ethanol precipitation were performed to treat the DNA fragment.

This DNA fragment was added to 50 mM Tris-HCl, 5 mM MgCl
₂ , 10mM DTT, 1mM dATP, dCTP, dGT
It was dissolved in 50 μl of a solution containing P and dTTP, and E. coli
DNA polymerase-Klenow fragment (Takara Shuzo)
5 μl was added and incubated at 14 ° C. for 2 hours to make a blunt end.

Approximately 5.8 Kb by 0.8% agarose gel electrophoresis
6 .mu.g of the fragment were recovered.

5 μg of the recovered DNA fragment was added again to 50 mM Tris-HCl.
(PH 7.6), 10 mM MgCl ₂ , 10 mM DTT, 1 mM AT
It was dissolved in 50 μl of a reaction solution containing P, 2 μg of HindIII linker (Takara Shuzo) and 100 units of T4 DNA ligase (Takara Shuzo) were added, and the mixture was reacted at 4 ° C. overnight. Then, after phenol treatment, ether treatment and ethanol precipitation, 10 mM Tris-HCl (pH 7.5), 7 mM Mg
It was dissolved in 30 μl of a solution of Cl ₂ and 60 mM NaCl, and incubated at 37 ° C. for 3 hours in the presence of 10 units of HindIII restriction enzyme. After treatment with T4 DNA ligase again, this DNA was treated with the rubidium chloride method (see "Molecular Clonin Cloning" above).
g))). E. coli DHI strain was transformed with ampicillin-resistant (Amp ^r ) colonies to obtain p.
EcoRI site of HGA410 plasmid is HindII
Bacteria carrying the plasmid replaced by I were selected.
The plasmid thus obtained was used as pHGA410.
It is named (H) (FIG. 9).

2). Construction of recombinant vector for expression pTN-G4 pHGA410 (H) (20 μg) obtained in 1) above was
10 mM Tris-HCl (pH 7.5), 7 mM MgCl ₂ , 175
50μ reaction solution consisting of mM NaCl, 0.2mM EDTA, 7mM 2-mercaptoethanol, 0.01% bovine serum albumin
20 units of restriction enzyme SalI (Takara Shuzo) was added, and the mixture was incubated at 37 ° C. for 5 hours. Next, after phenol treatment and ethanol precipitation, DNA polymerase K
Similar to the above reaction with lenow fragment (Takara Shuzo)
It was incubated at 14 ° C for about 2 hours to give a blunt end. The DNA fragment obtained by ethanol precipitation was treated with a restriction enzyme HindIII without being recovered by agarose electrophoresis, and 5 μg of a HindIII-SalI fragment of about 2.7 kb was recovered by 1% agarose gel electrophoresis.

On the other hand, bovine papilloma virus
(BPV)] containing the plasmid pdBPV-1 (Sarv
er, N.N. Sbyrne, J .; C & Howley, P.M. M. (1982) Pr
oc. Natl. Acad. Sci. USA 79 , 7147-7151; Dr. Howley
(Obtained from Fukunaga, Sokaw)
a, & Nagata, (1984) Proc, Natl. Acad. Sci. U SA81 Volume 5
086-5090) treated with HindIII and PvuII for 8.4
Obtain a kb DNA fragment.

This 8.4 kb DNA fragment and the above-mentioned approximately 2.7 kb H
The indIII-SalI DNA fragment was treated with T4D according to a conventional method.
After treatment with NA ligase, the above-mentioned "Molecular Clonin
by the rubidium chloride method described in "g." coliDH
I-strain and pHGA410-derived G-CSF c
E. coli carrying a plasmid with DNA E. coli colonies were selected. This plasmid is designated as pTN-G4 (Fig. 9).

On the other hand, adenovirus Type II [protein nucleic acid enzyme 27
December issue (1982), published by Kyoritsu Shuppan], VAI and VA
Fragments containing VAI and VAII were recovered from the plasmid ΔpVA containing the approximately 1700 bp SalI-HindIII fragment containing II. This fragment is the Hi of pTNG4 described above.
pTNG4VAα and pTN inserted at the ndIII site
G4VAβ was obtained (FIG. 9). In this plasmid, the expression efficiency of the transcription product from the SV40 early promoter was enhanced by the adenovirus VA gene.

Example 14 Transformation of C127 cells and expression thereof (+ V
SE) pTN-G4 obtained in Example 13 is treated with a restriction enzyme BamHI before being transformed into mouse C127 cells. That is p
20 μg of TN-G4 plasmid was added to 10 mM Tris-HC
1 (pH 8.0), 7 mM MgCl ₂ , 100 mM NaCl, 2
mM2-mercaptoethanol, 0.01% BSA mixed solution 10
It was dissolved in 0 μm and treated with 20 units of BamHI (Takara Shuzo), followed by phenol treatment, ether treatment, and ethanol precipitation.

Mouse C127I cells are grown in Dulbecoo's minimal essential medium containing 10% fetal bovine serum (GIBCO). C127I cells grown on a plate having a diameter of 5 cm were treated with the above-prepared DNA at a ratio of 10 μg per plate in a phosphate-calcium method (Haynes, J & Weissmann, C
(1983) Nucleic Acid Res. 11 ( 687-706), and after glycerol treatment, it was incubated for 12 hours at 37 ° C.

Then transfer the cells to three new 5 cm diameter plates,
The medium was exchanged twice a week. Fo on the 16th day
The portions in which ci (aggregates) were formed were transferred to new plates, subcultured in the above-mentioned medium, and clones having high G-CSF producing ability were selected. As a result, a level G-CSF production of ˜1 mg / was observed. As a result of further continuing cloning, production of G-CSF at a level of 10 mg / or higher was confirmed.

In addition to the above C127I cells, NIH3 is used as the host cell.
T3 cells can also be used.

Example 15 Expression of G-CSF by CHO cells (+ VS
E) 1). Construction of pHGG4-dhfr 20 μg of pHGA410 plasmid obtained in Example 12 was added to 10 mM.
Tris-HCl (pH 7.5), 7 mM MgCl ₂ , 175 mM
Dissolve in 100 μl of a solution containing NaCl, 0.2 mM EDTA, 0.7 mM 2-mercaptoethanol, 0.01% BSA,
After adding 20 units of restriction enzyme SalI (Takara Shuzo) and reacting overnight at 37 ° C., phenol treatment, ether washing and ethanol precipitation were performed.

Next, the obtained DNA precipitate was treated with 50 mM Tris-HCl,
5 mM MgCl ₂ , 10 mM DTT, 1 mM dATP, dC
Dissolve in 100 μl of a reaction solution consisting of TP, dGTP and TTP, E. coli DNA polymerase-Klenow
A fragment (10 μm, manufactured by Takara Shuzo) was added and reacted at 14 ° C. for 2 hours, followed by phenol treatment, ether washing and ethanol precipitation.

An EcoRI linker is added to this DNA. That is, the above DNA was added to 50 μm of 50 mM Tris-HCl (pH 7.4), 1
It was dissolved in a reaction solution consisting of 0 mM DTT, 0.5 mM spermidine, 2 mM ATP, 2.5 mM hexamine cobalt chloride, and 20 μg / ml BSA, added with EcoRI linker (Takara Shuzo), and added 200 units of T4 DNA ligase (Takara Shuzo) at 4 ° C., Reacted for 12-16 hours. After performing phenol treatment, ether washing, and ethanol precipitation according to a conventional method,
The DNA was partially digested with EcoRI and 3 μg of a fragment of about 2.7 kb was recovered by 1% agarose gel electrophoresis.

On the other hand, pAdD26SVpA plasmid (Kaufman,
R. G. & Sharp, P.S. A (1982) Mol. Cell Biol, Volume 2 13
04 to 1319) is treated with EcoRI and treated with bacterial alkaline phosphatase (BAP) to dephosphorylate. That is, pAdD26SVpA 20 μg and EcoRI20
The unit is reaction solution 50 mM Tris-HCl (pH 7.5), 7 mM
Add to a mixed solution 100μ of MgCl ₂ , 100mM NaCl, 7mM _2- mercaptoethanol, 0.01% BSA, and add 37 ° C 1
After reacting for 0 hour, 5 units of BAP was added to the above reaction solution and reacted at 68 ° C for 30 minutes.

After that, it is treated with phenol and electrophoresed into pAdD26.
The EcoRI fragment of SVpA was recovered (-5 μg).

0.5 μg each of the above-mentioned about 2.7 kb fragment and the pAdD26SVpA fragment was annealed. This plasmid was transformed into E. The Escherichia coli DHI strain is transformed by the rubidium chloride method, and colonies carrying the pHGG4-dhfr plasmid are selected. The resulting plasmid is pHGG
It was named 4-dhfr (Fig. 10a).

As an alternative to the above, the pHGG4 plasmid was replaced with Sa
treated with II and partially digested with EcoRI without adding an EcoRI linker to recover a fragment of about 2.7 kb,
E. The DNA fragment is treated with E. coli DNA polymerase-klenow fragment to blunt end the ends.

On the other hand, a blunt-ended EcoRI fragment of pAdD26SVpA was prepared in the same manner as described above, and both were treated with T4DN.
It is also possible to prepare pHGG4-dhfr by treating with A ligase.

The pHGA410 (H) obtained in 1) of Example 13 was treated with the restriction enzymes HindIII and SalI as described in 2) of Example 13 to blunt end the HindIII-SalI fragment into the pAdD26SVpA.
pHGG4-dhfr can also be obtained by ligation to coRI (Fig. 10b).

2). Construction of pG4DR1 and pG4DR2 10 μg of pAdD26SVpA described in 1) was added to 50 mM.
Tris-HC (PH7.5), 7 mM MgCl ₂ , 1
It was dissolved in 50 ml of a reaction solution containing 00 mM NaCl, 7 mM 2-mercaptoethanol and 0.01% BSA, and 10 units each of restriction enzymes EcoRI and BamHI were added, and 37
The reaction was performed at 10 ° C for 10 hours. Phenol treatment and ether washing were performed according to a conventional method. After recovering a DNA fragment of about 2 Kb by 1% low melting point agarose electrophoresis, it was blunt-ended with a DNA polymerase-Klenow fragment according to a conventional method, followed by phenol treatment, ether washing and ethanol precipitation.

On the other hand, pHGA410 (H) obtained in 1) of Example 13
10 μg to 10 mM Tris-HCl (PH7.5), 7 m
Reaction solution containing M MgCl ₂ and 60 mM NaCl 50μ
The solution was dissolved in, and 10 units of HindIII was added and reacted at 37 ° C for 6 hours. The DNA fragment was recovered by 1% low-melting point agarose electrophoresis according to a conventional method, further subjected to BAP treatment, and then K
It was blunt-ended with a lenow fragment. After treatment with phenol and washing with ether, blunt-end ligation was performed using the above-mentioned about 2 Kb DNA fragment and T4 DNA ligase. That is, 1 μg of each DNA fragment is 66 m
M Tris-HCl (pH7.5), 6.6 mM MgC
Reaction solution containing l ₂ , 5 mM DTT, 1 mM ATP 30
Dissolve in μg, add 50 units of T4 DNA ligase,
After reacting at 6 ° C. for 12 hours, E. E. coli strain DHI. Thus, the pG4D shown in FIG.
R1 and pG4DR2 were obtained.

3) Transformation and expression CHO cells (dhfr ^- strain, University of Columbia Dr. L. Chasin
Obtained from the above) was cultured and grown in a 9 cm diameter plate (manufactured by Nunc) in α minimum essential medium (α-MEM, adenosine, deoxyadenosine, thymidine added) containing 10% fetal calf serum, and the phosphate-calcium method ( Wigler et al., Cell 14
725 (1978)).

That is, pHGG4-dhfr plasmid 1 prepared in 1)
An appropriate amount of carrier DNA (calf thorax DNA) was added to μg and dissolved in TE solution 375μ to prepare 1M CaCl ₂ 125μ.
Add. Cool on ice for 3-5 minutes, 500μ of 2xH
BS (50 mM Hepes, 280 mM NaCl, 1.5 mM phosphate buffer) was added, and the mixture was ice-cooled again, mixed with 1 ml of the above CHO cell culture solution, added dropwise to the plate, and then cultured in a CO ₂ incubator for 9 hours. After removing the medium from the plate and washing with TBS (Tris-Buffered Saline),
After adding TBS containing% glycerol and washing again, a non-selection medium (α-MEM medium described above, with nucleotides added) was added and incubated for 2 days, and the cells were divided 1:10 with the selection medium (no nucleotides added). Then, the culture was continued while exchanging the medium with the selective medium every two days, and the resulting colonies were selected and transferred to a new plate.

0.02 μM methotrexate (MT
X) Proliferated in the presence of 0.05 μM and then 0.1 μM MTX
It was grown in the presence and cloned. CHO
Transformation of cells was performed by using pHGG4 and pAd for CHO cells.
Cotransformation of D26SVpA
Can also be done by doing (Scahill et al. Pro
c. Natl. Acad. Sci. USA 80 vol 4654-4658 (1983)).

CHO cells were also transformed by the method described below. That is, pG4D prepared in the above 2)
R1 or pG4DR2 is previously sal
A DNA fragment was obtained by treatment with I and KpnI, and 10 μg of the DNA fragment was transformed into CHO cells in the same manner as above. When the cells thus transformed were continued to be cultured in the selective medium as described above, 100 or more clear colonies appeared per plate on about the 7th day. When the colonies were transferred to a new plate again without selecting them one by one, and continued to be cultured in a selective medium in the presence of 0.01 μM MTX, dozens of colonies appeared. Furthermore, the concentration of MTX is 0.02 μM, 0.05 μM, 0.1
The colonies that survived were selected by raising the concentration to μM.
Moreover, even after selecting each of the obtained dozens of colonies and increasing the MTX concentration, similar colonies could be selected.

In addition, a recombinant vector having a so-called polycistronic gene was constructed, and this could be used to transform CHO cells. That is, pAdD26SVpA is set to Pst
I treatment, two fragments were recovered, and by ligating these fragments with the pBRG4-derived CSF cDNA fragment, adenovirus promoter, CSF cDNA, DHFR, SV
A recombinant vector having 40 poly A sites arranged in order was constructed and put into a CHO cell.

Example 16 G-CSF activity assay (+ VSE) of expressed substance C127 cells and CH obtained in Example 14 and Example 15
The culture supernatant of O cells was adjusted to pH 4 with 1N acetic acid,
After adding an equal volume of n-propanol, the resulting precipitate was removed by centrifugation, passed through an open column (1φ × 2 cm) packed with a C8 reverse phase carrier (Yamamura Chemical Co., Ltd.), and eluted with 50% n-propanol. Let After diluting the eluate twice with water,
30-60 containing 0.1% TFA by reverse phase high performance liquid chromatography using YMC-C8 column (manufactured by Yamamura Chemical Co., Ltd.)
Elution was performed with n-propanol having a linear concentration gradient of%. The fraction eluted at a position where the n-propanol concentration was around 40% was collected, freeze-dried, and dissolved in 0.1 M glycine buffer (pH 9). Through such a process, human G-CSF was concentrated about 20-fold from C127 and CHO cell supernatant.

As a control, human G-CS according to the method described above
After transforming the cells with the plasmid containing no FcDNA, the culture supernatant was concentrated. The obtained preparation was assayed for human G-CSF activity by a method based on "Method for measuring human G-CSA (a)" described in Reference Example. still,
When the expression efficiency is sufficiently high, the culture supernatant may be directly subjected to the assay. The results are shown here for concentrated examples. The results are shown in Table 1.

Example 17 Amino Acid Analysis and Sugar Analysis (+ VSE) 1) Analysis of Amino Acid Composition The crude CSF sample obtained in Example 16 was further purified according to the method of Example 2 (iii). The purified CSF sample was hydrolyzed by a conventional method, and the amino acid composition of the protein portion was analyzed by a special amino acid analysis method using a Hitachi 835 amino acid automatic analyzer (manufactured by Hitachi, Ltd.). The results are shown in Table-2. The hydrolysis conditions are as follows.

6N HCl, 110 ° C, 24 hours, 4N methanesulfonic acid + 0.2% 3- (2-aminoethyl) indole in vacuum, 110 ° C, 24 hours, 48 hours, 72 hours,
In vacuum The sample is dissolved in a solution (1.5 ml) containing 40% n-propanol and 0.1% trifluoroacetic acid, and then 0.1 ml of each is taken and dried with dry nitrogen gas, or the reagent is added and vacuumed. The tube was sealed and subjected to hydrolysis.

In the table, the actual measurement value is the average value of the 24 hour value and the 24, 48, 72 hour value in total of 4 times. However, Thr, Ser, 1 /
2Cys, Met, Val, Ile and Trp were calculated by the following method. (Biochemistry Laboratory, Protein Chemistry
II (see Tokyo Kagaku Doujin Shuppan) ・ Thr, Ser, 1 / 2Cys, Met are 24, 4
Extrapolated to zero time by taking the time-dependent change of 8,72 hours.

・ Val and Ile are 72-hour values.

・ Trp is the average of 24, 48 and 72 hours 2) Sugar composition analysis 200 ng of purified CSF sample used in the above amino acid composition analysis
After adding 25 nmol of inositol as an internal standard to
A methanol solution (500 μ) containing 1.5N HCl was added and the reaction was carried out at 90 ° C. for 4 hours in a sealed tube in which the atmosphere was replaced with nitrogen gas.
After opening the tube, the mixture was neutralized by adding silver carbonate (Ag ₂ CO ₃ ), shaken with 50 μl of acetic anhydride, and left overnight at room temperature in the dark. The upper layer was placed in a sample tube and dried with nitrogen gas. Methanol was added to the precipitate, which was washed and then centrifuged briefly, and the upper layer was added to the same sample tube and dried. 50μ to this
TMS reagent (mixed with pyridine: hexamethyldisilazane: trimethylchlorosilane = 5: 1: 1) was added, reacted at 40 ° C. for 20 minutes, and then stored in Deep Freezer. As a standard, galactose (Ga
l), N-acetylgalactosamine (GalNAc),
50 nmol of sialic acid and 25 nmo of inositol
1 was combined and the same operation was performed.

Gas chromatographic analysis was performed on this sample under the following conditions.

(Analysis conditions) Column: 2% OV-17VINport HP 60-80 mesh, 3 m, glass Temperature: 110 ° C-250 ° C up to 4 ° C / min Carrier gas: Initially 1.2-1.6 Kg / cm ² (nitrogen pressure) At the end, ^{2 to} 2.5 Kg / cm ² Sensitivity: 10 ³ MΩ range 0.1 to 0.4 V Pressure: Hydrogen gas 0.8 Kg / cm ² Air 0.8 Kg / cm ² Sample amount: 2.5 to 3.0 μ As a result of analysis, CSF of the present invention From this, galactose, N-acetylgalactosamine and sialic acid were confirmed.

Example 18. Preparation of pHGV2 vector (for animal cells,-
VSE system) E of the cDNA obtained in Example 10 and shown in FIG.
The coRI fragment was treated with the restriction enzyme DraI at 37 ° C. for 2 hours, and then treated with the Klenow fragment of DNA polymerase I (Takara Shuzo) to make the ends blunt. 1μ
AT of ggl BglII linker (8mer; manufactured by Takara Shuzo)
After phosphorylation with P, it was ligated with about 1 μg of the DNA fragment mixture obtained above. Then the restriction enzyme Bgl
After treatment with II, agarose gel electrophoresis was performed, and only the largest DNA fragment was recovered.

As shown in FIG. 6, this DNA fragment corresponded to about 700 base pairs containing the portion encoding the human G-CSF polypeptide. Vector pdKCR (Fukunaga et al .; Proc. Nat
l. Acad. Sci. USA; 81 : 5086 (1984)) with restriction enzyme B
After treating with amHI, the vector DNA obtained by dephosphorylating with alkaline phosphatase (Takara Shuzo) was used.
4G DNA ligase (Takara Shuzo) was added and ligated with the cDNA fragment to obtain pHGV2 (FIG. 11). As shown in FIG. 11, this plasmid contains the promoter of the SV40 early gene, the replication initiation region of SV40, a part of the rabbit β-globin gene, the replication initiation region of pBR322 and pB322.
It contains a β-lactamase gene (Amp ^r ) derived from R322, and human G is located downstream of the SV40 early gene promoter.
-The CSF gene is connected.

Example 19 Construction of recombinant vector for C127 cells (-V
SE) 1) Construction of pHGV2 (H) 20 μ of pHGV2 plasmid (FIG. 11) obtained in Example 18
Using g, a plasmid named pHGV2 (H) was obtained by the same method as described in 1) of Example 13 (FIG. 1).
2).

2) Recombinant expression vector pTN-V2 and pTN
Construction of VAα and pTNVAβ Using 20 μg of pHGV2 (H) obtained in 1) above,
PHGV2 was obtained in the same manner as described in Example 13-2).
E. coli carrying a plasmid containing the cDNA of the G-CSF derived from E. E. coli colonies were selected. The resulting plasmid is named pTN-V2 (Fig. 12).

On the other hand, adenovirus Type II [protein nucleic acid enzyme 27, 12
Monthly issue (1982), published by Kyoritsu Shuppan], VAI and VA
Fragments containing VAI and VAII were recovered from the plasmid ΔpVA containing the approximately 1700 bp SalI-HindIII fragment containing II. This fragment is the H of pTN-V2 described above.
pTNVAα and pTNV inserted into the indIII site
Aβ was obtained (Fig. 12). In this plasmid, the expression efficiency of the transcription product from the SV40 early promoter was enhanced by the adenovirus VA gene.

Example 20 Transformation of C127 cells and expression thereof (-
VSE) pTN-V2 obtained in Example 19 is treated with a restriction enzyme BamHI before being transformed into mouse C127 cells.

Then, mouse C127I cells were transformed with the above-prepared DNA for expression (see Example 14), and a clone having high G-CSF-producing ability was selected. As a result, a G-CSF production level of ˜1 mg / was observed.

By continuing the cloning, a clone having a G-CSF productivity of 10 mg / level could be selected. Similarly, pTNVAα and pT obtained in Example 19 were obtained.
C127 cells were respectively transformed with NVAβ, and G-C
As a result of selecting clones having high SF production ability, pTNVA
Regarding α, a high-producing clone of 20 mg / or higher could be obtained. Also, a clone having a productivity of several mg / can be obtained from pTNVAβ.

In addition to the above C127I cells, NIH is used as a host cell.
3T3 cells can also be used.

Example 21 Expression of G-CSF by CHO cells (-VS
E) 1) Construction of pHGV2-dhfr From 20 μg of pHGV2 plasmid obtained in Example 18 to Example
0.5 μg each of the approximately 2.7 kb fragment and the pAdD26SVpA fragment obtained by the method described in 1) of 15).
Each was annealed. This plasmid was transformed into E. coli
The DHI strain was transformed by the rubidium chloride method to pHG.
Colonies harboring the V2-dhfr plasmid were selected. The resulting plasmid was named pHGV2-dhfr (Fig. 13a).

As an alternative method to the above, the pHV2 plasmid was replaced with SalI.
Ec without treatment and without the addition of an EcoRI linker
It was partially digested with oRI and a fragment of about 2.7 kb was recovered.
The DNA fragment was treated with E. coli DNA polymerase-Klenow fragment to blunt end the ends.

On the other hand, a blunt-ended EcoRI fragment of pAdD26SVpA was prepared in the same manner as described above, and both were treated with T4DN.
It was also possible to prepare pHGV2-dhfr by treating with A ligase.

Also, pHGV2 (H) obtained in 1) of Example 19 was treated with the restriction enzymes HindIII and SalI as described in 2) of Example 13 and the HindIII-SalI fragment was blunt-ended with EcDd26SVpA.
It was also possible to obtain pHGV2-dhfr by ligation to the oRI fragment (Fig. 13b).

2). Construction of pV2DR1 and pV2DR2 10 g of pAdD26SVpA described in 1) was added to 50 mM.
Tris-HCl (PH7.5), 7mM MgCl 2, 1
It was dissolved in 50 ml of a reaction solution containing 00 mM NaCl, 7 mM 2-mercaptoethanol and 0.01% BSA, and 10 units each of restriction enzymes EcoRI and BamHI were added, and 37
The reaction was carried out at 10 ° C for 10 hours. Phenol treatment and ether washing were performed according to a conventional method. After recovering a DNA fragment of about 2 Kb by 1% low melting point agarose electrophoresis, it was blunt-ended with a DNA polymerase-Klenow fragment according to a conventional method, followed by phenol treatment, ether washing and ethanol precipitation.

On the other hand, pHGV2 (H) 10μ obtained in 1) of Example 19
g 10 mM Tris-HCl (PH7.5), 7 mM
It was dissolved in 50 μl of a reaction solution containing MgCl ₂ and 60 mM NaCl, 10 units of HindIII was added, and the mixture was reacted at 37 ° C. for 6 hours. The DNA fragment was recovered by 1% low-melting point agarose electrophoresis according to a conventional method, and further subjected to BAP treatment, and then Kl
Blunt-ended with the enow fragment. After phenol treatment and ether washing, D of about 2Kb as described above
Blunt end ligation was performed using the NA fragment and T4 DNA ligase. That is, 1 μg of each DNA fragment is 66 m
M Tris-HCl (pH7.5), 6.6 mM MgC
Reaction solution containing l ₂ , 5 mM DTT, 1 mM ATP 30
Dissolve in μ, add 50 units of T4 DNA ligase,
After reacting at 6 ° C. for 12 hours, E. E. coli strain DHI. Thus, the pV2D shown in FIG.
R1 and pV2DR2 were obtained.

3) Transformation and expression Using the pHGV2-dhfr plasmid prepared in 1) above, CH was prepared in the same manner as described in 3) of Example 15.
The O cell line was transformed and expressed.

In addition, the transformation of CHO cells is
Co-transformation of GV2 and pAdD26SVpA (Co tra
nsformation).

CHO cells were also transformed by the method described below. That is, pV2D prepared in the above item 2)
R1 or pV2DR2 are each Sal previously
A DNA fragment was obtained by treatment with I and KpnI, and 10 μg of the DNA fragment was transformed into CHO cells in the same manner as above. When the cells thus transformed were continued to be cultured in the selective medium as described above, 100 or more clear colonies appeared per plate on about the 7th day. When colonies were not individually selected but transferred to a new plate again and continued to be cultured in a selection medium in the presence of 0.01 μM MTX, dozens of colonies appeared. Furthermore, the concentration of MTX is 0.02 μM, 0.05 μM, 0.1
The colonies that survived were selected by raising the concentration to μM.
Moreover, even after selecting each of the obtained dozens of colonies and increasing the MTX concentration, similar colonies could be selected.

In addition, a recombinant vector having a so-called polycistronic gene was constructed, and this could be used to transform CHO cells. That is, pAdD26SVpA is set to Ps
By treating with tI, recovering the two fragments, and ligating these with the CSF cDNA fragment derived from pBRV2, adenovirus promoter, CSF, cDNA, DHF
A recombinant vector in which the poly A sites of R and SV40 were arranged in this order was constructed and put into a CHO cell to carry out.

Example 22 G-CSF activity assay (-VSE) of expressed substance C127 cells and C obtained in Example 20 and Example 21
Human G-CSF was obtained from the culture supernatant of HO cells in the same manner as in Example 16, and its human G-CSF activity was assayed. The results are shown in Table-3.

Example 23 Amino acid analysis and sugar analysis (-VSE) 1) Analysis of amino acid composition The crude CSF sample obtained in Example 22 was further purified according to the method of Example 2 (iii). This purified CSF sample was subjected to amino acid composition analysis by the method described in Example 17 1). The results are shown in Table-4.

2) Sugar composition analysis Using the purified CSF sample used in the above amino acid composition analysis, the sugar composition was analyzed by the same method and the same analysis conditions as described in 2) of Example 17. As a result of the analysis, galactose, N-acetylgalactosamine and sialic acid were confirmed from the CSF of the present invention.

Example 24 Construction of chromosome-derived gene-containing recombinant vector for COS cells The plasmid pBRCE3β containing the chromosomal gene shown in FIG. 5 obtained in Example 11 was treated with EcoRI. On the other hand, the pSVH ⁺ K ⁺ plasmid described in Banerji et al. (Cell 27, 299 (1981)) was treated with KpnI to remove the globin gene, and then partially digested with HindIII to partially digest the late gene of SV40. Was removed and then religated to prepare an expression vector pML-E ⁺ .

This vector was treated with a restriction enzyme EcoRI, and then dephosphorylated with alkaline phosphatase (Takara Shuzo), and the resulting vector DNA was ligated with the above chromosomal DNA fragment by adding T4 DNA ligase (Takara Shuzo) to obtain pML.
CE3α was obtained. As shown in FIG. 14, this plasmid contains SV40 gene enhancer, SV40 replication initiation region, pBR322 replication initiation region and pBR32.
A human G-CSF chromosomal gene is connected downstream of the enhancer of the SV40 gene in a plasmid containing the β-lactamase gene (Amp ^r ) derived from 2.

Example 25 Expression of Human G-CSF Chromosome Gene in COS Cells Using a DMEM (Nissui Pharmaceutical Co., Dulbecco's modified Eagle medium "Nissui") medium (10 ml) containing 10% fetal calf serum, Petri having a diameter of 9 cm was used. COS-1 cells (Cold Spring Harb, USA) grown to approximately 70% dense in a dish (Nunc)
or Institute Dr. Gluzman) (Gluzman) and the phosphate-calcium method (Wigler et al .; Cell 14 725 (1978)) and DEA.
E-dextran: chloroquine method (eg Gordon et al .;
Science 288 , p. 810 (1985)).

In the case of the phosphate-calcium method, it was carried out as follows.

Plasmid pMLCE3α 160 μg prepared in Example 24
Was dissolved in 320 μm of TE solution, 3.2 ml of distilled water was added, and then 504 μm of 2M CaCl ₂ was added.

Add 4 ml of 2 x HBS [50 mM Hepes, 28
0 mM NaCl, 1.5 mM phosphate buffer, (pH7.12)]
Was added and ice-cooled for 20 to 30 minutes, and then 1 ml was added dropwise to each Petri dish in which COS-1 cells were grown. CO at 37 ℃
After culturing for 4 hours in a ₂ incubator, the cells were washed with serum-free DMEM medium, then 5 ml of DMEM medium containing 20% glycerol was added and left at room temperature for about 3 minutes, and again with serum-free DMEM medium. Washed. Serum-free D
After removing the MEM medium, 10 ml of DMEM medium containing 10% fetal calf serum was added, and the cells were cultured overnight in a CO ₂ incubator, and the medium was exchanged again with the same medium for further 3 days of culture.

When the DEAE-dextran: chloroquine method is used, it is as follows.

Like the calcium phosphate method, COS-1 cells were cultivated to 70% confluence, and the cells were washed twice with serum-free DMEM medium. To this was added 250 μg / ml DEAE-dextran and 2 μg / ml plasmid pMLC prepared in Example 24.
A serum-free DMEM medium containing E3α was added, and the mixture was cultured at 37 ° C for 12 hours. Then, the cells were washed twice with serum-free DMEM medium, and DMEM containing 10% fetal calf serum and 1 mM chloroquine was added.
Culture was continued in the medium at 37 ° C. for 2 hours. Thereafter, the cells were washed twice with serum-free DMEM medium, DMEM medium containing 10% fetal calf serum was added, and the cells were cultured at 37 ° C. for 3 days.

The culture supernatant of COS-1 cells thus obtained was
The pH was adjusted to 4 with N acetic acid, an equal volume of n-propanol was added, the resulting precipitate was removed by centrifugation, and an open column (1φ × 2) packed with a C8 reverse phase carrier (manufactured by Yamamura Chemical Co., Ltd.)
cm) and eluted with 50% n-propanol. The eluate was diluted twice with water, and then subjected to reverse phase high performance liquid chromatography using a YMC-C8 column (manufactured by Yamamura Chemical Co., Ltd.) to obtain a linear concentration gradient n- of 30 to 60% containing 0.1% TFA.
Elute with propanol. n-propanol concentration is 40
The fraction eluted at a position of about% was collected, lyophilized and dissolved in 0.1 M glycidin buffer (pH 9).
Through such a process, human G-CSF was concentrated about 20 times from the COS-1 cell supernatant.

As a control, human G-CS according to the method described above
COS-1 cells were transformed with pML-E ⁺ containing no F chromosome gene, and the culture supernatant was concentrated.

The "human G-C" described in Reference Example for the obtained preparation was obtained.
Human GC by a method based on SA measurement method (a) ”
SF activity was assayed. The results are shown in Table-5.

Example 26 Expression of human G-CSF chromosomal gene by C127 cells The pMLCE3α plasmid obtained in Example 24 was transformed with Eco.
After treatment with RI, a fragment of about 4 Kb is recovered as a source of the G-CSF gene derived from the chromosome by the method described in Molecular Cloning, supra.

This is treated with DNA polymerase I-Klenow fragment to leave it as a blunt end (A).

On the other hand, SV4 was prepared from the pHGA410 plasmid prepared in Example 12 by the same molecular cloning method as above.
0 promoter part (EcoRI-about 0.4 of EcoRI
Kb fragment), and DNA polymerase I-Kle
Treat with now fragment (B).

Furthermore, the plasmid pdBPV-1 containing bovine papilloma virus (BPV) (Sarver, N., Sbyrne, JC & Ho.
wley, P.W. M. (1982) Proc. Natl. Acad. Sci. , USA 7
(Vol. 9, 7147-7151; obtained from Dr Howley) was treated with HindIII and PvuII to obtain a DNA fragment of about 8.4 Kb, which was treated with DNA polymerase I-Klenow fragment,
Dephosphorylate with bacterial alkaline phosphatase (C).

The above DNAs (A), (B) and (C) are each 0.1 μm
20μ reaction solution (50mM Tris-HCl (pH
7.6), 10 mM MgCl ₂ , 10 mM DTT, 1 mM
ATP), dissolved in 180 units of T4 DNA ligase, and reacted at 4 ° C. overnight.

Using this reaction solution, the pTNCE3α plasmid was obtained by the rubidium chloride method described in Molecular Cloning above.

As the source of the G-CSF gene derived from the chromosome, 20 μg of pMLCE3α was replaced with 10 mM instead of the above (A).
Tris-HCl (pH8.0), 7 mM MgCl ₂ , 100
mM NaCl, 7 mM 2-mercaptoethanol, 0.
After dissolving in 100 μl of 01% BSA mixture, adding 20 units of StuI and incubating at 37 ° C. for 5 hours, a DNA fragment of about 1.78 Kb was obtained by 1.2% agarose gel electrophoresis. Good.

The pTNCE3α plasmid thus obtained was used in the examples
Mouse C127I cells were transformed and expressed in the same manner as in 14, and a clone having high G-CSF producing ability was selected.

Example 27 Expression of human G-CSF chromosomal gene by CHO cells As in the case of C127 cells in Example 26, pMLCE3α was expressed.
The plasmid is treated with StuI to recover a DNA fragment of about 1.78 Kb, or treated with EcoRI to recover an EcoRI fragment of about 4 Kb as a source of the G-CSF gene derived from the chromosome.

This is treated with DNA polymerase I-Klenow fragment (a).

Also, as in Example 26, pHGA410 to SV40
The promoter part (EcoRI-EcoRI fragment) is cut out to obtain a fragment of about 0.4 Kb, which is similarly treated with DNA polymerase I-Klenow fragment (b).

On the other hand, pAdD26SVpA plasmid (Kaufman,
R. G & Sharp, P.G. A (1982) Mol. Cell. Biol, Volume 2 13
04-1319) is treated with EcoRI, then treated with DNA polymerase I-Klenow fragment, and subsequently treated with bacterial alkaline phosphatase to dephosphorylate (c).

0.1 μg each of (a), (b) and (c) above 2
0 μ reaction solution (50 mM Tris-HCl (pH 7.6),
10 mM MgCl ₂ , 10 mM DTT, 1 mM ATP)
It was then dissolved in, and 180 units of T4 DNA ligase was added, and the mixture was reacted overnight at 4 ° C.

Then, the reaction solution was treated with E. coli by the rubidium chloride method described in the above Molecular Cloning. E.coli DHI strain to obtain Tet ^r colonies, and to obtain plasmid p
Bacteria containing D26SVCE3α were selected.

The plasmid pD26SVCE3α is SV as shown in FIG.
The CSF gene is linked to the 40 early genes, and the DHFR gene is linked to the downstream of the adenovirus major late promoter.

On the other hand, a DNA fragment containing a DHFR gene of about 2 Kb obtained by treating pAdD26SVpA with EcoRI and BamHI as described in Example 15-2) and EcoRI-Sa of (a) and pHGA410 (H) described above.
The Amp ^r expression vector pDRCE3α (FIG. 15) was constructed by ligating with the II fragment.

This pD26SVCE3α plasmid and pDRCE
The 3α plasmid was transformed into CHO cells in the same manner as in Example 15, and MTX selection was repeated to obtain a G-CSF producing strain.

Example 28 G-CSF activity assay for expressed substances (human chromosome-derived gene) From the culture supernatants of C127 cells and CHO cells obtained in Examples 26 and 27, the same method as described in Example 25 was performed. Human G-CSF was obtained and its human G-CSF activity was assayed. The results are shown in Table-6.

Example 29 Infection protective effect of human G-CSF <Test method> 1. Pseudomonas ae
Ruginosa) Protective effect against infection 8 to 9-week-old (body weight 35.3 ± 1.38 g) IICR mouse (male) was intraperitoneally administered with 200 mg / Kg of endoxan (Shionogi Co., Ltd., trade name) 3 groups Divided into 2
Group G containing human G-CSF (25000u / mouse or 50000u / mouse) in a solvent (1% propanol in physiological saline, 0.
5% ((w / v) mouse serum albumin), and to another group, the solvent alone, was subcutaneously administered four times at 0.1 ml every 24 hours. Three hours after the fourth administration, Pseudomonas aeru
ginosa) GNB-139 (3.9 × 10 ⁵ CFU / mouse) was subcutaneously administered to infect. Twenty-one hours after the infection, a solvent containing human G-CSF (25000 u / mouse or 50,000 u / mouse) or only the solvent was subcutaneously administered to the corresponding groups.

The protective effect against infection was examined by the number of surviving mice up to 10 days after infection.

(Preparation of Bacterial Solution) Pseudomonas aeruginosa GNB-139 is shake-cultured overnight at 37 ° C. in a heart infusion liquid medium (manufactured by Difco, trade name). The culture solution was prepared by suspending it in physiological saline.

<Results> The same CHO used for the amino acid composition analysis of Example 17
The above test was performed on a purified human G-CSF sample (+ VSE) derived from cells.

The results are shown in Table-7.

In the same manner, the same protective results as above were obtained when the infection protection effect of the above test was examined using the same purified human G-CSF sample derived from C127 cells used for the amino acid composition analysis of Example 17.

The same CHO used for the amino acid composition analysis of Example 23.
The above test was performed on purified human G-CSF samples (-VSE) derived from cells.

The results are shown in Table-8.

In the same manner, the same protective results were obtained when the infection protection effect of the above-mentioned test was examined using the same purified human G-CSF sample derived from C127 cells used in the amino acid composition analysis of Example 23.

〔The invention's effect〕

The glycoprotein having human G-CSF activity of the present invention is extremely useful as an active ingredient such as a leukopenia therapeutic agent, a myelogenous leukemia therapeutic agent, a hematopoietic function recovery-promoting agent or an infection control agent.

Further, the establishment of a human G-CSF production method by gene recombination that embodies these long-awaited pharmaceuticals can be said to be a great achievement of the present invention.

[Embodiment]

1) A gene encoding a polypeptide having human granulocyte colony-stimulating factor activity is obtained, and then a recombinant vector incorporating the gene is prepared, and then a host cell is transformed with the vector, and then the obtained trait is obtained. A transformant strain is cultivated, and a target glycoprotein is collected from the culture, which has a polypeptide represented by the following amino acid sequence or a part thereof and a sugar chain portion, and has human granulocyte colony-stimulating factor activity. Method for producing glycoprotein.

Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Ph
e Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln
Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu (Val Ser Gl
u) _m Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Le
u Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Al
a Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu
Try Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pr
o Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp
Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Gl
u Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln
Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Ar
g Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser
Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Al
a Gln Pro (where m represents 0 or 1) 2 A gene encoding a polypeptide having human granulocyte colony stimulating factor activity was analyzed by sucrose density gradient centrifugation.
A method for producing a glycoprotein according to the first embodiment, which is a DNA complementary to a messenger RNA encoding a polypeptide having human granulocyte colony stimulating factor activity, which is obtained as a 15-17S fraction. .

3. The method for producing a glycoprotein according to item 1, wherein the gene encoding the polypeptide having human granulocyte colony stimulating factor activity encodes the polypeptide sequence shown below or a part thereof.

Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Ph
e Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln
Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu (Val Ser Gl
u) _m Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Le
u Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala
Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Al
a Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu
Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pr
o Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp
Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Gl
u Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln
Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Ar
g Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser
Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Al
a Gln Pro (where m represents 0 or 1) 4 A gene encoding a polypeptide having human granulocyte colony stimulating factor activity has a nucleotide sequence shown below or a part thereof: Embodiment 1 A method for producing the glycoprotein according to the item.

ACC CCC CTG GGC CCT GCC AGC TCC CTG CCC CAG AGC TT
C CTG CTC AAG TGC TTA GAG CAA GTG AGG AAG ATC CAG
GGC GAT GGC GCA GCG CTC CAG GAG AAG CTG (GTG AGT GA
G) _m TGT GCC ACC TAC AAG CTG TGC CAC CCC GAG GAG CT
G GTG CTG CTC GGA CAC TCT CTG GGC ATC CCC TGG GCT
CCC CTG AGC AGC TGC CCC AGC CAG GCC CTG CAG CTG GC
A GGC TGC TTG AGC CAA CTC CAT AGC GGC CTT TTC CTC
TAC CAG GGG CTC CTG CAG GCC CTG GAA GGG ATC TCC CC
C GAG TTG GGT CCC ACC TTG GAC ACA CTG CAG CTG GAC
GTC GCC GAC TTT GCC ACC ACC ATC TGG CAG CAG ATG GA
A GAA CTG GGA ATG GCC CCT GCC CTG CAG CCC ACC CAG
GGT GCC ATG CCG GCC TTC GCC TCT GCT TTC CAG CGC CG
G GCA GGA GGG GTC CTG GTT GCC TCC CAT CTG CAG AGC
TTC CTG GAG GTG TCG TAC CGC GTT CTA CGC CAC CTT GC
C CAG CCC (where m represents 0 or 1)

[Brief description of drawings]

FIG. 1 shows the sequences of probe (IWQ), probe (A) and probe (LC). FIG. 2 shows the nucleotide sequence of pHCS-1 insert. FIG. 3 (A) shows the nucleotide sequence of the pBRG4 cDNA insert. FIG. 3 (B) (I) shows the amino acid sequence of the human G-CSF precursor deduced from pBRG4 cDNA. FIG. 3 (B) (II) shows the amino acid sequence of human mature G-CSF deduced from pBRG4 cDNA. FIG. 4 (A) shows the nucleotide sequence of the pBRV2 cDNA insert. FIG. 4 (B) (I) shows the amino acid sequence of the human G-CSF precursor deduced from pBRV2 cDNA. FIG. 4 (B) (II) shows the amino acid sequence of human mature G-CSF deduced from pBRV2 cDNA. FIG. 5 shows the nucleotide sequence of a human chromosomal gene encoding human G-CSF. FIG. 6 shows human G-CSF derived from pBRG4 or pBRV2.
The restriction enzyme cleavage site of cDNA is shown. FIG. 7 shows a human chromosomal gene restriction enzyme cleavage site encoding human G-CSF. FIG. 8 shows a schematic structure of pHGA410. FIG. 9 shows recombinant vectors pTN-G4 and pTN-G for expression.
4 shows the construction process of 4VAα and pTN-G4VAβ. 10a and 10b show the construction process of pHGG4-dhfr. Figure 10c shows the construction process of pG4DR1 and pG4DR2. FIG. 11 shows a schematic structure of pHGV2. FIG. 12 shows recombinant vectors pTN-V2 and pTN-V for expression.
3 shows the construction process of Aα and pTN-VAβ. 13a and 13b show the recombinant vector pHGV2 for expression.
-Shows the construction process of dhfr. Figure 13c shows the construction process of pV2DR1 and pV2DR2. FIG. 14 shows a schematic structure of pMLCE3α. FIG. 15 shows a schematic structure of pD26SVCE3α and pDRCE3α.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location (C12P 21/02 C12R 1:91) (31) Priority claim number Japanese Patent Application No. 60-270839 (32) ) Priority date Sho 60 (1985) December 3 (33) Priority claiming country Japan (JP) Microbial accession number FERM BP-954 Microbial accession number FERM BP-955 Microbial accession number FERM BP-956

Claims (2)

[Claims] 1. A human granulocyte colony stimulating factor activity produced by culturing a mammalian cell transformed with a recombinant vector containing a gene derived from a human chromosome containing a nucleotide sequence encoding the following amino acid sequence, and then producing it. A method for producing a glycoprotein having a human granulocyte colony stimulating factor activity, which comprises isolating a glycoprotein having: Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Ph
e Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln
Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Th
r Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu
Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Se
r Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Le
u Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly
Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Ph
e Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly
Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pr
o Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly
Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Va
l Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 2. The method for producing a glycoprotein having human granulocyte colony stimulating factor activity according to claim 1, wherein the human chromosome-derived gene is a gene containing the following base sequence. ACC CCC CTG GGC CCT GCC AGC TCC CTG CCC CAG AGC TT
C CTG CTC AAG TGC TTA GAG CAA GTG AGG AAG ATC CAG
GGC GAT GGC GCA GCG CTC CAG GAG AAG CTG TGT GCC AC
C TAC AAG CTG TGC CAC CCC GAG GAG CTG GTG CTG CTC
GGA CAC TCT CTG GGC ATC CCC TGG GCT CCC CTG AGC AG
C TGC CCC AGC CAG GCC CTG CAG CTG GCA GGC TGC TTG
AGC CAA CTC CAT AGC GGC CTT TTC CTC TAC CAG GGG CT
C CTG CAG GCC CTG GAA GGG ATC TCC CCC GAG TTG GGT
CCC ACC TTG GAC ACA CTG CAG CTG GAC GTC GCC GAC TT
T GCC ACC ACC ATC TGG CAG CAG ATG GAA GAA CTG GGA
ATG GCC CCT GCC CTG CAG CCC ACC CAG GGT GCC ATG CC
G GCC TTC GCC TCT GCT TTC CAG CGC CGG GCA GGA GGG
GTC CTG GTT GCC TCC CAT CTG CAG AGC TTC CTG GAG GT
G TCG TAC CGC GTT CTA CGC CAC CTT GCC CAG CCC