JPH025395B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention is a novel and specific stimulating factor necessary for colony formation mainly of human granulocytic cells, that is, colony stimulating factor (hereinafter referred to as "CSF").
The present invention relates to a glycoprotein having activity (abbreviated as ) and a method for producing the same. [Conventional technology] When using a two-layer soft agar culture method with bone marrow cells as target cells in the upper layer and kidney cells or fetal cells in the lower layer, some of the cells in the upper layer proliferate and differentiate, resulting in neutrophil lineage. Since colonies consisting of granulocytes (hereinafter referred to as ``granulocytes'') and monocyte macrophages are formed, it was known that factors that promote colony formation exist in living organisms (Pluznik and Sach ；J.Cell.Comp.Physiol, Volume 66
319 pages (1965), Bradley and Metcalf; Aust.J.Exp.
Biol.Med.Sci. vol. 44 , p. 287 (1966)). This factor, collectively referred to as CSF, is known to be normally produced by cells that are widely distributed in the body, such as T cells, monocyte macrophages, fibroblasts, and endothelial cells. CSF contains granulocyte-monocyte macrophage stem cells that act on granulocyte-monocyte macrophage stem cells to stimulate their proliferation and induce differentiation, forming colonies consisting of granulocytes and monocyte macrophages in soft agar. Spherical macrophage CSF (GM
Monocyte macrophage CSF (abbreviated as M-CSF), which mainly acts to form colonies of monocyte macrophages, and pluripotent CSF (abbreviated as M-CSF), which acts on undifferentiated pluripotent stem cells
There are subclasses such as granulocytic CSF (abbreviated as G-CSF), or granulocytic CSF (abbreviated as G-CSF), which mainly acts to form granulocytic colonies as in the present invention, and each subclass affects the differentiation of target cells. It has come to be considered that there are different stages [Asano; Metabolism and
Disease, vol. 22 , p. 249 (1985), Yunis et al.;
“Growth and Maturation Factors”edited by
Guroff, John Wiley & Sons, NY, 1 volume,
209 pages (1983)]. Therefore, it is extremely important to purify individual subclasses and investigate their chemical and biological properties in more detail for the analysis of blood structure mechanisms and the pathology of various hematological diseases. Among the biological effects of G-CSF, induction of differentiation of myeloid leukemia cells and hyperfunction of mature granulocytes are attracting attention.
-The clinical usefulness of CSF has been highly anticipated. [Problems to be Solved by the Invention] Previous attempts to isolate and purify G-CSF have focused on extracting G-CSF from the culture supernatant using cell culture methods.
-This is a method for isolating CSF, but it has drawbacks such as the fact that only low concentrations of G-CSF are produced and that obtaining a trace amount of G-CSF from a large amount of culture solution requires a complicated purification process. Still large amounts of homogeneous G-CSF
I had not yet reached the point where I could obtain it. Therefore, recombinant
There has been a desire to produce G-CSF in large quantities using DNA technology. [Means for solving the problem] In this situation, the present inventors have developed human G-
We have successfully isolated a gene encoding a polypeptide with CSF activity and expressed the gene in host cells. The present invention has a base sequence encoding the amino acid sequence shown in Figure 3B or Figure 4B.
Human granulocyte colony stimulating factor activity is characterized by culturing mammalian cells transformed with a recombinant vector containing a cDNA fragment, and then degrading the produced glycoprotein having human granulocyte colony stimulating factor activity. The present invention provides a method for producing a glycoprotein having the following properties. The present invention will be explained in detail below. The gene encoding a polypeptide having human G-CSF activity used in the present invention includes Messenger gene encoding a polypeptide having human G-CSF activity, which is obtained as a 15-17S fraction by sucrose density gradient centrifugation. RNA (mRNA) has complementary DNA (cDNA). Applicants have obtained two series of such cDNAs.
One series of cDNAs has a gene encoding the polypeptide shown in Figure 3B or a portion thereof, and more specifically, the cDNA shown in Figure 3A.
Sequence from ATG at 32nd to 34th nucleotide position to CCC at 650th to 652nd nucleotide position from the 5′-end of the base sequence, ACC at position 122 to 124th to CCC at position 650 to 652nd
It has the sequence up to CCC or the sequence shown in FIG. 3A, or a part thereof. This series of cDNAs is called cDNA (+VSE). The other series of cDNAs have a gene encoding the polypeptide shown in Figure 4B or a part thereof, and more specifically, the cDNA contains the nucleotide sequence 640 to 640 from ATG at the 31st to 33rd nucleotide position from the 5'-end of the base sequence shown in Figure 4A. Sequence up to CCC at nucleotide position ~642,
It has the sequence from ACC at positions 121 to 123 to CCC at positions 640 to 642, or the sequence shown in FIG. 4A, or a portion thereof. This series of cDNAs is called cDNA(-VSE). The above gene is obtained by preparing mRNA encoding G-CSF from mammalian cells having the ability to produce a polypeptide having G-CSF activity, and then converting it into double-stranded cDNA by a known method.
It can be obtained by screening a collection of recombinants containing this DNA (hereinafter referred to as a cDNA library) using known methods. On the other hand, in the present invention, the mammalian cell serving as the source of the mRNA is cell line CHU-2 (collection Nationale De Culturer) derived from human oral floor cancer.
es De Microorganismes (CNCM) Deposit Number I
-483) However, it is not limited to tumor cell lines, but may also be cells that can be isolated from mammals or other established cell lines. In addition, mRNA can be prepared using methods that have already been used to clone the genes of several other physiologically active proteins, such as surfactant treatment and phenol treatment in the presence of a ribonuclease inhibitor using a vanadium complex. Do (Berger and
Biochemistry, Vol. 18, p. 5143 (1979)) or CsCl density gradient centrifugation after treatment with guanidine thiocyanate (Chirgwin
etc.; see Biochemistry Vol . 18 , p. 5294 (1979))
Possibly, after obtaining total RNA, oligo(dT)
-Poly(A ⁺ ) RNA by affinity column method or batch method using polyU-Sepharose etc. with cellulose or Sepharose 2B as a carrier.
(mRNA) can be obtained. Further, poly(A ⁺ ) RNA can be further fractionated by sucrose density gradient centrifugation or the like. The mRNA obtained as above is G-CSF
To confirm that the protein encodes an active polypeptide, methods such as translating the mRNA into a protein and examining its physiological activity, or identifying the protein using an anti-G-CSF antibody, are performed. That's fine. For example, Xenopus
(Gurdon et al.; see Nature, vol. 233 , p. 177 (1972)), or the rabbit reticulocyte system or wheat germ (Wheat germ).
Translation reactions using systems are being carried out (Schleif
and Wensin K; “Practical Methods in
Molecular Biology”, Springer-Verlag, NY,
(1981)). G-CSF activity can be assayed by applying a soft agar culture method using bone marrow cells. There is a review of these methods (Metcalf; “Hemopoietic
Colonies”, Springer-Verlag, Berlin,
Heideiberg, N.Y. (1977)). Using the mRNA obtained by the method described above as a template, 1
After synthesizing double-stranded cDNA, double-stranded cDNA is synthesized from this single-stranded cDNA, and a recombinant plasmid with an appropriate vector DNA is created. This is used to transform Escherichia coli, etc., and a DNA group (cDNA library) of the transformed strain is obtained. To obtain double-stranded cDNA from mRNA, for example,
Either an oligo (dT) complementary to the poly A chain at the 3'-end of mRNA is used as a primer and treated with reverse transcriptase, or an oligonucleotide corresponding to part of the amino acid sequence of G-CSF protein is synthesized. This is used as a primer and treated with reverse transcriptase to synthesize cDNA complementary to mRNA. double strand
cDNA is produced by decomposing and removing mRNA using alkaline treatment, and then converting the resulting single-stranded cDNA to reverse transcriptase or
DNA polymerase (e.g. Klenow fragment)
After treatment, it can be obtained by treating with SI nuclease, etc., or it can also be obtained by directly treating with RNase H and DNA polymerase (for example, Escherichia coli DNA polymerase, etc.) (for example, Naniatis et al.; Molecular cloning). ,Cold,
Spring Harborlaboratory (1982) and Gubler
and Hoffman; Gene 25 , p. 263 (1983)). The double-stranded cDNA thus obtained is transferred to an appropriate vector, such as pSC101, pDF41, ColE1,
EK type plasmid vectors such as pMB9, pBR322, pBR327, pACYC1, λgt.λc,
After integrating into phage vectors such as λgt10 and λgtWES, E. coli (X1776;
HB101; DH1, C600 strain, etc.), etc.
A cDNA library can be obtained (see, eg, "Molecular cloning", supra). To link double-stranded cDNA with a vector,
In order to attach a ligatable end to the DNA end, an appropriate chemically synthesized DNA fragment is added and added to the vector DNA, which has been previously cleaved using a restriction enzyme, in the presence of ATP.
This can be done by treatment with T4 phage DNA ligase. Alternatively, dG, dC-strands (or dA, dT-strands) are added to vector DNA and double-stranded cDNA that have been previously cleaved using restriction enzymes.
This can also be done, for example, by slowly cooling the solution containing both DNAs after adding
(see Molecular cloning). For example, when the host cell is E. coli, transformation of the host cell with the recombinant DNA obtained in this way is possible.
A method such as that described in detail by Hanahan (J.
Mol.Biol.; Vol . 166 , p. 557 (1983)), i.e.
This can be carried out by adding the recombinant DNA to competent cells prepared in the coexistence of CaCl ₂ , MgCl ₂ or RbCl. To search for cells harboring the gene of interest, the plus-minus method used in cloning interferon cDNA (Taniguchi et al.;
Proc.Jpn.Acad. Vol. 55 , Ser.B, p. 464 (1979)),
Hybridization-translation assay method (Nagata et al., Nature 284 , p. 316 (1980)), or colony or plaque hybridization using chemically synthesized oligonucleotide probes based on the amino acid sequence of the protein. Dyzation method (Wallace et al.; Nucleic
Acids Res. 9 , 879 (1981) and Benton.
Davise: Science, Vol. 196, p. 180 (1977), etc. may be used. Human G-CSF cloned in this way
Fragments containing genes encoding active polypeptides can be reincorporated into appropriate vector DNA to transform other prokaryotic or eukaryotic host cells. Furthermore, by introducing an appropriate promoter and expression-related sequences into these vectors, it is possible to express the gene in each host cell. Host cells derived from mammals include COS cells,
Chinese hamster ovary (CHO) cells, C
-127 cells, HeLa cells, etc. Vectors for transforming these cells include:
pSV2−gpt (Mulligan and Berg; Proc. Natl. Acad.
Sci.USA; Vol. 78, p. 2072 (see (1981)), etc. These vectors contain an origin of replication, a selection marker, a promoter located in front of the gene to be expressed, an RNA splice site, a polyadenylation signal, and the like. Promoters for gene expression in mammalian cells include retroviruses, polyomaviruses, adenoviruses, and simian viruses40.
A promoter such as (SV40) may be used. For example, when using the SV40 promoter,
This can be easily carried out according to the method of Mulligan et al. (Nature, Vol. 277 , p. 108 (1979)). As a replication origin, those derived from SV40, polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can be used, and as a selection marker, phosphotransferase can be used.
APH (3') or (neo) gene, thymidine kinase (TK) gene, E. coli xanthine-
A guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (DHFR) gene, etc. can be used. Using the above host-vector system, human G-
In order to obtain a polypeptide having CSF activity, after transforming a host cell with a recombinant DNA body in which the gene has been inserted into an appropriate site of the above-mentioned vector,
The obtained transformant may be cultured. Furthermore, the polypeptide can be separated and purified from cells or culture fluid using known means. In general, eukaryotic genes are thought to exhibit polymorphism (for example, as is known for the human interferon gene).
Nishi et al.; J.Biochem. 97 , p. 153 (1985)), this polymorphism may result in the substitution of one or more amino acids, or even if there is a change in the base sequence, the amino acid may not change at all. Also, for example, a polypeptide lacking or adding one or more amino acids in the amino acid sequence of FIG. 3B or FIG. 4B, or one or more amino acids replaced with one or more amino acids. Polypeptides may also have G-CSF activity. For example, it is already known that a polypeptide obtained by converting the base sequence corresponding to cysteine in the human interleukin-2 (IL-2) gene to a base sequence corresponding to serine retains interleukin-2 activity. (Wang et al.; Science, Vol. 224 , p. 1431 (1984)). Therefore, as long as those naturally occurring or artificially synthesized polypeptides have human G-CSF activity, animal cells transformed with recombinant vectors containing genes encoding those polypeptides (transgenic All glycoproteins obtained by culturing the transformant) are included in the present invention. A gene necessary for obtaining the glycoprotein having human G-CSF activity of the present invention, a recombinant vector containing the gene, a transformant containing the same, and the desired saccharide obtained by culturing this transformant. The outline of the manufacturing method for each protein is as follows. (1) Preparation of probe The amino acid sequence of the homogeneous human CSF protein obtained by purification from the culture supernatant of the tumor cell line CHU-2 was determined from the N-terminus, and the amino acid sequence was further fragmented by bromcyan digestion, trypsin treatment, etc. The amino acid sequences of the fragments were also determined [Example 3(i), (ii), (iii)]. Three types of nucleotide probes (A) corresponding to the sequence shown in Figure 1 from among the amino acid sequences,
Probe (LC) and probe (IWQ) were synthesized (Example 4). Probe (A) is a mixed type probe consisting of 14 consecutive nucleotides. The probe (IWQ) was used in the cloning of the human cholecystokinin gene (Takahashi et al.;
Proc. Natl. Acad. Sci., USA, Vol. 82 , p. 1931 (1985)) 30 consecutive nucleotides using deoxyinosine. The probe (LC) was synthesized from the portion corresponding to positions 32 to 39 from the N-terminus of the amino acid sequence shown in Example 3(i) based on the base sequence shown in Figure 3.
A probe consisting of 24 nucleotides. Chemical synthesis of nucleotides can be carried out by applying the improved phosphotriester method to the solid phase method, as described in the review by Narang (Tetrahedron, Vol . 39 , pp. 3-22 (1983)). The probe used may be based on an amino acid sequence at a position other than the probe used in the present invention. (2) Construction of cDNA library Add guanidine thiocyanate solution to CHU-2 cells, homogenize, and obtain total RNA by CsCl density gradient centrifugation. After selecting poly(A ⁺ ) RNA from this total RNA using an oligo-dT cellulose column, single-stranded cDNA was synthesized using reverse transcriptase, and RNase H
and using E. coli DNA polymerase.
Double-stranded cDNA was obtained. Obtained double-stranded cDNA
A dC chain was added to the Pst cleavage site, and the pBR322 vector was ligated to a dG chain added to the Pst cleavage site.
A library was constructed (Examples 5 and 6). Similarly, using the EcoRI linker, double-stranded
The cDNA was ligated with the λgt10 vector to construct a λ phage-based cDNA library (Example 7). (3) Screening The recombinant derived from the pBR322 cDNA library was immobilized on Watzmann 541 filter paper, and colony hybridization was performed using probe IWQ radiolabeled with ³² P. As a result, one clone was selected. did it. This clone was extracted using the Southern blotting method (Southern; J. Mol. Biol. 98
When further detailed examination was performed using the probe (Vol. 503, 1975), it was found that both probe A and probe A hybridized. The nucleotide sequence of this clone was determined by the dideoxy method (Sanger; Science Vol. 214 , p. 1205 (1981)). Figure 2 shows the nucleotide sequence of the obtained cDNA insert.
Shown below. As shown in Figure 2, this cDNA insert consists of 308 base pairs including probe IWQ and probe A, and has an open reading frame encoding 83 amino acids including the amino acid sequence shown in Example 3(iii). I found out that The pBR322-derived plasmid containing this 308 base pairs is hereinafter abbreviated as pHCS-1 (Example 8). Contains 308 base pairs obtained from pHCS-1
The DNA fragment was radiolabeled using the nick translation method (see above, Molecular Cloning), and this was used as a probe for the cDNA derived from λgt10.
The library was screened by plaque hybridization (Benton and Davis; Science 196, p. 180 (1977)) to obtain five clones, and the nucleotide sequence of the clones thought to contain cDNA was determined in the same manner as described above. (FIG. 3A). As shown in FIG. 3A, this cDNA insert has one large open reading frame. The amino acid sequence encoded by this cDNA can be mapped as shown in FIG. 3A. By comparison with the N-terminal amino acid sequence of G-CSF protein shown in 3(i), this
The cDNA consists of 32 to 34 nucleotides from the 5′-end.
A signal peptide encoded by 90 base pairs starting from the ATG sequence and ending with the GCC sequence at positions 119-121 and by 531 base pairs starting from the ACC sequence at positions 122-124 and ending with the CCC sequence at positions 650-652. It was found that it contained a base sequence corresponding to the encoded mature G-CSF polypeptide. Therefore, the polypeptide with the amino acid sequence shown in FIG. 3B consists of 207 amino acids,
Its molecular weight was calculated to be 22292.67 Daltons.
Similarly, the polypeptide of the amino acid sequence consisted of 177 amino acids, and its molecular weight was 18986.74 Daltons (Example 9). However, regarding the start site of the protein, 32~
ATG that ranks 34th or 68th to 70th could be considered as well. The E. coli strain X1776 carrying PBR322 with this cDNA (+VSE) inserted into the EcoR1 cleavage site has been deposited at the National Institute of Microbiology, Agency of Industrial Science and Technology (FERM BP
−954). FIG. 5 shows the oxygen-limiting cleavage site of the obtained gene. In addition, this cDNA was added to pBR327 [Soberon et al.;
Gene, Vol . 9 , p. 287 (1980)] at the EcoR site is called pBRG4. The pBRG4 obtained in this way is treated with the restriction enzyme EcoR to obtain approximately 1500 base pairs.
DNA fragments containing cDNA are subjected to nick translation method (see Molecular cloning above).
Using this as a probe, the cDNA library derived from λgt10 was again screened by plaque hybridization (see Benton and Davis, supra). At this time, two sheets of nitrocellulose filter paper with immobilized λ phage DNA were prepared at the same time, and similar plaque hybridization was performed using the aforementioned probe LC to select phages that were positive with both probes. Select clones that appear to be full length and use the dideoxy method to
The nucleotide sequence of the cDNA insert was determined and was as shown in Figure 4A. This cDNA has one large open reading frame, and the encoded amino acid sequence can be mapped as shown in Figure 4A. By comparison with the N-terminal amino acid sequence of G-CSF protein shown in Example 3(i), this
The cDNA consists of 31 to 33 nucleotides from the 5′-end.
A signal peptide encoded by 90 base pairs starting from the ATG sequence and ending with the GCC sequence at positions 118-120 and by 522 base pairs starting from the ACC sequence at positions 121-123 and ending with the CCC sequence at positions 640-642. It was found that it contained a base sequence corresponding to the encoded mature G-CSF polypeptide. Therefore, the polypeptide with the amino acid sequence shown in FIG. 4B consists of 204 amino acids,
Its molecular weight was calculated to be 21977.35 Daltons.
Similarly, a polypeptide with an amino acid sequence of 174
Consisting of amino acids, its molecular weight is 18671.42
Dalton (Example 10). However, regarding the start site of the protein, 31~
ATG in 58th to 60th or 67th to 69th place other than 33rd place
can be considered similarly. The E. coli X1776 strain carrying pBR327 with this cDNA (-VSE) inserted into the EcoR cleavage site has been deposited at the National Institute of Microbial Technology, Agency of Industrial Science and Technology (FERM BP-955). FIG. 5 shows the restriction enzyme cleavage sites of the obtained gene. Furthermore, a plasmid in which this cDNA was ligated to pBR327 at the EcoR site is called PBRV2. (4) Construction of recombinant vectors for animal cells Recombinant vectors when using C127 cells and NIH3T3 cells as host cells (BPV-derived)
and construction of recombinant vectors (containing DHFR) when using CHO cells +VSE system, -
Each test was carried out for VSE cDNA. Typical examples will be described here, but please refer to Examples for details. (A) Construction of +VSE-based recombinant vector The cDNA (+VSE) fragment obtained in (3) above was inserted into the vector pdKCR to create a pHGA410 plasmid (Example 11) (Fig. 6).
Partially digest with EcoR to make blunt ends. Hind to this DNA
After adding a linker, Hind treatment, and T4 DNA ligase treatment, the E. coli DH strain was transformed using the rubidium chloride method (see Molecular Cloning, supra). The obtained plasmid is named pHGA410(H) (Figure 7). pHGA410(H) is treated with Sal, then the ends are blunt-ended, and then treated with Hind again to recover Hind-Sal fragments. On the other hand, the pdBPV-1 plasmid containing the bovine papillomavirus transformation fragment was Hind
The larger DNA fragment is separated by treatment with Pvu and ligated to the previous Hind-Sal fragment. This was used to transform E. coli DH strain to obtain pTN-G4, a plasmid containing pHGG4-derived CFS-cDNA (FIG. 7) (Example 12). Meanwhile, pHGA410 plasmid or pHGA410
(H) Recombinant vector for CHO cells (+VSE) using plasmid and pAdD26SVpA plasmid
pHGG4-dhfr was constructed (Figures 8a and b) (Example 14). Furthermore, a DNA fragment of approximately 2 Kb containing the DHFR gene was extracted from pAdD26SVpA by EcoR and BamH.
Collected by treatment and Hind of pHGA410(H)
pG4DR1 and pG4DR2 were constructed by inserting into the site (Fig. 8c) (Example 14). (B) Construction of -VSE-based recombinant vector The cDNA (-VSE) fragment obtained in (3) above was inserted into the vector pdKCR to create a pHGV2 plasmid (Example 17), which was partially digested with EcoR and the ends blunt end
end). A Hind linker is added to this DNA, and then Hind treatment is performed to create T4DNA.
After ligase treatment, this was used using the rubidium chloride method (see Molecular Cloning, supra).
E. coli DH strain was transformed. The obtained plasmid is named pHGV2(H) (Figure 10). pHGV2(H) is treated with Sal, then the ends are blunt-ended, and then treated with Hind again to recover the Hind-Sal fragment, which also contains the transforming fragment of bovine papilloma virus.
The pdBPV-1 plasmid is treated with Hind and Pvu to separate the larger DNA fragment, which is ligated to the previous Hind-Sal fragment. This was used to transform E. Coli DHI strain and pHGV2
a plasmid carrying the CSF-cDNA derived from
pTN-V2 is obtained (FIG. 10) (Example 18). +pHGV2 plasmid or PH as well as VSE
A recombinant vector for CHO cells (-
VSE) pHGV2-dhfr was constructed (Figures 11a and b) (Example 20). Furthermore, a DNA fragment of approximately 2 Kb containing the DHFR gene was extracted from pAdD26SVpA by EcoR and BamH.
They were recovered by treatment and inserted into the Hind site of pHGV2(H) to construct pV2DR1 and pV2DR2 (Fig. 11c) (Example 20). (5) Character expression by animal cells Representative examples will be described here, but for other cases, please see each example. Expression in Mouse C127 Cells The pTN-G4 plasmid or pTN-V2 plasmid is treated with BamH, and used to transform C127 cells that have been cultured and grown using the phosphate-calcium method. The obtained transformed cells are cultured and clones with high CSF production ability are selected. The expressed G-CSF was recovered and purified from the culture medium of these transformed cells, and was confirmed to exhibit 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, the CSF samples used for amino acid analysis were subjected to amino sugar quantification using the Elson-Morgan method, neutral sugar quantification using the orcinol sulfuric acid method, and sialic acid quantification using the thiobarbituric method. The quantitative method is described in Chapter 13 of Biochemistry Experiment Course Volume 4, "Chemistry of Carbohydrates (Volume 2)" (Tokyo Kagaku Doujin). As a result of converting the weight% from each quantitative value, the obtained G
- The sugar content of CSF was distributed in the range 1-20 (% by weight). EXAMPLES The present invention will be described in detail with reference to Examples below, but before that, a method for measuring CSF activity will be explained using Reference Examples. Reference example Method for measuring CSF activity CSF activity used in the present invention (hereinafter referred to as
The method for measuring CSA (abbreviated as CSA) is as follows. “CSA measurement method” (a) When using human bone marrow cells: Method of Bradley TR, Metcalf D., etc. (Aust.J.Exp.Biol.Med.Sci. 44 , 287-300)
The culture was carried out using the monolayer soft agar culture method according to the method described in J. P., 1966). Namely, 0.2 ml of fetal bovine serum, 0.1 ml of test specimen, and 0.1 ml of human bone marrow non-adherent cell suspension.
(1 to 2 x 10 ⁵ nucleated cells), 0.2 ml of modified McCoy's 5A culture solution, and 0.4 ml of modified McCoy's 5A culture solution containing 0.75% agar were mixed and placed in a tissue culture plastic container with a diameter of 35 mm. After solidification, culture was performed at 37℃, 5% carbon dioxide/95% air, and 100% humidity, and the number of colonies formed after 10 days (one colony is a colony consisting of 50 or more cells). CSA was determined by counting the activity of forming one colony as one unit. (b) When using mouse bone marrow cells: 0.4 ml of horse serum, 0.1 ml of test sample, C3H/He
(Female) Mouse osteopathic cell suspension 0.1ml (0.5~
1 x 10 ⁵ nucleated cells), modified with 0.75% agar
After mixing 0.4ml of McCoy's 5A culture solution and solidifying it in a 35mm diameter plastic tissue culture dish, it was kept at 37℃, 5% carbon dioxide/95% air, and 100% humidity for 5 days. Cultivate, count the number of colonies formed (one colony is a colony consisting of 50 or more cells), and count the activity of forming one colony as one unit.
Asked for CSA. In addition, the "modification" used in methods (a) and (b) above
The McCoy's 5A culture solution and the human bone marrow non-adherent cell suspension used in (a) were prepared as follows. "Modified McCoy's5A culture solution (2x concentration)"
McCoy's5A culture solution (manufactured by GIBCO) 12g,
MEM amino acid vitamin medium (manufactured by Nissui Pharmaceutical Co., Ltd.)
After dissolving 2.55 g of sodium bicarbonate, 2.18 g of sodium bicarbonate, and 50,000 units of potassium penicillin G in 500 ml of twice-distilled water, the solution was filtered using a 0.22 μm Millipore filter. “Human non-adherent cell suspension” Bone marrow fluid obtained from healthy human sternal puncture.
Diluted 5 times with PRMI1640 culture medium, Ficol-
Layered with Paque liquid (manufactured by Pharmacia), 400
Centrifuge at ×g for 30 minutes at 25°C to collect the cell layer at the interface (specific gravity <1.077). After washing the cells, the concentration was adjusted to 5 × 10 ⁶ cells/ml with RPM1640 culture medium containing 20% fetal bovine serum, and 25
Place in a cm ² tissue culture plastic flask.
After incubating for 30 minutes in a carbon dioxide incubator, collect the non-adherent cells in the supernatant and re-incubate in a 25cm ²
After the cells were placed in a plastic flask and incubated for 2 hours and 30 minutes, the supernatant non-adherent cells were collected and used. Example 1 Establishment of "CHU-2" A tumor from a patient with oral floor cancer in which a marked increase in neutrophils was observed was transplanted into nu/nu mice. Approximately 10 days after transplantation, significant tumor growth and increased number of neutrophils were observed. Twelve days after transplantation, the tumor was excised aseptically and cut into 1-2 mm ^square pieces, which were cultured as follows. Place 10 to 15 pieces of the tumor mass cut into pieces into a 50 ml plastic centrifuge tube, add 5 ml of trypsin solution (containing 0.25% trypsin and 0.02% EDTA), and hold at 37°C.
After shaking in a hot bath for 10 minutes, the supernatant was discarded, 5 ml of the same trypsin solution was added again, and trypsin digestion was performed at 37° C. with stirring for 15 minutes. Collect the supernatant cell suspension and add 1 ml of fetal bovine serum.
ml to stop the action of trypsin, and then stored on ice. The above procedure was repeated to collect the cell suspension, and the cell suspension was combined with the previous suspension to obtain a cell pellet by centrifugation at 1500 rpm for 10 minutes. This cell pellet was mixed with F containing 10% fetal bovine serum.
After washing twice with -10, the cells were implanted into 25 cm ² plastic culture flasks at a cell concentration of 5 x 10 ⁶ cells/flask, and supplemented with 10% fetal bovine serum.
After incubating overnight in a carbon dioxide incubator (carbon dioxide concentration 5%, humidity 100%) using the F-10 culture solution containing F-10, the supernatant was removed together with non-adherent cells, and a new culture solution was added. In addition, culture was continued. Six days after the start of culture, the cells proliferated profusely, so at this point the culture medium was replaced with a new one. The next day, discard this culture solution and add anti-mouse red blood cell antibody (manufactured by Cappel) diluted 5 times with RPM1640.
2 ml of guinea pig complement (manufactured by Kyokuto Pharmaceutical Co., Ltd.) diluted 2.5 times with RPM1640 was added to the 2 ml, and the mixture was incubated at 37°C for 20 minutes. After incubation, wash twice with F-10 containing 10% fetal bovine serum.
Fibroblasts derived from nu/nu mice were stripped, followed by addition of F-10 nutrient solution containing 10% fetal bovine serum, and cultured for an additional 2 days. After that, a portion of the cells were removed and cloned by limiting dilution method. I went there. When the CSF activity of the 11 clones obtained was examined, a clone (CHU-2) was obtained that exhibited approximately 10 times higher activity than the others. Example 2 Isolation of CSF Cells were collected from two 150 cm ² culture flasks in which the cells established as described above had grown completely and densely, and cultured in F-10 containing 10% fetal bovine serum. After suspending it in 500ml of liquid, transfer it to a 1580cm ² glass roller bottle (manufactured by Belco) and rotate at 0.5rpm.
Rotary culture was performed at a speed of . When the cells had grown completely and densely on the inner wall of the roller bottle, the culture medium was changed to serum-free RPM1640, and after culturing for 4 days, the culture supernatant was collected, and 10% fetal bovine serum was added.
The culture is continued by adding F-10. After culturing for 3 days, the culture medium was changed to RPMI1640 that does not contain serum, and the culture supernatant was collected 4 days later. By repeating the same procedure, 500 ml of serum-free culture supernatant can be obtained from one bottle every week.
Moreover, by this method, it was possible to maintain cells for a fairly long period of time and recover the culture supernatant. The obtained culture supernatant 5 was made into one batch, and after adding 0.01% Tween 20 to it, Hollow Fiber
DC-4 and Amicon PM-10 (manufactured by Amicon)
After concentrating about 1,000 times by ultrafiltration using a filtrate, this was purified in the following order. (i) Using an Ultrogel AcA54 column (manufactured by LKB) with a diameter of 4.6 cm and a length of 90 cm, 0.15 M NaCl and 0.01
5 ml of the concentrated culture supernatant was gel-filtered using 0.01 M Tris-HCl buffer (PH7.4) containing % Tween 20 (manufactured by Hanui Chemical Co., Ltd.) at a flow rate of about 50 ml/hour. The column was pre-filled with bovine serum albumin (molecular weight 67000) and ovalbumin (molecular weight
45,000) and cytochrome C (molecular weight 12,400). After gel filtration, collect 0.1 ml from each fraction, dilute it 10 times, and proceed as described above in "CSA measurement method (b)".
The fractions showing activity were examined by As a result, we first found that the fraction with Ve = 400 to 700 ml showed macrophage-dominated CSA, and the fraction with Ve = 800 to 1200 ml showed granulocyte-dominated CSA.
The latter fractions were collected and concentrated to about 5 ml using an ultrafilter using PM-10 (manufactured by Amicon). (ii) 0.1% containing 30% n-propanol (manufactured by Tokyo Kasei Co., Ltd., for amino acid sequencing) in the above concentrated fraction
After adding a trifluoroacetic acid aqueous solution and leaving it on ice for about 15 minutes, the precipitate was removed by centrifugation at 15,000 rpm for 10 minutes. Next, after adsorption onto a μBondapak C18 column (manufactured by Waters, for semi-preparative use, 8 mm x 30 cm) equilibrated with an aqueous solution containing n-propanol and trifluoroacetic acid,
Contains n-propanol with a linear concentration gradient of 60%
It was sequentially eluted with 0.1% trifluoroacetic acid aqueous solution.
Absorption at 220 nm and 280 nm was measured simultaneously using a Hitachi 685-50 high performance liquid chromatography device and a Hitachi 638-41 detector (both manufactured by Hitachi, Ltd.) for detection. After elution, collect 10 μl from each fraction.
After diluting 100 times, use the method described above for measuring CSA.
(b)" fractions showing activity were examined. As a result, activity was observed in the peak eluted with n-propanol 40%, so this peak was collected and re-chromatographed under the same conditions, and CSA was examined in the same manner as above. Activity was observed in the peak at the % position, so this peak was collected (4 fractions = 4
ml) was lyophilized. (iii) Add 40% n-propanol to the above lyophilized powder.
The solution was dissolved in 200μ of a 0.1% trifluoroacetic acid aqueous solution, and subjected to high performance liquid chromatography (HPLC) using a TSK-G3000SW column (manufactured by Toyo Soda Co., Ltd., 7.5 mm x 60 cm). Elution was performed with the same aqueous solution at a flow rate of 0.4 ml/min, and fractions of 0.4 ml were collected using a fraction collector FRAC-100 (manufactured by Pharmacia). As a result of examining CSA in each separated fraction in the same manner as above, activity was observed in the fraction with a retention time of 37 to 38 minutes (equivalent to a molecular weight of approximately 20,000), so this fraction was collected. , and an analytical μBondapak C18 column (4.6mm
The main peak was collected and freeze-dried. The obtained product was assayed by the above-mentioned "CSA measurement method (a)" and was 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 digestion using a gas phase sequencer (manufactured by Applied Biosystems), and the obtained PTH amino acid was subjected to high performance liquid chromatography. Equipment (manufactured by Beckman Instruments) and
Analysis was carried out by a conventional method using an Ultrasphere-ODS column (manufactured by Beckman Instruments). Column (5 μm, diameter 4.6 mm, length 250 mm) was coated with starting buffer (15 mM sodium acetate buffer PH
4.5, an aqueous solution containing 40% acetonitrile), the sample (dissolved in 20 μl of starting buffer) was injected, and separation was performed by isocratic elution with the starting buffer. The flow rate is 1.4
ml/min, column temperature was maintained at 40°C. PTH
Amino acids were detected using ultraviolet absorption at 269 nm and 320 nm. 2 nmol of each standard PTH amino acid (manufactured by Sigma) was separated in advance in the same system to determine the retention time, and identification was performed from the retention time of the test analyte. As a result, the amino acid sequence from the N-terminus to the 40th residue was determined as follows. H ₂ N−Thr−Pro−Leu−Gly− Pro−Ala−Ser−Ser−Leu− Pro−Gln−Ser−Phe−Leu− Leu−Lys−Cys−Leu−Glu− Gln−Val−Arg−Lys− Ile− Gln−Gly−Asp−Gly−Ala− Ala−Leu−Gln−Glu−Lys− Leu−Cys−Ala−Thr−Tyr− Lys− (ii) Bromcyane decomposition The sample was dissolved in 70% formic acid and purified by sublimation. 200 equivalents of bromcyane were added and reacted overnight at 37°C. Next, after freeze-drying the reaction product, TSK
Using G3000SW column (manufactured by Toyo Soda)
It was fractionated by HPLC and four peaks were obtained. The peaks are CN-1, CN-2, CN- in descending order of molecular weight.
3. CN-1 with good yield, named CN-4;
The amino acid sequence of CN-2 was analyzed using an automatic gas phase sequencer (manufactured by Applied Biosystems) under the same conditions as (i). As a result, CN-1 is the N of G-CSF protein.
It was found that the peptide originates from the terminal end. Furthermore, CN-2 had the following amino acid sequence. Pro−Ala−Phe−Ala−Ser− Ala−Phe−Gln−Arg−Arg− Ala−Gly−Gly−Val−Leu− Val−Ala−Ser−His−Leu− Gln− (iii) Trypsin digestion. Dissolved in 0.1M Tris-HCl buffer (PH7.4) containing urea and 0.1M Tris-HCl buffer (PH7.4) containing 0.1% 2-mercaptoethanol.
was added to make the final concentration of 2M urea. The sample was then divided into enzymes at a ratio of 50:1.
Add TPCK-treated trypsin (trade name manufactured by Sigma) and react at 25°C for 4 hours, then add the same amount of TPCK-treated trypsin and incubate again at 25°C.
The reaction was allowed to proceed 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 is 0.1% TFA
The n-propanol concentration was increased linearly from 5% to 60%. Among the peaks obtained by measuring 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 is (ii) CN
It was found that the peptide had the following sequence containing a part of the -2 fragment. Gln−Leu−Asp−Val−Ala− Asp−Phe−Ala−Thr−Thr− Ile−Trp−Gln−Gln−Met− Glu−Glu−Leu−Gly−Met− Ala−Pro−Ala−Leu−Gln− Pro−Thr−Gln−Gly−Ala− Met−Pro−Ala−Phe−Ala− Ser− Example 4 Creation of DNA probe (i) Synthesis of probe IWQ Among the amino acid sequences obtained in Example 3 (iii) from
Ile−Trp−Gln−Gln−Met−Glu−Glu−Leu
Based on the sequence of 10 amino acids represented by -Gly-Met, 30 consecutive nucleotides were obtained (Figure 1). In the arrangement of Figure 1, for example, 5'-
The 9th nucleotide from the end indicates a mixture containing equal amounts of dA and dG. Dimers were mainly used as raw nucleotides, and mononucleotides were also used as needed. 20 mg of the starting material nucleotide resin Ap-d (G) (manufactured by Yamasa Soy Sauce Co., Ltd.) was placed in a column equipped with a glass filter, and after repeated washing with methylene chloride, the column was washed with a methylene chloride solution containing 3% trichloroacetic acid.
4,4'-dimethoxytrityl group is eliminated,
The column was then washed several times with 1 ml of methylene chloride. After washing with anhydrous pyridine and replacing the solvent, the nucleotide dimer (DMTr) ApTp
(NHR ₃ ) (manufactured by Nippon Zeon; NHR ₃ indicates triethylammonium, DMTr indicates dimethoxytrityl) 20 mg and 0.2 ml of pyridine were added, and the inside of the column was vacuum-dried using a vacuum pump. Next, after adding 20 mg of 2,4,6-trimethylbenzenesulfonyl-3-nitrotriazolide (MSNT, manufactured by Wako Pure Chemical Industries, Ltd.) and 0.2 ml of anhydrous pyridine, the inside of the column was purged with nitrogen gas, and the column was heated at room temperature. to
The nucleotide resin and dimer were condensed by shaking occasionally for 45 minutes. After the reaction was completed, the column was washed with pyridine, and then unreacted OH groups were acetylated with a pyridine solution containing excess acetic anhydride and 4-dimethylaminopyridine, and the column was washed again with pyridine.
Similarly, (DMTr)Ip(NHR ₃ ), (DMTr)
GpGp( _NHR3 ), (DMTr)Ip( _NHR3 ),
(DMTr)CpTp( _NHR3 ) and (MMTr)TpTp
Equal mixture of ( _NHR3 ), (DMTr)ApAp
A mixture of equal amounts of ( _NHR3 ) and (DMTr)ApGp( _NHR3 ), a mixture of equal amounts of (DMTr)ApGp( _NHR3 ) and (DMTr)GpGp( _NHR3 ),
(DMTr)GpAp( _NHR3 ), (DMTr)TpGp
( _NHR3 ), an equal mixture of (DMTr)ApAp( _NHR3 ) and (DMTr)GpAp( _NHR3 ),
(DMTr)CpAp( _NHR3 ), (DMTr)ApAp
Equivalent mixture of (NHR ₃ ) and (DMTr)ApGp(NHR ₃ ), (DMTr)GpGp(NHR ₃ ),
(DMTr)TpGp( _NHR3 ), (DMTr)Ip
(NHR ₃ ), (DMTr)ApTp (NHR ₃ ) [(DMTr)Ip (NHR ₃ ) is manufactured by Yamasa Soy Sauce Co., Ltd.
All others were manufactured by Nippon Zeon Co., Ltd.], and condensation was carried out by repeating the above-mentioned operations. After the final stage of the reaction, the resin was washed with pyridine, methylene chloride, and ether in this order without acetylation, and then dried. The dried resin was mixed with 1M tetramethylguanidine and 1Mα.
-Dioxane 1 containing picoline aldoxime
ml, suspended in 1.7 ml of a mixture of 0.5 ml of pyridine, and 0.2 ml of water, and left at room temperature overnight.
It was concentrated under reduced pressure to 200μ. After adding a small amount (2 to 3 drops) of pyridine to this concentrated solution, 2 to 3 ml of concentrated aqueous ammonia was added and the mixture was heated at 55°C for 6 hours.
Next, ethyl acetate was added for extraction and separation, and the resulting aqueous layer was concentrated under reduced pressure, then dissolved in 50mM triethylammonium acetic acid solution (PH7.0) and applied to a C-18 column (1.0 x 15cm, manufactured by Waters). The mixture was subjected to column chromatography. Elution was performed with acetonitrile in a linear concentration gradient of 10% to 30% in 50 mM triethylammonium acetic acid solution (PH 7.0), and the peak fraction eluted at a position where the acetonitrile concentration was around 25% was concentrated under reduced pressure. After adding 80% acetic acid to this concentrated solution and leaving it at room temperature for 30 minutes, ethyl acetate was added to extract and separate the resulting aqueous layer, which was concentrated under reduced pressure. The obtained concentrate was coated with a C18 column (manufactured by Sensyu Kagaku Co., Ltd.,
It was further purified by high performance liquid chromatography using SSC-ODS-272, 6φ x 200mm). Elution was performed using a linear concentration gradient of 10% to 20% acetonitrile in 50mM triethylammonium acetate solution (PH7.0), 10A ₂₆₀ units.
Synthetic DNA was obtained in the above yield. (ii) Synthesis of probe A From the amino acid sequence obtained in Example 3(iii)
Based on the 5 amino acid sequence shown as Met-Pro-Ala-Phe-Ala, 14 consecutive nucleotides were obtained (Figure 1). Synthesis was performed in the same manner as the probe IWQ, and (DMTr)CpAp( _NHR3 ); (DMTr) was added to the nucleotide resin AP-d(T) (manufactured by Yamasa Soy Sauce Co., Ltd.).
GpGp( _NHR3 ); (DMTr)CpAp( _NHR3 ),
(DMTr)CpTp( _NHR3 ), (DMTr)CpGp
Equivalent mixture of ( _NHR3 ) and (DMTr)CpCp( _NHR3 ); (DMTr)ApGp( _NHR3 ),
(DMTr)TpGp( _NHR3 ), (DMTr)GpGp
Equivalent mixture of ( _NHR3 ) and (DMTr)CpGp( _NHR3 ); (DMTr)ApAp( _NHR3 );
(DMTr)CpAp( _NHR3 ) and (DMTr)CpGp
Equivalent mixture of (NHR ₃ ); (DMTr)Gp
(NHR ₃ ) (both manufactured by Nippon Zeon) were mixed in this order to obtain about 10 A ₂₆₀ units of synthetic DNA. The base sequence of the obtained oligonucleotide
When examined by the Maxam-Gilbert method, it was confirmed that it had the base sequence shown in FIG. (iii) Synthesis of probe LC Applied Biosystems DNA synthesizer
Automatic synthesis was performed using 380A. This method is
The principle described by Caruthers et al. (J.Am.Chem.
Soc, Vol. 103 , p. 3185 (1981)).
It is called the phosphoramidite method. After condensing the phosphoramidite form of (DMTr)-dT previously activated with tetrazole to dG-S (S is the support) from which the 5' dimethoxytrityl group (DMTr) has been deprotected, the unreacted hydroxyl group is converted to acetyl. and then oxidized with iodine in the presence of water to yield a phosphate. Deprotect the DMTr group,
Thereafter, condensation was repeated in the same manner to synthesize 24 nucleotides having the sequence shown in FIG. After the obtained nucleotide was cleaved from the support and deprotected, a C18 column (manufactured by Sensyu Kagaku Co., Ltd.) was used.
It was purified by reversed-phase high performance liquid chromatography using SSC-ODS-272). Example 5 Cultivation of CHU-2 cells and purification of mRNA (1) Cultivation of CHU-2 cells and collection of cells After the established CHU-2 cells were grown completely and densely in two 150 cm ² culture flasks. , this was mixed with RPMI1640 culture medium containing 10% fetal bovine serum.
After suspending in 500 ml, transfer to a 1580 cm ² glass roller bottle (manufactured by Belco) and pump at 0.5 rpm.
Rotary culture was carried out for 4 days at a speed of . When the cells have grown completely and densely on the inner wall of the roller bottle, remove the culture medium from the roller bottle, add 100 ml of physiological saline containing 0.02% EDTA pre-warmed to 37°C, and incubate at 37°C for 2 minutes. Afterwards, the cells were detached 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 without EDTA and centrifuged at 1500 rpm for 10 minutes to obtain a cell pellet (wet weight approximately 0.8
g) The cells thus obtained are stored frozen at -80°C until the RNA extraction procedure is performed. (2) Purification of mRNA Isolation of mRNA from CHU-2 cells obtained as described above is essentially “Molecular
Coloning” [Maniatis et al., Cold, Spring
Harbor, p. 196 (1982)]. CHU that was cryopreserved
-2 cells (3.8 g wet weight) with 20 ml of 6M guanidine solution (6M guanidine thiocyanate, 5 m
Suspend in M sodium citrate (PH7.0), 0.1M β-mercaptoethanol, 0.5% sodium sarcosyl sulfate) and mix with a Vortex mixer for 2 hours.
After ~3 minutes of good mixing, inhalation and expulsion were repeated 10 times using a 20ml syringe fitted with an 18G needle. 6 ml in a polyallomer centrifuge tube that fits the Beckman SW40Ti rotor.
Add 5.7M CsCl - 0.1M EDTA (PH7.5) first, and add about 6 ounces of the above-mentioned guanidine solution, which has broken down and become viscous, so that the tube is filled.
ml was overlaid. The four centrifuge tubes thus prepared were centrifuged at 30,000 rpm and 20°C for 15 hours, and the resulting pellets were washed three times with a small amount of 70% ethanol. The pellets obtained from each tube were combined and dissolved in 550μ of water to adjust the NaCl concentration to 0.2M, and then treated with phenol-chloroform (1:1) and chloroform.
Total RNA was obtained by adding 2.5 times the volume of ethanol and performing ethanol precipitation (total RNA was obtained from 3.8 g of wet cells).
About 10.1 mg of RNA was obtained). Poly(A ⁺ )-RNA was purified from total RNA as follows. This method is affinity chromatography that utilizes the fact that mRNA has a polyA chain added to its 3' end. Oligo(dT)-cellulose (P-L Biochemicals Type 7) was used for adsorption, and total RNA was adsorbed in adsorption buffer (10mM Tris-HCl (PH7.5), 0.5M
After dissolving in NaCl, 1mM EDTA, 0.1% SDS solution) and heating at 65℃ for 5 minutes, it was passed through an oligo(dT)-cellulose column packed with the same solution, and elution was carried out with TE solution (containing 0.1% SDS solution). Ten
The test was carried out using mM Tris-HCl (PH7.5) containing 1 mM EDTA). The unadsorbed effluent was passed through the same column again, subjected to the same elution operation, and mixed with the first eluate. Using this procedure, 400 μg of poly(A ⁺ )-RNA was obtained. The mRNA prepared in this way was mixed with Schleif.
Wensink's Practical Metods in
Molecular Biology, Springer-Verlag;
New York, Heiderberg, Berlin, (1981))
Size fractionation was performed by sucrose density gradient centrifugation using a procedure similar to that described in . That is, a sucrose density gradient of 5% to 25% is created in the tube for the SW40Ti rotor (manufactured by Beckman). Sucrose solution is 0.1M NaCl, 10mM Tris-HCl (PH7.5), 1mM EDTA, 0.5%
5% and 25% in SDS solution, respectively.
Contains RNase-free sucrose (manufactured by Schwarz/Mann). 800 μg of mRNA (poly(A ⁺ )-RNA) prepared by the method described above was dissolved in 200 μl to 500 μl of TE solution, heated at 65°C for 5 minutes, and then rapidly cooled.
Place it on top of the sugar density gradient solution. After centrifugation at 30,000 rpm for 20 hours, collect fractions of 0.5 ml and measure the absorbance at 260 nm.
The size of the fractionated RNA was determined based on the position of the 5S ribosomal RNA) and the G-CSF activity of each fraction was determined based on the position of the Xenopus laevis (Xenopus).
The study was carried out using the oocyte system of A. laevis). Specifically, the mRNA of each fraction was prepared into an aqueous solution with a concentration of 1 μg/μ, and after injecting 50 ng of mRNA into one oocyte taken from a Xenopus laevis (approximately 1 year old), it was placed in a 96-well microtiter plate. Place 10 oocytes in each well and add 100μ of Barth medium (88mM NaCl, 1mM KCl, 2.4
mM _NaHCO3 , 0.82mM _MgSO4 , 0.33mM
Ca( _NO3 ) ₂ , 0.41mM _CaCl2 , 7.5mM Tris-
After culturing at room temperature for 48 hours in hydrochloric acid (PH 7.6), penicillin 10 mg/streptomycin sulfate 10 mg/), the supernatant is collected, concentrated and purified, and G-CSF activity is measured. As a result, G-CSF activity was observed in the 15-17S fraction. Example 6 Synthesis of cDNA (construction of pBR-based cDNA library) From poly(A ⁺ )-RNA obtained by the method described above
The method of Land et al. [Nucleic Acids Res., Vol . 9 , 2251]
cDNA was synthesized based on the method of Guber and Hoffman [Gene, Vol. 25 , p. 263 (1983)]. (1) Synthesis of single-stranded cDNA Add reagents in the following order into a 1.5 ml tube manufactured by Eppendorf. 80μ reaction buffer (500mM KCl, 50mM _MgCl2 , 250mM
M Tris-HCl, PH8.3), 20μ of 200mM dithiothreitol, 32μ of 12.5mM dNTPs
(12.5m each for dATP, dGTP, dCTP, and dTTP)
M), 10μ α- ^32P -dCTP (Amasyam, PB10205), 32μ oligo(dT) _12-18
(manufactured by PL Biochemicals, 500 μg/μ),
A total of 400 μ of a reaction solution consisting of 20 μ of poly(A ⁺ )-RNA (2.1 μg/μ) and 206 μ of distilled water is heated at 65° C. for 5 minutes and then at 42° C. for 5 minutes. 120 units of reverse transcriptase (manufactured by Takara Shuzo) was added to this reaction solution, and after further reaction at 42°C for 2 hours, an RNase inhibitor (Bethesda Research Laboratories) was added.
)2μ, 20μ TE solution, 16μ 100m
M sodium pyrophosphate and 48 units (4μ) of reverse transcriptase were added, and the reaction was then carried out at 46°C for 2 hours. 0.5M EDTA 8μ, 10% SDS 8μ
After stopping the reaction, phenol-chloroform treatment and ethanol precipitation (twice) were performed to obtain single-stranded cDNA. (2) Addition of dC-strand to single-stranded cDNA Dissolve the single-stranded cDNA obtained above in 60μ of distilled water, and then dissolve the single-stranded cDNA obtained above in 60μ of dC-strand addition buffer (400μ).
mM potassium cacodylate; 50mM Tris-HCl (PH6.9); 4mM dithiothreitol; 1m
MCoCl ₂ ; 1mM dCTP) and 5% at 37°C.
Warmed for minutes. This reaction solution was added with terminal transferase (27 units/μ, P-L).
Biochemicals) was added thereto and reacted for 2.5 minutes at 37°C, followed by phenol-chloroform treatment (once) and ethanol precipitation (twice), followed by dissolution in 40μ of TE solution containing 100mM NaCl. (3) Synthesis of double-stranded cDNA Add 4μ of oligo(dG) _12-18 (200μg/ml, P-L) to the above 40μ DNA solution.
Biochemicals) was added and heated at 65°C for 5 minutes, then at 42°C for 30 minutes, and then the reaction solution was kept at 0°C. Add 80μ of buffer (100mM Tris-HCl, PH7.5, 20mMgCl ₂ , 50mM
( _NH4 ) _2SO4 , _500mM KCl), 4mM of 4μ,
dNTPs (containing 4mM each of dATP, dCTP, dGTP, dTTP), 60μ of 1mM β-NAD and
210μ distilled water, 20μ E.coli DNA polymerase (Takara Shuzo Co., Ltd.), 15μ E. coli
DNA ligase (manufactured by Takara Shuzo Co., Ltd.), 15μ E,
After adding coli and RNase H (manufactured by Takara Shuzo Co., Ltd.) and reacting for 1 hour at 12℃,
MdNTP was added and the mixture was reacted at 25°C for 1 hour, followed by phenol-chloroform treatment and ethanol precipitation (once) to obtain about 8 μg of double-stranded cDNA. This double-stranded cDNA was dissolved in a TE solution,
1.2% agarose gel electrophoresis was performed, and a portion corresponding to a size of approximately 560 base pairs (bp) to 2 kilobase pairs (Kbp) was adsorbed onto Watzmann DE81 (manufactured by Watzmann Inc.) and eluted and recovered.
g was recovered. (4) Addition of dC-strand to double-stranded cDNA Add the double-stranded cDNA obtained as above to 40μ
Dissolve in TE solution, add 8μ of the dC-chain addition buffer described in section (2), and warm at 37°C for 2 minutes.
1μ of terminal transferase (27 units/μ) was added and reacted at 37°C for 3 minutes. The reaction solution was immediately cooled to 0°C and diluted with 0.5M
After stopping the reaction by adding 1μ of EDTA, phenol-chloroform treatment and ethanol precipitation were performed, and the resulting precipitate was suspended in 10μ of TE solution. (5) Construction of pBR-based cDNA library Commercially available oligo (dG) chain-added pBR322 vector (manufactured by Bethesda Research Laboratories,
10ng/μ) 4μ and the above dC-chain added double strand
2μ of cDNA was annealed in 75μ of TE solution containing 0.1M NaCl. Annealing at 65℃, 5
After heating for 2 minutes, the mixture was heated at 40°C for 2 hours, and then left to warm to room temperature. On the other hand, Naniatis et al.'s experimental book [Molecular
Cloning, Cold Spring Harbor, p. 249 (1982)], competent cells were prepared from E. coli strain X1776, and transformed with the above-annealed plasmid. body) was obtained. Example 7 cDNA synthesis (Construction of λ phage-based library) (1) Synthesis of single-stranded cDNA Oligo(dT) cellulose was extracted twice from 3.8 g of cryopreserved CHU-2 cells according to the method described in Example 5. Through column purification, 400 μg of poly(A ⁺ )-RNA was obtained. TE in which 12 μg of this poly(A ⁺ )-RNA was dissolved
After placing 10μ of the solution into a reaction tube containing 10μg of actinomycin D (manufactured by Sigma), the reagents were added in the following order: 20μ of reverse transcription buffer (250mM Tris-HCl (PH8.3), 40mM
_MgCl2 , 250mM KCl), 20μ 5mM
dNTP (containing 5mM each of dATP, dGTP, dCTP, dTTP), 20μl oligo(dT) _12-18 (0.2μ
g/ml PL Biochemicals), 1μ
1M dithiothreitol, 2μ 30unit/μ
RNasin (Promega Biotech), 10μ reverse transcriptase (10unit/μ Seikagaku Corporation),
1 μ of α-[ ₃₂ p] dATP (10 μCi manufactured by Amasyam) and 16 μ of water make a total reaction solution volume of 100 μ. After keeping the reaction solution at 42℃ for 2 hours, 5μ
The reaction was stopped by adding 0.5M EDTA and 1μ of 20% SDS. Approximately 4 μg of single-stranded cDNA was obtained by treatment with phenol-chloroform (100 μ) and ethanol precipitation (twice). (2) Synthesis of double-stranded cDNA The cDNA obtained as above was dissolved in 29μ TE solution, and the reagents were added in the following order to prepare a reaction solution; 25μ polymerase buffer (400μ
MHepes (PH7.6); 16mM _MgCl2 ; 63mM β-mercaptoethanol; 270mM KCl);
10μ of 5mM dNTP; 1.0μ of 15mM β
-NAD; 1.0 μ α-[ ³² p] dATP (10 μCi/
μ); 0.2μE.coli DNA ligase (60unit/
μ Takara Shuzo Co., Ltd.); 5.0 μ E. coli DNA polymerase (New England Biolabs Co., Ltd.);
10unit/μ); 0.1μ RNase H (60unit/μ);
(manufactured by μTakara 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 5μ 0.5M EDTA and 1μ
The reaction was stopped by adding 20% SDS, followed by phenol-chloroform treatment and ethanol precipitation. The obtained DNA was dissolved in 20μ of 0.5mM EDTA and 3μ of Klenow buffer (500mM
Tris-hydrochloric acid (PH8.0), 50mM _MgCl2 ), 3μ
After preparing a reaction solution by adding 5mM dNTP and 4μ of water, add 1μ of DNA polymerase (Klenow fragment) (manufactured by Takara Shuzo Co., Ltd.) and incubate at 30℃ for 15 minutes.
Incubate for 1 minute. Dilute this reaction by adding 70μ of TE solution, then add 5μ of 0.5M EDTA, 1μ of 20%
The reaction was stopped by adding SDS. The reaction solution was treated with phenol-chloroform and precipitated with ethanol to obtain about 8 μg of double-stranded cDNA. (3) Methylation of double-stranded cDNA 30μ aqueous solution of double-stranded cDNA synthesized in section (2)
, 40μ of methylation buffer (500mM Tris-HCl (PH8.0), 50mM EDTA), 20μ of SAM solution (800μM S-adenosyl-L-methylmethionine (SAM), 50mM β-mercaptoethanol), and 100μ of water were added. to the mixture
EcoRI Methylase (New England Biolabs
Co., Ltd., 20 units/μ) and add 15μ to the entire reaction solution.
200μ and incubated at 37°C for 2 hours.
After phenol treatment and ether treatment, ethanol precipitation was performed to recover DNA. (4) Addition of EcoRI linker Approximately 1.2 μg of the above methylated double-stranded DNA was added with ligase buffer (250 mM Tris-salt
7.5), 100mM _MgCl2 ) 1.5μ, prephosphorylated EcoRI linker 0.5μ (10mer
(manufactured by Takara Shuzo Co., Ltd.), 10m MATP of 1.5μ, 100mM
Add 1.5 μ of dithiothreitol and 2 μ of H ₂ O, make the reaction mixture 15 μ, add 0.7 μ of T4 DNA ligase (3.4 μ/μ, manufactured by Takara Shuzo Co., Ltd.)
After reacting overnight at 65°C, the ligase was inactivated by heating at 65°C for 10 minutes. Add this reaction solution further
100mM Tris-HCl (PH7.5), 5mM
After adjusting the total volume to 50μ with a concentration of MgCl ₂ , 50mM NaCl, and 100μg/ml gelatin, 3.5μ of EcoRI (10 units/μ) was added.
℃ for 2 hours. Then 0.5M
After adding 2.5μ of EDTA and 0.5μ of 20% SDS, phenol-chloroform treatment was performed and DNA was recovered by ethanol precipitation. After this Ultrogel
Unreacted EcoRI linkers were removed using AcA34 (manufactured by LKB) gel filtration or agarose gel electrophoresis, and the linker-added double-stranded cDNA was
0.5-0.7 μg was recovered. (5) Binding of double-stranded cDNA and λgt10 vector 2.4 μg of the above linker-added double-stranded cDNA was
EcoRI-treated λgt10 vector (Vector Cloning Systems), ligase buffer (250 m
Add 1.4μ of M Tris-HCl, 100mM MgCl ₂ ), and 6.5μ of distilled water and treat at 42°C for 15 minutes, then add 1μ of 10mM ATP, 1μ of 0.1M dithiothreitol, and 0.5μ of T ₄ DNA ligase to bring the total volume to 15μ. After that, the mixture was allowed to react at 12°C overnight. (6) In vitro packaging Approximately 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 library using probe IWQ
541 filter paper was placed on top and left at 37°C for 2 hours. below,
Taub and Thompson's method [Anal.Biochem. Volume 126
222 (1982)]. That is, after the colonies were transferred to 541 filter paper, they were transferred to an agar medium containing chloramphenicol (250 μg/μ), and further left at 37° C. overnight. 541After taking out the filter paper, 0.5N at room temperature
It was left on the filter paper soaked with NaOH solution for 3 minutes, and this was repeated twice. The same operation was carried out twice for 3 minutes using 0.5M Tris-HCl (PH8) solution, and then heated to 4°C with 0.05M Tris-HCl (PH8) solution.
1.5mg/ml lysozyme solution (0.05M
Tris-HCl (PH8) containing 25% sucrose)
for 1×SSC (0.15M NaCl and
0.015M sodium citrate) solution for 2 minutes,
with 1x SSC solution containing 200 μg/ml protease K.
30 minutes, then 95% for 2 minutes in 1x SSC solution at room temperature again.
After two 2 minute runs with the ethanol solution, the 541 filter paper was dried. The obtained dry 541 filter paper was mixed with phenol:chloroform:isoamyl alcohol (25:24:1, 100mM Tris-HCl (PH)) at room temperature.
8.5), 100mM NaCl, 10mM EDTA) solution for 30 minutes. Thereafter, the same operation was performed three times for 3 minutes with a 5×SSC solution, and then twice for 3 minutes with a 95% ethanol solution, and then the filter paper was dried. After radiolabeling the probe IWQ with ^32P according to standard methods (see Molecular cloning),
The method of Wallace et al. (Nucleic Acids Res. Vol. 9 , 879)
(1981)). 6×NET [0.9M NaCl,
0.09M Tris-HCl (PH7.5), 6mM EDTA]
5x Denhardt solution, 0.1% SDS 0.1mg/ml denatured
After prehybridization at 65°C for 4 hours in a hybridization buffer containing DNA (calf thorax DNA), the hybridization containing radiolabeled probe IWQ of 1×10 ⁶ cpm/ml was performed. Hybridization was carried out overnight at 56° C. using hybridization buffer. After the reaction was completed, the 541 filter paper was washed twice for 30 minutes with a 6x SSC solution containing 0.1% SDS at room temperature and for 1.5 minutes at 56°C, followed by autoradiography. After isolating plasmids from clones that produced signals, Southern prototyping was performed using probe IWQ. Hybridization and autoradiography were performed under the same conditions as described above. Southern blotting was performed in the same manner using probe A. Hybridization was carried out at 49°C for 1 hour using the above-mentioned hybridization buffer, and after slow cooling to 39°C, it was further carried out at 39°C for 1 hour. After the reaction is complete, soak the nitrocellulose filter in 6x SSC containing 0.1% SDS for 30 minutes at room temperature.
After washing twice and then at 390°C for 3 minutes, autoradiography was performed. As a result, one clone was obtained as a positive one, and the base sequence was determined by the dideoxy method, and as shown in Figure 2, the probe IWQ
Consisting of 308 base pairs including probe A portion and
The pBR322-derived plasmid containing this insert was named pHCS-1. Example 9 Screening of lambda phage-based library using pHCS-1-derived DNA probe Benton and Davis method [Science vol. 196 , p. 180;
(1977)]. pHCS-1 obtained in Example 8
About 600 base pairs after treatment with Sau3A and EcoRI
A DNA fragment was obtained, and this DNA fragment was radiolabeled by nick translation according to a conventional method. Place a nitrocellulose filter paper (S&S Co., Ltd.) on the agar medium with phage plaque and transfer the phage.
DNA was denatured with 0.5M NaOH, and filter paper was processed in the following order. 0.1M NaOH, 1.5M NaCl
for 20 seconds followed by 0.5M Tris-HCl (PH7.5), 1.5M
NaCl twice for 20 seconds, finally 120mM NaCl, 15mM
Sodium citrate, 13mM KH ₂ PO ₄ , 1mM
Treated with EDTA and PH7.2 for 20 seconds. The filter paper was then dried and heated at 80°C for 2 hours.
DNA fixed, 5x SSC, 5x Denhardt solution, 50mM phosphate buffer, 50% formamide,
0.25mg/ml denatured DNA (salmon testis DNA), and 0.1%
42 in hybridization buffer containing SDS
Perform prehybridization overnight at °C.
Radiolabeled by Nikk translation
Hybridization buffer ( ^5x SSC, 5x
Denhardt solution, 20mM phosphate buffer (PH6.0), 50
% formamide, 0.1% SDS, 10% dextran sulfate, and 0.1 mg/ml denatured DNA (salmon testis DNA)) at 42°C for 20 hours. The nitrocellulose filter paper was washed with 2x SSC containing 0.1% SDS at room temperature for 20 min, then at 44°C.
After washing with 0.1×SSC containing 0.1% SDS for 30 minutes and further washing with 0.1×SSC at room temperature for 10 minutes, detection was performed using autoradiography. As a result, five positive clones G1~
5 was obtained. Therefore, among the obtained clones, the DNA of the clone that seems to contain full-length cDNA
When the base sequence was examined using the dideoxy method, Figure 3A
The nucleotide sequence shown in was obtained. So this
Excise the cDNA from the λgt10 vector and create pBR327.
[Soberon et al.; Gene vol . 9 , p. 287 (1980)] and EcoRI
They were ligated at the site and prepared in large quantities as plasmids. This plasmid is called pBRG4. Example 10 pBRG4-derived DNA probe and probe LC
Screening of λ phage-based library by Plaque hybridization was performed according to the method of Benton and Davis (see the above-mentioned document) used in Example 9. Place nitrocellulose filter paper (manufactured by S&S) on the agar medium containing phage plaque.
Transfer the Phage with 0.5M NaOH.
The DNA was denatured and the filter paper was processed in the following order. 0.1M NaOH, 1.5M NaCl for 20 s, followed by
0.5M Tris-HCl (PH7.5), 1.5M NaCl twice for 20 seconds, and finally 120mM NaCl, 15mM sodium citrate, 13mM KH ₂ PO ₄ , 1mM EDTA, (PH
7.2) for 20 seconds. Then dry the filter paper and
The DNA was fixed by drying at ℃ for 2 hours. In this way, two identical filter papers were prepared, and pBRG4-derived
It was subjected to screening using DNA probe and probe LG, respectively. If using a pBRG4-derived DNA probe, treat pBRG4 with EcoRI to approx.
A 1500 base pair DNA fragment was obtained, and this DNA fragment was radiolabeled by nick translation according to a conventional method. Transfer the above filter paper to 5x SSC, 5x Denhardt
solution, 50mM phosphate buffer, 50% formamide,
0.25mg/ml denatured DNA (salmon testis DNA), and 0.1%
42 in hybridization buffer containing SDS
Prehybridization was carried out overnight at °C, and prehybridization buffer [ ^5x SSC, 5×
Denhardt solution, 20mM phosphate buffer (PH6.0),
Hybridization was performed at 42°C for 20 hours in a mixture of 50% formamide, 0.1% SDS, 10% dextran sulfate, and 0.1 mg/ml denatured DNA (salmon testis DNA). Place the nitrocellulose filter paper at room temperature.
Wash with 2x SSC containing 0.1% SDS for 20 min, then wash with 0.1x SSC containing 0.1% SDS for 30 min at 44 °C.
After further washing with 0.1% SSC at room temperature for 10 minutes,
Detected by autoradiography. For probe LC, filter paper was pretreated with 3x SSC containing 0.1% SDS at 65°C for 2 hours, then 6x
NET, 1× Denhardt solution, 100 μg/ml denaturation
Prehybridization was performed at 65° C. for 2 hours in a solution containing DNA (salmon testis DNA). Radiolabeled probe LC (2 x 10 ⁶ cpm/ml)
Hybridization buffer containing [6x
NET, 1x Denhardt solution, 100 μg/ml denatured
DNA (salmon testis DNA)] at 63°C overnight, the nitrocellulose filter paper was washed at room temperature with 6x SSC containing 0.1% SDS for 20 minutes, and this washing was repeated three times. After this, the cells were washed with 6x SSC containing 0.1% SDS at 63°C for 2 minutes. After drying the filter paper, it was detected by autoradiography. In the screening conducted in this way, clones that were positive for both probes were selected, and the nucleotide sequence of the clone that seemed to contain the full-length cDNA was examined using the dideoxy method, as shown in Figure 4A. A nucleotide sequence as shown below was obtained. Therefore, this cDNA was excised from the λgt10 vector and ligated to pBR327 at the EcoR site.
Plasmid PBRV2 was obtained. Example 11 Preparation of PHGA410 vector (for animal cells, +
VSE system) of the cDNA shown in Figure 3A obtained in Example 9.
The EcoR fragment was treated with the restriction enzyme Dra for 2 hours at 37°C, followed by DNA polymerase Klenow.
Fragment (manufactured by Takara Shuzo Co., Ltd.) was treated to make the ends blunt. After phosphorylating 1 μg of Bgl linker (8mer; manufactured by Takara Shuzo Co., Ltd.) using ATP, it was combined with about 1 μg of the DNA fragment mixture obtained above.
Next, the DNA fragments were treated with restriction enzyme Bgl and subjected to agarose gel electrophoresis, and only the largest DNA fragment was recovered. This DNA fragment is human G-
Approximately 710 containing the portion encoding the CSF polypeptide
It corresponds to a base pair. vectorpdKCR
(FuKunaga et al.; Proc. Natl. Acad. Sci. USA; vol. 81
5086 (1984)) with the restriction enzyme BamHI and then dephosphorylated with alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.), and the resulting vector DNA was transformed into T4DNA.
Ligase (manufactured by Takara Shuzo Co., Ltd.) was added to bind the cDNA fragment to obtain pHGA410 (Figure 6). As shown in Figure 6, this plasmid contains the promoter of the SV40 early gene, the replication initiation region of SV40, the rabbit β
- Contains part of the globin gene, the replication initiation region of pBR322, and the pBR322-derived β-lactamase gene (Amp ^r ), and the human G-CSF gene is connected downstream of the promoter of the SV40 early gene. Example 12 Construction of recombinant vector for C127 cells (+VSE) (1) Construction of pHGA410(H) 20 μg of pHGA410 plasmid (Figure 6) obtained in Example 11 was mixed with 50 mM Tri-HCl (pH 7.5),
7mM _MgCl2 , 100mM NaCl, 7mM2-
Restriction enzyme dissolved in mercaptoethanol and 0.01% bovine serum albumin (BSA) reaction solution
EcoR (manufactured by Takara Shuzo Co., Ltd., 10 to 15 units) was added and reacted for about 30 minutes at 37°C to perform partial digestion with EcoR. Next, the DNA fragments were treated by phenol-chloroform (1:1) treatment twice, ether treatment, and ethanol precipitation. This DNA fragment was dissolved in 50mM Tris-HCl for 5m
M _MgCl2 , 10mM DTT, 1mM dATP,
The mixture was dissolved in 50μ of a solution consisting of dCTP, dGTP, and dTTP, and 5μ of E. coli DNA polymerase-Klenow fragment (manufactured by Takara Shuzo Co., Ltd.) was added and incubated at 14°C for 2 hours to obtain blunt ends. From this, 6 μg of a fragment of about 5.8 Kb was collected by 0.8% agarose gel electrophoresis. 5μg of the recovered DNA fragment was added to 50mM Tris again.
-HCl (PH7.6), 10mM _MgCl2 , 10mM
DTT, dissolved in 50μ of a reaction solution consisting of 1mM ATP, and 2μ of Hind linker (manufactured by Takara Shuzo Co., Ltd.)
g, and T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) 100
The units were added and reacted overnight at 4°C. Next, after phenol treatment, ether treatment, and ethanol precipitation, 10mM Tris-HCl (PH7.5),
Dissolved in 30μ of a solution of 7mM MgCl ₂ and 60mM NaCl and incubated for 3 hours in the presence of 10 units of restriction enzyme Hind.
Incubated at 37°C. After treatment with T4 DNA ligase again, the DNA was transformed into E. coli DH strain by the rubidium chloride method (see "Molecular Cloning" above) to obtain ampicillin-resistant (Amp ^r ) colonies.
EcoR site of pHGA410 plasmid is Hind
Bacteria carrying the replaced plasmid were selected. The plasmid obtained in this way
Name it pHGA410(H) (Figure 7). (2) Construction of recombinant expression vector pTN-G4
10mM pHGA410(H) (20μg) obtained in (1)
Tris-HCl (PH7.5), 7mM _MgCl2 , 175m
Restriction enzyme Sal was dissolved in 50μ of a reaction solution consisting of
(manufactured by Takara Shuzo Co., Ltd.) 20 units were added and incubated at 37°C for 5 hours. Then phenol treatment,
After ethanol precipitation, DNA polymerase
The mixture was incubated at 14° C. for about 2 hours using Klenow fragment (manufactured by Hoshuzo Co., Ltd.) in the same manner as in the above reaction to obtain blunt ends. This was ethanol-precipitated DNA without being recovered by agarose electrophoresis.
The fragment was treated with the restriction enzyme Hind to obtain approximately 2.7kb.
5 μg of the Hind-Sal fragment was recovered by 1% agarose gel electrophoresis. On the other hand, bovine papillomavirus [bovine
plasmid containing papilloma virus (BPV)]
pdBPV-1 (Sarver, N., Sbyrne, J.C.
Howley, P. M. (1982) Proc. Natl. Acad. Sci.
USA 79 vol. 7147-7151; obtained from Dr. Howley,
ATCC37134…published in the 1985 ATCC catalog)
as in the method of Nagata et al. (Fukunaga,
Sokawa, & Nagata, (1984) Proc. Natl.
Acad.Sci.USA Vol. 81 5086-5090) Hind and
Treat with Pvu to obtain an 8.4kb DNA fragment. This 8.4kb DNA fragment and the approximately 2.7kb DNA fragment above
Hind-Sal DNA fragment according to standard method
Treated with T4 DNA ligase, transformed into E.Coli DH strain by the rubidium chloride method described in "Molecular Cloning" above,
E. coli colonies carrying a plasmid containing the pHGA410-derived G-CSF cDNA were selected.
This plasmid is named PTN-G4 (Figure 7). On the other hand, from Adenovirus Type [Protein Nucleic Acid Enzyme Vol. 27, December issue (1982), published by Kyoritsu Shuppan], approximately 1700 bp of Sal-
Plasmid △PVA containing Hind fragment VA
and fragments containing VA were recovered. This fragment was inserted into the Hind site of PTNG4 described above to obtain pTNG4VAα and pTNG4VAβ (FIG. 7). This plasmid uses the adeno VA gene to increase the efficiency of expressing transcripts from the SV40 early promoter. Example 13 Transformation of C127 cells and its expression (+VSE) Before transforming pTN-G4 obtained in Example 12 into mouse C127 cells, it is treated with restriction enzyme BamH.
That is, 20μg of pTN-G4 plasmid was added to 10mM Tris.
-HCl (PH8.0), 7mM _MgCl2 , 100mM,
NaCl, 2mM 2-mercaptoethanol, 0.01%
It was dissolved in 100μ of a BSA mixture and treated with 20 units of BamH (manufactured by Takara Shuzo), followed by phenol treatment, ether treatment, and ethanol precipitation. Mouse C127 cells were supplemented with 10% fetal bovine serum (GIBCO
The cells are grown in Dulbecco's minimal essential medium containing (manufactured by) Proliferated on a plate with a diameter of 5 cm.
Add the above prepared DNA per plate to C127 cells.
Calcium phosphate method (Haynes, J & Weissmann, C (1983) Nucleic
Acid Res. Vol. 11, 687-706) was used for transformation, followed by glycerol treatment and incubation at 37°C for 12 hours. Next, the cells were transferred to three new plates with a diameter of 5 cm, and the medium was exchanged twice a week. The portions that formed Foci (clumps) on the 16th day were each transferred to a new plate, subcultured in the above-mentioned medium, and clones with high G-CSF production ability were selected. As a result, G-CSF production at a level of ~1 mg/ml was observed. As a result of further cloning, 10
G-CSF production at a level of mg/mg or higher was confirmed. In addition to the above C127 cells, host cells include
NIH3T3 cells can also be used. Example 14 Expression of G-CSF by CHO cells (+VSE) (1) Construction of PHGG4-dnfr 20 μg of pHGA410 plasmid obtained in Example 11
10mM Tris-HCl (PH7.5), 7mM _MgCl2 ,
175mM NaCl, 0.2mM EDTA, 0.7mM
The mixture was dissolved in 100μ of a solution containing 2-mercaptoethanol and 0.01% BSA, 20 units of restriction enzyme Sal (manufactured by Takara Shuzo Co., Ltd.) was added, and the mixture was reacted overnight at 37°C, followed by phenol treatment, ether washing, and ethanol precipitation. Next, the obtained DNA precipitate was mixed with 50mM Tris-
HCl, 5mM _MgCl2 , 10mM DTT, 1m
Dissolve M in 100μ of a reaction solution consisting of dATP, dCTP, dGTP, and TTP, add E.Coli DNA polymerase-Klenow fragment (Takara Shuzo Co., Ltd., 10μ) and react for 2 hours at 14°C, followed by phenol treatment, ether washing, and ethanol precipitation. I went there. Add an EcoR linker to this DNA. That is, the above DNA was diluted with 50μ of 50mM Tris-HCl (PH
7.4), 10mM MDTT, 0.5mM spermidine, 2
mMATP, 2.5mM hexamine cobalt chloride,
Dissolved in a reaction solution consisting of 20 μg/ml BSA,
Add EcoR linker (manufactured by Takara Shuzo Co., Ltd.) and add 200 units of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.).
℃ for 12-16 hours. After phenol treatment, ether washing, and ethanol precipitation according to conventional methods, the DNA was partially digested with EcoR, and 1%
3 μg of a fragment of about 2.7 kb was recovered by agarose gel electrophoresis. On the other hand, the pAdD26SVpA plasmid (Kaufman, RG & Sharp, PA (1982) J.
Mol.Biol.159, 601-621 and Kaufman, R.G.
& Sharp, P.A. (1982) Mol.Cell Biol, 2 vols.
1304-1319) is treated with EcoR and treated with bacterial alkaline phosphatase (BAP) to perform dephosphorylation. That is,
Reaction mixture of 20 μg of pAdD26SVpA and 20 units of EcoR
50mM Ris-HCl (PH7.5), 7mM _MgCl2 , 100
Add to 100μ of a mixture of mM NACl, 7mM 2-mercaptoethanol, and 0.01% BSA and incubate at 37°C.
The reaction was allowed to proceed for 10 hours, and then 5 units of BAP were added to the above reaction solution, followed by reaction at 68° C. for 30 minutes. After that, it was treated with phenol and analyzed by electrophoresis.
The EcoR fragment of pAdD26SVpA was recovered (~
5μg). 0.5 μg each of the approximately 2.7 kb fragment described above and the pAdD26SVpA fragment were annealed. This plasmid is transformed into E. coli DH strain by the rubidium chloride method, and colonies carrying the pHGG4-dhfr plasmid are selected. The obtained plasmid was named pHGG4-dhfr (Fig. 8
a). As an alternative method to the above, the pHGG4 plasmid was treated with Sal, partially digested with EcoR without adding an EcoR linker, and a fragment of approximately 2.7 kb was recovered, and then digested with E. coli DNA polymerase-klenow.
The DNA fragment is treated with the fragment to make the ends blunt-ended. On the other hand, pHGG4-dhfr can also be prepared by preparing a blunt-ended EcoR fragment of pAdD26SVpA using the same method as described above and treating both with T4 DNA ligase. In addition, PHGA410(H) obtained in Example 12 (1) was treated with the restriction enzyme Hind as described in Example 12 (2).
The Hind-Sal fragment was treated with pAdD26SVpA and Sal to blunt-end the above pAdD26SVpA.
PHGG4-dhfr can also be obtained by ligation to EcoR (Fig. 8). (2) Construction of pG4DR1 and pG4DR2 10 μg of pAdD26SVpA mentioned in (1) was added to 50 m
M Tris-HC (PH7.5), 7mM _MgCl2 , 100
The mixture was dissolved in 50 ml of a reaction solution containing mM NaCl, 7 mM 2-mercaptoethanol, and 0.01% BSA, 10 units each of restriction enzymes EcoR and BamH were added, and the mixture was reacted at 37°C for 10 hours. Phenol treatment and ether washing were performed according to conventional methods. 1%
Approximately 2Kb was detected by low melting point agarose gel electrophoresis.
After collecting the DNA fragments, DNA polymerase
The Klenow fragment was blunt-ended according to a conventional method, followed by phenol treatment, ether washing, and ethanol precipitation. On the other hand, pHGA410(H) obtained in Example 12 (1)
10μg of 10mM Tris-HCl (PH7.5), 7mM
It was dissolved in 50μ of a reaction solution containing MgCl ₂ and 60mM NaCl, 10 units of Hind was added, and the mixture was reacted at 37°C for 6 hours. DNA fragments were collected by 1% low-melting-point agarose electrophoresis according to a conventional method, and then subjected to BAP treatment, blunt-ended with Klenow fragments, phenol treatment, and ether washing. DNA fragment and T4DNA
Blunt end ligation was performed using ligase.
That is, 1 μg of each DNA fragment was added to 66 mM Tris-
HCl (PH7.5), 6.6mM _MgCl2 , 5mM
Dissolve DTT in 30 μg of reaction solution containing 1 mM MATP, add 50 units of T4 DNA ligase, and add 50 units of T4 DNA ligase.
After reacting at ℃ for 12 hours, it was transformed into E.ColiDH strain. In this way, as shown in Figure 8c
pG4DR1 and pG4DR2 were obtained. (3) Transformation and expression CHO cells (dhfr ^- strain, Columbia University Dr.L.
(obtained from Chasin) and a 9cm diameter plate (Nunc
The culture was grown in α-minimum essential medium (α-MEM, supplemented with adenosine, deoxyadenosine, and thymidine) containing 10% calf serum (manufactured by Wigler et al., Cell 14, p. 725 (1978)). ) was transformed. That is, the pHGG4-dhfr plasmid prepared in (1)
Add an appropriate amount of carrier DNA (calf thorax DNA) to 1 μg, dissolve in 375 μg of TE solution, and make 1M.
Add 125μ of _CaCl2 . Chill on ice for 3-5 minutes
500μ of 2x HBS (50mM Hepes, 280mM
NaCl, 1.5mM phosphate buffer), cooled on ice again, and mixed with 1ml of the above CHO cell culture solution.
After dropping onto the plate, the mixture was cultured in a CO ₂ incubator for 9 hours. Remove the medium from the plate, wash with TBS (Tris-Buffered Saline), add TBS containing 20% glycerol, wash again, and add non-selective medium (α-MEM medium,
After incubation for 2 days, the cells were split 1:10 using a selective medium (no nucleotides added). Next, culturing was continued while replacing the medium with a selective medium every two days, and the resulting colonies were selected and transferred to a new plate. New plates were grown in the presence of 0.02 μM methotrexate (MTX), 0.05 μM, and
Cloning was performed by growing in the presence of 0.1 μM MTX. Note that CHO cell transformation can also be performed by co-transforming CHO cells with pHGG4 and pAdD26SVpA (Scahill et al. Proc. Natl.
(See Acad.Sci.USA, Vol. 80 , 4654-4658 (1983)). Also, depending on the method described below, CHO
Cell transformation was performed. That is, pG4DR1 or pG4DR2 prepared in section (2) above was treated with Sal and Kpn, respectively, and the DNA was
Obtain the fragments and add 10 μg of them in the same manner as above.
CHO cells were transformed. When the thus transformed cells were continued to be cultured in a selective medium as described above, more than 100 distinct colonies appeared on each plate in about 7 days. After transferring the colonies to a new plate again without selecting them one by one, culturing was continued on a selective medium in the presence of 0.01 μM MTX, and more than ten colonies appeared. Furthermore, by such a method
The concentration of MTX was increased to 0.02 μM, 0.05 μM, and 0.1 μM, and surviving colonies were selected.
Furthermore, after selecting each of the ten or so obtained colonies, similar colony selection could be performed even when the MTX concentration was increased. We also constructed a recombinant vector containing a so-called polycistronic gene, and used this to
We were able to transform CHO cells. That is,
By treating pAdD26SVpA with Pst, collecting the two fragments, and ligating these with the CSF cDNA fragment derived from pBRG4, the adenovirus promoter, CSF cDNA, DHFR, and polyA of SV40 were synthesized.
Construct a recombinant vector arranged in the order of the sites.
It was carried out in CHO cells. Example 15 G-CSF activity assay of expressed substances (+VSE) The culture supernatants of C127 cells and CHO cells obtained in Examples 13 and 14 were diluted to pH 4 with 1N acetic acid, respectively.
After adding an equal volume of n-propanol, the resulting precipitate was removed by centrifugation, and an open column (1φ
x 2 cm) and eluted with 50% n-propanol. After diluting the eluate 2 times with water, YMC-
30~ containing 0.1% TFA by reverse phase high performance liquid chromatography using a C8 column (manufactured by Yamamura Chemical Co., Ltd.)
Elution was performed with a 60% linear gradient of n-propanol. After collecting the fraction eluted at a position where the n-propanol concentration is around 40%, it is lyophilized and
It was dissolved in 0.1M glycine buffer (PH9).
Through this process, human G-
CSF was concentrated approximately 20 times from C127 and CHO cell supernatants. As a control, cells were transformed with a plasmid containing no human G-CSF cDNA according to the method described above, and the culture supernatant was concentrated. Regarding the obtained specimen, “Human G-
Human G
- CSF activity was assayed. In addition, if the expression efficiency is sufficiently high, the culture supernatant may be directly used for assay.
Here, results are shown for concentrated examples. The results were as shown in Table-1.

【table】

[Table] Example 16 Amino acid analysis and sugar analysis (+VSE) (1) Analysis of amino acid composition The crude CSF sample obtained in Example 15 was further analyzed in Example 2.
It was purified according to method (iii). This purified CSF
The 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℃, 24 hours in vacuum 4N methanesulfonic acid + 0.2% 3-(2-
aminoethyl) indole, 110℃, 24 hours,
48 hours, 72 hours in vacuum After dissolving the sample in a solution (1.5 ml) containing 40% n-propanol and 0.1% trifluoroacetic acid, take 0.1 ml of each and dry with dry nitrogen gas, or The reagent was added to the tube, the tube was vacuum-sealed, and the tube was subjected to hydrolysis. In the table, the actual measured values are 24, 48, and 24 hours, respectively.
This is the average value of a total of four 72-hour values. however,
Thr, Ser, 1/2Cys, Met, Val, Ile and
Trp was calculated using the following method. (Refer to Biochemistry Experiment Course, Protein Chemistry (Tokyo Kagaku Doujin Publishing)) ●Thr, Ser, 1/2Cys, Met are 24, 48, 72
Takes the change in time value and extrapolates it to zero time. ●Val and Ile are 72-hour values. ●Trp is the average value of 24, 48, and 72 hour values.

[Table] (2) Sugar composition analysis Purified CSF sample used in the above amino acid composition analysis
After adding 25 nmol of inositol as an internal standard to 200 ng, a methanol solution (500 μ) containing 1.5 N HCl was added, and the tube was replaced with nitrogen gas in a sealed tube.
The reaction was carried out at 90°C for 4 hours. After opening the tube, add silver carbonate (Ag ₂ CO ₃ ) to neutralize it, and then add 50μ of acetic anhydride.
After adding and shaking, the mixture was left in a dark place at room temperature overnight. The upper layer was taken into a sample tube and dried with nitrogen gas. Methanol was added to the precipitate, the precipitate was washed, and then briefly centrifuged, and the upper layer was added to the same sample tube and dried. After adding 50μ of TMS reagent (mixture of pyridine: hexamethyldisilazane: trimethylchlorosilane = 5:1:1) to this and reacting at 40°C for 20 minutes, it was stored in a deep freezer. Incidentally, as standards, 50 nmol each of galactose (Gal), N-acetylgalactosamine (GalNAc), sialic acid, etc. and 25 nmol of inositol were combined and the same operation was performed. Gas chromatography analysis was performed on this sample under the conditions shown below. (Analysis conditions) Column: 2% OV-17VINport HP60-80 mesh, 3m, glass Temperature: Increase temperature at 4°C/min from 110°C to 250°C Carrier gas (nitrogen pressure): Initially 1.2-1.6
Kg/cm ² 2 to 2.5 Kg/cm ² at end 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 Analysis results: Galactose, N-acetylgalactosamine and sialic acid were confirmed in the CSF of the present invention. Example 17 Preparation of pHGV2 vector (for animal cells, -VSE
system) of the cDNA shown in Figure 4A obtained in Example 10.
The EcoR fragment was treated with the restriction enzyme Dra for 2 hours at 37°C, followed by DNA polymerase Klenow.
Fragment (manufactured by Takara Shuzo Co., Ltd.) was treated to make the ends blunt. After phosphorylating 1 μg of Bgl linker (8mer; manufactured by Takara Shuzo Co., Ltd.) using ATP, it was combined with about 1 μg of the DNA fragment mixture obtained above.
Next, the DNA fragments were treated with restriction enzyme Bgl and subjected to agarose gel electrophoresis, and only the largest DNA fragment was recovered. This DNA fragment is human G-
Approximately 700 containing portions encoding CSF polypeptides
It corresponds to a base pair. vectorpdKCR
(Fukunaga et al.; Proc. Natl. Acad. Sci. USA; Volume 81
5086 (1984)) with the restriction enzyme BamH and then dephosphorylated with alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.), and the resulting vector DNA was transformed into T4DNA.
Ligase (manufactured by Takara Shuzo Co., Ltd.) was added to bind the cDNA fragment to obtain pHGV2 (Figure 9). As shown in Figure 9, this plasmid contains the SV40 early gene promoter, the SV40 replication initiation region, the rabbit β-
It contains part of the globin gene, the replication initiation region of pBR322, and the β-lactamase gene (Amp ^r ) derived from pBR322, and the human G-CSF gene is connected downstream of the promoter of the SV40 early gene. Example 18 Construction of recombinant vector for C127 cells (-VSE) (1) Construction of pHGV2(H) Using 20 μg of pHGV2 plasmid (Fig. 9) obtained in Example 17, the procedure was carried out in (1) of Example 12. A plasmid designated pHGV2(H) was obtained in the same manner as described (FIG. 10). (2) Recombinant expression vector pTN-V2 and
Construction of pTNVAα and pTNVAβ Using 20 μg of pHGV2(H) obtained in (1) above,
In the same manner as described in Example 12 (2)
E. coli colonies carrying a plasmid containing pHGV2-derived G-CSF cDNA were selected. The obtained plasmid is named pTN-V2 (Figure 10). On the other hand, adenovirus type [protein nucleic acid enzyme
From Volume 27, December issue (1982), published by Kyoritsu Shuppan] VA
and about 1700bp of Sal−Hind including VA
Plasmid △pVA containing the fragment VA and
Fragments containing VA were collected. Insert this fragment into the Hind site of pTN-V2 mentioned above.
pTNVAα and pTNVAβ were obtained (Figure 10). This plasmid is transferred to SV40 by the adeno VA gene.
This is designed to increase the efficiency of expression of transcripts from the early promoter. Example 19 Transformation of C127 cells and its expression (-VSE) Before transforming pTN-V2 obtained in Example 18 into mouse C127 cells, it is treated with restriction enzyme BamH. Next, mouse C127 cells were transformed with the above-prepared DNA and expressed (see Example 13), and clones with high G-CSF production ability were selected. The result~1
G-CSF production was observed at a level of mg/mg/ml. By continuing cloning further, 10
Clones having the ability to produce G-CSF at the mg/g level were selected. Obtained in the same manner in Example 19
As a result of transforming C127 cells with pTNVAα and pTNVAβ and selecting clones with high G-CSF production ability, we were able to obtain clones with high production of pTNVAα of 20 mg/or more. Also
A clone with a production capacity of several mg/ml could be obtained from pTNVAβ. In addition to the C127 cells mentioned above, host cells include
NIH3T3 cells can also be used. Example 20 Expression of G-CSF by CHO cells (-VSF) (1) Construction of pHGV2-dhfr The approximately 0.5 μg each of the 2.7 kb fragment and the pAdD26SVpA fragment were annealed. This plasmid was transformed into E. coli DH strain by the rubidium chloride method, and colonies harboring the pHGV2-dhfr plasmid were selected. The obtained plasmid was named pHGV2-dhfr (Fig. 11a). In addition, as an alternative method to the above, the pHV2 plasmid
Sal treatment, partial digestion with EcoR without addition of EcoR linker, recovery of approximately 2.7 kb fragment, and E. coli DNA polymerase-Klenow.
The DNA fragment was treated with the fragment to make the ends blunt-ended. On the other hand, pHGV2-dhfr could also be prepared by preparing a blunt-ended EcoR fragment of pAdD26SVpA using the same method as described above and treating both with T4 DNA ligase. In addition, the pHGV2(H) obtained in Example 18 (1) was treated with a restriction enzyme, Hind, as described in Example 12 (2).
The Hind-Sal fragment was treated with pAdD26SVpA and Sal to blunt-end the above pAdD26SVpA.
pHGV2-dhfr could also be obtained by ligation to the EcoR fragment (Fig. 11b). (2) Construction of pV2DR1 and pV2DR2 10g of pAdD26SVpA mentioned in (1) was added to 50mM.
Tris-HCl (PH7.5), 7mM _MgCl2 , 100m
The mixture was dissolved in 50 ml of a reaction solution containing M NaCl, 7 mM 2-mercaptoethanol, and 0.01% BSA, 10 units each of restriction enzymes EcoR and BamH were added, and the mixture was reacted at 37°C for 10 hours. Phenol treatment and ether washing were performed according to conventional methods. Approximately 2Kb of DNA was analyzed using 1% low melting point agarose gel electrophoresis.
After collecting the fragments, DNA polymerase
The Klenow fragment was blunt-ended according to a conventional method, followed by phenol treatment, ether washing, and ethanol precipitation. On the other hand, pHGV2(H) obtained in Example 19 (1)
10μg of 10mM Tris-HCl (PH7.5), 7mM
It was dissolved in 50μ of a reaction solution containing MgCl ₂ and 60mM NaCl, 10 units of Hind was added, and the mixture was reacted at 37°C for 6 hours. DNA fragments were collected by 1% low-melting-point agarose electrophoresis according to a conventional method, treated with BAP, and then blunt-ended with Klenow fragment. After phenol treatment and ether washing, the approximately 2Kb DNA fragment mentioned above and
Blunt end ligation was performed using T4 DNA ligase. That is, 1μg of each DNA fragment is 66
mM Tris-HCl (PH7.5), 6.6mM _MgCl2 ,
Reaction solution containing 5mM DTT and 1mM ATP
After dissolving in 30μ of E.
coliDH strain. In this way,
pV2DR1 and pV2DR2 shown in Figure 11c were obtained. (3) Transformation and expression Using the pHGV2-dhfr plasmid prepared in (1) above, a CHO cell line was transformed and expressed in the same manner as described in Example 14 (3). Note that transformation of CHO cells can also be performed by co-transforming CHO cells with pHGV2 and pAdD26SVpA (Co transformation). CHO cells were also transformed by the method described below. That is, pV2DR1 or pV2DR2 prepared in section (2) above was previously treated with Sal and Kpn to obtain DNA fragments, and 10 μg of the DNA fragments were injected in the same manner as above.
CHO cells were transformed. When the thus transformed cells were continued to be cultured in a selective medium as described above, more than 100 distinct colonies appeared on each plate in about 7 days. After transferring the colonies to a new plate again without selecting them one by one, culturing was continued on a selective medium in the presence of 0.01 μM MTX, and more than ten colonies appeared. Furthermore, by such a method
The concentration of MTX was increased to 0.02 μM, 0.05 μM, and 0.1 μM, and surviving colonies were selected.
Furthermore, after selecting each of the ten or so obtained colonies, similar colony selection could be performed even when the MTX concentration was increased. In addition, we constructed a recombinant vector containing a so-called polycistronic gene and used it to
We were able to transform CHO cells. That is,
By treating pAdD26SVpA with Pst, collecting the two fragments, and ligating them with the CSF cDNA fragment derived from pBRV2, the adenovirus promoter, CSF cDNA, DHFR, and SV40 polyA
Construct a recombinant vector arranged in the order of the sites.
It was carried out in CHO cells. Example 12 G-CSF activity assay (-VSE) of expressed substances From the culture supernatants of C127 cells and CHO cells obtained in Examples 19 and 20, human G - CSF was obtained and its human G-CSF activity was assayed. The results were as shown in Table 3.

[Table] Example 22 Amino acid analysis and sugar analysis (-VSE) (1) Analysis of amino acid composition The crude CSF sample obtained in Example 21 was further analyzed in Example 2.
It was purified according to method (iii). This purified CSF
The sample was subjected to amino acid composition analysis by the method described in Example 16 (1). The results are shown in Table 4.

[Table] (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 under the same analysis conditions as described in Example 16 (2). As a result of the analysis, galactose, N-acetylgalactosamine, and sialic acid were confirmed in the CSF of the present invention. Example 23 Infection-preventive effect of human G-CSF <Test method> 1 Protective effect against Pseudomonas aeruginosa infection Endoxan (Shionogi After intraperitoneal administration of 200mg/Kg
divided into groups, and two groups were given human G-CSF (25000u/
solvent (1% propanol in saline, 0.5% (W/V)) containing mouse or 50000u/mouse)
Mouse serum albumin (mouse serum albumin) was administered subcutaneously to the other group, and vehicle alone was administered subcutaneously to another group, each in an amount of 0.1 ml every 24 hours, four times. Three hours after the fourth dose, each group was given Pseudo monas aeruginosa GNB-139 (3.9
×10 ⁵ CFU/mouse) was subcutaneously administered for infection. 21 hours after infection, human G
-CSF (25000u/mouse or 50000u/mouse)
The solvent containing 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 cultured with shaking overnight at 37°C using Heart Infusion liquid medium (manufactured by Difco, trade name).
A culture solution was prepared by suspending it in physiological saline. <Results> The same purified human G-CSF sample (+
VSE). The results are shown in Table-5.

[Table] Purified human G derived from the same C127 cells as used for the amino acid composition analysis in Example 16
- When the infection prevention effect of the above test was investigated using a CSF sample, almost the same results were obtained. A purified human G-CSF sample derived from the same CHO cells as used for the amino acid composition analysis in Example 22 (-
VSE). The results are shown in Table-6.

〔Effect of the invention〕

The glycoprotein having human G-CSF activity of the present invention is a therapeutic agent for leukopenia, a therapeutic agent for myeloid leukemia,
It is extremely useful as an active ingredient in hematopoietic function recovery promoters, infection preventive agents, and the like. Furthermore, the establishment of a method for producing human G-CSF through genetic recombination, which has made these long-awaited pharmaceutical products a reality, can also be considered a major achievement of the present invention.

[Brief explanation of drawings]

Figure 1 shows the sequences of probe IWQ, probe A and probe LC. Figure 2 shows the base sequence of pHCS-1 insert. Figure 3A shows pBRG4
The base sequence of the cDNA insert is shown. Figure 3B,
shows the amino acid sequence of human G-CSF precursor derived from pBRG4 cDNA. Figure 3B, is
Human mature G-CSF derived from pBRG4 cDNA
The amino acid sequence of Figure 4A shows pBRV2
The base sequence of the cDNA insert is shown. Figure 4B,
shows the amino acid sequence of human G-CSF precursor derived from pBRV2 cDNA. Figure 4B,
Human mature G-CSF derived from pBRV2 cDNA
The amino acid sequence of Figure 5 shows pBRS4 or
Restriction enzyme cleavage sites of pBRV2-derived human G-CSF cDNA are shown. Figure 6 shows the schematic structure of pHGA410. Figure 7 shows recombinant expression vector pTN-G4,
The construction process of pTN-G4VAα and pTN-G4VAβ is shown. Figures 8a and 8b show pHGG4-
Showing the process of building a dnfr. Figure 8c shows the construction process of pG4DR1 and pG4DR2. Figure 9 is
The schematic structure of pHGV2 is shown. Figure 10 shows recombinant expression vectors pTN-V2, pTN-VAα and pTN
- Shows the construction process of VAβ. Figures 11a and 11b are expression recombinant vector pHGV2-dhfr.
The construction process is shown below. Figure 11c shows the construction process of pV2DR1 and pV2DR2.

Claims (1)

[Scope of Claims] 1. A mammalian cell transformed with a recombinant vector containing a cDNA fragment having a base sequence encoding the following amino acid sequence is cultured, and then a human granulocyte colony-stimulating factor having activity is produced. 1. A method for producing a glycoprotein having human granulocyte colony-stimulating factor activity, which comprises separating the glycoprotein. Met Ala Gly Pro Ala Thr Gln Ser Pro
Met Lys Leu Met Ala Leu Gln Leu Leu Leu
Trp His Ser Ala Leu Trp Thr Val Gln Glu
Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu
Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu
Gln Val Arg Lys Ile Gln Gly Asp Gly Ala
Ala Leu Gln Glu Lys Leu (Val Ser Glu) _n
Cys Ala Thr Tyr Lys Leu Cys His Pro Glu
Glu Leu Val Leu Leu Gly His Ser Leu Gly
Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro
Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu
Ser Gln Leu His Ser Gly Leu Phe Leu Tyr
Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr
Leu Gln Leu Asp Val Ala Asp Phe Ala Thr
Thr Ile Trp Gln Gln Met Glu Glu Leu Gly
Met Ala Pro Ala Leu Gln Pro Thr Gln Gly
Ala Met Pro Ala Phe Ala Ser Ala Phe Gln
Arg Arg Ala Gly Gly Val Leu Val Ala Ser
His Leu Gln Ser Phe Leu Glu Val Ser Tyr
Arg Val Leu Arg His Leu Ala Gln Pro (where m represents 0 or 1) 2 The manufacturing method according to claim 1, wherein the base sequence is the following base sequence. ATG GCT GGA CCT GCC ACC CAG
AGC CCC ATG AAG CTG ATG GCC CTG
CAG CTG CTG CTG TGG CAC AGT GCA
CTC TGG ACA GTG CAG GAA GCC ACC
CCC CTG GGC CCT GCC AGC TCC CTG
CCC CAG AGC TTC CTG CTC AAG TGC
TTA GAG CAA GTG AGG AAG ATC
CAG GGC GAT GGC GCA GCG CTC CAG
GAG AAG CTG (GTG AGT GAC) _n TGT
GCC ACC TAC AAG GTG TGC CAC CCC
GAG GAG CTG GTG CTG CTC GGA CAC
TCT CTG GGC ATC CCC TGG GCT CCC
CTG AGC AGC TGC CCC AGC CAG GCC
CTG CAG CTG GCA GGC TGC TTG AGC
CAA CTC CAT AGC GGC CTT TTC CTC
TAC CAG GGG CTC CTG CAG GCC CTG
GAA GGG ATC TCC CCC GAG TTG GGT
CCC ACC TTG GAC ACA CTG CAG CTG
GAC GTC GCC GAC TTT GCC ACC ACC
ATC TGG CAG CAG ATG GAA GAA
CTG GGA ATG GCC CCT GCC CTG CAG
CCC ACC CAG GGT GCC ATG CCG GCC
TTC GCC TCT GCT TTC CAG CGC CGG
GCA GGA GGG GTC CTG GTT GCC TCC
CAT CTG CAG AGC TTC CTG GAG GTG
TCG TAC CGC GTT CTA CGC CAC CTT
GCC CAG CCC (where m represents 0 or 1) 3 The base sequence is the following base sequence (a) or base sequence (b)
The manufacturing method according to claim 1, which is an example of the base arrangement shown in . Base arrangement examples (a) [Table] [Table] [Table] [Table] [Table] [Table]