JPS62236497A - Novel glycoprotein and production thereof - Google Patents

Abstract

PURPOSE:A gene coding a polypeptide having stimulation factor activity to form a human granulocyte colony is separated and is made to be manifested in a host cell. CONSTITUTION:A DNA necessary for forming human granulocyte cell colony and coding a polypeptide having specific stimulation factor activity is obtained as a 15-17S fraction by sucrose density gradient centrifugation. The DNA has an amino acid sequence of formula. A host cell is transformed with a recombinant vector integrated with said DNA. The objective glycoprotein having human granulocyte colony stimulation factor activity can be separated from the culture product of the obtained transformed strain.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention utilizes 17 specific stimulating factors, mainly colony stimulating factors (hereinafter referred to as rcsFJ), necessary for stimulating colony formation of human granulocytic cells. The present invention relates to a glycoprotein having activity (abbreviated as ) and a method for producing the same.

[Conventional technology]

When cultured using the two-layer soft agar culture method, with bone marrow cells as target cells in the upper layer and kidney cells and fetal cells in the lower layer, some of the cells in the upper layer proliferate and differentiate, forming neutrophilic granulocytes (hereinafter referred to as neutrophil-type granulocytes). Colonies consisting of granulocytes (referred to as granulocytes J), monocytes and macrophages are formed,
It was known that factors that promote colony formation exist in living organisms (Pluznik and 5ach: J, C
e11. Comp, Physiol, vol. 66, 319
Page (1965), Bradley and Hetcalf
: Au5t, J, EXI).

8io1. Hed, Sci, vol. 44, p. 287 (196&)).

This factor, collectively referred to as C8F, normally differentiates widely in the body, such as cells, monocytes, macrophages,
It is known to be produced by fibroblasts, endothelial cells, etc. C3F is a granulocyte that acts on granulocyte/monocyte/macrophage stem cells, stimulates their proliferation, induces their differentiation, and forms colonies consisting of granulocytes and monocytes/macrophages in soft agar. -monocyte macrophage C3
F (abbreviated as GM-C3F) is a pluripotent C8F (abbreviated as M-C8F) that acts on undifferentiated pluripotent stem cells, which mainly acts to form colonies of monocytes and macrophages. mu I t
There are subclasses such as granulocyte C3F (abbreviated as G-C3F), which mainly acts to form granulocytic colonies as in the present invention, and each subclass has a specific effect on target cells. It has come to be considered that the differentiation stages are also different [ASa
nO: Metabolism-Hctabolisn+ and Dis
ease, vol. 22, p. 249 (1985).

Yunis et al.; “Growth and Hatura
tion Factors”edited by Gu
roff, John Wiley & 5ons, N
Y, vol. 1, p. 209 (1983)].

Therefore, it is extremely important to purify each subclass and investigate its chemical and biological properties in more detail for the analysis of the hematopoietic mechanism and the pathology of various hematological diseases.

Among the biological effects of G-C3F, induction of differentiation of myeloid leukemia cells and hyperfunction of mature granulocytes are attracting attention, and the clinical usefulness of G-C3F in the treatment and prevention of leukemia is highly anticipated. It has been.

[Problem that the invention seeks to solve]

Conventional attempts to isolate and purify G-C3F involve isolating G-C3F from the culture supernatant using cell culture methods, but G-C8F is only produced in low concentrations. In particular, it has not yet been possible to obtain a large amount of homogeneous G-C3F due to difficulties such as the need for a complicated purification process to extract 1 q of trace amounts of G-C3F from a large amount of culture fluid. Therefore, it has been desired to produce G-C3F in large quantities using recombinant DNA technology.

[Means for solving problems]

Under such circumstances, the present inventors succeeded in isolating a gene encoding a polypeptide having human G-C3F activity and expressing the gene in host cells.

The present invention provides a glycoprotein having human granulocyte colony-stimulating factor activity, which has a polypeptide represented by the amino acid sequence set forth in claim 1 or a portion thereof and a sugar chain moiety.

The present invention also provides a method for producing the above-mentioned glycoprotein, that is, obtaining a gene encoding a polypeptide having human granulocyte colony-stimulating factor activity, and then producing a recombinant vector incorporating this into vA, and then producing the vector. A method of producing a glycoprotein having human granulocyte colony-stimulating factor activity as described above, which is characterized in that a host cell is transformed using the above-mentioned human granulocyte colony-stimulating factor. A manufacturing method is provided.

The present invention will be explained in detail below.

The gene encoding the polypeptide having human G-C3F activity used in the present invention includes human (~G
-Messenger RNA (mRNA) encoding a polypeptide with C3F activity
DNA).

Applicants have obtained two series of such CDNAs.

One of the series of CDNAs has a gene encoding polypeptide A or ■ in Figure 3(B) or a part thereof, and more specifically, it has a gene encoding the polypeptide A or ■ in Figure 3(B), or a part thereof, and more specifically, it has a gene encoding the polypeptide A or ■ in Figure 3(B), or a part thereof. ATG at nucleotide positions 32-34
to CCC at nucleotide positions 650-652, from ACC at nucleotide positions 122-124 to CCC at nucleotide positions 650-652
It has the arrangement up to CC or the arrangement shown in FIG. 3(A), or a part thereof.

The Kono series (7) CDNA is referred to as CDNA (+VSE).

Other series of CDNAs have the gene encoding polypeptide A or ■ in Figure 4(B) or a part thereof, and more specifically, the nucleotide sequence from 31 to 33 from the 5'-end of the base sequence in Figure 4(^). 640~ from nucleotide position ATG
Sequence up to CCC at nucleotide position 642, 121-1
It has the sequence from ACC at position 23 to CCC at positions 640 to 642, the sequence shown in FIG. 4(A), or a part thereof.

This series of cDNAs is called CDNA (-VSE).

The above gene can be obtained from mammalian cells having the ability to produce polypeptides having G-C3F activity, for example.
After preparing mRNA encoding C3F, it is converted into double-stranded CDNA by a known method, and rescreened from a collection of recombinants containing this DNA (hereinafter referred to as a C[)NA library) by a known method. obtained by

Furthermore, the gene used in the present invention may be a gene derived from a human chromosome that encodes a polypeptide having human G-C3F activity. This gene contains a base sequence involved in transcriptional regulation, and specifically has the base sequence shown in FIG. 5 or a portion thereof.

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

In this case, as a source of human chromosomal genes, any human cells can be used, including the liver.

Extracted cells such as kidney cells, cultured cells such as tumor cells, etc. can be used.

In addition, to prepare a human chromosomal gene library from human cells, known methods [Haniatis et al.: Ce1
Vol. 115, p. 687 (1978) and) laniati
s etc.; Molecular cloning, Co1d
Spring Laborator
For example, human chromosomal DNA is extracted from human fetal liver with phenol, etc., and the resulting DNA is partially or completely digested with restriction enzymes. , obtain DNA fragmented to an appropriate length, and convert this fragmented DNA into T4 DNA.
Using ligase etc., EC0RI is added to the grains as necessary.
A linker containing a cleavage site such as the following is added and inserted into the λ phage vector DNA fragment. Lambda phage particles are then obtained by in vitro packaging methods and can be made by transforming host cells such as E. coli.

The λ phage used as the above vector is, for example, Cha
Examples include rOn4A and EMBL-3,4.

On the other hand, in the present invention, the mammalian cell serving as the source of the mRNA is the cell line CHU-2 derived from hydrocaval carcinoma.
(Cot Iection Nationale De
Cultures De )ficroorgan
ismes (C, N, C, M) Deposit number l-
483>, but 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, the preparation of mRNA is carried out using a method that has already been used to clone the genes of several other physiologically active proteins.
For example, surfactant treatment and phenol treatment are performed using a vanadium complex etc. in the presence of a ribonuclease inhibitor (Ber(ler and Birkenmeier:
Biochemistry18i5143 pages (197
9)) or after treatment with guanidine thiocyanate, C
Perform 3CI density gradient centrifugation (Chir (Win et al.:
Biochemistry Kaimaki 5294 pages (1979)
After obtaining the total RNA by
Poly(A”) RNA (mRNA
A) can be obtained by (7). Poly(A) RNA can also be further fractionated by sucrose density gradient centrifugation or the like.

In order to confirm that the mRNA obtained as described above encodes a polypeptide with G-C3F activity, it is necessary to translate the mRNA into protein and examine its physiological activity, or to use anti-G-C3F A method such as identifying the protein using an antibody may be used. For example, mRNA was injected into the oocytes of the African frog (Xen0pus 1aevis) and translated (GLIrdOn et al.: Nature, Vol. 233, p. 177 (1972)).
), or rabbit reticulocytes (see
Translation reactions are being carried out using the cyte and wheat germ systems (Schleif and Wens in: “Practical Method
ds in Molecular Biology”,
Springer-Verlag, NY, (19
81)).

G-C3F activity can be assayed by applying a soft agar culture method using bone marrow cells. There is a review article on those methods.
cColonies”, Springcr-Ver
lag, Berlin, Heldeibero,
N Y (1977)).

Single-stranded cD was generated using the mRNA obtained by the method described above as a template.
After synthesizing NA, double-stranded CD is extracted from this single-stranded CDNA.
Synthesize NA and create a recombinant plasmid with appropriate vector DNA. Now Escherichia
a coli) etc. to obtain a DNA group (cDNA library) of the transformed strain.

To obtain double-stranded CDNA from mRNA, for example, mRNA
An oligo (d
T> is used as a primer and treated with reverse transcriptase, or an oligonucleotide corresponding to a part of the amino acid sequence of G-C3F protein is synthesized, and this is used as a primer and treated with reverse transcriptase to generate cDNA complementary to mRNA. Synthesize.

Double-stranded CDNA is prepared by decomposing and removing mRNA by alkaline treatment, and then treating the resulting single-stranded CDNA with reverse transcriptase or D
It can be obtained by treating with NA polymerase (e.g., +enow fragment, etc.) and then with St nuclease, or by directly treating with RNASe H and DNA polymerase (e.g., Escherichia coli DNA polymerase, etc.). Can be done (for example, Haniati
s et al.: HOleClJIar cloning,
Col1d Spring 1larborLabora
tory (1982) and Gubler and Hoffm.
an; see Gene vol. 25, p. 263 (1983))
.

The double-stranded CDNA thus obtained is transferred to an appropriate vector, such as pscroll, pDF41. Co
l El, pMB9. pBR322, pBR327
.

After integrating into EK-type plasmid vectors such as DACYCl or phage vectors such as λat, λC1λgtio, λgtWEs, etc., E. coli (X1776: HB 101: DH1, C6
00 strain, etc.) to create a CDNA library (7) (for example, the above-mentioned “HOIeCLIf strain”).
ar cloning”).

To ligate double-stranded CDNA to a vector, use an appropriate chemical synthesis method to attach ligatable ends to the ends of the DNA.
This can be carried out by adding the A fragment and treating with T4 phage DNA ligase in the presence of ATP and vector DNA that has been previously cleaved using a restriction enzyme. or,
dG, dC-strand (or dA
.

dT-chain) and then slowly cooling the solution containing both DNAs (Mo1
(see ecular cloning).

Transformation of a host cell with the thus obtained recombinant DNA can be carried out, for example, when the host cell is E. coli.
The method as described in detail by J.N.

l, Biol, 1 volume 557 pages (198
3)), i.e., CaCl, MOCI2 or R
This can be carried out by adding the recombinant DNA body to competent cells prepared in the coexistence of bC+.

To search for cells harboring the gene of interest, the plus-minus method used in cloning interferon CDNA (Daniguchi et al.; proc,
Jpn, ACad, Volume 55 Ser, [3,464
(1979)) and the hybridization-translation assay (Na (lata et al.: Na
ture 284, p. 316 (1980)), or colony or plaque hybridization method using oligonucleotide probes chemically synthesized based on the amino acid sequence of the protein (Wallace et al.
etc.: NuCIeiCAc1ds Res, Volume 9, 87
9 pages (1981) and BentOn and Dav ise
: 5science vol. 196 p. 180 (1977)
) etc. may be used.

The thus cloned fragment containing the gene encoding the polypeptide having human G-C3F activity is reincorporated into an appropriate vector DNA to transform other prokaryotic or eukaryotic host cells. be able to. Furthermore, by introducing an appropriate promoter and expression-related sequences into these vectors, it is possible to express the gene in each host cell.

Examples of host cells derived from mammals include CO8 cells, Chinese hamster ovary (CHO) cells, C-127 cells, and He1-a cells, and vectors for transforming these cells include pSV2-gpt ( M
uligan and Berg; Pr0C, Natl, Ac
ad, sci, UsA; vol. 78, p. 2072 ((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.

As a promoter for gene expression in mammalian cells, promoters such as retrovirus, polyomavirus, adenovirus, and simian virus 40 (SV40) may be used. For example, when using the SV40 promoter, the method such as ) fulligan (N
ture 2771108 (1979)).

As a replication origin, those derived from SV40, polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can be used, and as a selection marker,
Phosphotransferase APH (3') II or neo i gene, thymidine kinase (TK) m gene, Escherichia coli chirintin-guanine phosphoribosyltransferase (ECOClt) gene, dihydrofolate reductase (DHFR) i gene etc. can be used.

In order to obtain a polypeptide having human G-C3F activity using the above-mentioned host-vector system, after transforming a host cell with a recombinant DNA in which the gene has been inserted into an appropriate site of the 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 exhibit polymorphism (polymorphism), as is known for the human interferon gene.
hysm) (for example, N15hi et al.: J, Biochem, Vol. 97, p. 153 (198
5)), this polymorphism may result in the substitution of one or more amino acids, or may result in a change in the base sequence but no change in the amino acid.

Also, for example, a polypeptide lacking or adding one or more amino acids in the amino acid sequence of FIG. 3 (8) or FIG. 4 (8), or one or more amino acids having one or more amino acids A polypeptide substituted with an amino acid may also have G-C3F activity.

For example, it is already known that a polypeptide obtained by converting the base sequence corresponding to cysteine in human interleukin 2 (L-2) W Buddha to a base sequence corresponding to serine retains interleukin 2 activity. It has become (
Wan (1st prize: 5science, vol. 224, 14
31 (1984)).

Therefore, as long as these naturally occurring or artificially synthesized polypeptides have human G-C3F activity, animal cells transformed with recombinant vectors containing genes encoding those polypeptides (transformed All glycoproteins obtained by culturing the human body) are included in the present invention.

A gene necessary for obtaining the glycoprotein having human G-C3F 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 ItI! from the culture supernatant of tumor cell line CHU-2! The amino acid sequence of the homogeneous human C3F protein obtained by I.I was determined from the N-terminus, and the amino acid sequence of the fragment was also determined after rough fragmentation by bromcyanide digestion, trypsin treatment, etc. [Example 3 (i) , (ii)
, (iii) ].

Among the amino acid sequences, three types of nucleotide probes (8) and probes (LC
) and probe (IWQ>) were synthesized (Example 4).

Probe (A) is a mixed type probe consisting of 14 consecutive nucleotides. The probe (IWQ) is
As 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) is a probe consisting of 24 nucleotides synthesized based on the base sequence shown in FIG. It is.

Chemical synthesis of nucleotides can be carried out by applying a modified phosphotriester method to a 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 C)-I Add guanidine thiocyanate solution to homogenize U-2 cells, 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 transcription Vf and double-stranded using RNase H and E, Co I 1 DNA polymerase. Strand cDNA@l. A dC chain was added to the obtained double-stranded cDNA, and a dG chain was added to the pst cleavage site. B
Combined with R322 vector, E. coli X1776
The strain was transformed and a ρBR322-based cDNA library was constructed (Examples 5 and 6).

Similarly, the double-stranded CDNA was ligated to the λgtio vector using the EC0RI linker to construct a λ phage-based cDNA library (Example 7).

(3) Screening The recombinant derived from the pB R322-based CDNA library was immobilized on Whatman 541 filter paper, and colony hybridization was performed using a probe (IWQ) radiolabeled with 32P. As a result, one clone was selected. did it. This clone was cloned using the Sanambu atuting method (Sout
hern: J, ) fol, Biol, vol. 98 5
03 (1975)), it was found that it also hybridized with probe (A).

The nucleotide sequence of this clone was determined using the dideoxy method (sanger
: 5science vol. 214 p. 1205 (198
1)).

The base sequence of the obtained CDNA insert is shown in FIG.

As shown in Figure 2, this cDNA insert consists of 308 base pairs including probe (IWQ) and probe (A), and is an open reading sequence encoding 83 amino acids including the amino acid sequence shown in Example 3(iii). It turns out that it has a frame.

The pBR322-derived plasmid containing this 30B base pair is hereinafter abbreviated as pHC3-1 (Example 8).

A DNA fragment containing 308 base pairs obtained from pHcs-1 was subjected to nick translation method (mentioned above, MOleCL
radiolabeled with IIarCl0nin (see J),
Using this as a probe, a GDNA library derived from λgtio was subjected to plaque hybridization (BentO
n and DaViS: 5CienCe Volume 19B 18
0 (1977), and 5 clones were selected.The nucleotide sequences of the clones thought to contain cDNA were determined in the same manner as described above (Fig. 3 (
A)).

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

The amino acid sequence encoded by this CDNA is shown in Figure 3.
It can be performed as shown in (A).

Comparison with the N-terminal amino acid sequence of the G-C3F protein shown in Example 3(i) reveals that this CDNA is 5'
- a signal peptide encoded by 90 base pairs starting from the ATG sequence 32-34 nucleotides from the end and ending with the CCC sequence 119-121 and 12
It was found that it contains a base sequence corresponding to the mature G-C8F polypeptide encoded by 531 base pairs starting from the ACC sequence at positions 2 to 124 and ending at the CCC sequence at positions 650 to 652. Therefore, the polypeptide with the amino acid sequence shown in FIG. 3(B) consisted of 207 amino acids, and its molecular weight was calculated to be 22,292.87 daltons. Similarly, the polypeptide with the amino acid sequence ■ is 177
Consisting of amino acids, its molecular weight is 18986.74
Dalton (Example 9).

However, regarding the start site of the protein, ATG at positions 32-34 or 68-70 can be considered as well.

EC0R1 cleavage site Nico (7) cDNA (:+-VS
The Escherichia coli (E. coli) X1776 strain carrying pB R322 inserted with E) has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology (FERM
BP-954).

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

The plasmid ligated with Mata, Kono cDNA@pBR327 [5oberon et al.; Gene 9, p. 287 (1980)] at the EcoRI site is referred to as pBRG4.

The thus obtained pBRG4 was treated with the restriction enzyme EC0
A DNA fragment containing cDNA of approximately 1500 base pairs obtained by treatment with RI was radiolabeled using the nick translation method (see Molecular' cloning above), and using this as a probe, the cDNA library derived from λgt10 was again used. Plaque hybridization (see Benton and Davis, supra)
Screened by. At this time, two sheets of nitrorelulose filter paper with immobilized λ phage DNA were prepared at the same time, and similar plaque hybridization was performed using the '>'tf-solid probe (LG) first, resulting in positive results with both probes. Phages were selected. Select clones that appear to be full-length, and extract cDNA using the dideoxy method.
The nucleotide sequence of the insert was determined and was as shown in FIG. 4(A).

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

N of the G-C3F protein shown in Example 3(i)
By comparison with the terminal amino acid sequence, this C[)NA is 5′
- a signal peptide encoded by 90 base pairs starting from the ATG sequence at nucleotide positions 31-33 from the end and ending with the CCC sequence at positions 118-120;
It was found that it contains a base sequence corresponding to the mature G-C3F polypeptide encoded by 522 base pairs starting from the ACC sequence at positions 1 to 123 and ending at the CCC sequence at positions 640 to 642. Accordingly, the polypeptide having the amino acid sequence (■) shown in FIG. 4(B) consisted of 204 amino acids, and its molecular weight was calculated to be 21,977, 35 Daltons. Similarly, the polypeptide with the amino acid sequence (2) consisted of 174 amino acids, and the distance between the molecules was 18671.42 daltons (Example io).

However, regarding the start site of the protein, in addition to positions 31-33, ATG at positions 58-60 or 67-69 may also be considered.

Escherichia coli (E,
coIi) strain X1776 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (FERMBP-95
5>.

Figure 6 shows the restriction enzyme cleavage sites of the (q) gene.

Furthermore, a plasmid in which this CDNA was ligated to pBR327 at the EC:ORI site is referred to as pBRV2.

(4) Screening of human chromosomal gene libraries The method described by Haniatis et al. (cited above) 1o1e
A human chromosomal gene library prepared according to the method (see cularcloning) was screened using pHcs-1 as described above.

As a probe used for screening, pHcs-
In addition to the 308 base pair DNA fragment derived from 1, the approximately 1500 base pair DNA fragment derived from pBRG4, the approximately 1500 base pair DNA fragment derived from pBRV2, or these DNAs.
A DNA fragment of an appropriate length containing a part of the A fragment, and the aforementioned oligonucleotide probes [(IWQ), (A
), (LC)].

The DNA fragment derived from pHC3-1 was subjected to the nick translation method [Roop et al.; Ce1l vol. 15, p. 431 (19
78)], and using this as a probe, a human chromosome-derived gene library was subjected to plaque hybridization (BentOn described above).
Davis et al.) and screened 17 clones.

After recovering DNA from the obtained clone, a known method [Frltsch et al.;
A restriction enzyme map was created according to [0) ].

Next, using the above DNA probe) J-tJ' blotting (see the above-mentioned 5OUTHER reference) was performed, and human G-C3F polypeptide was added to the approximately 4 kilobase pair DNA fragment excised with EC0RI and XhoI. It was found that there may be a region encoding Therefore, we created a DNA fragment of approximately 4 kilobase pairs as pBR327.
pBRcE3β was obtained by inserting the pBRcE3β into the ECoRI site using the ECoRI linker. This plasmid was used as DNA for determining the base sequence, and approximately 3
The kilobase pair base sequence was determined to be human G-C3F.
It was determined that this gene encodes a polypeptide (Figure 5).

The E. cot 1X1776 strain carrying plasmid pBR327 (pBRCE3β) in which this approximately 4 kilobase pair DNA fragment was inserted into the ECoRl site has been deposited at the National Institute of Microbial Technology, Agency of Industrial Science and Technology. (FERM B
P-956).

Figure 32 A comparison with the pBRG4 cDNA insert and pBRV2 cDNA insert shown in Figure 4 revealed that this DNA fragment contains 5 exons and encodes the amino acid sequence derived from pBRG4 and pBRV2. It turned out to be.

In addition, this DNA fragment contains the front region to be transcribed into human G-C3F mRNA, that is, the intrachromosomal gene of human G-C3F, and furthermore, it has a base sequence involved in transcriptional regulation [Benoist and Chamb
on; Nature vol. 290, p. 304 (1981) and 8reathnaCk and Chambon;
, Rev. Biochem.

349 pages (1981)].

(5) Construction of recombinant vectors for animal cells Recombinant vectors (derived from BPV) when using C127 cells and NIH3T3 cells as host cells, and recombinant vectors when using C[]G0 cells (containing DHFR) ) construction was carried out for +VSE system, -VSE system CDNA, and chromosome-derived genes, respectively. A similar recombinant vector was also constructed for CoS cells. 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 used as vector pdKC.
After integration into R to create a pHGA410 plasmid (Example 12) (FIG. 8), this was partially digested with EC0RI to make the ends blunt. A HindI[I linker was added to this DNA, and then H
After indI [lff1 procedure and T4 DNA ligase treatment, this was used for the rubidium chloride method (Mo1ecular described above).
Cloning) using E, Co11[))-I
The J strain was transformed. The obtained plasmid was transformed into pl-(G
Name it A410()(> (Figure 9).

pHGA410 (H) was treated with 5alI, then the terminal was converted to plant-end, and then treated with Hind■ again.
Recover the indI[l-3a lI fragment.

On the other hand, pd containing a transforming fragment of bovine papillomavirus
The BPV-1 plasmid is treated with 1-1ind[[, PVU] to separate the larger DNA fragment, which is ligated to the previous 1indl[[-8a1I fragment.

This was used to transform E. coli DHI strain and the pH
Plasmid containing C3F-cDNA derived from GG4, p
TN-G4 (FIG. 9) (Example 13).

On the other hand, 1IGA410 plasmid or pHGA410(H
) pH which is a recombinant vector (+VSE) for CHO cells using the plasmid and pAdD26SvpA plasmid.
GG4-dhfr was constructed.
m1-1 ■Recovered by treatment, pHGA410 ()
・Insert into the 1indI [1 site of pG4DR1]
and pG4DR2 was constructed (Figure 10c) (Example 15)
.

CB) Construction of -VSE-based recombinant vector The cDNA (-VSE) fragment generated in (3) above was used as vector
After integrating into KCR to obtain a pHGV2 plasmid (Example 18), this was partially digested with ECORI to make the ends blunt. this DNA
Add a l-1indIII linker to the l-1i
After ndl treatment and T 4 DNA ligase treatment, the rubidium chloride method (Mo1ecular
Cloning) using E, coli [)H
The I strain was transformed. The obtained plasmid was transformed into pHGV2
(H>, !: Naming Suru (Figure 12).

pHGV2 (H> was treated with 5alI, then the end was converted to plant-end and then treated with HindIII again)-1
pdBPV- carrying the transforming fragment of bovine papillomavirus while recovering the indlll-3a l I fragment.
1 plasmid was treated with Hind[[, pvu[, the larger DNA fragment was separated, and this and the previous HindlI
Join the I-3a II fragment. Using this, E, C
01iDHI strain was transformed with pHGV2-derived C3F-
A plasmid containing cDNA, DTN-V2 (
Figure 12) (Example 19).

+VSE as well as pHGV2 plasmid or p t-IG
Recombinant vector for CHO cells (-VSE
) is DHGV2-dhfr￥! - Constructed (Fig. 13
a and b) (Example 21).

Furthermore, a DNA fragment of approximately 2 Kb containing the DHFR5 gene was extracted from pAdD26SVDA with EC0RI and BamHI91.
The Hin of pHGV2 (t-1) was collected by
pV2DR1 and pv2DR2 were inserted into the dI [1 site] (Fig. 13C) (Example 21).

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

On the other hand, the literature by Baner'j et al. (Cell vol. 27, 2
psVHK described on page 99 (1981))
The plasmid was treated with Kpnl to remove the globilin gene, and then partially digested with t-1indI[l to generate SV4.
After removing a part of the late gene of 0, it was recombined to prepare an expression vector pML-E.

This vector was treated with the restriction enzyme EC0RI, and then dephosphorylated with alkaline phosphaterpi (manufactured by Takara Shuzo Co., Ltd.), and the resulting vector DNA was added with T4 DNA ligase (manufactured by Mitsu Shuzo Co., Ltd.) to form the above-mentioned chromosomal DNA fragment. Combined with CO8
The cell recombinant vector pMLCE3α was used as j9 (Example 24). As shown in Figure 14, this plasmid
40 gene enhancer-1 SV40 replication initiation region,
A plasmid containing the replication initiation vA region of pBR322 and the β-lactamase gene (Amp') derived from pBR322, with human G-lactamase downstream of the enhancer of SV40M Buddha.
The C3F chromosome gene is connected.

Construction of an expression vector for C127 cells was performed as follows.

That is, a DNA fragment containing the chromosome-derived C3Fi gene was cut out from the expression vector pMLCE3α in CO8 cells using restriction enzymes, and T4 DNA was added to the DNA fragment containing the replication origin of bovine papilloma virus (BPV) and the DNA fragment containing the SV40 early promoter. Ligation was performed using ligase. The obtained pTNCE3α is an expression vector in which the chromosome-derived C3F gene is linked downstream of the SV40 early promoter and contains 65% of BPV.

On the other hand, the expression vector for CHO cells is a DNA fragment containing the same chromosome-derived C8F3tm gene and a part of the SV40 early promoter as above, and a DNA fragment derived from ρAdD26SVpA.
14 DNA fragments containing the I-I FR gene
These are linked using ligase.

4% pT26SVCE3ff is 5V407C1-
This is an expression vector that has a chromosome-derived C8F gene downstream of the E-tar and a Df-IFR return gene downstream of the adenovirus main late promoter.

(6) Expression of traits using animal cells Representative examples will be described here, but for other cases, please see each Example.

(A) Expression of pTN-G4 by mouse C127 cells
”! Bam Lasmid or DTN-V27' Lasmid
C127 cells, which have been treated with HT and grown in culture, are transformed using the HT-calcium method. The obtained transformed cells are cultured and clones with high C3F production ability are selected.

The expressed G-C3F was recovered and purified from the culture medium of these transformed cells, and it was confirmed that it exhibits human 〇-C3F activity. The target glycoprotein was also confirmed by amino acid analysis and sugar content analysis of the sample.

Regarding sugar content analysis, C3 used for amino acid analysis
Sample F was subjected to amine axis quantification using the Erlan-Morgan method, neutral sugar quantification using the Orcinol fiiRf2 method, and sialic acid quantification using the Thiopal-Gotzu method.

The quantitative method is described in Biochemistry Experiment Course Volume 4 “Chemistry of Carbohydrates (Volume 2)”
” (Tokyo Kagaku Doujin), Chapter 13. As a result of converting each quantitative value into weight%, the sugar content of the obtained G-C3F was 1 to 1, depending on the expression vector, culture conditions, etc.
It was distributed in a range of 20 (wt%).

(B) Character expression by CO3 cells G-C3F derived from human chromosomes obtained in (5)-(C) above
The vector pMLcE3α containing iButsuko is a monkey CV-
CO8 cells derived from 1 cell and transformed with a replication origin-deficient mutant of SV40 and expressing the large T antigen of 5v40 (Gluzman et al.; Ce1132 Vol. 1
75 (1981)) as a host cell, it was found to exhibit human G-C8F activity (Example 25).

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

〔Example〕

The present invention will be described in detail below with reference to Examples, but before that, a method for measuring C3F activity will be explained using Reference Examples.

Reference Example> Method for Measuring C8F Activity The method for measuring C3F activity (hereinafter abbreviated as C3A) used in the present invention is as follows.

rcsA measurement method" (a) When using human bone marrow cells: method of Bradley T, R, Hctcalf D, et al. (^ust, J, Exp, Biol, He
d, Sci, Vol. 44, pp. 287-300, 196
The culture was carried out using the monolayer soft agar culture method according to the method described in 1996). Namely, fetal bovine serum 0.2mi, test specimen 0.1d, human bone marrow non-adherent cell suspension o, 1rn1 (1-2X105 nucleated cells), modified McCoy's 5A culture solution 0.21
d, modified HcCoy' s5A containing 0.75% agar
After mixing 0.4 d of nutrient solution and solidifying it in a tissue culture plastic dish with a diameter of 35 mm, the mixture was heated to 37°C.
, 5% carbon dioxide / 95% air, 100% humidity conditions, count the number of colonies formed after 10 days (one colony is a colony consisting of 50 or more cells),
C8A was determined by setting the activity of forming 1 colony as 1 unit.

(b) When using mouse bone marrow cells: Horse serum 0.4d, test sample 0.1nj! , C31-
1/He (female) mouse bone marrow cell suspension 0.1 d (0
, 5-1 x 105 nucleated cells), modified with 0.15% agar) fccoy' S 5A culture medium 0. /7 was mixed and placed in a plastic dish for tissue culture with a diameter of 35 mm, and after solidifying, it was cultured for 5 days at 37°C, 5% carbon dioxide/95% air, and 100% humidity to form a tissue culture dish. The number of colonies (a colony consisting of 50 or more cells is considered as one colony) was counted, and the activity of forming one colony was calculated as 1.
C3A was determined as a unit.

The modified HcCoy's 5A culture solution used in methods (a) and (b) above and the human bone marrow non-adherent cell suspension used in (a) were prepared as follows.

"Modified HcCoy's 5A culture solution (2x yA degree)"
HcCoy'S 5A! Nutrient solution (manufactured by GIBCO) 12
g.

MEM amino acid vitamin medium (manufactured by Hakusui Pharmaceutical Co., Ltd.) 2.55
After dissolving 2.18 g of sodium bicarbonate and 500 units of penicillin G potassium in 500 rIIl of distilled water, filtration sterilization was performed using a 0.22 μm Millipore filter.

"Human bone marrow non-adherent cell suspension" RPM1164G bone marrow fluid obtained from healthy human sternum puncture
Diluted 5 times with culture solution, layered on Ficol-Paque solution (manufactured by Pharmacia), and incubated at 400×g for 30 minutes.

Centrifuge at 25°C, and the cell layer at the interface (specific gravity 1.07)
7) Collect. After washing the cells, they were incubated at 5×106C in RPM I 1640 culture medium containing 20% fetal bovine serum.
After adjusting the m degree of el l/rI to 11 m, put it in a 25 cut plastic tissue culture flask and incubate for 30 minutes in a carbon dioxide gas incubator, collect the non-adherent cells in the supernatant, and put it again in a 25 CIi plastic flask. After incubating for 2 hours and 30 minutes, the supernatant of non-adherent cells was collected and used.

Example 1 Establishment of rcHU-2J A tumor from a patient with oral floor cancer in which a marked increase in neutrophils was observed was transplanted into nLJ/nu mice. This tumor is transplanted approximately 10 times
A day later, clear tumor growth and an increase in neutrophils were observed.

This tumor was removed aseptically 12 days after transplantation, and
It was cut into cubes and cultured as follows.

50/i! Place the 5r111 trypsin solution (
After adding 0.25% trypsin and 02% EDTA O, and photographing for 10 minutes in a 37°C hot bath, the supernatant was discarded, and 5 Ini of the same trypsin solution was added again.
Trypsin digestion was performed with stirring for 15 minutes at °C.

Collect the supernatant cell suspension and add 1r111 fetal bovine serum.
In addition, after stopping the action of trypsin, they were stored in water.

The above operation was repeated to collect the cell suspension, which was combined with the previous suspension and centrifuged at 1.50 Or, p, m for 10 minutes to obtain a cell pellet.

This cell pellet was mixed with 10% fetal bovine serum [-10
After washing twice with water, the cells were implanted into a 25 clft plastic culture flask at a density of 5 x 10 6 cells/flask. Using F-10 culture solution containing 10% fetal bovine serum, a portion of the culture solution was incubated in a carbon dioxide incubator (5% carbon dioxide concentration, 100% humidity), and the supernatant was removed together with non-adherent cells. Culture was continued by adding new culture medium. The cells proliferated rapidly on the 6th day after the start of culture, so at this point the culture medium was replaced with a new one. The next day, discard this culture solution and use RPM I
Anti-mouse red blood cell antibody (Cap
Guinea pig complement (manufactured by Kyokuto Pharmaceutical Co., Ltd.) 2d diluted 2.5 times with RPM I 1640
was added and incubated at 37°C for 220 minutes. F-10 containing 10% fetal bovine serum after incubation
The fibroblasts derived from n IJ/nu mice were removed by washing twice with F containing 10% fetal bovine serum.
After adding 7-10 culture solution and culturing for another 2 days, a portion of the cells were taken out and cloned by the limiting dilution method. (When the C8F activity was examined for the 11 clones identified, 17 clones (CHU-2>) were identified that exhibited approximately 10 times higher activity than the others.

Example 2 Isolation of CSF The cells established as described above grew completely and densely.
Cells were collected from two 50 cIi culture flasks and mixed with 500ml of F-10 culture medium containing 10% fetal bovine serum.
After floating in 1580 ci glass roller bottle (manufactured by Betco),
Rotary culture was performed at a speed of , m. When the cells grew completely and densely on the inner wall of the roller bottle, the culture medium was changed to serum-free RPM I 1640, cultured for 4 days, the culture supernatant was collected, and F containing 10% fetal bovine serum was used. -10 and continue culturing.

After culturing for 3 days, use RPM1164 without serum again.
Wave sound was performed at 0, and the culture supernatant was collected 4 days later.

By repeating the same procedure, 50 (7!) serum-free culture supernatants can be obtained from one bottle every week.
Moreover, this method made it possible to maintain cells for a fairly long period of time and collect the culture supernatant. 5 g of the obtained culture supernatant was made into one batch, and after adding 0.01% Tween 20, 11o11ow Fiber DC
-4 and 8m1con PM-10 (Amicon Co., Ltd.) were concentrated approximately 1000 times by ultrafiltration, and then purified in the following order.

(i) Old trog with a diameter of 4.6c and a length of 90c.
el ACA54 column (manufactured by LKB), 0.1
0.01M Tris-HCl buffer (D)-1 containing 5M NaCl and 0.01% Tween 20 (Hanka Kagaku Co., Ltd.)
7, 4) at a flow rate of about 5 d to the concentrated culture supernatant 5d.
0mf! /hour. The column was pre-prepared with bovine serum albumin (molecular weight 67,000), ovalbumin (molecular weight 45,000), and cytochrome C (molecular weight 45,000).
Sharp represion was performed at a molecular weight of 12.400>. O,17 from each fraction after gel filtration! After collecting each sample and diluting it 10 times, the fraction showing activity was examined using the rcsA measurement method (b) described above. As a result, both Ve = 400-7007 showed macrophage-dominated C3A, and Ve = 800-1200 m
It was found that both fractions showed granulocyte-dominated C3A, so the latter two fractions were collected and concentrated to about 5 d using an ultrafilter using PM-10 (manufactured by Amicon).

(ii) A 0.1% trifluoroacetic acid aqueous solution containing 30% n-propertool (manufactured by Tokyo Kasei Co., Ltd., for amino acid sequencing) was added to the concentrated fraction, and after standing in water for about 15 minutes, 15. The precipitate was removed by centrifugation at 000 r, p, m for 10 minutes. Then, u equilibrated with an aqueous solution containing the above n-propanol and trifluoroacetic acid.
Bondapak C18 column (manufactured by Waters,
For semi-separation, 30-60% after adsorption to 8 shoes x 30cm>
The mixture was sequentially eluted with a 0.1% aqueous trifluoroacetic acid solution containing n-propatool with a linear concentration gradient of . The high performance liquid chromatography device is Hitachi 685-50, and the detection is Hitachi 638-41.
Using a type detector (both manufactured by Hitachi), 220 nm
and 280nl11 absorption were measured simultaneously. After elution, 10 μg was taken from each fraction and diluted 100 times, and both fractions showing activity were examined by the above-mentioned "Measurement method for C3A (b)". As a result, activity was observed in the peak eluted at 40% of 1-propatool, so this peak was collected and re-chromatographed under the same conditions.
When we investigated this, we found activity at the peak at 40% n-propatool, so we collected this peak (
4 fractions = 4rIJl) lyophilized.

(iii) Add 40% of n-propertool to the above freeze-dried powder.
The solution was dissolved in 200 μg of a 0.1% trifluoroacetic acid aqueous solution containing 0.1% trifluoroacetic acid and subjected to high performance liquid chromatography (HPLC) using a TSK-G300O3W column (manufactured by Toyo Soda Co., Ltd., 7.5 #1IIi x 60Cgt). Elution was performed with the same aqueous solution at a flow rate of 0.4 ml/min, and fractions of 0.4 d were collected using a fraction collector FRAC-100 (manufactured by Pharmacia). C3 for each separated fraction
As a result of examining A in the same manner as above, the retention time was 37-3
Activity was observed in the 8 minute fraction (corresponding to a molecular weight of approximately 20,000), so both fractions were collected and further analyzed using μBOndapa.
k C18 column (4,6#!IIIX 30cm)
After purification, the main peak was collected and lyophilized. The obtained specimen was assayed by the above-mentioned "C8A measurement method (a)" and was found to have human G-C3F 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 jS
The PTI-1 amino acid obtained was collected using a high performance liquid chromatography device (manufactured by Beckman Instruments) and
Itraphcre-0DS column (Beckman
(manufactured by Instruments Inc.) using a conventional method.

Column (5 μm, diameter 4.6 mm, length 250 m) was prepared with starting buffer (15 mM vinegar M phthorium buffer pt-14,5
After equilibration with an aqueous solution containing 40% acetonitrile), the sample (dissolved in 20 μg of starting buffer) was injected and separated by isocratic elution with the starting buffer. The flow rate was 1.4 nf/min and the column temperature was maintained at 40°C.

PTH amino acid was detected using ultraviolet absorption at 269 nm and 320 nm. Each 2 nmo+ of standard PTH amino acids (manufactured by Sigma) was separated in advance in the same system, the retention time was determined, and identification was performed from the retention time of the test specimen.

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

H2N Thr Pro L eLJ GIV-Pro
-A I a-3e r-3e r-L eu -P
ro-Gl n-3er-Phe-Leu-Leu-L
'l/5-CVS-Leu -G I LJ -G I
n-Va l-Arg-Lys -I I e -G
l n-GI V-AS p-GI V-AI a-A
l a-Leu -G l n-G I u-L y
s-Leu-Cys-AI a-Thr-Tyr-1
j/5- (ii) A decomposed sample of bromic cyanide was dissolved in 70% formic acid, 200 equivalents of bromic cyanide purified by sublimation were added, and the mixture was reacted overnight at 37°C.

Next, the reaction product was freeze-dried and then fractionated by HPLC using a TSK G300O3W column (manufactured by Toyo Soda Co., Ltd.) to obtain four peaks. The peaks are arranged in descending order of molecular weight: CN-1,
Named CN-2, CN-3, and CN-4, C
The amino acid sequences of N-1 and CN-2 were analyzed using an automatic gas phase sequencer (manufactured by Applied Biosystems) under the same conditions as in (i).

As a result, it was found that CN-1 is a peptide from the N-terminus of G-C8F protein. Furthermore, CN-2 had the following amino acid sequence.

Pro-Alia-Phe-Alia-8e r
-Al a-Ph e-G In-A r g-
A r g-A I a-G ly-G I y-V
a I -Leu -Va l -A l a-3e
r-His-1eu-1n- (iii) The i-lipsin degradation sample was dissolved in 0.1M Tris-HCl buffer containing 8M urea (11
7.4)) and added 7 JO of 0.1M Tris-HCl buffer (pH 7,4) containing 0.1% 2-mercaptoethanol to adjust the final concentration to 2M urea.

Next, add PCK-treated trypsin (trade name, manufactured by Sigma) so that the ratio of sample and enzyme is 50=1, and incubate at 25°C for 4 hours.
After reacting for an hour, the same amount of TPCK-treated trypsin was added, and the reaction was carried out again at 25° C. 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%
R)-propatool containing TFA was used and the n-propatool concentration was increased linearly from 5% to 60%. 28
Among the peaks obtained by measuring the ultraviolet absorption of 0n... and 1q, 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, it was found that the main peak was a peptide having the following sequence containing a part of the CN-2 fragment (ii).

G l n-1eu-ASp-Va l -A I a
-ASp-Phe -A I a-Th r-Th
r -1l e-Tr p-G l n-G I n-
Met-Glu-Glu-Leu-G
IV-Met-A1a-Pro-A1a
-L eu-G l n -Pro-Th r-G l
n-Gly-A Ia-Met-pro-A
I a-Phe-Ala-er- Example 4 Preparation of DNA probe (i) Synthesis of probe (IWQ) Example 3. Ile-'rrp-Gln-Gln-Me from the amino acid sequence obtained in (iii)
t-Glu-Glu-Leu-GIy
Based on the sequence of 10 amino acids indicated by -Met, 30 consecutive nucleotides were obtained (Figure 1). In the sequence of FIG. 1, for example, the nucleotide at position 9 from the 5'-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. Nucleotide resin Ap as a starting material is placed in a column with a glass filter.
-d (G) (manufactured by Yamana Shiyu Co., Ltd. > 2 omg) was added, and after repeated washing with methylene chloride, the 4,4'-lame 1-xytrityl group was removed with a methylene chloride solution containing 3% trichloroacetic acid. The column was washed several times with 1d methylene chloride. After washing with anhydrous pyridine and replacing the solvent, the nucleotide dimer (DMTr) ApTp
(NHR3) (manufactured by Japan L-ON Co., Ltd.; N [•IF5 indicates triethylammonium, DMTr indicates dime] to oxytrityl) 20 mg and 0.27 hours of pyridine were added, and the inside of the column was vacuum-dried using a vacuum pump. Next, 2, 4.
After adding 6-trimethylbenzenesulfonyl-3-nitrotriazolide (MSNT, Iu > 20 rng from Wako Pure Chemical Industries, Ltd.) and 0.2 d of anhydrous pyridine, the inside of the column was purged with nitrogen gas and the column was shaken occasionally for 45 minutes at room temperature. The nucleotide resin and dimer were condensed by boiling. After the reaction, the column was washed with pyridine, and unreacted OH
After the group was acetylated with a pyridine solution containing excess acetic anhydride and 4-dimethylaminopyridine, the column was washed again with pyridine. Similarly, (DHTr)It)
(NHR3) , (DHTr)Gl)Gl)(
NtlR3) , (DHTr)It) (NHR
3) , (DHTr)Cpdingp (NHR3
) and (D)lTr)DTI)(NtlR3), ([)ITr)ApAD(NtlR3)
and (DHTr)ApGI) (NHR3),
(D)lTr)ApGp(NHR3) and ([)HTr
') Gt) Homogenous mixture of Gp(NtlR3).

(DHTr)Gt)AI)(NtlR3), (
DHTr) DingpGp(NHR3), (D)tT
r)ADAi)(NHR3) and (DHTr)Gl)AI
) (NtlR3), (DHTr
)C1)Ap(NHR3), (DI-ITr)At
) AI) (NHR3) and (DMTr) ApGI) (Nl
lR3) in an equi-N mixture.

(DHTr)Gl)CI)(NHR3), (
DHTr)TpGD(NHR3).

(DHTr)Ip(NtlR3), (DHTr)8pr
D(NHR3) [(DH[r)II) (NIIR3) is manufactured by Yamasa Oil Co., Ltd., and all others are manufactured by Nippon Zeon Co., Ltd.], and condensation was carried out by repeating the above-mentioned operation in this order. After the final step of the reaction, the resin was washed with pyridine, methylene chloride, and ether in this order without acetylation.
Dry. The dried resin was suspended in 1.7 ml of a mixture of 1 ml of dioxane containing 1 M tetramethylguanidine and 1 M α-picoline aldoxime, 0.5 d of pyridine, and 0.2 ml of water, and then left at room temperature overnight. ,
It was concentrated under reduced pressure to 100-200 μg.

After adding a small amount (2-3 drops) of pyridine to this concentrate,
2nd to 3rd portions of concentrated ammonia water were added and 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, and then dissolved in a 50 mM t'ethylammonium acetic acid solution (DH7.0) and loaded onto a C-18 column (1, OX 15 cm, Water (manufactured by rS)
It was subjected to column chromatography using . The elution is
50mM triethylammonium acetate solution (pH 7,
A linear concentration gradient of acetonitrile from 10% to 30% in 0.0% was used, 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 allowing it to stand 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 Center 1-Kagaku Co., Ltd., 5SC-〇DS-272, 5φ
Roughly purified by high performance liquid chromatography using 200m x 200m>.

Elution was performed using 50mM triethylammonium acetate solution (pH
10A 2601Jn! [Synthesized NA was obtained in a yield of 3 or higher.

(ii) Synthesis Example 3 of probe (A). Among the amino acid sequences obtained in (iii), Met-Pro-Ala-Phe-Ala
We obtained 14 consecutive nucleotides based on the sequence of 5 amino acids shown in (Figure 1).

Synthesis was performed in the same manner as the probe (IWQ) using nucleotide resin AP-d (T) (manufactured by Yamasa Akane Oil Co., Ltd.) km
(DHTr) CpAri (NHR3): (DHTr
)GDGI)(NHR3): (DHTr)CpAp
(NHR3), (DHTr)Cpdingp(Ntl
R3).

(DHrr)DpGp(NHR3) and (DHTr)
homogeneous mixture of (CpCI) (NtlR3); (DHTr
)ApGI)(NHR3), (DMTr)Tl)G
l)(NtlR3), (DHTr)hh(NHR3
) and (DHTr)CpGp(NHR3); ([))lTr)^DAp(NIIR3):
(DHTr)CpAp(NHR3) and (DHTr)Cp
Homogenous mixture of (Gl) (NHR3): (DHTr)Go
(NHR3) (both manufactured by Nippon Pion Co., Ltd.) in order of condensation and approximately 10A 260 un! A synthetic NA of Is was obtained.

HaXam the base sequence of the oligonucleotide
- When examined by Gilbert's method, it was confirmed that it had the base sequence shown in FIG.

(iii) Synthesis of probe (LC) Automated DNA synthesis was performed using Applied Biosystems' 380A. This method is described by Caruther
The principle described by S et al. (J, Am, Chem.

Soc, Vol. 103, p. 3185 (1981)), and is called the phosphoramidite method.

After condensing the phosphoramidite form of (DMTr)-dT previously activated with tetrazole to dG-3 (S is the support) from which 5' dimethoxytrityl W (DMTr> has been deprotected), the unreacted hydroxyl group is converted to acetyl. and then iodine oxidation in the presence of water to give a phosphate form.The DHTr group was deprotected and condensation was repeated in the same manner to synthesize 24 nucleotides with the sequence shown in Figure 1. After the nucleotides are cleaved from the support and deprotected, C.
18 columns (manufactured by Senshu Kagaku Co., Ltd. 5SC-ODS-27
It was purified by reverse phase high performance liquid chromatography using 2).

Example 5 CHU-2 cell culture and mRNA purification 1) C)-ILJ-2 cell culture and cell recovery Established CHU-2 cells were grown to complete density in two 150 cm culture flasks. After that, add 10% of this to fetal bovine serum.
After suspending it in 500 volumes of RPM I 1640 culture solution containing 0.5% RPM I, it was transferred to a 1580CIA glass roller bottle (manufactured by 3elco), and 0.5r, p,
Rotary culture was performed for 4 days at a speed of m. When cells have grown completely and densely on the inner wall of the roller bottle, remove the culture medium from the rotor bottle and pre-warm at 37°C.
Add 100 d of physiological saline containing 0.02% DTA,
After heating at 7°C for 2 minutes, cells were detached by pipetting. (Photographed cell suspension at 150 or p.
m, cell pellet by centrifugation for 10 min (7 min).

The cells were resuspended in 5 ml of saline without EDTA and centrifuged at 1500 r, p, m for 10 min to pellet the cells (wet weight approx. RNA output operation up to 180°
Store frozen at C.

2) Purification of mRNA mRNA from CHU-2 cells obtained as above
The isolation of is essentially ') molecular cloni
ng ”[)ianiatis et al., Co1d Spr.
ing labor, p. 196 (1982)]
It was carried out as described.

Cryopreserved CHU-2 cells (wet weight 3.89)
20d of 6M guanidine solution (6M guanidine thiocyanate, 5mM sodium citrate (pH 7,0),
0. Suspended in IM β-mercaptoethanol, 0.5% sodium sarcosyl sulfate) and mixed well with VO ('teX mixer for 2 to 3 minutes, then injected 10 times using a 20d syringe equipped with an 18G needle) Inhalation and expulsion were repeated. 5.7M of 6rIJl was placed in a polyaromatic centrifuge tube that fits a Beckman 5W40Ti rotor.
CsCl-0, IM EDTA, (pH 7
, 5) first, and then add 6 m of guanidine solution, which has broken down the cells and become viscous, to fill the tube.
layered. Centrifuge tube 4 prepared in this way
3 (), ooor, l) 9m, 15 at 20℃
After centrifugation for an hour, the resulting pellet was washed three times with a small amount of 70% ethanol.

Combine the pellets from each tube to 550μ
After adjusting the NaCl concentration to 0.2M in water, phenol-chloroform (1:1) was added.
After treatment and chloroform treatment, 2.5 times the volume of ethanol was added to perform ethanol precipitation to obtain total RNA (approximately 10.1 g of RNA was obtained from 3.8 g of wet cells).

Purification of poly(A)-RNA from total RNA was performed as follows. This method is affinity chromatography that utilizes the fact that mRNA has a polyA chain added to its 3' end. Oligo(dT)-Se)Lt loose (P
For adsorption, total RNA was mixed with adsorption buffer (10mM Tris-L Biochemicals arype7).
Hydrochloric acid (pH 7.5), 0.5M NaCl, 1mM
EDTA, containing 0.1% SDS solution),
After heating at 5°C for 5 minutes, the same solution was passed through an oligo(dT)-cellulose column packed, and elution was carried out using a TE bath solution of 10mM Tris-HCl (p)-17,5>
, containing 1mM EDTA). The unadsorbed effluent was passed through the same column again, subjected to the same elution operation, and mixed with the first eluate.

Using such operations, poly(A>-RNA40
0 μq was obtained.

The mRNA thus prepared was mixed with 5chlcif and W
ens ink's experimental technology @ (Practical
)tcthods inMolecular Bio
logy, Springer-Verlag, Ne
wYork, Heiderberg, Berlin
JJ-Is fractionation was performed by sucrose density gradient centrifugation in a manner similar to that described in , (1981)).

That is, a 5% to 25% sucrose density gradient is created in a tube for a 5W40Ti o-tar (manufactured by Beckman).

The sucrose solution is 0. IM NaCl, 10mM Tris-
Hydrochloric acid (pH 7.5), 1mM EDTA, 0.5%S
RNas at a rate of 5% and 25%, respectively, in a solution of DS.
Contains e-free sucrose (Schwarz/)iann).

mRNA (poly(A
)-RNA > 800μ9 to 200μ9 to 500μ
Dissolve g in TE bath solution, heat at 65°C for 5 minutes, rapidly cool, and place on top of the sucrose density gradient solution.

0.5 after centrifugation for 20 hours at 30000 r, p, m.
The absorbance at 260 nm was measured by collecting ml fractions, and the size of the fractionated RNA was determined based on the position of the standard RNA (28S, 18S, 53 ribosomal RNA), which was carried out in the same way. G-C3F activity was investigated using the African frog (Xenopus Iaevis) oocyte system.
Prepare an aqueous solution with a concentration of μg/μg and apply 50 lq mR to one oocyte taken from a Xenopus laevis (approximately 1 year old).
After injecting NA at the same ratio, 10 oocytes were placed in each well of a 96-well microtiter plate, and 100 μm of Pers's medium (88 mH NaCI, 1 m
HKCI, 2.4mM NaHCO3.

0.82mM MClSO4, 0.33mHCa
(NO3) 2.

0.41 ml CaCI2. 7.5m) f Tris-
Salt Wi (pH 7,6), penicillin 10ml/fl
After culturing at room temperature for 48 hours in 10 mg/liter of streptomycin sulfate, the supernatant was collected, concentrated and purified, and G-
Measure C3F activity.

As a result, G-C3F activity was observed in the 15-17S fraction.

Example 6 CDNA synthesis (construction of pBR-based CDNA library) 1-
[Nucleic Ac1ds Res
,, Vol. 9, p. 2251 (1981)], Gub.
ler and Hoffman's method [Gene, 25
vol. 263 (1983)], cDNA was synthesized.

1) Synthesis of single-stranded CDNA Place the reagents in the following order into a 1.5rIIN (manufactured by Eppendorf) tube. 80 μg reaction M'tX
i solution (500mM KCfl, 50mM MQ (
j! 2, 250mM Tris-Salt W, pH 8,3), 2
0μ, 200mM dithiothreitol, 32μ fJ
12.5 m of g) l d N T P (dATP, d
Contains 12.5mM each of GTP, dCTP, and dTTP)
, 10μ, 1! of α-3”P-dCTP (manufactured by Amajam, PB 10205), 32 μg of oligo (dT
) (manufactured by PL Biochemicals.

500μg/IIJ1), 20μg poly(A
>-RNA (2.1 μg/μg) and 206 μg of distilled water, a total of 400 μg of the reaction solution, was heated at 65°C for 5 minutes, then
Warm at ℃ for 5 minutes. To this reaction solution, 120 units of reverse transcriptase (manufactured by Takara Shuzo) were added, and after further reaction at 42°C for 2 hours, RNaSe inhibitor (Bethesda R
Manufactured by e5earch Laboratories>2μu
, 20 μJ of TE bath solution, 16 μg of 100 mM sodium pyrophosphate, and 48 units (4 μg) of reverse transcriptase were added, and the mixture was reacted at 46° C. for 2 hours. 0.5M
EDTA 8μβ.

After stopping the reaction by adding 8 μg of 10% SDS,
Single-stranded CDNA was obtained by phenol-chloroform treatment and ethanol precipitation (twice).

2) Addition of dC-strand to single-stranded CDNA After dissolving the single-stranded CDNA obtained above in 60 μp of distilled water, 60 μg of dC-strand addition buffer (400 mM potassium cacodylate; 50 mM Tris-HCl (pH 6) ,9)
: 4mM dithiothreitol: 1mHCoCI2:1
mM dCTP> and heated at 37°C for 5 minutes.

Add terminal transferase (27un) to this reaction solution.
it/μ, Q, P-L Biochemicals
After adding 3 μg of the product (manufactured by Seiko Co., Ltd.) and reacting at 37°C for 2.5 minutes,
Phenol-chloroform treatment (once) and ethanol precipitation (twice) were performed, and the product was dissolved in 40 μg of TE solution containing 100 mM NaCl2.

3) Synthesis of double-stranded CDNA Add 4 μg of oligo(dG) 1 to the above 40 μj DNA solution.
2-13 (200μ(J/ml, P-L Bio
Chemicals) was heated at 65°C for 5 minutes, then at 42°C for 30 minutes, and then the reaction solution was kept at 0°C. To this reaction solution was added a buffer solution of 80μρ (100mM Tris-HCl, pt-17,5,20mM MqC12,50
mM (NH4)2304, 500mM KCI
), 4 μJ) of 4 mM dNTPs (dATP, dCT
(containing 4mM each of P, dGTP, and dTTP), 60μ
, 1mM β-NAD in Q and 210μm distilled water,
20 μg of E, coli DNA polymerase ■ (manufactured by Takara Shuzo Co., Ltd.), and 15 μp of E and G.

1i DNA ligase (manufactured by Takara Shuzo Co., Ltd.), 15 μg of E, c
Add oli RNase H (manufactured by Takara Shuzo Co., Ltd.) to 12
After reacting for 1 hour at °C, an additional 4μρ of 4mM dN
Add TP and react at 25°C for 1 hour to convert phenol-
Chloroform treatment, ethanol precipitation 111ffi (1 time)
Approximately 8 μg of double-stranded CDNA was obtained. This double-stranded CDNA was dissolved in a TE bath solution, subjected to 1.2% agarose gel electrophoresis, and a portion corresponding to the large serrations of approximately 560 base pairs (bp) to 2 kilobase pairs (Kbp) was isolated using Whatman DE81 (Whatman Approximately 0.2 µg was recovered by adsorption and elution using a gel (manufactured by Co., Ltd.).

4) Addition of dC-strand to double-stranded CDNA.
dC-chain addition buffer 8 described in section 2).
After adding μg and heating at 37°C for 2 minutes, 1 μ, Q terminal transferase (27 units/μfJ>
was added and reacted at 37°C for 3 minutes. The reaction solution was immediately cooled to O'C and 1 μg of 0.5M EDTA was added to stop the reaction, followed by phenol-chloroform treatment and ethanol precipitation.
suspended in.

5) Construction of pBR-based cDNA library 4 μg of commercially available oligo (dG) chain-added pBR322 vector (manufactured by Bethesda Reach Laboratories, 10 μg/μg)
and 75 μg of 2 μN of the above dC-stranded double-stranded CDNA. Annealed in TE bath solution containing IM NaCl. Annealing was performed at 65°C for 5 minutes, then at 40°C for 2
The mixture was heated for an hour and then left to stand until it reached room temperature.

On the other hand, Maniatis et al.'s experiment l[)tolecul
ar cloning.

Cold Spring Harbor, p. 249 (1982)], competent cells were prepared from E. coli strain X1776, and transformed with the above annealed plasmid. )was gotten.

Example 7 cDNA synthesis (construction of λ phage-based library) 1) Synthesis of single-stranded CDNA 3.8 g of cryopreserved CH according to the method described in Example 5
400 μq of poly(A+)-RNA was obtained from U-2 cells by purification using an oligo(dT) cellulose column twice.

After placing 0 μg of the warm TE solution in which 12 μq of poly(A+)-RNA had been dissolved into a reaction tube containing 10 μg of actinomycin D (manufactured by Sigma), reagents were added in the following order: 20 μρ of reverse transcription buffer; (250mM Tris-Salt M (pH 8,3>, 40mM MCJC12,2
50mv t<cl>, 20μ, 5mM dN of Q
TP (dATP, dGTP, dCTP.

(containing 5mM each of dTTP), 20 μm ori (dTTP)
) (0,2μ(J/rail P-L
Biochemica 13), 1 μg of 1M dithiothreitol, 2 μN of RN at 30 units/μjJ
as in (Promega Biotic Co., Ltd.), 10 μg of reverse transcriptase (10 units/μ, Q Seikagaku Corporation!u>
, 1ufl of (X-[32pldA-P (10μCi manufactured by Amaziyam), 16μp of water makes a total reaction solution of 100μg. After keeping the reaction solution at 42°C for 2 hours, 5μg of 0.5M EDTA and 1 μg 20% S
The reaction was stopped by adding O3. Phenol-chloroform (100μg) treatment, ethanol precipitation i! '! i (twice) to obtain approximately 4 μQ of single-stranded CDNA.

2) Synthesis of double-stranded CDNA 29 μg of the CDNA obtained above was dissolved in a TE bath solution, and the reagents were added in the following order to prepare a reaction solution;
ρ polymerase buffer (400mM
(pH 7,6): 16mM MciCl2
:63mM β-mercaptoethanol: 27
0mM KCI); 10d of 5mM dNTP; 1
．． 15mM β-NAD at 0μ, Q: 1. OuN a-[32pl dATP (10μCi/μfl)
: 0.2μ! JE, caliDNA rigger:
(60 units/manufactured by QQ Takara Shuzo Co., Ltd.); 5.0 μg of l:
, coli DNA polymerase engineering (New En
grand Biolabs, 10u
nit/μM); 0.1 μ, Q of RNA
se H (60 units/μ, manufactured by Q Takara Shuzo): 28
．． 7 μg distilled water.

After incubating the reaction solution at 14° C. for 1 hour, it was returned to room temperature and incubated in a colander for 1 hour. Then 5 μg of 0.5M EDTA and 1 μg of 20% SDS
was added to stop the reaction, followed by phenol-chloroform treatment and ethanol precipitation.

The obtained DNA was diluted with 0.5mM EDTA20. cz,
l! Dissolve in 3μ2+enow buffer (50
0mM Tris-HCl (DH8,0), 50mM
MCJCI 2>.

After preparing a reaction solution by adding 3μg of 5mM dNTP and 4μρ of water, 1μg of DNA polymerase (Kl
enow fragment) (manufactured by Takara Shuzo Co., Ltd.) and incubated at 30'C for 15 minutes.

Add 70 μg of TE bath solution to this reaction solution, dilute it, add 5 μg of 0.5M EDTA, 1 μg of 20% SD
The reaction was stopped by adding S. The reaction solution was treated with phenol-chloroform and precipitated with ethanol to yield approximately 8 μ9 of 2
The heavy chain cDNA is 1q.

3) Methylation of double-stranded CDNA 30 μg of the aqueous solution of double-stranded CDNA synthesized in section 2),
Methylation buffer (500mM l'-liss-hydrochloric acid (pt.
18.0), 50mM EDTA) 40μ, l! ,
EC was added to a mixture of SAM solution (800 μM MS-adenosyl-monomethylmethionine (SAM), 50 mM β-mercaptoethanol >20 μM, and 100 μM water).
0RI methylase (New England 8iol
abs, 20unit/μ, IJ) 15μj!
was added to bring the total reaction solution to 200 μp, and incubated at 31° 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-HCl (lI 7.5),
100mM MgC12)L 5μl, pre-phosphorylated EC0RI linker-0.5μN (
10mar, Takara Shuzo), 1.5μ, l! 10
mMATP, 100mM dithi A threitol 1
．． 5/, (41, 2 μg of H2O was added and the reaction solution was diluted to 15
T4 DNA ligase (3,4u/ufl,
After adding 0.7 μg (manufactured by Takara Shuzo Co., Ltd.) and reacting at 4°C overnight, the gauze was heated at 65°C for 10 minutes to inactivate it. This reaction solution was roughly mixed with 100mM Tris-HCl (pH
7,5).

5mM MgCl2.50mM NaCl, 100
After preparing the total liquid volume to 50 μm with a gelatin concentration of μa/1rdl, EcoRI (10 units/
μ, 1! >3.5 μg was added and reacted at 37° C. for 2 hours. then 0.5 M EDTA 2.5 μj), 20%
After adding 5 μg of SDS O, phenol-chloroform treatment was performed, and DNA was recovered by ethanol precipitation.

After that, the unreacted EcoRI linker was removed by gel filtration using Ultrogel^cA34 (manufactured by LKB) or agarose gel electrophoresis, and the linker-added 2-terminal C
Approximately 0.5-0.7 μQ of DNA was recovered.

5) Binding of double-stranded cDNA and λgtio vector The above linker-added double-stranded cDNA was added to
C0R1-treated λgtlo vector (Vector Cloning Systems), ligase buffer (250mM Tris-HCl, 100mM MgCI2>1.4μg, 6.5μp of distilled water) were added, and 15 molecules were treated at 42°C, followed by 10mM ATPlμm.

Add 1 μl of 0.1 M dithiothreitol and 0.5 μg of T4 DNA ligase to bring the total amount to 15 μg, and then incubate at 12°C.
The mixture was allowed to react overnight.

6) In vitro packaging Approximately 173 of the recombinant DNA obtained in 5) above was added using an in vitro packaging kit (Promega Biotique).
A phage plaque was obtained by package jig using

Example 8 Screening of pBR library using probe (IWQ) Whatman 541 filter paper was placed on the agar medium on which colonies had grown and left at 37°C for 2 hours. Below, Taub and Th
ompson's method [Anal, Biochem, 1
261,222 (1982)].

That is, after the colonies were transferred to 541'a paper, they were transferred to an agar medium containing chloramphenicol (250 μg/μp) and allowed to stand overnight at 37°C.

After taking out the 541ira paper, apply 0.5N at room temperature.
Leave it on the filter paper soaked with NaOH solution for 3 minutes, and then soak it for 2 minutes.
Repeatedly. The same operation was then carried out twice for 3 minutes using 0.5M Tris-HCl (pH 8> solution), and the temperature was further increased at 4°C.
At the bottom, add 0.05M Tris-hydrochloric acid (pH 8> solution for 3 minutes, L5my/ml lysozyme solution (0.05M Tris-
Hydrochloric acid (pH 8>, containing 25% sucrose) for 10 min, then 2 min at 37°C with 1X5SC (0,15M NaCI and 0.015M sodium citrate) solution for 20 min.
3 with 1x SSC solution containing 0 μg/ml protease K.
After 0 minutes, the 541'a paper was dried at room temperature again for 2 minutes with the 1X5SC solution and 2 minutes with the 95% ethanol solution. The obtained dry 541 filter paper was mixed with phenol:chloroform:isoamyl alcohol (25:
24:1, 100mM Tris-Salt M (pH 8,
5), 100mM NaCl.

The cells were soaked in a solution equilibrated with IGmM EDTA for 30 minutes. Repeat the same operation for 3 minutes with 5XSSC solution.
After three passes and then two passes of 3 minutes with 95% ethanol solution, the filter paper was dried.

The probe (IWQ) was prepared using a conventional method (Molecular cl
After radiolabeling with 32p according to the method of Wallace et al.
Colony hybridization was performed according to 1ds Res, p. 9W879 (1981).
ET[0,9M NaC! .

0.09M Tris-salt M (pH 1,5>, 6m
MEDTA], 5x Denhardt solution, 0
．． After prehybridization at 65°C for 4 hours in a hybridization buffer containing 1% SDS and 0.1 mg/rnll denatured DNA (calf thymus DNA), radiolabeled probe (IWQ) IXIO6C
Hybridization was performed overnight at 56° C. using the above hybridization buffer containing Dm/ml. After the reaction was completed, the 541'a paper was treated with a 6xssc solution containing 0.1% SDS at room temperature twice for 30 minutes and once at 56°C.
After washing for 5 minutes, autoradiography was performed.

After isolating the plasmid from the clone that gave the signal,
Saturday plotting was performed using probe (IWQ). Hybridization and autoradiography were performed under the same conditions as described above.

Southern blotting was similarly performed using the probe (A>. Hybridization was performed at 49°C for 1 hour using the above-mentioned hybridization buffer, and then at 39°C.
After cooling slowly until temperature reached 39° C., the test was carried out for 1 hour. After the reaction was completed, the nitrocellulose filter was washed twice with 6X SSC containing o, i% SDS at room temperature for 30 minutes, and then
After washing for 3 minutes at °C, autoradiography was performed.

As a result, one clone was obtained as a positive clone, and the base sequence was determined by the dideoxy method, as shown in Figure 2, probe (IWQ) and probe (A
The pBR322-derived plasmid containing this insert was named pHC3-1.

Example 9 Screening of λ)7-di-based library using pI-IC3-'l-derived DNA probe Benign and DaVis method [5science
196i, p. 180, (1977)]. Example 8
pl-IC3-1 obtained in 5au3A and EC0
A DNA fragment of about 600 base pairs was obtained by treatment with RI, and this DNA fragment was radiolabeled by nick translation according to a conventional method. A nitrohirulose filter paper (S&S Co.) was placed on the agar medium on which the phage plaques had formed, and the fercilla was transferred, the DNA was denatured with 0.5M Na0I-1, and the filter paper was treated in the following order. 0.1M NaOH,
1,5M NaCl for 20 seconds followed by 0.5M Tris-
Salt 1 (DH7,5>, 20 in 1.5M NaCl
2 times per second, and finally 120mM NaCl, 15
mM Sodium Citrate, 13 mM KH2PO4,1
Treat for 20 seconds with mM EDTA, pH 7.2.

The filter paper was then dried and heated at 80° C. for 2 hours to fix the DNA. 5xSSC, 5x[)cnhardt solution,
50mM phosphate buffer, 50% formamide.

0.25111! J/1all denatured DNA (salmon testis DNA) and pHC8, which was prehybridized overnight at 42°C in a hybridization buffer containing 0.1% SDS and radiolabeled by nick translation. -110-bu4X105CplII
/d hybridization buffer (5XS S
C, 5x Denhardt solution, 20mM J acid buffer (pH 8,0>, 50% formamide, 0.
1% SDS.

Hybridization was performed at 42° C. for 20 hours with a mixture of 10% dextran sulfate and o, img, /i denatured DNA (salmon testis DNA).

The nitrocellulose filter paper was washed with 2X SSC containing 0.1% SDS at room temperature for 20 minutes, and then at 44°C with 0.1% SDS.
0.0% containing SDS. After washing with 1×5SC for 30 minutes, wash at room temperature for 10 minutes with 1×SSC, and detect by autoradiography.

As a result, 5 positive loans (G'1 to 5)
was gotten. Therefore, among the clones obtained, full-length c
When the DNA base sequence of the clone thought to contain DNA was examined by the dideoxy method, the base sequence shown in FIG. 3(A) was obtained. Therefore, this cDNA was converted into λCJtl
It was excised from the O vector and pB R327[5ober
on et al.: Gene 9, p. 287 (1980)] and E.
It was ligated at the C0RI site and prepared in large quantities as a plasmid. This plasmid is called pBRG4.

Example 10. Screening of a λ Phage Library Using DBRG4-Derived DNA Probes and Probes (LC) Plaque hybridization was performed according to the method of BentOn and Davis (see the above-mentioned literature) used in Example 9. Nitrocellulose filter paper (manufactured by S&S) was placed on the agar medium on which phage plaques had formed, and the phages were transferred, the DNA was denatured with 0.5 M NaOH, and the filter paper was treated in the following order.

0.0, IM NaOH, 1.5 M NaCl for 20 s, followed by 0.5 M Tris-HCl (pH 7.5>, 1.5
MNaCl twice for 20 seconds and finally 120mM NaC
l, 15mM sodium citrate, 13mM KH2PO
4.1mM EDTA, (pH 7.2> treated for 20 seconds).

The filter paper was then dried and heated at 80° C. for 2 hours to fix the DNA. In this way, two identical filter papers were made, and p
Each sample was subjected to screening using a BRG4-derived DNA probe and a probe (LC). pBRG4-derived D
When using an NA probe, pBRG4 is treated with EC0RI to obtain a DNA fragment of about 1500 base pairs, and this DNA
The A fragment was radiolabeled by nick translation according to a conventional method. The above filter paper is 5XSSC15x Denh
ardt solution, 501118 phosphate buffer, 50% formamide, 0.25 my/d denatured DNA (salmon testis D
Prehybridization was performed overnight at 42°C in a hybridization buffer containing NA) and o, i% SO8, and the radiolabeled approximately 1500 base pair DNA probe described above (approximately 1
) containing prehybridization buffer [5XSS
C15x Denhardt solution, 20mM phosphate buffer (p) -16°O), 50% formamide, 0.1% S
DS, 10% dextran sulfate, 0.1 ffilf/r
denatured DNA (salmon testis DNA)]
Hybridization was performed at ℃ for 20 hours. Nitrocellulose filter paper was placed at room temperature with 2 containing 0.1% SDS.
Washed with XSSC for 20 minutes, then at 44°C with 0.1%
0.0 containing SDS. After washing with 1X5SC for 30 minutes and 0.1% SSC at room temperature for 10 minutes, the cells were detected by autoradiography.

For probe (LC), filter paper was pretreated with 3XSSC containing 0.1% SO8 at 65°C for 2 hours, and then
XNET, I XDenhardt solution, 100μ (I
In a solution containing t/d denatured DNA (salmon testis DNA),
Prehybridization was performed at 65°C for 2 hours.

Radiolabeled probe (LG> (2xlO6cpm
/d) in hybridization buffer [6X N
E T , 'l X 0enhardt solution, 10
0 μ0 /d denatured DNA (salmon testis DNA)] at 63°C
After hyperdivision overnight at room temperature, the nitrocellulose filter paper was soaked in 6
After washing with SC for 20 minutes and performing this washing three times, 0.
Washing was performed at 63°C for 2 minutes in 6XSSC containing 1% SDS.

After drying the filter paper, it was detected by autoradiography.

In the screening conducted in this way, we selected clones that were positive for both of the two probes, and examined the nucleotide sequences of clones that were thought to contain the full-length CDNA using the dideoxy method, as shown in Figure 4 ( A base sequence as shown in A) was obtained. So this cDNA
was cut out from the λgtio vector, and pBR327 and E
It was ligated at the CoRI site to obtain plasmid pBRV2.

Example 11 Screening of human chromosomal gene library 1) Construction of human chromosomal gene library Human chromosomal gene library was manufactured by Haniatis (Harvar
d University), and was created as follows.

Total chromosomal DNA is extracted from human fetal liver using phenol, etc., and partially digested with restriction enzymes 1-1ae[I and AIuI. Among the DNA fragments obtained in this way, the chain length is 18~
The approximately 25 Kb fragment was concentrated by sucrose density gradient centrifugation, and then isolated from Escherichia coli phage λCha via a short synthetic nucleotide with a restriction enzyme EC0RH cleavage site.
connects to the arm DNA of ron4A and is infectious) 7
-Creating a di-DNA recombinant. Next, in order to increase the infectivity, complete fur 92 was removed using a packaging method.
It's grainy. The human gene library created in this way is, in principle, considered to be a collection of recombinants containing human DNA with a chain length of 18 to 25 Kb, including almost all human genes.

2) Screening of human chromosomal gene library using pHC3-1-derived DNA probe BentOn and Davis method [5CienCe
Plaque hybridization was performed according to Vol. 196, p. 180 (1977)]. pHcs-1 obtained in Example 8 was treated with 3au3A# and EcoRI to obtain a DNA fragment of about 600 base pairs, and this DNA fragment was released by nick translation according to a conventional method.
I did W&. Nitrocellulose filter paper (S&S Co., Ltd.) was placed on the agar medium-L on which phage plaques had formed, and the phages were transferred, the DNA was denatured with 0.5M NaOH, and filter paper 89!1 was placed in the following order. 0. IM NaOH.

1.5M NaC,Q for 20 seconds, followed by 0.5M
Tris salt M (17,5>, 1.5M NaC, l! twice for 20 seconds.

Finally 120mM NaCρ, 15mM Sodium Citrate, 13mM KH2PO4, 1mM EDT
A.

(Processed at pH 7.2> for 20 seconds.

The filter paper was then dried and heated at 80°C for 2 hours to remove the DNA.
was fixed. 5X SSC, 5x Denhardt solution,
50mM phosphate buffer, 50% formamide, 0.25
Denatured DNA of mFJ/r111 (salmon testis DNA).

and pHC3-, which was radiolabeled by nick translation and hybridized overnight at 42°C in a hybridization buffer containing 0.1% SO3.
Hybridization buffer [5X SSC, 5x Denhardt
Solution, 20mM phosphate buffer (DH6,0), 50%
Denatured DNA (salmon testis DNA) in formamide, 0.1% SDS, 10% dextran sulfate, o, img, /mi
) Hybridization was performed at 42° C. for 20 hours.

The nitrocellulose filter paper was washed with 2X SSC containing 0.1% SDS at room temperature for 20 minutes, then at 44°C with 0.1% SDS.
0.0 containing SDS. IX5SC for 30 minutes, then rinse at room temperature for 0. After washing with IX5SC for 10 minutes, detection was performed by autoradiography.

As a result, several positive loans were obtained.

Recombinant DNA from these clones was extracted by Haniati.
By the method of s (Cell Vol. 15, p. 687 (1978))
Prepared by

The jqed DNA is EcoRI and BamHI.

After treatment with restriction enzymes such as Bgl It, the samples were analyzed by agarose gel electrophoresis, and a restriction enzyme map was created according to the method of Frltsch et al. (see the above-mentioned document).

Radiolabeled pHC3- used in the above screening
Saturday hybridization was performed using a probe of the DNA fragment derived from pBR327, and as a result, a DNA fragment of about 8 kilobase pairs that had been cleaved with ECoRI was selected from among those that hybridized with the probe, and the ECoRI fragment of pBR327 was selected.
The site was nubcloned.

Roughly, this subcloned DNA was treated with restriction enzymes as described above, and Southern hybridization was repeatedly performed to generate a DNA fragment of approximately 4 kilobase pairs excised with EC0RI and XhoI, which contained the human G-C3F polypeptide. It was discovered that there is a gene that codes for

When the sequence of about 3 kilobase pairs of this DNA fragment was investigated using the deoxy method, the base sequence shown in FIG. 5 was obtained.

Further, the restriction enzyme cleavage site of this DNA fragment was as shown in FIG.

In addition to the probes mentioned above, DNA derived from pBRG4 and DNA derived from 1RV2 were used as probes for screening human chromosomal genes.

For both DNAs, the 1500 base pair DNA fragment treated with EC0RI was directly radiolabeled using the nick translation method as described above, or the 1500 base pair DNA fragment was treated with EC0RI and then radiolabeled [)r.
A DNA fragment of about 700 base pairs obtained by the A step (7) was radiolabeled in the same manner, and clones were selected by plaque hybridization under the same conditions as described above, and then subjected to unhybridization. Through analysis, we were able to obtain a DNA fragment having the base sequence shown in Figure 5.The resulting plasmid was transformed into pBRCE3β.
It was named.

Example 12. Preparation of pHGA 410 vector (for animal cells, +VSE system) E of the cDNA shown in Figure 3(A) obtained in Example 9
The coRI fragment was treated with restriction enzyme [)ra for 2 hours at 37°C, and then treated with DNA polymerase KleinOW.
Fragment (manufactured by Takara Shuzo Co., Ltd.) was treated to make the ends blunt.

1μq of BQII [linker (8mer: manufactured by Takara Shuzo Co., Ltd.)
After phosphorylating with ATP, approximately 1 obtained above
It was combined with a DNA fragment mixture of μq. Next, the DNA fragments were treated with restriction enzymes BgI II and subjected to agarose gel electrophoresis, and only the largest DNA fragment was recovered.

As shown in FIG. 6, this DNA fragment corresponded to approximately 710 base pairs including a portion encoding human G-C5F polypeptide. Vector pdKCR (Fukunaoa et al.;
Proc, Nat 1. Acad, Sci,
USA; Vol. 81, p. 5086 (1984)) with restriction enzyme B.
After treatment with amHI, the resulting vector DNA was dephosphorylated with alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.).
4DNA Rigavi (manufactured by Takara Shuzo Co., Ltd.) was added and bound to the CDNA fragment to obtain 11GA410 (FIG. 8). As shown in Figure 8, this plasmid contains the promoter of the SV40 early gene, the replication initiation region of SV40, part of the rabbit β-globin gene, the replication initiation region of pBR322, and the β-lactamase gene derived from pBR322 (Amp
>, and the human G-C5F gene is connected downstream of the promoter of the SV40 early gene.

Example 13. Construction of recombinant vector for Cl27 cells (+
VSE) 1), Construction Example 12 of pHGA 410 (1-1>)
The pHGA410 plasmid obtained in (Figure 8 > 20
μG to 50 mHTTri 5-HCI (pH 7,5
> ,7 m) f IVLQC12,100mHNaC
Dissolved in a reaction solution of 1, 7 mN 2-mercaptoethanol and 0, oi% bovine serum albumin (BSA), added restriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd., 10-15 units), and allowed to react at 37°C for about 30 minutes. Partial digestion with EC0RI was performed. Then phenol-chloroform (1
:1) The DNA fragments were processed by performing two treatments: ether treatment and ethanol precipitation.

This DNA fragment was treated with 501118 Tri 5-HCl.
, 5mHMgC12, 10mM DTT, 1m)i dATP.

Dissolved in a 50 μm solution consisting of dCTP, (jGTP, dTTP, E, Gol 1 DNA polymerase-Kle
Add 5 μg of now fragment (manufactured by Takara Shuzo Co., Ltd.) and incubate for 2 hours at 14'C.
d).

From this, approximately 5.
8) 6 μq of fragment (b) was collected.

5μa of the recovered DNA fragment was again diluted with 50mHTris-1cI (pH 7,6>, 10mHMOCI2).
, 10 mHDTT, and IIIHATP, 2 μql of HindI[I linker (manufactured by Takara Shuzo Co., Ltd.) and 100 units of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.] were added, and the mixture was reacted overnight at 4°C.

Next, after phenol treatment, ether treatment, and ethanol precipitation, 10 mHT r i 5-HCI (pH 7,
5), 7 mHMgC12, 60 mH NaCl solution 3
It was dissolved in 0u, Q and incubated at 37°C for 3 hours in the presence of 110 units of restriction enzyme Hindl. T4DN again
After treatment with A ligase, this DNA was processed using the l'-Molecular Cloni chloride method (described above).
ng J) into the E. coli DHI strain to obtain ampicillin-resistant (Amp') colonies, and the ECoRI site of the pHGA410 plasmid was transformed to l-1.
Bacteria carrying a plasmid that replaced indI[[ were selected. The plasmid thus obtained was
It is named A410 (t-1) (Figure 9).

2) Construction of recombinant vector pTN-G4 for expressing 0
) obtained in 10 mHTri 5-HCl (pf-i7.5>
, 7mHMgC12,175+nHNaCI, 0.2
mHEDTA17 mN 2-mercaptoethanol, 50μ reaction solution consisting of 0.01% bovine serum albumin
20 units of restriction enzyme 5alI (manufactured by Takara Shuzo Co., Ltd.) was added, and the mixture was incubated at 37°C for 5 hours.

Then, after performing a fail treatment and ethanol precipitation, the DNA polymerase was incubated with +enow fragment (manufactured by Takara Shuzo Co., Ltd.) at 14° C. for about 2 hours in the same manner as in the previous reaction, and then used as a plant end. The DNA fragments were precipitated with ethanol without being collected by agarose electrophoresis and then treated with the restriction enzyme Hin.
Processed with dl[I, approximately 2.7 kb of l-i in
5 μq of the dlI[-3a II fragment was recovered by 1% agarose gel electrophoresis.

On the other hand, bovine papillomavirus [bovine papillomavirus]
Plasmid pdBPV-1 (Sarver, N., 5byrn
e, J., C. & Howley, P. M. (1982) P.
roc, Natl, Acacl, Sci, USA 79 Vol. 7147-7151; Dr.

(obtained from t+ow+ey) using the method of Nagata et al. ((rukunaga, Sokawa, & Nagat
a, (1984) Proc, Natl, Acacl,
Sci, UsA Vol. 81, 50136-5090) Hi
nd m and pvu [8.4 kb DNA
Get the pieces.

This 8.4kb DNA fragment and the approximately 2.7kb @
The NA fragment from the indIu-sa l was treated with T4 DNA ligase according to a conventional method, and then
E. coli strain DHI was transformed by the rubidium chloride method described in Cular Cloning J.
E, Go II colonies harboring a plasmid containing the cDNA of ptoIGA410 Yamaki G-C3F were selected. This plasmid is named pTN-G4 (Figure 9)
.

On the other hand, from adenovirus Type II (Protein Nucleic Acid Enzyme Vol. 27, December issue, 1982, published by Imori Publishing), Salnie H of approximately 1700 bp containing VAI and VAn was obtained.
VAI from plasmid ΔDVA containing the indlII fragment
and a fragment containing VAII was recovered. This fragment was inserted into the l-1indI11 site of pTNG4 described above to obtain pTNG4VAα and pTNG4VAβ (Fig. 9
). This plasmid is SV4 by Azone's VA gene.
This is designed to increase the efficiency of expression of transcripts from the early promoter of 0.

Example 14 Transformation of C127 cells and its expression (
+VSE) pTN-G4 obtained in Example 13 is treated with restriction enzyme 3amt-II before being transformed into mouse C127 cells.

That is, 20μq of pTN-04 plasmid was added to 10mM T.
ris-HCI (pH 8,017m) l M(J
CI2, 100mH NaCl, 2mM 2-mercapdolethanol, o, oi% BSA mixture 100
μg was treated with 20 units of U Shime 3amHI (manufactured by Takara Shuzo Co., Ltd.), followed by phenol treatment, ether treatment, and ethanol precipitation.

Mouse C1271 cells were incubated with 10% tallow serum (GIBCO
Dulbecco's minimal
Avoid growing in essential medium. C127I cells grown on plates with a diameter of 5 cm were plated or treated with 10 μq of the above-prepared DNA using the calcium phosphate method (Tlaynes, J & Weissmann, C.
1983) Nucleic Ac1d Res, 11
Transformation was carried out using the following method (see Vol. 687-706), and after glycerol treatment, the cells were incubated at 37° C. for 12 hours.

Next, the cells were transferred to three new 5 cm diameter plates, 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-C3F production ability were selected. As a result ~1mg/,
l! A level of G-C3F production was observed. As a result of further cloning, G- levels of over 10 my/u were detected.
C3F production was confirmed.

In addition to the above C127I cells, the host cells include NII-
(3T3 cells can also be used.

Example 15 Expression of G-C3F by CHO cells (+V
SE) 1) Construction of DI-IGG4-d b fr 20 μci of pHGA410 plasmid obtained in Example 12 was
m) tTri 5-HCl (DH7,5),
7111HIVFC12, 175mHNaC+, 0.
2mHEDTA, 0.7m) f2-mercaptoethanol, o,oi% Dissolved in 100 μg of a solution containing BSA, added restriction enzyme 5alI (manufactured by Takara Shuzo Co., Ltd. >20 units), and reacted at 37°C overnight, treated with phenol, and treated with ether. Washing and ethanol precipitation were performed.

Next, the obtained DNA precipitate was treated with 50 mt4 T r
i 5-HCfI, 5 m) f MQCI2.10m
) fDTT, 1 mM dATP, dCTP, dGTP
, a reaction solution consisting of TTP 100/! Dissolved in ρ, E, c
oliDNA polymera-2-Klenow fragment (10 μg, manufactured by Takara Shuzo Co., Ltd.) was added, followed by reaction at 14° C. for 2 hours, followed by phenol treatment, ether washing, and ethanol precipitation.

Add an EC0RI linker to this DNA.

That is, the above DNA was 50 μm thick with 50 mHTri 5-
HCl (pH 7,4), 10 mM DTT, 0
．． 5 m)l spermidine, 2 mW ATP,
2.5 mM hexamine cobalt chloride, 20μ (J
/dBsA, EC0RI linker (manufactured by Takara Shuzo Co., Ltd.) was added, 200 units of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) was added, and the mixture was reacted at 4°C for 112 to 16 hours. After phenol treatment, ether washing, and ethanol precipitation according to conventional methods, the DNA was partially digested with EC0RI, and 3 μq of a fragment of approximately 2.7 kb was recovered by 1% agarose gel electrophoresis.

On the other hand, pAdD26SVpA plasmid (Kaufma
n, R, G, & 5harp, P, A (1982)) io
1, cell 8io1.201304-1319) is treated with ECORI and treated with bacterial alkaline phosphatase (BAP) to perform dephosphorylation. That is, pA
dD26SVl) 20 μq of A and 20 units of EcoRI were added to the reaction solution of 50 mHTri 5-HCl (pH 7,5)
, 7 mHMgC12, 100mM NaC
l, 7mM 2-mercaptoethanol, 0.01%
Add 100 μg of BSA mixture and react at 37°C for 10 hours, then add 5 units of BAP to the above reaction solution and incubate at 68°C.
The reaction was carried out for 30 minutes.

After that, pAdD26 was treated with phenol and electrophoresed.
SVpA (7) EcoRI fragment collection (~5μq)
.

About 2. [piece of zkb and pAdD26SVpA
0.5 μq of each fragment was annealed. This plasmid is transformed into E. coli DHI strain by the rubidium chloride method, and colonies carrying the pHGG4-dhfr plasmid are selected.

The obtained plasmid was named pHGG4-dhfr (Fig. 10a).

In addition, as an alternative method to the above, the 1IGG4 plasmid is
ll treatment and partial digestion with EC0RI without adding an EC0RI linker to recover a fragment of approximately 2.7 kb.
The DNA fragment is treated with E.coli DNA polymerase-klenOW fragment to make the ends into plant-ends.

On the other hand, a plant-ended EC0RI fragment of pAdD26SVpA was prepared in the same manner as described above, and both were added to T4DN.
pHGG4-dhfr can also be prepared by A ligase treatment.

In addition, pHGA410 ([''
) as described in Example 13, 2), restriction enzyme 1:
Treated with Hindl[ and 5alI to produce Hindl[l-
Even if the 3a l ■ fragment is ligated to the plant-ended EC0RI of the above pAdD26SVpA, pHGG4-d
hfr can be (q) (Fig. 1ob).

2), Construction of pG4DR'l and pG4DR2 1)
pAdD26SVpAIOμ described in section 50mM
Tri 5-HC (PH7,5>, 7mMMg
CfI2.100mM NaCf1,7mM 2-
Restriction enzymes [:coRI and Bam] were dissolved in 50ml of reaction solution containing mercaptoethanol and 0.01% BSA.
(10 units of each I was added and reacted for 10 hours at 37°C. Phenol treatment and ether washing were performed according to the usual method. Approximately 2 Kb DNA
After collecting the A fragment, DNA polymerase-KlenO
The W fragment was converted into a plant end according to a conventional method, and subjected to phenol treatment, ether washing, and ethanol precipitation.

On the other hand, 1IGA410 (H
) 10μ9 to 10mM Tris-1-104)
(PH7,5>, 7mM MgCG 2.60mM
NaC,l! Hin
dl[[10 units were added and reacted at 37°C for 6 hours.

DNA fragments were collected by 1% low-melting-point agarose electrophoresis according to a conventional method, further treated with BAP, and then converted into plant-ends using K1enow fragments. After phenol treatment and ether washing, plant-end ligation was performed using the approximately 2 Kb DNA fragment described above and T4 DNA ligase. That is, 1μf of each DNA fragment was added to 66mM Tr.
i 5-H (j! (pH 7,5), 6.6mM
MgCN was dissolved in 30μ7 of a reaction solution containing 2.5mM DTT and 1mM ATP, and T4DNA ligapi 50
After adding the units and reacting at 6°C for 12 hours, E, Co
It was transformed into the l1DHI strain. In this way, pG4 DR1 and pG4DR2 shown in FIG. 10G were obtained.

3) Transformation and expression CHO cells (dhfr-strain, Columbia University Or, L
.

(obtained from ChaSin) is 9C#! Diameter plate (Nt
alpha minimal essential medium (manufactured by JnC) containing 10% calf serum (
α-MEM, adenosine, deoxyadenosine, and thymidine were added to culture and proliferate using the phosphate-calcium method (
Wigler et al., Ce1l 14, 725 (1978)
) was transformed.

That is, pHGG4-dhfr plasmid 1 prepared in 1)
Add an appropriate amount of carrier DNA (calf trunk line DNA) to μQ, dissolve it in 375μJ of TE solution, and add 1M CaCl2.
Add 125 μN. Chill on ice for 3-5 minutes,
11 of 2xHBS (50mHHepes, 280
After adding mHN aCl , 1.5 mH phosphate buffer and cooling with water again, the mixture was mixed with the above CHO cell culture solution 1d, dropped onto a plate, and then cultured in a CO2 incubator for 9 hours. Remove medium from play 1~ and T 133
After washing with (Tris-[3uffered 5aline), TBS containing 20% glycerol was added, and after washing again, a non-selective medium (alpha-MEM medium, supplemented with nucleotides) was added and incubated for 2 days. Cells were split 1:10 (no additives).

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.

0.02MM methotrexate (M
TX), grown in the presence of 0.05 MM, then 0.1 MM
Cloning was performed by growing in the presence of MTX.

In addition, the transformation of CHO cells is performed using p for C)-to cells.
HGG4 and pAdD26SVpA were co-transformed (Co
transformation) (Scahill et al., Pr0C,
Natl, ACad, SCi, IJSA Volume 80 4654
-4658 (1983)).

CHO cells were also transformed by the method described below. That is, pG4D prepared in section 2) above
5al of R1 or pG4DR2 in advance.
DNA fragments were obtained by treatment with I and KpnI, and 10 μJ of them were transformed into CHO cells in the same manner as above. 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. When the colonies were transferred to a new plate again without being selected one by one and cultured on a selective medium in the presence of 0.01 MM MTX, several colonies appeared. Furthermore, using this method, the concentration of MTX was reduced to 0.02MM, 0.
．． The concentration was increased to 0.05 μM and 0.1 μM, and surviving colonies were selected. Further, after selecting several obtained colonies, it was possible to perform similar colony selection even when the MTX concentration was increased.

In addition, we constructed a recombinant vector containing the so-called polycistronitus gene, and were able to transform CHOIII cells using this vector. That is, pAdD26SVpA is PstI5I! 1, collect the two fragments and combine them with p
By joining the C3F cDNA fragment derived from BRG4, the adenovirus promoter, C3F cDNA, D
A recombinant vector arranged in the order of HFR and SV40 polyA sites was constructed and carried out in CHO cells.

Example 16 G-C3F activity assay of expressed substances (10 VS
E) The culture supernatants of C127 cells and CHO cells obtained in Example 14 and Example 15 were adjusted to pH 4 with 1N acetic acid, and an equal volume of n-propatool was added, and the resulting precipitate was removed by centrifugation. and passed through an open column (1φ x 2cm) packed with a C8 reverse phase carrier (manufactured by Yamamura Chemical Co., Ltd.) to reduce 50%
Eluted with n-propatool. After diluting the eluate twice with water, it was purified with 0.1% TEA by reverse phase high performance liquid chromatography using a YMC-C8 column (manufactured by Yamamura Chemical Co., Ltd.).
Elution was performed with a 30-60% linear concentration gradient of n-propatool containing . After separating both fractions eluted at a position where the n-propertool concentration is around 40%, they are lyophilized and
．． It was dissolved in 1M glycine buffer (pH 9). Through this process, human G-C3F was dissolved in C1
27 and CHO cell supernatants approximately 20 times.

As a control, human G-C3 was added according to the method described above.
After cells were transformed with a plasmid containing no F cDNA, the culture supernatant was concentrated. [Method for measuring human G-C3A (a)] described in the reference example for the obtained specimen
Human G-C3F activity was assayed using a method based on . 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.

(Left below) Example 17 Amino acid analysis and sugar analysis (+VSE
)1) Analysis of amino acid composition The crude C3F sample obtained in Example 16 was further analyzed in (ii) of Example 2.
Purified according to method i). This purified IC3F sample was hydrolyzed by a conventional method, and the amino acid composition of its 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 Ftc+, IIO℃, 24 hours, in vacuum ■
4N methanesulfonic acid + 0.2% 3-(2-7minoethyl)indole, 110°C, 24 hours.

For 48 h and 72 h, the samples were dissolved in a solution (1.5 m) containing 40% n-propanol and 0.1% trifluoroacetic acid, then o,
After taking 1rnl and drying it with dry nitrogen gas,
Alternatively, the reagent (2) was added, the tube was vacuum-sealed, and the tube was subjected to hydrolysis.

In the table, the actual measured value is the 24-hour value in ■ and 24.48.72 in ■.
This is the average value of a total of four time values. However, Thr.

Ser, 1/2Cys, Met, Val, Ile
and Trp were calculated by the following method. (Refer to Biochemistry Experiment Course, Protein Chemistry ■ (Tokyo Kagaku Doujin Publishing)) ・Thr, Ser, 1/2Cys, and Met are 24 in ■
．． 48. Take the change over time of the 72-hour value and extrapolate it to zero time.

-Val and lie are the 72-hour values of ■.

・Trp is the average value of the 24.48.72 hour values of
g and inositol 25 n m as an internal standard.

After adding 1.1 methanol solution (500 μl) containing 1.5N HCl, in a sealed tube purged with nitrogen gas,
The reaction was carried out at 90°C for 4 hours. After opening the tube, silver carbonate (Ag2
After neutralization by adding CO3>, 50 μg of acetic anhydride was added, shaken, and left overnight at room temperature. 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. Add 50 μg of TMS reagent (mixture of pyridine: hexamethyldisilazane: trimethylchlorosilane = 5:1:1) and hold at 40°C.
After reacting for 20 minutes, the mixture was stored in a Deep Freezer. In addition, galactose (Gal
), N-acetylgalactosamine (Gal NAC)
, 50 nmol each of sialic acid, etc. and inositol 2
5nm01 was combined and the same operation was performed.

Gas chromad analysis was performed on this sample under the conditions shown below.

(Analysis conditions) Column: 2% OV-17VINpOrt HP60-8
0 mesh, 3 m, glass temperature: 4°C/min heating from 110'C to 250°C Carrier gas: 1.2 to 1.6 K'j/ci (nitrogen pressure) at the beginning, 2 to 2 at the end .5 Kg/crA sensitivity:
103MΩ range 0.1 to 0.4 V pressure: Hydrogen gas 0.8 K'J/crit Air 0.8 K'
j/cti sample weight: 2.5 to 3.0 μg As a result of the analysis, galactose, N-acetylgalactosamine, and sialic acid were confirmed from the C3F of the present invention.

Example 1B, Preparation of pHGV2 Vector (!!JJ
(for physical cells, -VS2 system) C[)NA shown in FIG. 4(A) obtained in Example 10
The EC0RI fragment was digested with restriction enzyme [)ra at 37℃ for 2 hours.
After treatment with DNA polymer-1m'Kle
It was treated with nOW fragment (manufactured by Takara Shuzo Co., Ltd.) to make the ends blunt. 1 μq of Bgl II clinker (8mer; manufactured by Takara Shuzo Co., Ltd.) was phosphorylated using ATP, and then combined with the approximately 1 μq DNA fragment mixture obtained above. Next, the DNA fragments were treated with restriction enzyme BIII and subjected to agarose gel electrophoresis, and only the largest DNA fragment was recovered.

As shown in FIG. 6, this DNA fragment corresponded to approximately 700 base pairs including a portion encoding human G-C3F polypeptide. Vector DdKCR (Fukunaga et al.;
Proc, Natl, Acad, Sci, USA,
Volume 81, page 5086 (1984)) Restriction enzyme BamHI
The vector DNA obtained by treatment with T4 DNA and dephosphorylation with alkaline phosphatase (manufactured by Takara Shuzo)
Add ligase (manufactured by Takara Shuzo Co., Ltd.) and combine with the cDNA fragment. 1) HGV2 i? J (Figure 71), shown in Figure 11c
5, this plasmid contains the promoter of the SV40 early gene, the replication initiation region of SV40, part of the rabbit β-globin gene, the replication initiation region of pBR322, and the β-lactamase gene derived from pBR322 (Amp
), and the human G-C3Fi gene is connected to the downstream of the promoter of SV40 early Dobutsu child.

Example 19 Construction of recombinant vector for C127 cells (
-VSE) 1) Construction of pHGV2 (H) pHGV2 plasmid obtained in Example 18 (Figure 11)
A plasmid named pHGV2 (H) was obtained using 20Ii9 in the same manner as described in Example 13, 1) (FIG. 12).

2) Recombinant vectors for expression pTN-V2 and pTN
Construction of VAα and pTNVAβ Using the above 1) HGV2 (H) 20μ9, LTllGV2Yamaki G-C
E,co carrying a plasmid carrying the cDNA of 3F
li colonies were selected. The obtained plasmid is pTN
−V2 (Figure 12).

On the other hand, adenovirus Type II (protein nucleic acid enzyme 2
VA from Volume 7, December issue (1982), published by Imori Publishing
Approximately 1 yoobp of Sa I l containing I and VAI [
- HlndlI [V from plasmid ΔpVA containing the fragment
Fragments containing AI and VAI were recovered. This fragment was inserted into the Hin dl [1 site of pTN-V2 described above to obtain pTNVAα and pTNVAβ (Fig. 12).
. This plasmid is SV40 by Azone's vA gene.
This is designed to increase the efficiency of expression of transcripts from the early promoter.

Example 20 Transformation of 0127 cells and their expression (
-VSE) pTN-V2 obtained in Example 19 is treated with restriction enzyme 3amHI before being transformed into mouse C127 cells.

Next, mouse Cl27I cells were transformed with the above-prepared DNA and expressed (see Example 14), and clones with high G-C3F production ability were selected. As a result, G-C8F production at the level of 11Q/1 was observed.

By continuing cloning further, 10m1/
Clones having fl level G-C3F production ability were selected. Similarly, C127 cells were transformed with pTNvAα and pTNVAβ obtained in Example 19,
As a result of selecting clones with high G-C3F production ability, pT
Regarding NVAα, we were able to obtain clones with high production of 20 mg/ρ or more.

Furthermore, a clone with a production capacity of several 1 n'j/fl could be obtained from pTNVAβ.

In addition to the C127I cells mentioned above, the host cells include NIH
3T3 cells can also be used.

Example 21 Expression of G-C3F by CHO cells (-V
SE) 1) l) HGV2-dhfr (7) Construction of the approximately 2.7 kb fragment obtained from pf-(GV2 plasmid 20μ3 obtained in Example 18 by the method described in Example 15-1) and the pAdD26sVpA fragment. 0.5μq each
Annealed each. This plasmid was used in E. coli
DHI strain was transformed by the rubidium chloride method and the pH
Colonies carrying the GV2-dhfr plasmid were selected. The obtained plasmid was named DHGV2-dhfr (Fig. 13a).

In addition, as an alternative method to the above, the pHV 2 plasmid is
I processing and E without adding the EC0RI linker.
Partially digested with C0RI, a fragment of approximately 2.7 kb was recovered,
E. coli DNA polymerase-1<+enow
The DNA fragment was treated with the fragment to make the ends into plant ends.

On the other hand, a plant-ended EC0RI fragment of pAdD26SVpA was prepared in the same manner as described above, and both were added to T4DN.
pHGV2-dhfr could also be prepared by A ligase treatment.

In addition, the obtained 11GV2 (H) in 1) of Example 19 was treated with restriction enzyme 1(ind) as described in 2) of Example 13.
HindlII-3
Even if the al■ fragment is ligated to the plant-ended EC0RI fragment of pAdD26SVpA, pHGV2-dh
It was possible to obtain fr (Fig. 13b).

2), pV2DR1cLLCFpV2DR2 (7) Construction 10g of pAdD26sVpA mentioned in section 1)
mM Tri 5-HCj! (PH7,5
>, 7mMMCJCI 100mM Na
Cj! , 7mM 2-mercaptoethanol, o,
Reaction solution 5 (7) containing oi% BSA and restriction enzyme E
10 units each of C0RI and Bam1-tI were added and reacted at 37°C for 10 hours. Fail treatment and ether cleaning were performed according to conventional methods. After recovering a DNA fragment of about 2 Kb by 1% low melting point agarose electrophoresis, the DNA
Plant-endization was performed using polymerase using the 1enow fragment according to a conventional method, followed by washing with phenol ff11, ether washing, and ethanol precipitation.

On the other hand, pHGV2 (H) obtained in Example 19 1)
10μ57 to 10mM Tri 5-1-1cf1
(PH7,5>, 7mM MgC42, 60mM
Dissolved in 50 μg of reaction solution containing NaCl, 1-(ind
1110 units of I1 were added and 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 converted into plant-ends using KlenOW fragments. After phenol treatment and ether washing, plant-end ligation was performed using the approximately 2 Kb DNA fragment described above and T4 DNA ligase. That is, 1μ9 of each DNA fragment was mixed with 66mM T.
r i 5-)-1cd! (1) H7,5>, 6.
6mM MgC,l! 2.5mM DTT, 1m
T4DN was dissolved in 30 μg of reaction solution containing MATP.
After adding 50 units of A. ligature and reacting at 6° C. for 12 hours, the mixture was transformed into E. col 1Df-tI strain.

In this way, tpV2DR1 and pV2DR2 shown in FIG. 130 were obtained.

3) Transformation and expression Using the pHGV2-dhfr plasmid prepared in 1) above, C
The HO cell line was transformed and expressed.

In addition, for the transformation of CHO cells, 1) H
GV2 and 1) AdD26SVpA were co-transformed (Co
This can also be done by (transformation).

CHO cells were also transformed by the method described below. That is, in the above section 2), ML, pV2
DR1, G, and tpv2DR2 were clarified and treated with Sal and KpnI to obtain 1q of DNA fragments, of which 10. U9 was transformed into CHO cells as described above. 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 after about 71 cells. 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 a dozen colonies appeared. Furthermore, by such a method, the concentration W of MTX can be reduced to 0.
02μM10.05μM10.1μM,! : rise u
1. The surviving colonies were selected. In addition, after selecting each of the more than ten colonies that were destroyed, MT
．． Similar selection of colonies was possible even when the temperature was raised to 8 degrees.

Furthermore, we constructed a recombinant vector containing a so-called polydystronic gene, and were able to transform CHO cells using this vector. 1) Treat AdD26SVl)A with PstI, collect two fragments, and combine these with pBRV.
By joining the C3F cDNA fragments derived from 2, the adenovirus promoter, C3F cDNA,
A recombinant vector in which the poly8 sites of Df-IFR and SV40 were arranged in the order was constructed and carried out in CHO cells.

Example 22 G-C3F activity assay (-VSE) of expressed substances From the culture supernatants of C127 cells and σCHO cells obtained in Examples 20 and 21, human G -C3F was obtained, and the human G-C3F
Activity was assayed. The results were as shown in Table-3.

Margin Table-3 Human G-C3F Activity Assay Example 23 Amino Acid Analysis and Sugar Analysis (-VSE) 1)
Analysis of Amino Acid Composition The crude C3F sample obtained in Example 22 was further analyzed in Example 2 (ii
Purified according to method i). This purified C3F sample was subjected to amino acid composition analysis by the method described in Example 17, 1). The results are shown in Table 4.

(Leaving space below) Table 4 (Amino acid analysis table) 2) Sugar composition analysis The purified C3F sample used in the above amino acid composition analysis was obtained, and the sugar The composition was analyzed.

As a result of the analysis, galactose, N-acetylgalactosamine, and sialic acid were confirmed from the C3F of the present invention.

Example 24 Construction of a recombinant vector containing a chromosome-derived gene for CO3 cells The plasmid pBRCE3β containing the chromosomal gene shown in FIG. 5 obtained in Example 11 was treated with EC0RI.

On the other hand, the literature of Banerj et al. (Cel12γ volume 299
1 (1981)) pSvH+ described in
The K plasmid was treated with KpnI to remove the globin gene, further partially digested with HindlII to remove part of the SV40 late gene, and then religated to prepare the expression vector pML-E.

This vector was treated with the restriction enzyme EC0RI, and then dephosphorylated with alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.), and the resulting vector DNA was ligated with the above chromosomal DNA fragment by adding T4 DNA ligavi (manufactured by Takara Shuzo Co., Ltd.) to form pML.
I jqed CE3α. As shown in FIG.
replication initiation region, pBR322 replication initiation region and pBR
β-lactamase gene derived from 322 (Arr+p'
), and the human G-C5F chromosomal gene is connected downstream of the enhancer of the SV40 gene.

Example 25 Expression of human G-C8F chromosomal gene in CO3 cells DMEM containing 10% calf serum (manufactured by Nippon Pharmaceutical Co., Ltd.).

Dulbetsko's modified Eagle medium ``Nitsui'') medium (io
A 9cm diameter Vetri dish (manufactured by NUnC)
CO3-1 cells grown to approximately 10% confluency (Col. Spring Harbor Laboratory DI', USA)
, received from GIUZman) using the phosphate-calcium method (WiCILQr et al.; Ce1l Vol. 14, p. 725 (19
γ8)) and the DEAE-dextran:chloroquine method (see, eg, Gordon; 5science 22 Shoe Volume 810 (1985)).

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

Plasmid pMLcE3α160 prepared in Example 24
[mu]J was dissolved in 320 [mu]J of TE solution, 3.2-distilled water was added, and then 504 [mu]g of 2MCaCu2 was added.

To this solution was added 4rIdl of 2Xl-IBs [50mM
Hepes, 280mM NaQ, Il, 1.5m
After adding M phosphate buffer, U)t-17,12') and cooling with water for 20 to 30 minutes, 1 d of the solution was added dropwise to each vetri dish in which CO3-1 cells had grown. 37℃C
After culturing in a Q2 incubator for 4 hours, the cells were washed with serum-free DMEM medium, and then 5 g 1N of DMEM medium containing 20% glycerol was added and incubated at room temperature for about 3 hours.
The plate was left for a minute and washed again with serum-free DMEM medium.

After removing the serum-free DMEM medium, 10 ml of DMEM medium containing 10% calf serum was added and cultured overnight in a CO2 incubator.The medium was replaced with the same medium again and cultured for approximately 3 days.

DEAE-Dextran: When using the chloroquine method, the procedure is as follows.

CO3-1 cells were cultured to 70% confluency in the same manner as the calcium phosphate method, and the cells were washed twice with serum-free DMEM medium. To this was added serum-free DMEM medium containing 250 μg/d DEAE-dextran and 2 μg/ml plasmid pMLcE3α prepared in Example 24, and
The cells were cultured at 7°C for 12 hours. Cells were then treated with serum-free DME.
The cells were washed twice with M medium, and cultured at 37° C. for 2 hours in DMEM medium containing 10% calf serum and 1 mM chloroquine. After that, the cells were washed twice with serum-free DMEM medium, and then DMEM medium containing 10% calf serum was added and incubated at 37°C.
The cells were cultured for 3 days.

The culture supernatant of CO3-1 cells obtained in this way was
After adjusting the pH to 4 with N acetic acid and adding an equal volume of ")-propatool," the resulting precipitate was removed by centrifugation, and an open column (1φ
x2cm> and eluted with 50% n-propertool. After diluting the eluate twice with water, it was subjected to reverse phase high performance liquid chromatography using a YMC-08 column (manufactured by Yamamura Kagaku Co., Ltd.) to obtain a linear 11 degree gradient of 30 to 60% containing o, i% TFA. It was eluted with n-propatool. Both fractions eluted at a position where the concentration of n-propertool was around 40% were separated, lyophilized, and added to 0.1M glycicin buffer (p
H9>. Through this process, human G-C3F is extracted from CO3-1 cell supernatant by approximately 20%.
concentrated twice.

As a control, human G-C3 was added according to the method described above.
After transforming CO3-1 cells with DML-E'' which does not contain the F chromosome gene, the culture supernatant was concentrated.

Regarding the obtained specimen, “Human G-C” described in the reference example
3A measurement method (a)"
8F activity was assayed. The results were as shown in Table-5.

(Space below) Table 5 Example 26 Expression of human G-C3F chromosomal gene by C127 cells The pMLCE3α plasmid 1q generated in Example 24 was
Processed with C0RI, the aforementioned Molecular C1o
By the method described in ninQ, an approximately 4 Kb fragment is recovered and used as the source of the chromosome-derived G-C3F gene.

This is treated with DNA polymerase I-KIenOW fragment to form plant ends (A).

On the other hand, from the pHGA410 plasmid prepared in Example 12, iolecLIIar Cl0ni (same as above)
A portion of the SV40 promoter (ECO
The approximately 0.4 Kb fragment of RI-ECORI) was excised and D
Treat with NA polymerase I-Klenow fragment (B
).

In addition, plasmid pdspv-1 (Sarver, N., 5byrne) harboring bovine papilloma virus (BPV)
.

J, C & tlowley, P, H, (1982) Pro.
c, Natl, Acad, Sci.

USA Volume 79 7147-7151: Or tlo
wiey) from Hindl[I and pvu[
A DNA fragment of approximately 8.4 Kb was obtained, which was transformed into a DNA fragment.
After processing the polymerase Klenow fragment, dephosphorylate it with bacterial alkaline riftase (C)
.

0.1 each of the above (A>, (B) and (C) DNA)
20 μn of each μ9 reaction solution (50 mM Tri 5
-HCu (pH 7,6>, 10mM MgCl2
, 10mM DTT, 1mM ATP>, 180 units of T4 DNA ligase was added, and the mixture was reacted at 4°C overnight.

Using this reaction solution, the above-mentioned) molecular Cl
pTN by the rubidium chloride method described in
A CE3α plasmid was obtained.

In addition, as a source of the chromosome-derived G-C3F gene, instead of the above (A), pMLcE3α2Gμ9 is used as a source of 10 m
M Tri 5-HCl t:pt-1
a, o).

7mM McJC12, 100mM NaGfl
, 7mM 2-mercaptoethanol, 0.01% B
Dissolved in 100 μg of SA mixture, added 5tu120 units, incubated at 37°C for 5 hours, and then added 1.2
% agarose gel electrophoresis of approximately 1.78 Kb D
An NA fragment may be obtained and used.

Mouse C127I cells were transformed with the DTNCE3α plasmid thus obtained in the same manner as in Example 14,
The clones were expressed and clones with high G-C5F production ability were selected.

Example 27 Expression of human G-C3F chromosomal gene by CHO cells pMLCE3 as in the case of 0127 cells in Example 26
α plasmid was treated with 3tu engineering to produce approximately 1.78 Kb D
Collect the NA fragments or process them with ECOR and
The EC0RI fragment of Kb was recovered and the chromosome-derived G-C8
It is considered as the source of Fm gene.

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

Also, as in Example 26, from pHGA410 to SV4
A portion of the 0 promoter (EcoRI-ECORI fragment) was excised to obtain a fragment of approximately 0.4 Kb, and the DNA was similarly cut out.
Polymerapi I-KIenOW fragment processing (b
).

On the other hand, pAdD26SVpA plasmid (Kaufma
n, R, G & 5harp, t',^ (1982)
Ho1. Ce11. Biol, vol. 2, 1304-131
After treating 9) with EC0RI, DNA polymerase I
- treatment with KlenOW fragment followed by bacterial alkaline phosphatase treatment to dephosphorylate (G).

0.1 μt each of the above (a>, (b) and (C)) was added to 20 μfl of the reaction solution (50 mM Tri S-H
C,+! (pH7,8), 10mlv1
0m1vi2, iomMDT7.1mM
ATP>, 180 units of T4 DNA ligase was added, and the mixture was reacted at 4°C overnight.

Next, the reaction solution was mixed with the above-mentioned Molecular Cl.
By the rubidium chloride method described in Oning, E,
G.

+: Transformed into DHr strain to obtain Tet' colonies,
Bacteria containing plasmid pD26SVCE3α were selected.

Plasmid pD26SVCE3α is S as shown in Figure 15.
The C3FJ gene is linked to the V40 early gene, and DHFRi Butsuko is further linked downstream of the adenovirus main late promoter.

On the other hand, pAdD26sVpA was treated with ECOR and BamHI (7) in Example 15, 2) to obtain a DNA fragment containing the approximately 2 Kb DHFI gene and the DNA fragments of (a) and DHGA410 (H) described above. EcoRI-3
Amp expression vector pDRCE3α (FIG. 15) was constructed by ligation with the a II fragment.

This pD26SVCE3α plasmid and pDRCE
The 3α plasmid was transformed into CHO cells in the same manner as in Example 15, and MTX selection was repeated to obtain a G-C3F producing strain.

Example 28 G-C3F activity assay of expressed substance (human chromosome-derived gene) From the culture supernatants of C127 cells and CHO cells obtained in Examples 26 and 27, the same method as described in Example 25 was carried out. Human G-C3F was obtained, and the human G-C3
F activity was assayed. The results were as shown in Table-6.

Margin Table-6 Testing Example 29 of Human G-C3F Activity Test Method for Infection Prevention Effect of Human G-C3F Endoxan (manufactured by Geotsugi Co., Ltd., trade name > 200 my/Ks) was administered intraperitoneally, then divided into 3 groups, and 2 groups were treated with a vehicle (in physiological saline) containing human G-C5F (25,000 U/mouse or 50,000 u/mouse). 1%70H/-)Lt, 0.5%((W/V
) mouse serum albumin), and one group received vehicle only, each at 0.005 mg every 24 hours. IIIf was administered subcutaneously four times each. Three hours after the fourth dose, each group was treated with Pseudomonas aeruginosa (Pseud).

monas aeruginosa) GNB-13
9 (3,9xlO" CFU/mouse) was subcutaneously administered. Twenty-one hours after infection, human G-C3F (25,000 u/v mouse or 50,000 u/mouse) was administered once again.
/mouse) or only the vehicle 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) Heart infusion liquid medium ([)ifc.

Pseudomonas overnight at 31°C using
A. aeruginosa GNB-139 is cultured with shaking. A culture solution was prepared by suspending it in physiological saline.

<Results> ■ The same C used for the amino acid composition analysis in Example 17
) Purified human G-C3F samples derived from 10 cells (10 VSE)
The above test was conducted for.

The results are shown in Table-7.

Table 7 Effect on Pseudomonas aeruginosa When the infection prevention effect in the above test was investigated using the same C127 cell-derived purified human G-C3F sample used for the amino acid composition analysis in Example 17, it was found that Similar results were obtained.

■ The same C used for the amino acid composition analysis in Example 23.
The above test was conducted on a purified human G-C3F sample (-VSE) derived from HO cells.

The results are shown in Table-8.

Table 8 In the same manner, the infection prevention effect of the above test was investigated using the same purified human G-C3F sample derived from C127 cells used for the amino acid composition analysis in Example 23, and the results were similar to that of Habo. Obtained.

〔Effect of the invention〕

The glycoprotein having human G-C8F activity of the present invention can be used as a leukopenia therapeutic agent, a myeloid leukemia therapeutic agent, a hematopoietic function recovery promoter, or ll! It is extremely useful as an active ingredient in dye repellents and the like.

Furthermore, the establishment of a method for producing human G-C3F 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 the probe (IWQ), probe (A>, and probe (LC). Figure 2 shows the base sequence of pHcs-1 inner. Figure 3 (A) shows the base sequence of the cDNA insert of pBRG4. Figure 3(B)(I> shows the amino acid sequence of human G-C3F precursor extracted from pBRG4 cDNA. Figure 3(B)(n) shows the amino acid sequence of human mature G-C3F extracted from pBR04cDNA. Figure 4 (A) shows the nucleotide sequence of the CDNA insert of pBRv2. Figure 4 (B) (I> shows the amino acid sequence of the human G-C8F precursor extracted from pBRV2 CDNA. 4 (8) (II) shows the amino acid sequence of human mature G-C3F derived from pBRV2 cDNA. Figure 5 shows the base sequence of the human chromosomal gene encoding human G-C5F. Figure 6 HapB RG 4 t tc HapB RV 2
Restriction enzyme cleavage sites of the derived human G-C3F CDNA are shown. Figure 7 shows the restriction enzyme cleavage site of the human chromosomal gene encoding human G-C3F. FIG. 8 shows the schematic structure of pHGA410. Figure 9 shows the expression recombinant vector pTN-G4. pTN-
The construction process of G4VAcr and pTN-G4VAβ is shown. 10a and 10b are 1) HGG4-d h f
The construction process of r is shown. Figure 10c shows the construction of pG4DR1 and pG4DR2. FIG. 11 shows the schematic structure of L)HGV2. Figure 12 shows the recombinant expression vector pTN-V2. ρTN−
HypTN-VAB(7) construction 7'O in VA(rct:L
Indicates t-s. Figures 13a and 13b are expression recombinant vector pHG.
The construction process of V2 dhfr is shown. Figure 13c shows the pV2()R1# and pv2DR2(7) construction process. Figure 14 shows the schematic structure of pMLcE3α. Figure 15 does not show the schematic structures of pD26SVCE3cr and pDRCE3(2).

Claims (1)

[Scope of Claims] 1. A glycoprotein having human granulocyte colony-stimulating factor activity, which has a polypeptide represented by the following amino acid sequence or a portion thereof and a sugar chain moiety. [There is an amino acid sequence] (where m represents 0 or 1) 2. Obtain a gene encoding a polypeptide having human granulocyte colony stimulating factor activity, then prepare a recombinant vector incorporating this, and then Transforming host cells using the vector, then culturing the resulting transformed strain,
A method for producing a glycoprotein having human granulocyte colony-stimulating factor activity and having a polypeptide represented by the following amino acid sequence or a part thereof and a sugar chain moiety, the method comprising collecting the target glycoprotein from the culture. [There is an amino acid sequence] (where m represents 0 or 1) 3. A gene encoding a polypeptide having human granulocyte colony stimulating factor activity is obtained as a 15-17S fraction by sucrose density gradient centrifugation. 3. The method for producing a glycoprotein according to claim 2, wherein the DNA is complementary to messenger RNA encoding a polypeptide having human granulocyte colony stimulating factor activity. 4. The method for producing a glycoprotein according to claim 2, wherein the gene encoding the polypeptide having human granulocyte colony stimulating factor activity is a gene derived from a human chromosome. 5. The method for producing a glycoprotein according to claim 4, wherein the human chromosome-derived gene contains a base sequence involved in transcriptional regulation. 6. The method for producing a glycoprotein according to claim 2, wherein the gene encoding a polypeptide having human granulocyte colony stimulating factor activity encodes the polypeptide sequence shown below or a part thereof. [There is a polypeptide acid sequence] 7. According to claim 2, the gene encoding a polypeptide having human granulocyte colony stimulating factor activity encodes the polypeptide sequence shown below or a part thereof. A method for producing the described glycoprotein. [There is a polypeptide acid sequence] (where m represents 0 or 1) 8. The gene encoding a polypeptide having human granulocyte colony stimulating factor activity has the base sequence shown below or a part thereof. A method for producing a glycoprotein according to claim 2. [There is a base sequence] (where m represents 0 or 1) 9. A patent claim in which a gene encoding a polypeptide having human granulocyte colony-stimulating factor activity has the base sequence shown below or a part thereof A method for producing the glycoprotein according to item 2. [There is a base acid sequence] (where m represents 0 or 1) 10 A gene encoding a polypeptide having human granulocyte colony stimulating factor activity has the base sequence shown in Figure 3 (A) or a part thereof A method for producing a glycoprotein according to claim 2. 11. The method for producing a glycoprotein according to claim 2, wherein the gene encoding a polypeptide having human granulocyte colony stimulating factor activity has the base sequence shown in FIG. 4(A) or a part thereof. . 12. Production of the glycoprotein according to claim 2, wherein the gene derived from a human chromosome encoding a polypeptide having human granulocyte colony stimulating factor activity has the base sequence shown in FIG. 5 or a part thereof. Method. 13. The method for producing a glycoprotein according to claim 2, wherein the host cell is an animal cell.