JPH08325294A - Human hematopoietic cell growth potentiating factor - Google Patents

Abstract

PURPOSE: To obtain the subject new factor, which is a polypeptide having a specific amino acid sequence, activities in proliferating a hematopoietic cell, especially a bone marrow cell and further immunomodulating and hematopoietic regulating functions and useful for treatment, etc., of immunodeficiency, autoimmune diseases, infectious diseases, cancers, etc. CONSTITUTION: This new human hematopoietic cell growth potentiating factor is a polypeptide, having an amino acid sequence represented by any of formulas I to III and capable of manifesting activities in proliferating a human hematopoietic cell, especially a bone marrow cell. The factor has immunomodulating and hematopoietic regulating functions, can be applied to treatment, etc., of immunodeficiency, infectious diseases, cancers, hepatitises, nephritides, bone marrow transplantation, etc., and is further effective in potentiating effects and reduction, etc., in adverse effects of other medicines by using thereof with other immunologically active substances, immunotherapeutic agents, lymphokines, cytokinins, interferons, cell growth factors, chemotherapeutic agents, antibiotic substances or antiviral agents in combination. This factor is obtained by isolating an mRNA from a monocytic cell derived from a human peripheral blood, preparing a cDNA library, screening the resultant cDNA library and expressing the resultant gene in a host cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a factor (hereinafter, referred to as H) for enhancing the activity of proliferating hematopoietic cells, especially bone marrow cells.
CGPF: Hematopoietic cell g
rowh potentiating facto
r) Polypeptides.

This HCGPF alone, even in bone marrow cells,
It promotes the growth of thymocytes and embryonic hepatocytes, but when used in combination with other hematopoietic cell growth factors (HCGF), the proliferation of these hematopoietic cells is significantly enhanced. Not known at all.

The present inventors have already identified the gene of a factor that induces the proliferation of mouse IL-3-dependent proliferating cell line FDC-P cells (Japanese Patent Application No. 61-2633). This factor was successfully produced as a single cytokine using this gene, was purified to a single protein, and was the first substance identified as a substance. It has been confirmed that the compound has a general effect on hematopoietic cells such as hepatocytes, and when it is used in combination with other factors to be called HCGF, it induces more prominent proliferation of hematopoietic cells, thus completing the present invention. That is why.

The present HCGPF has a wide range of immunoregulatory and hematopoietic regulating functions based on the above-mentioned actions in the human body, and has immunodeficiency, autoimmunity, infectious diseases, hepatitis, nephritis, cancer,
It is strongly expected to be useful in fields such as bone marrow transplantation. At the same time, other immunoactive substances, immunotherapeutics, lymphokines, cytokines, interferons,
When used in combination with cell growth factors, chemotherapeutic agents, antibiotics and antiviral agents, it is also effective in enhancing the effect and reducing the side effects of other agents.

[0005] The novel substance having the above-mentioned usefulness is not limited to the usefulness in the pharmaceutical field, and the present substance, the antibody against this substance, and the receptor molecule of this substance are expected to be useful as diagnostic agents. To be done.

[0006]

2. Description of the Related Art Recently, three types of factors, which are collectively referred to as colony stimulating factors and directly act on hematopoietic cells and proliferate, have been reported for humans, and their genes have been cloned. That is, Goush, N .; M et al. (198
4) Nature 309 , 763 Kawasak
i, E. S. , Et al. (1985) Science
230 , 291 Nagata, S .; et al. (1
986) Nature 319 , 415. These are each
They are abbreviated as GM-CSF, M-CSF, and G-CSF. In addition, multi-CSF as a colony stimulating factor that acts widely on hematopoietic cells is known in mice, and gene cloning has already been performed (Fung, MC et al. (1984) Nat).
ure, 307 , 233 Yokota, T .; et a
l. (1984) Proc. NAS, USA 81 , 10
70).

[0007] In addition, undifferentiated mouse T
A lymphokine having a specific action of inducing 20α-hydroxysteroid dehydrogenase (hereinafter referred to as “20αSDH”) to act on cells and differentiating into mature T cells has been found. It is named. Mouse IL-3 is considered to be involved not only in the differentiation and proliferation of cells such as granulocytes, megakaryocytes, mast cells, and basophils, but also to extend its biological activity to the metabolism of sex hormones. There is great interest in its wide range of biological activities (Immunol
optical Rev, (1982) 63 , 5-3.
2). From this, mouse IL-3 can be expressed in mouse multi.
-Also called CSF.

Due to such biological activity, this substance is expected to be generally useful for immunologically deficient or abnormal diseases and useful for clinical diagnosis of immune system diseases. It is also useful as a research reagent in general medicine.

However, the gene and protein structure of mouse IL-3 has only been elucidated, and human IL-3 has only been elucidated.
Does not have a suitable activity assay system, human IL
That no human I-producing cell is known
Even the presence of L-3 has not been confirmed.

These human G-CSF, M-CSF, GM
-CSF, mouse IL-3 (multi-CSF),
It is well known that stem cells in the bone marrow act at the stage of differentiation and maturation to form colonies of hematopoietic cells, and their usefulness is being studied in the area of bone marrow transplantation and the treatment of immunodeficiency disorders. .

In administering such a factor to a living body, it is most desirable to apply it within the physiological concentration range originally possessed by the living body, and administering a large amount to the human body from the outside unnecessarily causes only side effects. However, it is clear from the clinical cases of human interleukin 2 (IL-2) that it is often difficult to apply it as a medicine.

The novel technique to be completed by the present invention has an effect of synergistically enhancing the proliferation of hematopoietic cells when used in combination with these factors having a colony forming effect on hematopoietic cells, and the novel technique itself is used alone. The purpose of the present invention is to identify and provide substances exhibiting the action. Therefore, this factor is human IL-
It also has the aspect of having three kinds of activities.

[0013]

The factors involved in the proliferation of hematopoietic cells (interleukin 3 = multi)
i CSF, etc.) or by independently identifying a factor that enhances the growth of human hematopoietic cells (hereinafter referred to as HCGPF) as a substance and introducing a gene encoding this factor into cells such as microorganisms. To be produced
This is an essential means of making the present HCGPF useful. Accordingly, an object of the present invention is to identify and provide the present HCGPF as a novel substance.

[0014]

The present invention provides a human hematopoietic cell growth-enhancing factor having the amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence listing. (HCGPF).

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors prepared human peripheral blood-derived monocytic cells by a conventional method, and
RNA was extracted from the activated lymphocytes by conventional phenol extraction, precipitated with ethanol, and then mRNA was collected by affinity chromatography using oligo dT-cellulose.
The oligo dT-cellulose chromatography was further repeated to improve the purity of the product and to perform size fractionation of mRNA.

On the other hand, cDN in monkey cells (COS cells)
An A expression vector pDE-2 was constructed (Japanese Patent Application No. 59-2).
08645, 59.10.4 application). That is, this pD
E-2 is pML-R11G (Nature 293, 7
9 (1981)) and pKCR (Proc. Natl. A.
cad. Sci. , U. S. A. , 78, 1527 (1
981)) and constructed as shown in FIG.
The DNA can be sandwiched between the SV40 early promoters in both orientations, and E. It can be replicated in E. coli and can be selected for ampicilly resistance.

The pDE-2 obtained by C-tailing and the above-mentioned G-tailed double-stranded cDNA were mixed and annealed. This annealed DNA was transformed into competent E. coli MC1061
(J.Mol.Biol., 1 38, 179-207
(1980)). The L medium was smeared on an agar plate to allow colonies to appear.

The colonies thus obtained were divided into 192 groups each of 32 colonies, mixed and cultured, and 32 types of mixed plasmid DNAs were purified by a conventional method.

This plasmid DNA mixture (32 kinds) was used for monkey cells (COS-7) (Cell 23 , 175-182).
(1981)), and after chloroquine treatment and washing, 4
After culturing for 8 hours, the FDC-P cell proliferation activity of the culture supernatant was measured, and a group of active DNA mixtures was identified. The DNA plasmids of the group containing the DNA mixture in which activity was recognized here were purified as 32 single plasmid DNAs, re-introduced into COS cells, and the culture supernatant was subjected to the same activity assay to obtain FDC-P.
Clone p4-15, which exhibited cell proliferation activity, ie, IL-3-like activity, was identified.

Then, the cDNA contained in this clone was d
ideoxy chain termination method (Sanger et al., Proc. Natl. Acad. S.
ci, U. S. A. 74 , 5463-5467 (197)
7)) and the method of Maxam-Gilbert (Me.
th. Enzym, 65 , 510-580 (198
0)) and analyzed the whole base sequence. This cDNA was found to encode a secretory protein having a base peptide of about 900 bp and having a signal peptide. The structure of this cDNA, as well as the polypeptide deduced therefrom, has not been found to date and is therefore entirely new. Since the messenger RNA corresponding to this cDNA is specifically expressed in human lymphocytes activated by TPA and PHA,
It is considered to be a novel lymphokine.

A cleavage diagram of the cDNA insert with a restriction endonuclease is shown in FIG. As shown in FIG. 1, this cDNA has a structure that is cleaved by restriction endonucleases AvaI, PstI, and BanI. The nucleotide sequence of cDNA is shown in SEQ ID NO: 9 in the sequence listing.

The DNA sequence of this cDNA insert carries one large open reading frame. ATG sequence predicted to be a read initiation sequence for eukaryotes (Kozak, M. Cell. 15 , 1109-112).
3 (1978)) is located 64 to 66 nucleotides from the 5'-end, and the 196 codons at nucleotide positions 655 to 657 where the read-end codon TGA is present
It is connected to TG. 3'-poly of mRNA
(A) A linkage corresponding to the terminus is found at the cDNA terminus and consists of the six nucleotides AATAAA (883-88) normally found in most eukaryotic mRNAs.
(8th position) is located first (Prodfoot, NJ.
& Brownlee C.E. G. , Nature 26
3 , 211-214 (1976)). See SEQ ID NO: 9 in the Sequence Listing.

The amino acid sequence encoded by the cDNA can be performed as shown in SEQ ID NO: 3 in the sequence listing, and the polypeptide of SEQ ID NO: 3 consists of 197 amino acids, and is almost all of the secretory proteins known to date. As reported in (Blobel G. et a
1, Sym. Soc. Exp. Med. , 33 , 9-3
6 (1979)), the N-terminal region of the described HCGPF polypeptide is also hydrophobic. This region is mature HC
It may play the role of a signal peptide that is cleaved upon secretion of GPF. Cleavage: Ser and T at positions 18-19
hr or truncated between positions Gly-Ile between positions 30-31, respectively.
To produce a polypeptide having This is because similar cleavage sites are often found in other secreted proteins known so far (Blobel, G. et a.
l. , Symp. Soc. Exp. Med. , 33 , 9
-36 (1979)).

Accordingly, the mature HCGPF polypeptide is 17
It is calculated to consist of 9 to 167 amino acids. In addition, as shown in Example 4, DN starting from the ACC codon at nucleotide positions 118 to 120 of SEQ ID NO: 9 in the sequence listing.
It was confirmed that the code for the A fraction, ie, the polypeptide starting from Thr located at position 19, expressed a polypeptide having HCGPF activity (SEQ ID NO: 1 in the sequence listing). Nucleotide sequence 154-15 of SEQ ID NO: 9 in Sequence Listing
The DNA fraction starting from ATC at position 6, that is, the DNA fraction encoding the polypeptide starting from Ile at position 31, may express a polypeptide having HCGPF activity as shown in Example 5. It was confirmed (SEQ ID NO: 2 in the sequence listing).

Eukaryotic genes are known to exhibit polymorphism, as is also known for the human interferon gene (Taniguchi et al., Gene 10 , 11-15 (19).
80), Ohno, Taniguchi. , Proc. Natl. Aca
d. Sci. USA, 77 , 5305-5309 (19
81): Gray et al, Nature 29
5 , 501-508 (1981)). Due to this polymorphism, some of the amino acids in the protein product may be substituted, or there may be changes in the nucleotide sequence but no change at all.

As is clear from the above description, the gene of the present invention starts from the ATG sequence at positions 64-66 of DNA having the nucleotide sequence shown in SEQ ID NO: 9 in the sequence listing, and 6
DNA having a continuous base sequence at least from position 52-654 to the GCC sequence, DNA having a continuous base sequence starting from the ACC sequence at positions 118 to 120 and extending from the ACC sequence to at least the GCC sequence, and positions 154 to 156 DNA having a continuous base sequence from the ATC sequence to at least the GCC sequence.

The gene of the present invention also terminates at the GCC sequence at positions 652-654 and begins at G at position 1, 64-,
DNA beginning with ATG at position 66, A at positions 118-120
DNA starting with CC sequence or ATC at positions 154-156
It includes DNA starting with the sequence. Furthermore, the gene of the present invention comprises a DNA ending with T at position 909, starting with A at position 1, 6
DNA starting with ATG at positions 4-66, DNA starting with ACC at positions 118-120, or AT at positions 154-156
Includes DNA that begins with the C sequence.

The gene of the present invention may also comprise a DNA ending with poly (A) and beginning with the ATG codon at positions 64-66.
DNA starting from ACC sequence 118-120 or 1
It includes DNA starting with the ATC sequence at positions 54-156. The present invention also includes a gene having a base sequence corresponding to the amino acid sequence described in SEQ ID NO: 1, 2, or 3 in the sequence listing.

A polypeptide lacking one or more amino acids in the amino acid sequence of SEQ ID NO: 3 in the sequence listing, or one or more amino acids in the amino acid sequence of SEQ ID NO: 3 in the sequence listing Polypeptides substituted with one or more amino acids may also have HCGPF activity, and thus genes encoding such polypeptides can be used as the gene of the present invention.

Similarly, a gene in which one or more bases capable of expressing one or more amino acids with respect to the amino acid sequence described in SEQ ID NO: 1, 2, or 3 in the sequence listing may be added. As long as the added amino acids do not interfere with the expression of HCGPF activity of the polypeptide, they are included in the present invention. Even a modified region having an additional amino acid sequence that inhibits the polypeptide function as HCGPF can be used as the gene of the present invention as long as the newly added region can be easily removed.

The same applies to SEQ ID Nos. 1, 2 or 3 in the sequence listing.
The DNA encoding an amino acid addition at the C-terminus of each amino acid sequence of the gene corresponding to the described amino acid sequence is 3'-
The same can be said for the DNA additionally bound to the end. Accordingly, the use of genes encoding such polypeptides is encompassed by the present invention.

Recombinant DNA bodies that produce HCGPF in living cells can be produced by the following various methods. For example, HCG
The sequence encoding the PF cDNA is inserted downstream of the promoter sequence of the expressing vector. Alternatively, a cDNA fragment having a promoter sequence can be inserted upstream of the sequence encoding HCGPF before or after insertion of the cDNA into the expression vector.

Expressing HCGPF cDNA, HCGPF
The method for constructing a eukaryote or a prokaryote that produces a polypeptide is described in detail below.

HCGPFcDN in Escherichia coli
To express A, cDNA is first linked to various bacterial promoters, and then a hybrid plasmid containing cDNA downstream of the promoter is prepared. This plasmid was used to infect Escherichia coli HB101,
Bacteria that biosynthesize a protein having human HCGPF activity are cloned. HCGPF as long as any bacterial promoter is properly connected to cDNA
Express the cDNA. Examples of such cDNA expression will be shown in the Examples.

HCGPF cDNA can also be expressed in yeast by incorporating it into an appropriate expression vector and introducing it into a host cell. Various shuttle vectors for the expression of foreign genes in yeast have already been reported several (Heitzman et Nature 293, 7
17-722 (1981) Miyashita et al., Proc. Nat
l. Acad. Sci. USA, 80 , 1-5 (198
3)). These vectors are capable of growing in both E. coli and yeast hosts. Basically, all expression vectors containing a yeast gene promoter sequence can be used for expression of HCGPF cDNA. The use of yeast as compared to the use of animal cells and bacteria makes it possible to increase the level of HCGPF-producing ability, and it is also possible to produce HCGPF containing sugar chains, which is useful for improving the solubility of HCGPF. It is.

The yeast-Escherichia coli shuttle vector pAT77, pAM82 was prepared by Miyashita et al.
tl. Acad. Sci. USA 80 1-5 (19
83)). Vector pAM82
Is a derivative of pAT77, both of which have an arsl marker (Stinchcomb DT et al.
Nature 282 , 39-43 (1979), 2
μm ori (Broach JR et al, G
ene, 8 , 121-133 (1979), leu2
(Ratzkin B. et al Proc. Nat
1, Acad. Sci. USA, 7 4, 474-491
(1979)) Yeast acid phosphatase (APas
Has a promoter for e). PBR3 with ampicillin resistance marker (APr) for both vectors
It has 22 DNA fractions consisting of 3,700 bases and an amplified origin. The APase promoter is induced only when a high concentration of phosphate is shifted to a low concentration in a culture solution.

To insert the amplified DNA into other eukaryotic cells, a vector suitable for the host cell may be ligated to the cDNA insert that has been cut and separated from the prokaryotic cell. Then, the nucleated cells are infected with the synthesized vector and cultured.

Cells in which the recombinant DNA body has been inserted are cultured to amplify the recombinant DNA body or produce the HCGPF polypeptide. This cultivation is performed by a usual method. For example, the infected yeast is subjected to aerobic conditions in a medium containing carbon sources, nitrogen sources, inorganic salts and, if necessary, organic nutrients such as vitamins and amino acids in the range of 27 ° C to 37 ° C, pH 4 to 7. To do. Transformed prokaryotes such as Escherichia coli or Bacillus subtilis can also be cultured by conventional methods.

The HCGPF obtained here was a mouse IL-
It is also presumed that it is a human-derived lymphokine corresponding to 3, but the presence of human IL-3 is currently unknown, and human IL-3
Due to the absence of the authoritative activity assay system of -3, it cannot be concluded that it is of human origin corresponding to mouse IL-3. In fact, the structure of this cDNA is mouse IL
-3 which has no similarity to that of cDNA and has the same anc
It is hard to imagine that both branches off from the estral gene.

FDC-P cells are widely used in Japan and overseas as IL-3-dependent cell lines, and it has been confirmed that they do not proliferate in the presence of other lymphokines. For example, interleukin 2, interleukin 1, and interferon do not have these activities.
The protein having the amino acid sequence encoded by the gene sequence obtained heretofore unknown and has human G-CSF, M-CSF, GM-CSF showing an activity slightly similar to IL-3.
Both have different structures and are different from so-called selective colony stimulating factor (CSF). Therefore, this gene (c
It is clear that the activity of the novel putative lymphokines expressed by (DNA) exhibits at least part of the human IL-3 activity described above.

The human IL-3-like protein obtained here was subjected to salting out, gel filtration, affinity chromatography, ion exchange chromatography, isoelectric focusing,
It is easily purified by high performance liquid chromatography and other known methods for separating and purifying polypeptides alone or in combination.

A large amount of this IL-3 like protein was expressed in COS cells, and its activity was assayed. As a result, it showed growth-inducing activity on mouse bone marrow cells, human bone marrow cells, thymocytes and mouse embryonic hepatocytes. It has also been confirmed that, apart from the activity intensity, it exhibits multi-CSF-like activity. Furthermore, in a system using mouse bone marrow cells as target cells, the expressed protein synergistically induced significant proliferation in the presence of mouse IL-3.

From the above, it can be seen that the present human IL-3-like, mul
It has a ti-CSF-like activity and acts synergistically with IL-3 to produce hematopoietic cells (Hematopoietic Cell).
This novel lymphokine polypeptide was designated as HCGPF as a factor for proliferating Iss).

The HCGPF thus obtained shows the same biochemical and biological behavior as HCGPF produced from mammalian cells and shows HCGPF activity. HCGPF activity was measured at 56 ° C3 even after DNase and RNase treatment.
It is stable even after heat treatment for 0 minutes, and the activity is stable at pH 4-9.

[0045]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail based on embodiments.

Example 1 (1) The method of Falkoff et al. (J. of Immun)
ol. Method. , 50 , 39 (1982)) and the method of Taniguchi et al. (Proc. Natl. A.).
cad. Sci. U. S. A. 78 , pp3469-3
472 (1981)). And PHA 5 μg / ml and TPA 5 ng / m
peripheral blood-derived cells (3.4 × 10 1
0) is TBS (10 mM Tris-HCl, pH 7.
(5, 10 mM NaCl, 1.5 mM MgCl2). The cells were washed twice by centrifugation, and 200 m of a TBS solution containing 10 mM of ribonucleoside-vanadyl complex which is a nuclease inhibitor.
resuspended in 1 l.

Further, the surfactant NP-40 was added so that the final concentration was 0.1%, the mixture was slowly mixed, and the cell nuclei were removed by centrifugation at 3000 rpm for 5 minutes at 4 ° C.
The supernatant was SDS (0.5%) and EDTA (5 mM)
After adding, an equal amount of water-saturated phenol was added for extraction.
After three extractions with phenol, the RNA was 2
The precipitate was precipitated with twice the volume of cold ethanol, and the precipitate was collected by centrifugation and dissolved in a 10 mM Tris-HCl, pH 7.5 solution. The RNA obtained was 40 mg.

Fractionation of mRNA was performed by affinity chromatography with oligo dT-cellulose (PL Biochemicals, Type 7).
The adsorbent was 20 mM Tris-HCl, 0.5 M Na.
P containing Cl, 1 mM EDTA and 0.5% SDS
It is a solution of H7.5, and elution is performed with a buffer solution (20 m
M Tris-HCl, pH 7.5, 0.5M NaC
After washing well with 1,1 mM EDTA, 10m
Alternately with M Tris-HCl, pH 7.5. The mRNA obtained by elution was 0.9 mg based on 33 mg of RNA used.

To further enhance the purification of mRNA, affinity chromatography with oligo dT-cellulose was repeated in the same manner to obtain 0.45 mg of the mRNA standard.
I got

(2) Next, a cDNA library was prepared from the present mRNA sample as follows and used for cDNA cloning. The method of cDNA synthesis is basically La
nd et al. (Nucleic Acids Re
s. , Vol 9, P2551 (1981)).

(A) 50 mM Tris-HCl, pH
8.3, 10 mM MgCl2, 0.1 M KCl, 1
0 mM dithiothreitol (DTT), 0.5 mM of each dATP, dGTP, dCTP, dTTP (dCTP is
32P radiolabeled), 0.375 OD units of oligo dT10, 50 μg mRNA and 50 units AMV
Reverse transcriptase (manufactured by Life Science) was mixed and reacted at 41 ° C. for 90 minutes. After the completion of the reaction, phenol treatment was performed, and mRNA-cD
The NA hybrid was recovered and dissolved in 0.3N NaOH solution. Let stand at room temperature for 15 hours, then pH 7.5
Was neutralized by adding an equal volume of a 2M Tris-HCl solution, and the cDNA was recovered through a Sephadex G-50 column. The recovered single-stranded cDNA was about 10 μg.

(B) 0.1 M potassium cacodylate (pH
7.2) 10 mM DTT, 2 mM CoCl2,
0.5 mM 32 P-dCTP (specific activity 1 × 10 6 cpm
/ Nmole), 10 μg of cDNA and 60 units of deoxynucleotidyl terminal transferase (BRL) were mixed, incubated at 24 ° C. for 23 minutes, and then treated with phenol to give Sephadex G-5.
The cDNA fraction was collected through a 0 column to obtain 9.5 μg of dC-tailed cDNA as an ethanol precipitate. This cDNA had about 20 dCMP residues added to the 3 'end.

(C) Next, a 50 mM potassium phosphate buffer (pH 7.5), 10 mM MgCl 2, 10 mM D
TT, 1 mM each of dATP, dGTP, dCTP, dT
TP (dCTP includes those labeled with 3H), 7.2
μg oligo dG10, 9.5 μg single-stranded cDNA and 5 μg
0 units of DNA polymerase I (Klenow) (BR
L) was mixed and the reaction was carried out at 15 ° C. for 6 hours. After completion of the reaction, phenol treatment was performed, and the double-stranded cDNA fraction was collected through a Sephadex G-50 column, and about 16 μg of DNA was recovered as an ethanol precipitate.

(D) Next, 33 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 10 mM
DTT, 66 mM potassium acetate, 0.1 mM each dA
TP, dGTP, dTTP (excluding dCTP), 16μ
g of double-stranded cDNA and 22.5 units of T4 DNA
A polymerase (Takara) was mixed and reacted at 37 ° C. for 15 minutes to remove dC-tail attached to the 31 terminal. After the reaction, phenol treatment is applied to Sephadex G-
The DNA fraction was collected through 50 columns and 15.5 μg of DNA was recovered as an ethanol precipitate.

(E) Next, 50 mM sodium acetate (pH 4.
5), 0.2M NaCl, 1mM ZnCl2 and 1M
5.5 μg of double-stranded cDNA was preincubated at 37 ° C. for 20 minutes, 3 units of nuclease S1 (manufactured by Sankyo) was added, and the mixture was further incubated at 37 ° C. for 15 minutes. After completion of the reaction, phenol treatment was performed twice, and 15 μg of double-stranded cDNA was obtained through Sephadex G-50.

(F) 15 μg of the obtained double-stranded cDNA was subjected to sucrose density gradient centrifugation (50 mM Tris-HCl, 1
Fractionation was carried out in a solution containing mM EDTA, pH 7.5 by sucrose density gradient 5-25%, 40,000 rpm, 4 ° C. for 13 hours), and a part thereof was analyzed by autoradiogram by agarose gel electrophoresis. And double-stranded cd
Fractions having an NA size of 600 bp or more were collected and collected by ethanol precipitation. The recovered double-stranded cDNA is about 7
It was μg.

(G) 0.1 M potassium cacodylate (pH 7.2 with Tris Base) 10 mM DT
T, 2mM CoCl2, 0.5mM 32P-dGTP
(Specific activity 1 × 10 6 cpm / nmole), 7 μg double-stranded cDNA and 50 units of deoxynucleotidyl terminal transferase (BRL) were mixed,
After incubation at 20 ° C for 20 minutes, phenol treatment was performed and cDNA was passed through a Sephadex G-50 column.
Fractions were collected and 6 μg dG-t as ethanol precipitate
Ailed cDNA was obtained. This cDNA had about 13 dGMP residues added to both 3 'ends.

(H) Next, dG-tailed cDNA
6 μg was subjected to 1% agarose gel electrophoresis to size fractionate. That is, the cDNA fraction of about 700 bp or more
Adsorbed on EAE-cellulose paper, then 1.5
20 mM Tris-HCl containing M NaCl, pH
It was recovered by eluting with a 7.5 solution and further by phenol treatment, chloroform treatment and ethanol precipitation,
About 1.9 μg of dG-tailed cDNA was obtained.

(3) On the other hand, as shown in FIG. 2, a cDNA expression vector pDE-2 in monkey cells (COS cells) was constructed. pDE-2 is capable of sandwiching the cDNA into the bidirectional SV40 early promoter; coli
And can be selected as ampicillin resistant.

This pDE-2 was cut with EcoRI to give D
After filling the cohesive ends with NA polymerase I (Klenow), dGta was added to both ends of the 3 'end of the ds-cDNA.
The dC tail is set to 13 just like attaching il.
Around one piece was given. Next, this dC-tailed pDE
-2 100 ng and dG-tailed ds-cDNA
20 ng of 50 mM Tris-HCl, pH7.
5,0.1 M NaCl, 1 mM EDTA, mixed at 65 ° C. for 2 minutes, then at 45 ° C. for 60 minutes,
Incubated at 37 ° C. for 60 minutes and at room temperature for 60 minutes. Then, the annealed DNA was treated with competent E. coli. coli MC1061. Then M
The method for preparing and introducing the competent cells of C1061 is described below.

E. 100 ml of E. coli MC1061
Medium (2% tryptone, 0.5% yeast extract, 0.1%
5% MgSO 4 .7H 2 O, pH 7.6), and cultured with shaking at 37 ° C. until the absorbance of the culture solution was about 0.3 to 0.5 at 550 nm. After completion of the culture, the culture was maintained at 0 ° C. for 5 minutes, and the cells were collected by centrifugation.
I (30 mM potassium acetate, 100 mM RbCl, 1
Suspended in 40 ml of 0 mM CaCl2, 50 mM MnCl2, 15% glycerin, pH 5.8).
Let stand for minutes.

The bacterial cells were collected again by centrifugation, and TfbII
(10 mM MOPS or PIPES, 75 mM Ca
Cl2, 10 mM RbCl, 10% glycerin, pH
The suspension was suspended in 4 ml of 6.5) and allowed to stand at 0 ° C. for 15 minutes.
This suspension was dispensed and stored at -70 ° C.

Next, 100 μl of the competent cells thus prepared was kept at 0 ° C. for 15 minutes, into which the dG-tailed pDE-2 vector and dC-ta
10 μl of a sample annealed with iled cDNA and a solution 9 of 50 mM MgCl 2 and 10 mM CaCl 2
Mix with 0 μl and let stand at 0 ° C. for 20 minutes. Then 3
After heat treatment at 7 ° C for 60 seconds, hold at 0 ° C for 1 to 2 minutes,
To this, 7 ml of Ψ medium was added, and the mixture was cultured at 37 ° C. for 60 minutes with shaking. This culture was mixed with 25 μg / ml of ampicillin and 2
L medium containing 5 μg / ml streptomycin (1%
Tryptone, 0.5% yeast extract, 0.5% NaCl)
When smeared on the agar plate of No. 1 and incubated at 37 ° C. overnight, colonies appeared.

(4) 19 colonies each of which appeared 32
Divided into two groups, 25 μg / ml ampicillin and 2
Medium 100 containing 5 μg / ml streptomycin
The mixture was inoculated in a volume of 5 ml and cultured with shaking at 37 ° C for 5 to 7 hours. Next, 100 ml of a new Ψ medium containing chloramphenicol was added to a final concentration of 170 μg / ml, followed by shaking culture overnight.

The plasmid DN thus amplified
A was purified as follows. The culture was collected by centrifugation, and only the cells were collected and 50 mM Tris-HCl, pH 7.5.
Suspended in 5 ml of lysozyme, frozen at -80 ° C, thawed, then added lysozyme (final concentration, 2 mg / ml) and added at 0 ° C for 10
Allow to stand for 10 minutes, add EDTA (final concentration 0.1M), and let stand at 0 ° C for 10 minutes. Then, TritonX
Add -100 (final concentration 0.1%) and let stand at 0 ° C for 60 minutes. Then, ultracentrifugation is performed at 30,000 rpm for 30 minutes, and the supernatant is treated with an equal amount of water-saturated phenol. The aqueous layer was further treated with an equal volume of chloroform, the aqueous layer was extracted, RNase was added to this to a final concentration of 20 μg / ml, and the mixture was incubated at 37 ° C. for 60 minutes.

Then, 0.2 volume of 5M NaCl and ３
Volume of polyethylene glycol was added, and the mixture was allowed to stand at 0 ° C. for 60 minutes and then centrifuged at 10,000 rpm for 20 minutes to give D
Collect the NA precipitate. Next, this precipitate was dissolved in 3.8 ml of water, 4 g of CsCl was added and dissolved, and then 10 mg / m
Add 200 μl of EtBr and add 40,000 rpm
Ultracentrifugal fractionation is performed at 20 ° C. for 16 hours.

After the centrifugation, the plasmid DNA fraction was extracted and extracted with 1 to 2 volumes of water-saturated n-butanol to remove EtBr. After that, dialysis was performed in H 2 O to remove CsCl. 1/10 volume of 3M sodium acetate pH 5.6 was added and 2 volumes of cold ethanol was added,
Let stand overnight at -20 ° C. The ethanol precipitate was collected by centrifugation, washed with an 80% ethanol-water solution, dried well, and the precipitate was washed with 10 mM Tris-HCl, pH
Dissolve in 50 μl of 7.5 to use as a sample for monkey cell transfection.

Example 2 (1) Method of Infecting Monkey COS-7 Cells with Plasmids COS-7 cells were cultivated in 10% to 1 × 10 5 cells / ml.
Suspend in DMEM containing fetal bovine serum, and add 8 ml of this to 10
37% in a 5% carbon dioxide gas incubator in a cm dish
C. overnight. The culture supernatant was removed, 5 ml of fresh DMEM containing 10% fetal calf serum was added, and the cells were cultured at 37 ° C. in a 5% carbon dioxide gas incubator for 4 hours. After the culture, the supernatant was removed, and TBS (25 mM Tris-HCl, p
H7.5, 130 mM NaCl, 5 mM KCl,
Washed once with 5 ml of 0.6 mM Na2 HPO4).

TBS (+) (0.7 mM Ca in TBS
Cl2, 0.5 mM MgCl2) 2.5
ml, plasmid DNA 5 μg and 10 mg / ml
Add 128 μl of DEAE-dextran, 37 ° C
After incubating for 1 hour in a 5% CO 2 incubator, removing the supernatant, washing and removing with 5 ml of TBS,
10% FCS DMEM containing 0 μM chloroquine 5 ml
Was added. 3 in a 5% CO2 incubator at 37 ℃
After incubation for 1 hour, the supernatant was removed, and 5 ml of TBS was added.
Washed twice. 8 ml of DMEM containing 10% fetal bovine serum was added, and the cells were cultured overnight at 37 ° C. in a 5% carbon dioxide gas incubator. After removing the supernatant, 8 ml of the same DMEM was added, and 37
The cells were cultured in a 5% carbon dioxide gas incubator for 2 days.
The culture supernatant was spun down and the supernatant was used as a sample for measuring IL-3-like activity.

(2) Assay method and results of assay The measurement of the proliferation activity of FDC-P cells was carried out according to the following method. In each well of a 96-well tissue culture plate, FDC-
100 μl of COS cell culture supernatant to be assayed for P cell proliferation activity was added, and RPMI1640 plus 10%
Repeat 2-fold dilution in FBS medium.

Next, FDC, which is an IL-3 dependent cell line
-Add 100 μl of P cells to each well at a cell density of 1 × 10 4 cells / 100 μl. 18 at 37 ° C, 5% CO2
After culturing for 1 hour, 1 μCi of tritium thymidine was added and pulsed for 6 hours. Then, cells were collected on a glass fiber filter paper according to a method well known in the art, and scintillator was added, and β-radiation incorporated into the cells The quantity was measured. Since the amount of tritiated thymidine incorporated into cells increases as the amount of FDC-P cell growth activity increases, the amount of FDC-P cell growth factor produced in the COS cell culture supernatant can be easily quantified.

According to this method, the above plasmid mixture was
An activity assay was performed on 192 culture supernatants obtained by transfection into OS cells, and the results shown in Table 1 were obtained. The presence of significant activity was confirmed in the groups indicated by 3-11.

[0073]

[Table 1]

(3) Group 3-11 is a mixture of 32 clones. Next, 32 clones were separately cultured, and a plasmid was prepared by the same method as that used in (4) of Example 1 to prepare the plasmid described above. When COS cells were transfected by the same method as in (1) above, and the FDC-P cell proliferative activity of the culture supernatant was measured, the proliferative activity was observed in one clone (15th, named p4-15) ( FIG. 3), it was identified that this clone has a cDNA showing IL-3-like activity.

(4) EcoR from plasmid p4-15
CDNA was cut out by I and did using M13
oxy chain termination method and Ma
When its nucleotide sequence was determined using the method of xam-Gilbert, this IL-3 cDNA was found to be approximately 900 bp.
It is estimated that it encodes a precursor HCGPF protein consisting of p base pairs and consisting of about 197 amino acids or 83 amino acids from its open reading frame. Base sequence of this cDNA (SEQ ID NO: 9 in Sequence Listing)
And deduced amino acid sequences (SEQ ID NOS: 3 and 10 in the sequence listing).

That is, the present cDNA is composed of 909 bases, and the amino acid sequence to be encoded may be any of those shown in SEQ ID NOs: 3 and 10 in the sequence listing.

Example 3 (1) In order to express the human HCGPF gene more efficiently, polyGC t contained on the 5 'side of cDNA was used.
The plasmid excluding ail was constructed as follows (FIG. 4). CDNA was excised from the plasmid p4-15 constructed in Example 1 with the restriction enzyme EcoRI, and a part thereof was cut with the restriction enzyme BglI. The 396 base pair BglI-BglI fragment thus obtained was designated as T4.
After blunting both ends with polymerase, Hin
Mix with dIII linker at a molar ratio of 1: 1 and mix with T4
Ligation was performed using DNA ligase. After the bound fragment was recovered, it was digested with restriction enzymes HindIII and SphI,
An indIII-SphI fragment was obtained.

One of the cDNAs was digested with SphI to obtain a SphI-EcoRI fragment. Furthermore, the plasmid p
SP62-PL (NEN) is cut with restriction enzymes HindIII and EcoRI to obtain a DN containing SP6 promoter.
A fragment was obtained.

The three fragments obtained as described above were mixed at a molar ratio of 1: 1: 1 and ligated using T4 DNA ligase. The recombinant DN thus obtained
A was introduced into Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected. A plasmid was prepared from the isolated strain, and a cleavage test with a restriction enzyme and determination of the nucleotide sequence near the binding site were performed to obtain pSP6-
Bacteria retaining 4-15 were selected.

Next, the plasmid obtained from this bacterium was digested with the restriction enzymes HindIII and EcoRI to recover the cDNA insert. This cDNA was blunt-ended at both ends with T4 polymerase, then mixed with a BamHI linker at a molar ratio of 1: 1 and ligated with T4 DNA ligase. The ligated fragment was digested with the restriction enzyme BamHI.
PKCR cleaved at (Glutzman, Y. Cell
23 , 175-182 (1981)) and ligated with T4 ligase.

The recombinant DNA thus obtained was introduced into the Escherichia coli HB101 strain to select a strain having ampicillin resistance. A plasmid was prepared from the isolated strain and subjected to a restriction enzyme cleavage test to confirm that the restriction sites on the vector and cDNA were EcoR.
Plasmid p into which cDNA has been inserted in the form of I-BamHI-SphI-BglI-BamHI
Bacteria retaining KCR-4-15 were selected (pKCR-
4-15 / HB101). This pKCR-4-15 / HB
101 was cultured in a conventional manner to obtain a plasmid pKCR-4-15 according to the method described in Example 1, section (4).

Example 4 (1) The vector DNA used for expressing the human HCGPF gene in prokaryotes was constructed as follows (FIG. 5). First, each of the DNA fragments (a) to (l) having the DNA sequence shown in FIG. 6 was synthesized by the solid phase phosphate triester method. Except for (a) and (g), the 5'end was phosphorylated using T4 polynucleotide kinase and ATP.

Next, (a) to (l) were mixed, annealed, and then double-stranded synthetic DNA was prepared using T4 DNA ligase.
(A) was formed. On the other hand, pT9-11 (Japanese Unexamined Patent Publication No.
-88882) was cut with restriction enzymes HpaI and XbaI, and a large DNA fragment was separated by agarose gel electrophoresis. Next, the obtained pT9-11 fragment and the synthetic DNA
(A) was mixed and ligated using T4 DNA ligase. The obtained recombinant DNA was used for Escherichia coli H
The strain was introduced into the B101 strain and a strain having ampicillin resistance was selected. From the isolated strain, a plasmid DNA was obtained, a digestion test with a restriction enzyme and a nucleotide sequence were determined to select a bacterium having pT13S (Nco).

(2) HCGP using plasmids pT13S (Nco) and p4-15 and N-terminal at Thr19
Recombinant DNA expressing F was constructed as follows (FIG. 7).

I) The plasmid pT13S (Nco) was digested with restriction enzymes ClaI and PvuII, and a large DNA fragment was isolated and purified by agarose gel electrophoresis.

On the other hand, plasmid p4-15 was replaced with restriction enzyme E
Cleavage with coRI and agarose gel electrophoresis
The GPF cDNA insert was recovered. And the Eco
Replace RIHCGPF cDNA insert with restriction enzyme BalI
And complete digestion with HgiAI, and a large DNA fragment was recovered by agarose gel electrophoresis. Then, a pT13S (Nco) fragment containing a tryptophan promoter / operator (trpP / O); a HgiAI-BalI fragment containing HCGPF cDNA and a synthetic DNA (I).
[SEQ ID NOS: 17 and 18 in the sequence listing] were mixed, and
Ligation was performed using ligase.

The obtained recombinant DNA was used for E. coli
The strain was introduced into E. coli HB101 strain and a strain having ampicillin resistance was selected. By obtaining a plasmid from the isolated strain and performing a cleavage test with a restriction enzyme and determining the nucleotide sequence near the binding site, a bacterium holding pTHCGPF-19 was selected (pTHCGPF-19 / HB101,
FERM BP-1246).

Ii) pTHCGPF-19 / HB101 was converted to 25 μg / ml streptomycin and 25 μg / m
The cells were grown overnight at 37 ° C. in 10 ml of an LG medium (1% bactotryptone, 0.5% yeast extract 0.5% NaCl, 0.1% glucose, pH 7.5) containing 1 ampicillin. Then, 5 ml of the culture suspension was added to an M9-casamino acid medium (0.6% Na2 HPO4.12H2 O, 0.3% KH
2 PO4, 0.05% NaCl, 0.1% NH4 Cl,
0.05% MgSO4.7H2O, 0.00147% C
aCl2, 0.2% glucose, 0.2% casamino acid,
0.02% L-leucine, 0.02% L-proline,
0.0002% thiamine hydrochloride, pH 7.4) and cultured at 28 ° C. for 3 hours.

Thereafter, 3-tondoleacrylic acid (IAA) was added to a concentration of 25 μg / ml.
Induction culture was performed for hours. The cultured cells are centrifuged, and 20 mM Tris-hydrochloric acid (containing pH 7.5 and 30 mM NaCl)
, And suspended in 8 ml of the same buffer. The protein thus produced in the cells was extracted by digestion with 1% sodium dodecyl sulfate (SDS) or 1 mg / ml lysozyme in the presence of 50 mM EDTA, followed by sonication (50 W, 30 seconds). As shown in FIG. 8, the culture extract showed HCGPF activity.

Example 5 (1) Plasmid pT13S (N
co) and p4-15, a recombinant DNA expressing HCGPF with the N-terminus at Ile31 was constructed as follows (FIG. 9).

I) The plasmid pT13S (Nco) was cleaved with the component enzymes NcoI and BamHI, treated with DNA polymerase I (Klenow), and a larger DNA fragment was recovered by agarose gel electrophoresis. On the other hand, after cutting plasmid p4-15 with restriction enzyme ApaI, T4
After treatment with DNA polymerase, small DNA fragments were recovered by agarose gel electrophoresis. Both of these fragments
Ligation was performed using 4 DNA ligase. The obtained recombinant DNA was introduced into Escherichia coli HB101 strain,
A strain having ampicillin resistance was selected. By obtaining a plasmid from the isolated strain and performing a cleavage test with a restriction enzyme, a bacterium carrying pTHCGPF-01 was selected (pTHCGPF-01 / HB101).

Ii) The plasmid pTHCGPF obtained in i)
After completely digesting -01 with the restriction enzyme ClaI, it was partially digested with AvaI and treated with alkaline phosphatase. Thus prepared DNA fragment and synthetic DNA (I
I) [Sequence ID Nos. 11 and 12 in the Sequence Listing] are mixed, and T4D
It was ligated using NA ligase. Obtained recombinant D
NA was introduced into Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected. By obtaining a plasmid from the isolated strain and carrying out a restriction enzyme digestion test and determination of the nucleotide sequence near the binding site, pTHC
Bacteria carrying GPF-31 were selected (pTHCGPF
-31 / HB101, FERM BP-1247).

Iii) A cultured cell extract was obtained from pTHGPPF-31 / HB101 according to Example 4ii). As shown in FIG. 8, the culture extract showed HCGPF activity.

Example 6 (1) Plasmid pT13S (N
co) and pTHCGPF-0 constructed in Example 5
HIL-2 protein fusion N-terminal was used as Thr19
Recombinant DNA expressing CGPF was constructed as follows (FIG. 10).

I) The plasmid pT13S (Nco) was digested with restriction enzymes SacI and BamHI, and then a larger DNA fragment was recovered by agarose gel electrophoresis. On the other hand, the plasmid pTHCGPF-01 was digested with restriction enzymes EcoRI and BamHI, and a smaller DNA fragment was recovered by agarose gel electrophoresis. The DNA fragment was converted into HgiA
After complete digestion with I, a large D
The NA fragment was recovered.

The DNA fragment thus prepared and synthetic D
NA (III) [SEQ ID NO: 13 and SEQ ID NO: 14 in the sequence listing] were mixed and ligated using T4 DNA ligase. The obtained recombinant DNA was introduced into Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected.
By obtaining plasmid DNA from the isolated strain and performing a cleavage test with a restriction enzyme and determining the nucleotide sequence, pT
13SΔHIL2 (53) -HCGPF-19 (pT1
3SΔHIL2 (53) -HCGPF-19 / HB10
1, FERM BP-1248) was selected.

(2) Obtaining the product pT13SΔHIL2 (53) -HCGPF-19 / H
B101 was cultured according to Example 4, and granules generated in the cells were extracted by the following procedure. The bacterial cells were collected by centrifugation, and 20 mM Tris-HCl buffer solution (pH 7.5) containing 30 mM NaCl was added so as to be 10-fold concentrated. After suspension, 1 mg / ml of lysozyme and EDTA were added thereto.
After adding 0.05 M and stirring, the mixture was left in ice for 1 hour. Next, the cells were disrupted by sonication, and
The granules were collected by centrifugation at 0 rpm for 5 minutes.

The granules were solubilized with 6M guanidine hydrochloride. The solubilized material was HCGP as shown in FIG.
It showed F activity. In addition, reverse phase HPLC (AP
(312, Yamamura Kagaku) fraction also showed HCGPF activity (FIG. 12). Further, the 6 M guanidine hydrochloride solubilized product was used when the ΔHIL2 (53) -HCGPF-19 protein concentration was 1%.
The concentration was adjusted to be 00 mg / ml and a 2M guanidine hydrochloride solution, and oxidized glutathione 1
mM and reduced glutathione 10 mM, pH8.
0 and left at room temperature for 10-16 hours.

Next, guanidine hydrochloride was removed by gel filtration using Sephadex G-25, and at the same time, ΔHIL2 (53) -H, which became a kallikrein reaction buffer solution, was obtained.
A fraction corresponding to the CGPF-19 protein was obtained. The substance has a molecular weight almost identical to the value calculated from the amino acid composition by SDS polyacrylamide gel electrophoresis, and the amino acid sequence on the N-terminal side was assayed by a protein sequencer. confirmed.

(3) Cleavage with Kallikrein 50 mM Tris-H containing 113 mM NaCl
The resulting ΔHIL2 (5 in Cl buffer, pH 7.8)
3) After reacting 80 µg of -HCGPF-19 protein with human plasma kallikrein at 37 ° C for 15 hours, a fraction corresponding to HCGPF-19 protein was collected by reverse phase HPLC. This was analyzed using a protein sequencer to analyze the amino acid sequence near the N-terminus. As a result, ΔHIL2 (53) -HCGPF-19
It was confirmed that the protein was quantitatively converted to HCGPF-19 protein. The HCGPF-19 protein is HCGPF
It showed activity (FIG. 13).

Example 7 (1) HCG using the plasmids pT13S (Nco) and pTHCGPF-01 constructed in Examples 4 and 5, respectively, with the N-terminal of the HIL-2 protein fused to Ile31
Recombinant DNA expressing PF was constructed as follows (FIG. 14).

I) Plasmid pT13S (Nco) was cleaved with restriction enzymes SacI and BamHI, and then a larger DNA fragment was recovered by agarose gel electrophoresis. On the other hand, the plasmid pTHCGPF-01 was completely digested with the restriction enzyme BamHI, then partially digested with AvaI, and HCGPFcDN.
The DNA fragment containing the A insert was recovered by agarose gel electrophoresis.

DNA fragment and synthetic DNA thus prepared
(IV) [SEQ ID NOS: 15 and 16 in the sequence listing] were mixed, and T4
Ligation was performed using DNA ligase. The obtained recombinant DNA was introduced into Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected. By obtaining a plasmid DNA from the isolated strain and carrying out a cleavage test with a restriction enzyme and determination of the nucleotide sequence, pT13SΔH
A bacterium carrying IL2 (53) -HCGPF-31 was selected (pT13SΔHIL2 (53) -HCGPF-3.
1 / HB101, FERM BP-1251).

(2) Obtaining the product pT13SΔHIL2 (53) -HCGPF-31 / H
B101 was cultured according to Example 4, and the granules formed in the cells were extracted according to Example 6, and the granules were solubilized with 6M guanidine hydrochloride. As shown in FIG. 15, the solubilized product is H
It showed CGPF activity. Furthermore, ΔHIL2 (53)-
HCGPF-31 protein concentration of 100 μg / ml, and 2
The concentration was adjusted so as to obtain a M guanidine hydrochloride solution, to which 1 mM of oxidized glutathione and 10 mM of reduced glutathione were added, and the mixture was allowed to stand at pH 8.0 at room temperature for 10 to 16 hours.

Next, guanidine hydrochloride was removed by gel filtration using Sephadex G-25, and at the same time, ΔHIL2 (53) -HC which became a buffer solution for kallikrein reaction was obtained.
A fraction corresponding to the GPF-31 protein was obtained. The molecular weight of this substance was almost the same as the value calculated from the amino acid composition by SDS polyacrylamide gel electrophoresis, and the N-terminal side amino acid sequence was assayed by a protein sequencer. Was confirmed.

(3) Cleavage by Kallikrein 50 mM Tris-H containing 113 mM NaCl
ΔHIL2 (5) obtained in Cl buffer, pH 7.8
3) 80 µg of -HCGPF-31 protein and human plasma kallikrein were reacted at 37 ° C for 15 hours, and then a fraction corresponding to HCGPF-31 protein was collected by reverse phase HPLC. As a result of analyzing the amino acid sequence near the N-terminus with a protein sequencer, ΔHIL2 (53) -HCGPF-31
It was confirmed that the protein was quantitatively converted to HCGPF-31 protein. The HCGPF-31 protein is HCGP
F activity was shown (FIG. 13).

Example 8 (1) i) Plasmid pT13S Constructed in Example 6
ΔHIL2 (53) -HCGPPF-19 is a restriction enzyme Bg
cut with lII and XbaI to give DNA polymerase I
After treatment with (Klenow), they were ligated with T4 DNA ligase (FIG. 16). The obtained recombinant DNA was introduced into Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected. Plasmid D from the isolated strain
By obtaining NA and performing a cleavage test with a restriction enzyme and determining a base sequence near the binding site, pT13SΔHI
A bacterium carrying L2 (20) -HCGPF-19 was selected (pT13SΔHIL2 (20) -HCGPF-19.
/ HB101, FERM BP-1249).

(2) Obtaining the product pT13SΔHIL2 (20) -HCGPF-19 / H
B101 was cultured according to Example 4, and the granules formed in the cells were extracted according to Example 6, and the granules were solubilized with 6M guanidine hydrochloride. As shown in FIG. 11, the solubilized material is H
It showed CGPF activity. Further, ΔHIL2 (20) −
HCGPF-19 protein concentration of 100 μg / ml, and 2
The concentration was adjusted so as to obtain a M guanidine hydrochloride solution, to which 1 mM of oxidized glutathione and 10 mM of reduced glutathione were added, and pH was adjusted to 10 to 1 at room temperature.
It was left for 6 hours.

Next, guanidine hydrochloride was removed by gel filtration using Sephadex G-25, and at the same time, ΔHIL2 (20) -H, which became a buffer solution for kallikrein reaction, was obtained.
A fraction corresponding to the CGPF-19 protein was obtained. The substance has a molecular weight almost identical to the value calculated from the amino acid composition by SDS polyacrylamide gel electrophoresis, and the N-terminal side amino acid sequence was assayed by a protein sequencer. confirmed.

(3) Cleavage with kallikrein 50 mM Tris-H containing 113 mM NaCl
HCGPF-1 obtained in Cl buffer, pH 7.8
9 protein and human plasma kallikrein at 37 ° C
After the reaction for 5 hours, a fraction corresponding to the HCGPF-19 protein was collected by reverse phase HPLC. This was analyzed by using a protein sequencer to analyze the amino acid sequence near the N-terminus.
2 (20) -HCGPF-19 protein is quantitatively HCGP
It was confirmed that the protein was converted to F-19 protein. The HCG
The PF-19 protein showed HCGPF activity.

Example 9 (1) i) Plasmid pT13S constructed in Example 7
ΔHIL2 (53) -HCGPF-31 is replaced with restriction enzyme Bg
cut with lII and XbaI to give DNA polymerase I
After the (Klenow) treatment, binding was performed using T4 DNA ligase (FIG. 17). The obtained recombinant DNA was introduced into Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected. PT13SΔH was obtained by obtaining plasmid DNA from the isolated strain and carrying out a cleavage test with a restriction enzyme and determination of the nucleotide sequence near the binding site.
A bacterium retaining IL2 (20) -HCGPF-31 was selected (pT13SΔHIL2 (20) -HCGPF-3).
1 / HB101, FERMBP-1252).

(2) Obtaining the product pT13SΔHIL2 (20) -HCGPF-31 / H
B101 was cultured according to Example 4, and the granules formed in the cells were extracted according to Example 6, and the granules were solubilized with 6M guanidine hydrochloride. As shown in FIG. 15, the solubilized product is H
It showed CGPF activity. Further, ΔHIL2 (20) −
HCGPF-31 protein concentration of 100 μg / ml, and 2
Adjust the concentration so that it becomes M guanidine hydrochloride solution,
Oxidized glutathione 1 mM and reduced glutathione 10 mM were added thereto, and the mixture was allowed to stand at pH 8.0 at room temperature for 10 to 16 hours.

Next, guanidine hydrochloride was removed by gel filtration using Sephadex G-25, and at the same time, ΔHIL2 (20) -H, which became a kallikrein reaction buffer solution, was obtained.
A fraction corresponding to CGPF-31 protein was obtained. The molecular weight of this substance was almost the same as the value calculated from the amino acid composition by SDS polyacrylamide gel electrophoresis, and the N-terminal side amino acid sequence was assayed by a protein sequencer. Was confirmed.

(3) Cleavage by kallikrein 50 mM Tris-H containing 113 mM NaCl
ΔHIL2 (2 in Cl buffer, pH 7.8)
0) -HCGPF-31 protein (80 μg) and human plasma kallikrein were reacted at 37 ° C. for 15 hours, and then H was measured by reverse phase HPLC.
A fraction corresponding to the CGPF-31 protein was collected. As a result of analyzing the amino acid sequence near the N-terminal with a protein sequencer, it was confirmed that ΔHIL2 (20) -HCGPF-31 protein was quantitatively converted to HCGPF-31 protein. The HCGPF-31 protein showed HCGPF activity.

Example 10 (1) Plasmid pT13S (N
co) and the plasmid pT13 constructed in Example 6.
HIL using SΔHIL2 (53) -HCGPF-19
Recombinant DNA expressing HCGPF in which the N-terminal of the -2 protein fusion was Thr19 was constructed as follows (FIG. 18).

I) Plasmid pT13S (Nco) was digested with restriction enzyme PstI, treated with T4 DNA polymerase, further digested with restriction enzyme PvuI, and the second largest DNA fragment was recovered by agarose gel electrophoresis. On the other hand, plasmid pT13SΔHIL2 (53) -HCG
After cleaving PF-19 with restriction enzyme XbaI, it is treated with DNA polymerase I (Klenow), and further, restriction enzyme Pv
cleaved with uI and larger D on agarose gel electrophoresis
The NA fragment was recovered.

These two fragments were ligated using T4 DNA ligase. The obtained recombinant DNA was introduced into Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected. PT13SΔHIL2 (11) was obtained by obtaining a plasmid from the isolated strain and carrying out a restriction enzyme digestion test and determination of the nucleotide sequence near the binding site.
-A bacterium holding HCGPF-19 was selected (pT13
SΔHIL2 (11) -HCGPF-19 / HB10
1, FERM BP-1250).

(2) Acquisition of product pT13SΔHIL2 (11) -HCGPF-19 / H
B101 was cultured according to Example 4, and the granules formed in the cells were extracted according to Example 6, and the granules were solubilized with 6M guanidine hydrochloride. As shown in FIG. 11, the solubilized material is H
It showed CGPF activity. Further, ΔHIL2 (11) −
The concentration was adjusted so that the concentration of HCGPF-19 was 100 μg / ml and a 2M guanidine hydrochloride solution.
Oxidized glutathione 1 mM and reduced glutathione 10 m
M was added and left at pH 8.0 and room temperature for 10-16 hours.

Next, guanidine hydrochloride was removed by gel filtration using Sephadex G-25, and at the same time, ΔHIL2 (11) -H, which became a kallikrein reaction buffer solution, was obtained.
A fraction corresponding to the CGPF-19 protein was obtained. The molecular weight of this substance was almost the same as the value calculated from the amino acid composition by SDS polyacrylamide gel electrophoresis, and the N-terminal side amino acid sequence was assayed by a protein sequencer. Was confirmed.

(3) Cleavage with kallikrein 50 mM Tris-HC containing 113 mM NaCl
The resulting ΔHIL2 (11) in 1 buffer pH 7.8
-HCGPF-19 protein (80 µg) was reacted with human plasma kallikrein at 37 ° C for 15 hours, and then HCG was detected by reverse phase HPLC.
A fraction corresponding to PF-19 protein was collected. The amino acid sequence near the N-terminus was analyzed using a protein sequencer, and it was confirmed that ΔHIL2 (11) -HCGPF-19 protein was quantitatively converted to HCGPF-19 protein. The HCGPF-19 showed HCGPF activity.

Example 11 HC using mouse bone marrow cells
GPF activity assay (1) COS cell culture supernatant containing human HCGPF was prepared as follows. Plasmid pKC obtained in Example 3
5 μg of R-4-15 was added to monkey C according to the method of Example 2.
OS-7 cells were infected to obtain a culture supernatant, which was concentrated 5-fold using an Amicon ultrafiltration membrane (YM10), and human H
This was used as a CGPF activity measurement sample.

(2) Preparation of Escherichia coli extract containing human HCGPF and its cleavage with kallikrein were performed as described in Examples 6 to 10. Each of the obtained culture extract, 6M Guanidine hydrochloride-soluble substance, its purified fraction by reverse phase HPLC (AP-312, Yamamura Chemical) and cut kallikrein were each 1 μg / m 2.
and diluted with RPMI medium containing 20% FBS to obtain a human HCGPF activity measurement sample.

(3) Assay Method A human HCGPF assay using mouse bone marrow cells was performed as follows. The femur of a 5-9 week old DBA / 2 female mouse (Charles River Japan Co., Ltd.) was taken out, and both epiphyses were excised, followed by 1 ml of 10% (volume ratio) fetal bovine serum (FBS). The bone marrow cells were extruded into a plastic tube by injecting the RPMI medium from one side into a syringe with a needle attached. Further, the bone marrow cell suspension was repeatedly made into single cells by repeatedly sucking and blowing with a syringe barrel having a needle attached thereto. R with 10% FBS
After washing 3 times with PMI medium, the cells were suspended in RPMI medium containing 20% FBS so that the concentration became 1 × 10 6 cells / ml.

H in individual wells of 96-well tissue culture plates
100 μl of the sample whose CGPF activity is to be measured
, And a 2-fold dilution was repeated in an RPMI medium containing 10% FBS. Then, according to the method of Example 2,
50 μl of culture supernatant containing mouse IL-3 obtained by infecting monkey COS cells with 5 μg of plasmid pQRMIL3 containing mouse interleukin 3 (IL-3) cDNA was added to each well.

When measuring the growth stimulation of HCGPF directly on bone marrow cells, 20% was used instead of the main culture supernatant.
Add 50 μl of RPMI containing FBS.

Finally, the mouse bone marrow cell suspension prepared as described above was added in an amount of 50 μl / well, and the mixture was added at 37 ° C. and 5% CO 2.
After culturing in the presence of 2 days for 5 days, tritiated thymidine 1 μCi
, And pulsed for 8 hours, and then the amount of β-radiation taken into the cells was measured according to a conventional method.

Among the bone marrow cells, cells proliferating in response to HCGPF can easily quantify the amount of tritium thymidine incorporated in the COS cell supernatant and the amount of Escherichia coli extract and the like produced. According to this method, it was revealed that the COS supernatant contained weak activity to grow bone marrow cells alone as shown in FIG. 19 and activity to grow bone marrow cells in cooperation with mouse IL-3. No activity was observed in the COS-7 cell supernatant obtained by infecting the plasmid pKCR containing no cDNA used as a control.

Also, Escherichia coli extract, 6M
Guanidine hydrochloric acid solubilized product, said extract reverse phase HPL
The activity was similarly observed in the C fraction and also in the kallikrein digest (FIG. 8, FIG. 11, FIG. 12, FIG. 13, FIG. 15).

Example 12 Activity Assay Using Human Bone Marrow Cells and Thymocytes H
CGPF activity was measured. Human bone marrow cells (2 × 10 5 cells / well) or thymocytes (5 × 1) obtained from a healthy person.
(0 5 / well) was suspended in RPMI medium containing 10% FBS, and the diluted HC containing 10% FBS was double-diluted.
After adding 100 μl of each well of a 96-well tissue culture plate containing 100 μl of the sample to be measured for GPF activity, the cells were cultured for 5 days at 37 ° C. in 5% CO 2, and then 1 μC
i tritiated thymidine was added to each well and pulsed for 8 hours. After harvesting the cells according to a conventional method, the incorporated β-radioactivity was measured. As shown in Table 2, significant growth induction was observed although it was weak.

[0130]

[Table 2]

[0131]

EFFECTS OF THE INVENTION Since HCGPF of the present invention has an immunoregulatory function and a hematopoietic control function, it can be applied to the fields of immunodeficiency, infectious diseases, cancer, hepatitis, nephritis, bone marrow transplantation and the like. Also, this H
When CGPF is used in combination with other immunoactive substances, immunotherapeutic agents, lymphokines, cytokinins, interferons, cell growth factors, chemotherapeutic agents, antibiotics and antiviral agents, it is considered to be effective in enhancing the effect and reducing side effects of other agents. To be

[0132]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 179 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence: Thr Leu Leu Lys Arg Leu Leu Gln Glu His Ser Gly Ile Phe Gly Phe 1 5 10 15 Ser Val Ser His Thr Thr Arg Asn Pro Arg Pro Gly Glu Glu Asn Gly 20 25 30 Lys Asp Tyr Tyr Phe Val Thr Arg Glu Val Met Gln Arg Asp Ile Ala 35 40 45 Ala Gly Asp Phe Ile Glu His Ala Glu Phe Ser Gly Asn Leu Tyr Gly 50 55 60 Thr Ser Lys Val Ala Val Gln Ala Val Gln Ala Met Asn Arg Ile Cys 65 70 75 80 Val Leu Asp Val Asp Leu Gln Gly Val Arg Asn Ile Lys Ala Thr Asp 85 90 95 Leu Arg Pro Ile Tyr Ile Ser Val Gln Pro Pro Ser Leu His Val Leu 100 105 110 Glu Gln Arg Leu Arg Gln Arg Asn Thr Glu Thr Glu Glu Ser Leu Val 115 120 125 Lys Arg Leu Ala Ala Ala Gln Ala Asp Met Glu Ser Ser Lys Glu Pro 130 135 140 Gly Leu Phe Asp Val Val Ile Ile Asn Asp Ser Leu Asp Gln Ala Tyr 145 150 155 160 Ala Glu Leu Lys Glu Ala Leu Ser Glu Glu Ile Lys Lys Ala Gln Arg 165 170 175 Thr Gly Ala

SEQ ID NO: 2 Sequence length: 167 Sequence type: amino acid Topology: linear Sequence type: protein Sequence: Ile Phe Gly Phe Ser Val Ser His Thr Thr Arg Asn Pro Arg Pro Gly 1 5 10 15 Glu Glu Asn Gly Lys Asp Tyr Tyr Phe Val Thr Arg Glu Val Met Gln 20 25 30 Arg Asp Ile Ala Ala Gly Asp Phe Ile Glu His Ala Glu Phe Ser Gly 35 40 45 Asn Leu Tyr Gly Thr Ser Lys Val Ala Val Gln Ala Val Gln Ala Met 50 55 60 Asn Arg Ile Cys Val Leu Asp Val Asp Leu Gln Gly Val Arg Asn Ile 65 70 75 80 Lys Ala Thr Asp Leu Arg Pro Ile Tyr Ile Ser Val Gln Pro Pro Ser 85 90 95 Leu His Val Leu Glu Gln Arg Leu Arg Gln Arg Asn Thr Glu Thr Glu 100 105 110 Glu Ser Leu Val Lys Arg Leu Ala Ala Ala Gln Ala Asp Met Glu Ser 115 120 125 Ser Lys Glu Pro Gly Leu Phe Asp Val Val Ile Ile Asn Asp Ser Leu 130 135 140 Asp Gln Ala Tyr Ala Glu Leu Lys Glu Ala Leu Ser Glu Glu Ile Lys 145 150 155 160 Lys Ala Gln Arg Thr Gly Ala 165

SEQ ID NO: 3 Sequence length: 197 Sequence type: amino acid Topology: linear Sequence type: protein Sequence: Met Ser Gly Pro Arg Pro Val Val Leu Ser Gly Pro Ser Gly Ala Gly 1 5 10 15 Lys Ser Thr Leu Leu Lys Arg Leu Leu Gln Glu His Ser Gly Ile Phe 20 25 30 Gly Phe Ser Val Ser His Thr Thr Arg Asn Pro Arg Pro Gly Glu Glu 35 40 45 Asn Gly Lys Asp Tyr Tyr Phe Val Thr Arg Glu Val Met Gln Arg Asp 50 55 60 Ile Ala Ala Gly Asp Phe Ile Glu His Ala Glu Phe Ser Gly Asn Leu 65 70 75 80 Tyr Gly Thr Ser Lys Val Ala Val Gln Ala Val Gln Ala Met Asn Arg 85 90 95 Ile Cys Val Leu Asp Val Asp Leu Gln Gly Val Arg Asn Ile Lys Ala 100 105 110 Thr Asp Leu Arg Pro Ile Tyr Ile Ser Val Gln Pro Pro Ser Leu His 115 120 125 Val Leu Glu Gln Arg Leu Arg Gln Arg Asn Thr Glu Thr Glu Glu Ser 130 135 140 Leu Val Lys Arg Leu Ala Ala Ala Gln Ala Asp Met Glu Ser Ser Lys 145 150 155 160 Glu Pro Gly Leu Phe Asp Val Val Ile Ile Asn Asp Ser Leu Asp Gln 165 170 175 Ala Tyr A la Glu Leu Lys Glu Ala Leu Ser Glu Glu Ile Lys Lys Ala 180 185 190 Gln Arg Thr Gly Ala 195

SEQ ID NO: 4 Sequence length: 194 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence: Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Pro Arg Phe Arg Thr 1 5 10 15 Leu Leu Lys Arg Leu Leu Gln Glu His Ser Gly Ile Phe Gly Phe Ser 20 25 30 Val Ser His Thr Thr Arg Asn Pro Arg Pro Gly Glu Glu Asn Gly Lys 35 40 45 Asp Tyr Tyr Phe Val Thr Arg Glu Val Met Gln Arg Asp Ile Ala Ala 50 55 60 Gly Asp Phe Ile Glu His Ala Glu Phe Ser Gly Asn Leu Thr Gly Thr 65 70 75 80 Ser Lys Val Ala Val Gln Ala Val Gln Ala Met Asn Arg Ile Cys Val 85 90 95 Leu Asp Val Asp Leu Gln Gly Val Arg Asn Ile Lys Ala Thr Asp Leu 100 105 110 Arg Pro Ile Tyr Ile Ser Val Gln Pro Pro Ser Leu His Val Leu Glu 115 120 125 Gln Arg Leu Arg Gln Arg Asn Thr Glu Thr Glu Glu Ser Leu Val Lys 130 135 140 Arg Leu Ala Ala Ala Gln Ala Asp Met Glu Ser Ser Lys Glu Pro Gly 145 150 155 160 Leu Phe Asp Val Val Ile Ile Asn Asp Ser Leu Asp Gln Ala Tyr Ala 165 170 175 Glu Leu L ys Glu Ala Leu Ser Glu Glu Ile Lys Lys Ala Gln Arg Thr 180 185 190 Gly Ala

SEQ ID NO: 5 Sequence length: 203 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence: Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Pro Arg Phe Arg Thr Leu Leu Lys Arg Leu Leu Gln 20 25 30 Glu His Ser Gly Ile Phe Gly Phe Ser Val Ser His Thr Thr Arg Asn 35 40 45 Pro Arg Pro Gly Glu Glu Asn Gly Lys Asp Tyr Tyr Phe Val Thr Arg 50 55 60 Glu Val Met Gln Arg Asp Ile Ala Ala Gly Asp Phe Ile Glu His Ala 65 70 75 80 Glu Phe Ser Gly Asn Leu Tyr Gly Thr Ser Lys Val Ala Val Gln Ala 85 90 95 Val Gln Ala Met Asn Arg Ile Cys Val Leu Asp Val Asp Leu Gln Gly 100 105 110 Val Arg Asn Ile Lys Ala Thr Asp Leu Arg Pro Ile Tyr Ile Ser Val 115 120 125 Gln Pro Pro Ser Leu His Val Leu Glu Gln Arg Leu Arg Gln Arg Asn 130 135 140 Thr Glu Thr Glu Glu Ser Leu Val Lys Arg Leu Ala Ala Ala Gln Ala 145 150 155 160 Asp Met Glu Ser Ser Lys Glu Pro Gly Leu Phe Asp Val Val Ile Ile 165 170 175 Asn Asp S er Leu Asp Gln Ala Tyr Ala Glu Leu Lys Glu Ala Leu Ser 180 185 190 Glu Glu Ile Lys Lys Ala Gln Arg Thr Gly Ala 195 200

SEQ ID NO: 6 Sequence length: 236 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence: Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Ser Arg Phe Arg Thr Leu Leu Lys Arg Leu Leu 50 55 60 Gln Glu His Ser Gly Ile Phe Gly Phe Ser Val Ser His Thr Thr Arg 65 70 75 80 Asn Pro Arg Pro Gly Glu Glu Asn Gly Lys Asp Tyr Tyr Tyr Phe Val Thr 85 90 95 Arg Glu Val Met Gln Arg Asp Ile Ala Ala Gly Asp Phe Ile Glu His 100 105 110 Ala Glu Phe Ser Gly Asn Leu Tyr Gly Thr Ser Lys Val Ala Val Gln 115 120 125 Ala Val Gln Ala Met Asn Arg Ile Cys Val Leu Asp Val Asp Leu Gln 130 135 140 Gly Val Arg Asn Ile Lys Ala Thr Asp Leu Arg Pro Ile Tyr Ile Ser 145 150 155 160 Val Gln Pro Pro Ser Leu His Val Leu Glu Gln Arg Leu Arg Gln Arg 165 170 175 Asn Thr G lu Thr Glu Glu Ser Leu Val Lys Arg Leu Ala Ala Ala Gln 180 185 190 Ala Asp Met Glu Ser Ser Lys Glu Pro Gly Leu Phe Asp Val Val Ile 195 200 205 Ile Asn Asp Ser Leu Asp Gln Ala Tyr Ala Glu Leu Lys Glu Ala Leu 210 215 220 Ser Glu Glu Ile Lys Lys Ala Gln Arg Thr Gly Ala 225 230 235

SEQ ID NO: 7 Sequence length: 191 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Pro Arg Phe Arg Ile Phe Gly Phe Ser Val Ser His 20 25 30 Thr Thr Arg Asn Pro Arg Pro Gly Glu Glu Asn Gly Lys Asp Tyr Tyr 35 40 45 Phe Val Thr Arg Glu Val Met Gln Arg Asp Ile Ala Ala Gly Asp Phe 50 55 60 Ile Glu His Ala Glu Phe Ser Gly Asn Leu Tyr Gly Thr Ser Lys Val 65 70 75 80 Ala Val Gln Ala Val Gln Ala Met Asn Arg Ile Cys Val Leu Asp Val 85 90 95 Asp Leu Gln Gly Val Arg Asn Ile Lys Ala Thr Asp Leu Arg Pro Ile 100 105 110 Tyr Ile Ser Val Gln Pro Pro Ser Leu His Val Leu Glu Gln Arg Leu 115 120 125 Arg Gln Arg Asn Thr Glu Thr Glu Glu Ser Leu Val Lys Arg Leu Ala 130 135 140 Ala Ala Gln Ala Asp Met Glu Ser Ser Lys Glu Pro Gly Leu Phe Asp 145 150 155 160 Val Val Ile Ile Asn Asp Ser Leu Asp Gln Ala Tyr Ala Glu Leu Lys 165 170 175 Glu Ala L eu Ser Glu Glu Ile Lys Lys Ala Gln Arg Thr Gly Ala 180 185 190

SEQ ID NO: 8 Sequence length: 224 Sequence type: amino acid Topology: Linear Sequence type: Protein Sequence: Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Ser Arg Phe Arg Ile Phe Gly Phe Ser Val Ser 50 55 60 His Thr Thr Arg Asn Pro Arg Pro Gly Glu Glu Asn Gly Lys Asp Tyr 65 70 75 80 Tyr Phe Val Thr Arg Glu Val Met Gln Arg Asp Ile Ala Ala Gly Asp 85 90 95 Phe Ile Glu His Ala Glu Phe Ser Gly Asn Leu Tyr Gly Thr Ser Lys 100 105 110 Val Ala Val Gln Ala Val Gln Ala Met Asn Arg Ile Cys Val Leu Asp 115 120 125 Val Asp Leu Gln Gly Val Arg Asn Ile Lys Ala Thr Asp Leu Arg Pro 130 135 140 Ile Tyr Ile Ser Val Gln Pro Pro Ser Leu His Val Leu Glu Gln Arg 145 150 155 160 Leu Arg Gln Arg Asn Thr Glu Thr Glu Glu Ser Leu Val Lys Arg Leu 165 170 175 Ala Ala A la Gln Ala Asp Met Glu Ser Ser Lys Glu Pro Gly Leu Phe 180 185 190 Asp Val Val Ile Ile Asn Asp Ser Leu Asp Gln Ala Tyr Ala Glu Leu 195 200 205 Lys Glu Ala Leu Ser Glu Glu Ile Lys Lys Ala Gln Arg Thr Gly Ala 210 215 220

SEQ ID NO: 9 Sequence length: 909 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to mRNA Sequence: GGATGCTGCG GCGCCCGCTG GCCGGGCGGC TGCGGCCGCC CTGGCCGGGC CCCACCGGAC 60 GGC ATG TCG GGC CCC AGG CCT GTG GTG CTG AGC GGG CCT TCG GGA GCT 108 Met Ser Gly Pro Arg Pro Val Val Leu Ser Gly Pro Ser Gly Ala 1 5 10 15 GGG AAG AGC ACC CTG CTG AAG AGG CTG CTC CAG GAG CAC AGC GGC ATC 156 Gly Lys Ser Thr Leu Leu Lys Arg Leu Leu Gln Glu His Ser Gly Ile 20 25 30 TTT GGC TTC AGC GTG TCC CAT ACC ACG AGG AAC CCG AGG CCC GGC GAG 204 Phe Gly Phe Ser Val Ser His Thr Thr Arg Asn Pro Arg Pro Gly Glu 35 40 45 GAG AAC GGC AAA GAT TAC TAC TTT GTA ACC AGG GAG GTG ATG CAG CGT 252 Glu Asn Gly Lys Asp Tyr Tyr Phe Val Thr Arg Glu Val Met Gln Arg 50 55 60 GAC ATA GCA GCC GGC GAC TTC ATC GAG CAT GCC GAG TTC TCG GGG AAC 300 Asp Ile Ala Ala Gly Asp Phe Ile Glu His Ala Glu Phe Ser Gly Asn 65 70 75 CTG TAT GGC ACG AGC AAG GTG GCG GTG CAG GCC GTG CAG GCC ATG AAC 348 Leu Tyr Gly Thr Ser Lys Val Ala Val Gln Ala Val Gln Ala Met Asn 80 85 90 95 CGC ATC TGT GTG CTG GAC GTG GAC CTG CAG GGT GTG CGG AAC ATC AAG 396 Arg Ile Cys Val Leu Asp Val Asp Leu Gln Gly Val Arg Asn Ile Lys 100 105 110 GCC ACC GAT CTG CGG CCC ATC TAC ATC TCT GTG CAG CCG CCT TCA CTG 444 Ala Thr Asp Leu Arg Pro Ile Tyr Ile Ser Val Gln Pro Pro Ser Leu 115 120 125 CAC GTG CTG GAG CAG CGG CTG CGG CAG CGC AAC ACT GAA ACC GAG GAG 492 His Val Leu Glu Gln Arg Leu Arg Gln Arg Asn Thr Glu Thr Glu Glu 130 135 140 AGC CTG GTG AAG CGG CTG GCT GCT GCC CAG GCC GAC ATG GAG AGC AGC 540 Ser Leu Val Lys Arg Leu Ala Ala Ala Gln Ala Asp Met Glu Ser Ser 145 150 155 AAG GAG CCC GGC CTG TTT GAT GTG GTC ATC ATT AAC GAC AGC CTG GAC 588 Lys Glu Pro Gly Leu Phe Asp Val Val Ile Ile Asn Asp Ser Leu Asp 160 165 170 175 CAG GCC TAC GCA GAG CTG AAG GAG GCG CTC TCT GAG GAA ATC AAG AAA 636 Gln Ala Tyr Ala Glu Leu Lys Glu Ala Leu Ser Glu Glu Ile Lys Lys 180 185 190 GCT CAA AGG ACC GGC GCC TGAGGCTTGC TGTC TGTTCT CGGCACCCTG 684 Ala Gln Arg Thr Gly Ala 195 GGCCCATACA GGACCAGGGC AGCAGCATTG AGCCACCCCC CTTGGCAGGC GATACGGCAG 744 CTCTGTGCCC TTGGCCAGCA TGTGGAGTGG AGGAGATGCT GCCCCTGTGG TTGGAACATC 804 CTGGGGTGAC CCCCGACCCA GCCTCGCTGG GCTGTCCCCT GTCCCTATCT CTCACTCTGA 864 ACCCAGGGCT GACATCCTAA TAAAATAACT GTTGGATTAG AAACT 909

SEQ ID NO: 10 Sequence length: 83 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence: Met Leu Arg Arg Pro Leu Ala Gly Arg Leu Arg Pro Pro Trp Pro Gly 1 5 10 15 Pro Thr Gly Arg His Val Gly Pro Gln Ala Cys Gly Ala Glu Arg Ala 20 25 30 Phe Gly Ser Trp Glu Glu His Pro Ala Glu Glu Ala Ala Pro Gly Ala 35 40 45 Gln Arg His Leu Trp Leu Gln Arg Val Pro Tyr His Glu Glu Pro Glu 50 55 60 Ala Arg Arg Gly Glu Arg Gln Arg Leu Leu Leu Cys Asn Gln Gly Gly 65 70 75 80 Asp Ala Ala

SEQ ID NO: 11 Sequence length: 48 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence: CGATAAGCCA TGATCTTTGG CTTCAGCGTG TCCCATACCA CGAGGAAC 48

SEQ ID NO: 12 Sequence length: 50 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence: TCGGGTTCCT CGTGGTATGG GACACGCTGA AGCCAAAGAT CATGGCTTAT 50

SEQ ID NO: 13 Sequence length: 42 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: ATCTAGATTC CGCACCCTGC TGAAGAGGCT GCTCCAGGAG CA 42

SEQ ID NO: 14 Sequence length: 42 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence: CCTGGAGCAG CCTCTTCAGC AGGGTGCGGA ATCTAGATAG CT 42

SEQ ID NO: 15 Sequence length: 49 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: ATCTAGATTC CGCATCTTTG GCTTCAGCGT GTCCCATACC ACGAGGAAC 49

SEQ ID NO: 16 Sequence length: 57 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: TCGGGTTCCT CGTGGTATGG GACACGCTGA AGCCAAAGAT GCGGAATCTA GATAGCT 57

SEQ ID NO: 17 Sequence length: 41 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: 35 CGATAAGCCA TGACCCTGCT GAAGAGGCTG CTCCAGCAGC A 41

SEQ ID NO: 18 Sequence length: 35 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence: CCTGGAGCAG CCTCTTCAGC AGGGTCATGG CTTAT 35

[Brief description of drawings]

FIG. 1 is a cleavage diagram of a cDNA insert with a restriction endonuclease.

FIG. 2 is a construction diagram of the expression vector pDE-2.

FIG. 3 shows FDC-P proliferation-inducing activity of a culture supernatant of COS cells into which p4-15 clone has been introduced. □-□ p4-15 clone (4-fold concentration) ○-○ p4-15 clone (stock solution) ●-● control (gene-introduced COS supernatant) ----- activity assay medium

FIG. 4 is a construction diagram of plasmid pKCR-4-15.

FIG. 5 is a construction diagram of a vector pT13S (Nco).

FIG. 6 shows a DNA fragment of human IL-2.

FIG. 7 is a diagram showing the construction of plasmid pTHCGPF-19.

FIG. 8 shows HCGPF activity of culture extracts of Escherichia coli infected with various plasmids.

FIG. 9 shows a construction diagram of plasmid pTHCGPF-31.

FIG. 10. Plasmid pT13SΔHIL2 (53)-
The construction drawing of HCGPF-19 is shown.

FIG. 11: 6 of Escherichia coli culture extract granules infected with various plasmids with and without addition of mouse IL-3
3 shows HCGPF activity of M guanidine hydrochloride lysate.

FIG. 12 shows the HCGPF activity of the reverse phase HPLC-purified fraction of ΔHIL2 (53) -HCGPF-19 protein.

FIG. 13 shows the HCGPF activity of a kallikrein cleavage product of the HIL-2 fusion HCGPF protein.

FIG. 14: Plasmid pT13SΔHIL2 (53)-
The construction drawing of HCGPF-31 is shown.

FIG. 15 shows the HCGPF activity of 6 M guanidine hydrochloride lysate of various plasmid-infected Escherichia coli culture extract granules.

FIG. 16: Plasmid pT13SΔHIL2 (20)-
It is a construction drawing of HCGPF-19.

FIG. 17: Plasmid pT13SΔHIL2 (20)-
It is a construction diagram of HCGPF-31.

FIG. 18 shows a plasmid pT13SΔHIL2 (11)-.
It is a construction drawing of HCGPF-19.

FIG. 19: C with and without mouse IL-3
4 shows HCGPF activity of OS cell culture supernatant.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1:91) (72) Inventor Shinsuke Taki 1-1 Central Research Institute, Ajinomoto Co., Inc. 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa (72) Inventor Yu Matsui 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Central Research Institute (72) Inventor Shinichi Kashima 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Central Research Laboratory

Claims (1)

[Claims] 1. A human hematopoietic cell growth-enhancing factor having the amino acid sequence of any of SEQ ID NO: 1 in the sequence listing, SEQ ID NO: 2 in the sequence listing, or SEQ ID NO: 3 in the sequence listing.