JPS6259299A - Novel csf and production thereof - Google Patents

Abstract

NEW MATERIAL:The CSF having the following physical and chemical properties. Molecular weight, 19,000+ or -1,000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis; isoelectric point, PI=5.5+ or -0.1, 5.8+ or -0.1 or 6.1+ or -0.1; ultraviolet absorption, absorption peak at 280nm and minimum absorption at 250nm; amino acid sequence, 21 amino acids from N-terminal are shown in the formula. USE:Remedy and clinical examination reagent for hypoleukocytosis, etc. PREPARATION:The objective CSF can be produced e.g. by culturing a cell strain capable of producing human marrow cell differentiation and proliferation promoting factor (CSF) in a serum-free culture liquid, subjecting the supernatant liquid of culture product to gel-filtration, adsorbing the obtained fraction to reverse-phase high-performance liquid chromatography carrier and purifying the eluted fraction by high-performance molecular sieve chromatography and isoelectric electrophoresis, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a factor that promotes the differentiation and proliferation of bone marrow cells (Colo
ny Stimulating Factor) (
In particular, the present invention relates to a novel CSF that has an effect of promoting the differentiation and proliferation of human neutrophils (hereinafter referred to as rCSFJ) and a method for obtaining the same.

[Industrial Application Fields] The CSF of the present invention is expected to be effective as a therapeutic agent for leukopenia, etc., and can also be used as a reagent for clinical tests and research.

[Prior Art] CSF is a substance that acts on animal bone marrow cells to promote differentiation and proliferation into macrophages or granulocytes, and several types of CSF have been reported so far. For example 5tanley
E, R, etc. are glycoproteins with a molecular weight of 45,000 obtained from healthy human urine and exhibit colony formation promoting activity against mouse bone marrow cells, but no colony formation promoting activity is observed against human bone marrow cells. reported that CSF was purified (Fed, Proc, Vol. 35, pp. 2272-2278,
(1975). Also Burgess A, W.

etc. from human placenta (Blood Vol. 49, 573-5
83.1977) (Blood Vol. 54, 614-6
27, 1979), 5hah R.

G1, etc. are human peripheral blood monocytes, PHA-stimulated lymphocytes, etc. (Blood Vol. 50, p. 811, 1977),
Fojo S, S, etc. were obtained from human lung culture supernatant (Bi
ochemistrV Volume 17 3109-3116
(Page, 1978) have reported that they have partially purified CSF that is effective for humans. These CSFs are all glycoproteins with molecular weights ranging from 25,000 to 41,000, and directly act on human bone marrow non-adherent cells to stimulate neutrophils,
Forms colonies of macrophages and eosinophils. However, in either case, there are restrictions on the materials, so completely purified CSF has not yet been obtained.

The above-mentioned CSF is derived from human normal tissue, but in recent years, it has been reported that certain human tumor cells produce CSF. For example, AsanoS.

et al. from nude mouse transplanted lung cancer cells (Blood Vol. 49, pp. 845-852, 1977), Okabe T.
, etc. are derived from mandibular squamous cell carcinoma cell lines and globus cancer cell lines (Cancer Res.

38, pp. 3910-3917, 1978) (JNC
I Vol. 89, pp. 1235-1243, 1982) (
J, Ce1l PhVsiol, vol. 110, 43-4
9, 1982), WuM.

C0 etc. were obtained from pancreatic cancer cell lines (J, Biol
.

Chem, vol. 254, pp. 8226-6228, 1979
) (J, Cl1n, Invest, Vol. 65, 772~
775 pages, 1980), Dipersio J.
F, etc. are Malignant Hi s t tocy
G CT Ce1l established from toma patient
From Line (Blo.

d, Volume 51, Page 1068, 1978) (Bl.

od 56, pp. 717-727, 1980), and Golde D, W, etc. are Hairy Ce11
MOCe1 established from Leukemia patients
l From Line (Blood Volume 52 1068
~1072 pages, 1978) (Blood Volume 57
13-21, 1981), each of whom reported that they obtained CSF. In either case, CSF is effective for humans.
is a glycoprotein with a molecular weight in the range of 27.000 to 34.000 and an isoelectric point of pI = 4.5 to 5.7. Among these, GCT Ce
11 The CSF obtained from the culture supernatant of Line has a specific activity of 1.12×106U/-glMOCel 1Lin
The CSF from the culture supernatant of E. has been purified to a purity of 3.5×10 6 U/■, but at present none of the CSF, including the other CSF mentioned above, has yet been completely purified.

[Disclosure of the Invention] The present inventors have succeeded in establishing a cell line that has extremely high C3F production ability and exhibits good proliferation ability from tumor cells of a patient with floor of mouth cancer. This cell line was named rCHU-IJ and was published in the Pasteur Institute Collection Nationale de Carlares de Microorganismes (COLLECTION NATIO) in France.
NALEDE CULTURES DE MIC
ROORGANISMES) (C,N,C,M) 19
It has been deposited with accession number rl-315J dated July 11, 1984.

The present inventors cultured this CHU-1 in vitro,
The culture supernatant showed human neutrophil colony formation promoting activity, with a molecular weight of approximately 18,000 (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis).
We succeeded in obtaining extremely pure CSF with a specific activity of 3.94×107 U/+ng≦. That is, the present invention is a novel CSF having the following physical and chemical properties, which has not yet been published in any literature.

"Physical and chemical properties" 1) Molecular weight: 19.000 ± 1,00 as measured by sodium nidodecyl sulfate-polyacrylamide gel electrophoresis 11) Isoelectric point: 1) I = 5.5 ± 0.1, pI = 5° 8
It has at least one of three isoelectric points of ±0.1 and pI=6.1±0°1.

iii) Ultraviolet absorption: maximum absorption at 280 nm, 2
It has a minimum value at 50 nm.

iv) The amino acid sequence from the N-terminus to the 21st residue is as follows.

Furthermore, the present invention provides human CS established from hydrocavitary carcinoma cells.
A cell line with F-producing ability is cultured in a serum-free culture medium, and the culture supernatant is subjected to the following treatments (1) to (3), and further treated in (4) or (5) as necessary. This is a method for obtaining CSF having the above-mentioned physical and chemical properties.

■ The culture supernatant was subjected to gel filtration using a gel with an effective fractionation range of 5,000 to 70,000 daltons to determine neutrophil-dominated colony formation promoting activity (hereinafter referred to as "CSA").
Collect fractions with .

(2) The fraction (2) is adsorbed onto a reversed-phase high performance liquid chromatography carrier, and eluted with a concentration gradient of a mixture of water and organic solvent to collect a fraction containing CSA dominated by neutrophils.

(2) The fraction (2) is subjected to high-performance molecular sieve chromatography to collect a fraction containing CSA dominated by neutrophils.

■ Fractions of ■ were subjected to isoelectric focusing, and neutrophil-dominated C
Collect the fractions with SA.

(2) After performing a sialic acid removal operation on the fraction (2), a fraction containing neutrophil-dominated CSA is collected.

A concrete outline of the above acquisition method is as follows, for example.

rCHU-IJ is suspended in F-10 culture medium containing 10% fetal bovine serum, and cultured by rotation at a constant speed in a glass roller bottle. When rCHU-IJ had grown completely and densely on the inner wall of the roller bottle, the culture medium was replaced with RPMII640, which does not contain fetal bovine serum, and after culturing for 4 days, the culture supernatant was collected, and then the fetal bovine serum was added again. Add F-10 culture solution containing F-10 and culture for 3 days. The culture medium was changed again to RPM11640 culture medium that does not contain fetal bovine serum, and after culturing for 4 days, the culture supernatant was collected. By repeating "Culture-Collection of serum-free culture supernatant" according to such a schedule, a culture supernatant of rC (U-IJ) is obtained.

The culture supernatant obtained in this way was filtered through an ultrafilter to give approximately 100%
After concentrating 0 to 2000 times, gel filtration is performed to collect a fraction containing CSA dominated by neutrophils. This fraction is then repeatedly purified by high-performance liquid chromatography, and then a portion containing neutrophil-dominated CSA is collected and freeze-dried.

The CSA measurement method used in the present invention is as follows.

rCSA measurement method” (a) When using human bone marrow cells: Bradley
T, R, +MetcalfDl et al. method (Aus
t, J, Exp, Bi.

1, Med, sci, Vol. 44, pp. 287-300,
(1966) using the monolayer soft agar culture method. That is, 0.2 ml of fetal bovine serum, 0-1 m of subject
l, human bone marrow non-adherent cell suspension 0.1ml (1
~2X105 nucleated cells), modified Me Co V's
Modified Mc containing 0.75% of 5A culture solution 0.2ml11 agar
Mix 0.4 ml of CoV's5A culture solution and
After solidifying in a tissue culture plastic dish, 37°C, 5% carbon dioxide/95% air, 10
Culture is carried out under 0% humidity conditions, and the number of colonies formed after 10 days (one colony is a colony consisting of 50 or more cells) is counted, and the activity of forming one colony is calculated as 1.
CSA was determined as a unit.

(b) When using mouse bone marrow cells: horse serum 0.
4ml + test sample 0.1ml + C3H/He (female)
Mouse bone marrow cell suspension 0.1ml (0.5~lXI
O3 nucleated cells).

Mix 0.4 ml of modified McCoV's5A culture solution containing 0.75% agar and make a 35 m diameter! After solidifying in a tissue culture plastic dish (1), heat at 37°C.
, 5% carbon dioxide/95% air.

Cultivate for 5 days under 100% humidity conditions, count the number of colonies formed (one colony is a colony consisting of 50 or more cells), and count the activity to form one colony as 1 unit. The CSA was calculated as follows.

In addition, the "modified M" used in the methods (a) and (b) above
The cCoY's5A culture solution and the human bone marrow non-adherent cell suspension used in (a) were prepared as follows.

"Modified McCoy's5A culture solution"McCoV's5A culture solution (manufactured by GIBCO) 12 g,
MEM amino acid vitamin medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) 2.55
g sodium bicarbonate 2.18 g.

5000 units of penicillin G potassium in 500 units of double distilled water
After dissolving in m1, filtration sterilization was performed using a 0.22 μm Millipore filter.

"Human bone marrow non-adherent cell suspension" Bone marrow fluid obtained by sternal puncture of a healthy person is used as RPM11640.
Diluted 5 times with culture solution and diluted with Ficol-PaQue solution (
(manufactured by Pharmacia), 400Xg, 30 minutes, 2
Centrifuge at 5°C to remove the physical cell layer (specific gravity <1.07).
7) Collect. After washing the cells, they were incubated at 5X106C in RPMIIE340 culture medium containing 20% fetal bovine serum.
The cells were adjusted to a concentration of e l l/ml, placed in a 25 CITF tissue culture plastic flask, and incubated in a carbon dioxide gas incubator for 30 minutes. The non-adherent cells in the supernatant were collected and placed in a 25 CITF plastic flask again. After the cells were placed in a flask and incubated for about 2 hours and 30 minutes, the supernatant of non-adherent cells was collected and used.

When the CSF of the present invention was applied to mouse bone marrow cells and human bone marrow cells as described in Example (7) below, promotion of neutrophil colony formation was observed in both cases. From this, it is clear that the CSF of the present invention is a type of CSF that promotes the differentiation and proliferation of bone marrow cells into neutrophils.

Next, the present invention will be explained in detail by way of examples.

[Examples] Example (1) Establishment of rCHU-IJ (1) Tumors of oral floor cancer patients in which a marked increase in neutrophils was observed were transplanted into nu/nu mice. Approximately 10 days after transplantation, significant tumor growth and increased number of neutrophils were observed in this tumor. This tumor was removed aseptically 12 days after transplantation, and
It was cut into 2 mm square pieces and cultured as follows.

(11) Primary culture Put 10 to 15 pieces of the tumor mass cut into pieces into a 50ml plastic centrifuge tube, add 5ml of trypsin solution (containing 0.25% trypsin, EDTAo, 02%),
After shaking for 10 minutes in a hot bath at 37°C, the supernatant was discarded, 5 ml of the same trypsin solution was added again, and trypsin digestion was performed at 37°C for 15 minutes with stirring. The supernatant cell suspension was collected, 1 ml of fetal bovine serum was added to stop the action of trypsin, and then stored in water.

The above operation was performed again to collect the cell suspension, which was then centrifuged at 1.50 Or, p, m for 10 minutes to obtain a cell pellet. After washing the cell pellet twice with F-10 containing 10% fetal bovine serum,
Cell concentration 5X10 in a plastic culture flask
They were implanted at 6 cells/flask. Using F-10 culture solution containing 10% fetal bovine serum, use a carbon dioxide incubator (carbon dioxide concentration 5%, humidity 100%)
After incubation overnight, the supernatant was removed together with non-adherent cells, fresh culture medium was added, and culture was continued. The cells proliferated rapidly on the 6th day after the start of culture, so
At this point, the culture medium was replaced with a fresh one. The next day, discard this culture solution and add 2 ml of anti-mouse red blood cells (manufactured by Cappe 1) diluted 5 times with RPMIIE340.
2 ml of guinea pig complement (manufactured by Kyokuto Pharmaceutical Co., Ltd.) diluted 2.5 times with 1840 was added and incubated at 37°C for 20 minutes. After incubation, add 10% fetal bovine serum.
Fibroblasts derived from nu/nu mice were removed by washing twice with F-10 containing F-10, and then washed with fetal bovine serum 10
% F-10 culture solution was added and cultured.

(III) Subculture When the cells had grown completely and densely in the primary culture, the medium was changed again at point 17 with F-10 culture medium containing 10% fetal bovine serum, and subculture was carried out the next day. After removing the culture solution with a Komagome pipette, add 2 ml of physiological saline containing 0.02% EDTA pre-warmed to 37°C, heat on a hot plate at 37°C for 2 minutes, and remove the cells by pipetting. It was peeled off. After adding 0.5 ml of fetal bovine serum, transfer the cell suspension to a 15 ml centrifuge tube,
Cell pellets were obtained by centrifugation at 0 Or, p, m for 10 minutes. Suspend cells in 1 ml of F-10 culture solution,
Passage was carried out by dividing the cells into 1/10 pieces. Thereafter, subculture was performed at intervals of 4 to 5 days by the same operation. In order to examine the proliferation ability of the cells thus obtained, 5×10 Ce
Prepare a cell suspension of l l s/ml and make a cell suspension with a diameter of 3.5
The cells were implanted in 1 ml portions (20 sheets) into m+ plastic push plates and cultured in a carbon dioxide gas incubator. After a certain period of time, dishes were taken out, adherent cells were collected, and the number of cells was calculated. The results are shown in FIG.

Proliferation begins approximately 20 to 24 hours after cell implantation.
The average doubling time (PDT) was approximately 20 hours.

Example (2) Isolation of CSF Cells established as described above were completely and densely proliferated.
Cells were collected from two 50c+ culture flasks, and were added to 500ml of F-10 culture medium containing 10% fetal bovine serum.
After floating in m1, a 1580cm glass roller bottle (Belc.

(manufactured by S.A., Inc.), and rotational culture was performed at a speed of 0.5 r, pom. When the cells have grown completely and densely on the inner wall of the roller bottle, pour the culture medium into serum-free RPM11640.
After culturing for 4 days, the culture supernatant is collected, F-10 containing 10% fetal bovine serum is added, and the culture is continued. After 3 days of culture, re-serum-free RPM1
Exercises were performed on 1e40, and culture supernatants were collected 4 days later.

By repeating the same procedure, 500ml of serum-free culture supernatant 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.

The obtained culture supernatant 5k was made into one batch, and 0.01
HollowFiber after adding %Tween 20
After concentrating about 1000 times by ultrafiltration using DC-4 and Amlcon PM-10 (manufactured by Amicon), it was purified in the following order.

(1) Ultr with a diameter of 4.8cm and a length of 90cIl
.

Using a gel AcA 54 column (manufactured by LKB), 0.0. OIM) 5 ml of the concentrated culture supernatant using Lis-HCl buffer (pH 7,4)
was gel-filtered at a flow rate of about 50 ml/hour. The column was pre-filled with bovine serum albumin (molecular weight 87,000),
Calibration was performed using ovalbumin (molecular weight 45.000) and cytochrome C (molecular weight 12.400). After gel filtration, 0.0% was collected from the gold fraction. 1m
1 of each sample, diluted 10 times, and then added the rcs described above.
Fractions exhibiting activity were examined by the measurement method (b) of A.

As a result, first, the fraction with Ve=400-700ml showed CSA dominated by macrophages, and the fraction with Ve=800-120ml showed CSA dominated by macrophages.
Since the 0ml fraction was found to exhibit granulocyte-dominated CSA, the latter fraction was collected using PM-10 (manufactured by Amicon).
It was concentrated to about 5 ml using an ultrafilter.

(+1) A 0.1% trifluoroacetic acid aqueous solution containing 30% n-propatool (manufactured by Tokyo Kasei Co., Ltd., for amino acid sequencing) was added to the above concentrated fraction, and after standing in water for about 15 minutes, 0OOOr, p.

The precipitate was removed by centrifugation for 10 minutes. then the previous n
- μBondapak C18 column equilibrated with an aqueous solution containing propatool and trifluoroacetic acid (Wa
After adsorption onto a semi-preparative device (manufactured by TERS Corporation, 8 Dragon x 30 cm), the mixture was sequentially eluted with a 0.1% aqueous trifluoroacetic acid solution containing n-propatool with a linear concentration gradient of 30 to 60%. High performance liquid chromatography equipment is Hitachi E! 85-50 type and a Hitachi 638-41 type detector (both manufactured by Hitachi Seisakusho) for detection, the absorption at 220 nm and 280 nm was simultaneously measured at a constant rate of 21 nm. After elution, 10 μl of each fraction was diluted 100 times, and the fractions showing activity were examined by the rCSA measurement method (b) J described above. As a result, activity was observed in the peak eluted at 40% n-propanol, so this peak was collected and re-chromatographed under the same conditions, and CSA was investigated in the same manner as above. Activity was observed at the peak at the 40% position, so
This peak was collected (4 fractions = 4 ml) and lyophilized.

(iii) The above freeze-dried powder was mixed with 200μ of a 0.1% trifluoroacetic acid aqueous solution containing 40% n-propanol.
1 and subjected to high performance liquid chromatography using a TSK-G3000SW column (manufactured by Toyo Soda Co., Ltd. + 2.5 mm x 60 cm). Elution is 0.4m with the same aqueous solution.
carried out at a flow rate of l/min and fraction collector FRA
0.4 ml portions were collected using C-100 (manufactured by Pharmacia). As a result of examining CSA in each separated fraction in the same manner as above, activity was observed in the fraction with a retention time of 37 to 38 minutes (corresponding to a molecular weight of about 20,000), so this fraction was collected. After further purification using an analytical μBondapak C18 column (4, Etm+aX 30 cm), the main peak was collected and freeze-dried.

Example (3) Physical and chemical properties In order to examine the physical and chemical properties of the CSF of the present invention obtained in Example (2), the following analyzes and tests were conducted.

(1) Molecular weight Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. The electrophoresis device is PROTEANTH 1μcm (manufactured by Bio-Rad)
A polyacrylamide slab gel (140 mm x 160 mm x 1.5 mm) with T = 15% and C = 2.6% and a concentrated gel (T = 3% and C = 20%) were used. 4 μg of the sample was used, which had been denatured by boiling for 3 minutes in a solution of 0.64 M 2-mercaptoethanol and sodium dodecyl sulfate added to a concentration of 2%. After 4 hours of electrophoresis at a constant current of 30 mA, the gel was taken out and 0.25% Coomassie Brilliant Blue R250 was added.
The band was detected by staining with (manufactured by Sigma). Phosphorylase B (Phospho) was used as a molecular weight marker.
ry1aseB1 molecular weight 92.500), bovine serum albumin (BSA, 87,000), ovalbumin (OV
A, 45.000), carbonic anhydrase (
C, arbonic Ambydrase+31.0
00), soy bean trypsin inhibitor (So
ybean Trypsin Inhibitor
, 2L 500), lysozyme CLysozVme+
14+400) was treated in the same manner and used. As a result, a single band with a molecular weight of about 19,000 was observed. The measurement results are shown in Figure 1.

When measuring the molecular weight of a protein such as the one of the present invention, regardless of the measurement method, the value generally differs by a certain range, and the human CS of the present invention
For F, the result of 5 measurements using the method described above is 1
It was 9,000±1,000.

(II) Isoelectric focusing flatbed type isoelectric focusing device FBE-3000
(manufactured by Pharmacia) was used. Ph of pH 4-6.5
Polyacrylamide gel (T = 5%, 623%, 115 x
After performing electrophoresis for 2 hours at 30W constant power (maximum voltage 2000V) at 230mm, 30% methanol/10%
It was fixed with trichloroacetic acid 735% sulfosalicylic acid, and then stained with Coomassie brilliant blue R-250. Low) IK as an isoelectric point marker
It pH 2.5 to 6.5 (manufactured by Pharmacia) was used.

When separation was investigated between pH 4 and 6.5, pI = 5.
Three bands, 52, 5, 80, and 6.13, were recognized. Among these, 1) two bands of I=5.52 and 5.80 were the main components.

The results are shown in FIG. C3FQ, CSF, in the figure.

CS F2 indicates a CSF of the present invention having a different isoelectric point.

(As a result of separately measuring the isoelectric point of the CSF of the present invention five times according to the method described above, the value was pI = 5.5 ±
0.1. pI=5.8±0.1゜pI=8.1±0.1
Met. ) In order to see the correlation between these three bands and CSA, Example (2) - (TSK-G3 in [1)
The CSF purified by the sw column was separated using a preparative isoelectric focusing device (FBE-3000, manufactured by Pharmacia). The separation conditions are as follows.

Data: 500μg of lyophilized powder containing 4M urea
．． Dissolve in 1 ml of 05N phosphoric acid.

Support: 225 ml of double-distilled water containing 4M urea and 0.1% Tween 20 was added to 15 g of 5ephadex-IEF (manufactured by Pharmacia), and then 12 ml of Pharmalite 4-8.5 (manufactured by Pharmacia) was added. ) was added and left to swell at room temperature overnight. After swelling, the mixture was sufficiently degassed using a suction bottle, and then a uniform gel layer with a thickness of 5 cm was created on a 230 mm x 230 mm glass plate.

Select the most uniform area on the plate and place 50 mm x 2
30 mm of gel was left and the remaining gel was removed.

Electrode solution: (anode) 0.1M phosphoric acid, (cathode) 0°1M
Electrode strips (6×10 strips, manufactured by Pharmacia) are impregnated with NaOH and placed parallel to and in contact with both ends of the gel. Constant power was supplied using a power supply (ECPS 2000/300° manufactured by Pharmacia).

Pre-phoresis: 8W electrophoresis was performed for 45 minutes.

Sample addition: At a position 5 cm from the anode, a 1 cm width of gel was scraped off, mixed with the sample solution, and then returned to the original position.

Main electrophoresis: Using the power supply mentioned above, 50W constant power 4
Time migration was performed.

After the electrophoresis is completed, the gel plate is taken out and divided into 26 fractions using a fractionation grid. After measuring the pH of each fraction, the gel of each fraction is scraped off and placed in a polypropylene mini column (Muromax, manufactured by Muromachi Kagaku Co., Ltd.). The mixture was transferred to a medium and extracted with 4 mL of a 0.1% aqueous trifluoroacetic acid solution containing 4M guanidine hydrochloride. A 5 μm sample was taken from each extracted fraction and diluted with 2 ml of RPM11640 culture solution containing 1% bovine serum albumin, and CSA was examined according to the rCSA measurement method (b) described above. As a result, there were three active peaks in all the eluted fractions, and the position of each active peak was at the isoelectric point p as described above.
It was in good agreement with I=5.52.5.80.6°13. In order to clarify whether the difference in isoelectric point observed here is due to the peptide moiety or the sugar chain (particularly the number of sialic acid additions), we investigated whether the difference in isoelectric point was caused by the neuraminidase-treated sample and the untreated sample. When isoelectric focusing was performed on the latter, three bands were observed in the latter, whereas only a band with pI=8.13 was observed in the former. In addition, instead of neuraminidase treatment, the sample was dissolved in a 6M guanidine hydrochloric acid aqueous solution, the pH was adjusted to 1.5 with IN hydrochloric acid, and then the sample was treated at 80°C for 120 minutes. Isoelectric focusing was performed in the same manner. However, a similar movement of bands was observed. Note that CSA was not impaired at all even after neuraminidase treatment. From this result, it is estimated that the difference in isoelectric point is due to the difference in the number of added sialic acids.

(Image I) The ultraviolet absorption sample was prepared using 0.1% trifluoroacetic acid containing 40% n-propanol as a reference.
As a result of examining the ultraviolet absorption using a spectrophotometer, as shown in FIG. 3, maximum absorption was observed at <280 nm and minimum value was observed at 250 nm.

(1v) Amino acid composition of protein part A sample was hydrolyzed by a conventional method, and the amino acid composition of the protein part was determined using Hitachi 8
Analysis was performed by a special amino acid analysis method using a 35 amino acid automatic analyzer (manufactured by Hitachi, Ltd.). Table this result -■
It was shown to.

The hydrolysis conditions are as follows.

■ E3N MC1, 110°C, 24 hours, in vacuum ■ 4N methanesulfonic acid + 0.2% 3-(2-aminoethyl)indole, 110°C, 24 hours, 48 hours, 72 hours, ゛Sample in vacuum: After dissolving in a solution (1.5 ml) containing % n-propatool and 0.1% trifluoroacetic acid, each
．． After taking 1 ml 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, Se
t+ 1/2Cys, Met+ Val+11e and Trp were calculated by the following method. (Biochemistry Experiment Course,
(See Protein Chemistry ■ (Tokyo Kagaku Doujin Publishing)) ・T h r + S e t + 1/2
CV s; M e t takes the change over time of the 24.48.72-hour value of ■, and extrapolates it to zero time. Val, lle is the 72-hour value of ■. Trp is the 24.48.72-hour value of ■. The number of amino acid residues in the average value table is a predicted value calculated assuming that Leu is 33. Generally, the amino acids that require correction as described above are either partially or significantly destroyed during hydrolysis, or
Alternatively, it is difficult to undergo hydrolysis, and furthermore, Pro has a low color development rate, so the actual measured value of such amino acids (
nm.

l), therefore the number of residues calculated therefrom tends to show a lower value than the actual value (see, for example, the above-mentioned biochemistry experiment course).

Table I (v) 1-g of temperature-stable sample (lyophilized powder) was mixed with 40 g of n-propertool.
Dissolve in 4 ml of 0.1% trifluoroacetic acid containing
OIM) was diluted with 10 ml of lithium-hydrochloric acid buffer (pH 7,4) to give a CSF concentration of 25 ng/ml. This sample was heated to 0°C and 37°C.

After treatment at 45°C, 56°C, 65°C and 100°C for 40 minutes, the remaining CSA was examined. As a result, it was stable at 0 to 45°C and deactivated at 56°C.

(vl) 1 mg of pH stable sample (lyophilized powder) was mixed with 40 mg of n-propatool.
Dissolve in 4 ml of 0.1% trifluoroacetic acid containing
, 3, 5, 7, 9, 11, and 13, and treated in water for 24 hours at a CSF concentration of 25 ng/ml. After processing, 2 each
ml was dialyzed against 0.01 M) IJS hydrochloric acid buffer (pH 7,4) to restore the pH, and then residual CSA was examined.

As a result, it was stable over a wide pH range of 1 to 11.

(vii) Stability against enzymes The sample (lyophilized powder) was dissolved in 0.1% trifluoroacetic acid containing 40% n-propanol, and 0.67 μg was dissolved in 0.05 M) Lis-HCl buffer (pH 8.0). ) to bring the total volume to 1 ml, and the same <0.67μ
One sample was prepared by adding 0.05 M acetate buffer (pH 5,0) to make a total volume of 1 ml, and each sample was
s e, ) Trypsin and 1 μg of Pronase were added, and the remaining one was used as a control. The latter includes neuraminidase (Neura
minidase) t μg was added thereto, and the mixture was reacted at 37° C. for 2 hours. After the reaction was completed, 1 ml of each sample was taken and diluted with 1 ml of RPMI 1640 culture solution containing 1% bovine serum albumin, and the CSA of each sample was examined. As a result, the CSF of the present invention was not inactivated by RNase and neuraminidase, but was inactivated by trypsin and pronase. (vNI) Sugar composition sample 11 nmol and inositol 2 as an internal standard.
After adding 5 nmol, a methanol solution (500 μm) containing 1.5N HCl was added, and the mixture was reacted at 90° C. for 4 hours in a sealed tube purged with nitrogen gas.

After opening the tube, the tube was neutralized by adding recycled acid silver (Ag2 CO3), then 50 μl of acetic anhydride was added, shaken, and left in a dark place at room temperature overnight. The upper layer was taken into a sample tube and dried with nitrogen gas. After adding methanol to the precipitate and washing it, it was briefly centrifuged, and the upper layer was added to the same sample tube and dried. A 50 μm TMS reagent (a mixture of pyridine: hexamethyldisilazane: trimethylchlorosilane = 5:1:1) was added to this, and the mixture was reacted at 40°C for 20 minutes, followed by De
Saved in ep Freezer. As a standard, 50 nmol each of various sugars such as galactose (Gal), N-acetylgalactosamine (GalNAc), and sialic acid and 25 nmol of inositol were combined and the same operation was performed.

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

(Analysis conditions) Column = 2% 0V-17 Vinport HP60~
80 mesh, 3m, glass temperature = 4℃/min heating from 110℃ to 250℃ Carrier gas: 1.2 to 1.8 kg/ctl (nitrogen pressure) at the beginning, 2 to 2.5 kg/ at the end Cze sensitivity = 103MΩ Range 0.1~0.4v pressure:
Hydrogen gas 0.8kg/cITI', air 0.8kg/C
Sample amount: 2.5 to 3.0μ1 Analysis results The constituent sugars of the CSF of the present invention are galactose,
It was found that there are three types: N-acetylgalactosamine and sialic acid.

(1x) Determination of amino acid sequence The sample was subjected to Edman digestion using a gas-phase sequencer (manufactured by Applied Biosystems), and the resulting PTH amino acid was analyzed using a high-performance liquid chromatography device (manufactured by Beckman Instruments). and Ult.
Analysis was performed using a rophere-ODS column (manufactured by Beckman Instruments) in a conventional manner. column(
5 μm.

Diameter 4.6. ,, length 25 om) with starting buffer (15
After equilibration with mM sodium acetate buffer (pH 4, 5, aqueous solution containing 40% acenitrile), the sample (20μ
1 of the starting buffer) was injected, and separation was performed by incratic elution with the starting buffer. The flow rate is 1
．． 4 ml/min, column temperature was maintained at 40°C. PTH
Amino acids were detected using ultraviolet absorption at 289 nm and 320 nm. 2 nmol of each standard PTH amino acid (manufactured by Sigma) was separated in advance in the same system to determine the retention time, and identification was performed from the retention time of the test specimen.

As a result, the amino acid sequence from the N-terminus to the 21st residue was determined as follows.

The specific activity value of the CSF of the present invention obtained in Example (2) against human bone marrow cells was measured according to the above-mentioned method for measuring rCSA (a), and the result was 3.94×10 7 U/g≦.

Example (5) The lyophilized powder of CSF obtained by the method of Example (2) was subjected to preparative isoelectric focusing under the following conditions to separate CSF based on differences in isoelectric point.

Apparatus: FBE-3000 (manufactured by Pharmacia) Sample: 10 g of freeze-dried powder was mixed with 0.0 g of lyophilized powder containing 4 M urea.
Dissolve in 2 ml of 05N phosphoric acid.

Support: 225 ml of double distilled water containing 4M urine L 0.1% Tween 20 was added to 15 g of 5ephadex-IEF (manufactured by Pharmacia), and then 12 ml of Pharmalite 4-6.5 (manufactured by Pharmacia) was added. ) was added and left to swell at room temperature overnight. After swelling, the gel was sufficiently degassed using a suction bottle, and then a uniform gel crumb of 5 mm thick was prepared on a 230 mm x 230 mm glass plate.

Electrode solution = (anode) 0.1M phosphoric acid, (cathode) 0°1M
Electrode strips (6 x 10 mm, manufactured by Pharmacia) are impregnated with NaOH and placed parallel to and in contact with both ends of the gel.

Constant power using a power supply (ECPS 2000/300, manufactured by Pharmacia).

Pre-phoresis: 8W electrophoresis was performed for 45 minutes.

Sample addition: At a position 5 cm from the anode, a 1 cm wide piece of gel was scraped off, mixed with the sample solution, and then returned to its original position.

Main electrophoresis: Using the power supply mentioned above, 50W constant power, 4
Time migration was performed.

After the electrophoresis, the gel plate was taken out and divided into 26 fractions using a fractionation grid. After measuring the pH of each fraction, the gel of each fraction was scraped off and placed in a polypropylene mini column (Muromac,
(manufactured by Muromachi Kagaku Co., Ltd.) and extracted with 10 ml of a 0.1% aqueous trifluoroacetic acid solution containing 4M guanidine hydrochloride. Take 5 μl from each extracted fraction and add 1% of IEiml.
After diluting with RPMIIθ40 culture solution containing bovine serum albumin, rcsA measurement method (b) described above.
As a result of investigating CSA according to the following, three isoelectric points (pI = 5
．． 52, 5, 80, and 8.13) were observed. Each active fraction was then loaded onto a μBondapak C18 column (Wate) equilibrated with an aqueous solution containing n-propanol and trifluoroacetic acid.
After adsorption with a 30-60% linear concentration gradient of 0.1% trifluoroacetic acid aqueous solution containing n-propatool,
The peaks eluted with 40% n-propatool were collected and lyophilized. Regarding these, Example (3) (
As a result of examining the amino acid composition and amino acid sequence according to methods iv) and (1x), both results were consistent with the results of Example (3).

Example (6) 10 mg of lyophilized CSF powder obtained in Example (2) was
ml O, 1M sodium carbonate-sodium bicarbonate (p
After dissolving in H9,0), the pH was adjusted to 5 using IN hydrochloric acid.
．． Adjusted to 0. After adding 100 μg of neuraminidase and reacting at 37°C for 2 hours, the reaction solution was loaded onto a μBondapak C18 column (Wate) equilibrated with an aqueous solution containing n-propanol and trifluoroacetic acid.
After adsorption to rs (for semi-preparative use, 8mm x 3Qcm),
It was eluted with a 0.1% aqueous trifluoroacetic acid solution containing n-propanol with a linear concentration gradient of 30 to 60%, and the peak eluted at 40% n-propanol was collected and lyophilized. A portion of this was taken and subjected to analytical isoelectric focusing, and as a result, it was confirmed that it was a single band with an isoelectric point pI = 6.13.

Example (7) Classification of Colonies Colonies formed based on the aforementioned rcsA measurement method (a) were classified using the method of Kubota et al. (Exp, H.
emat, *vol. 8, pp. 339-344, 1980)
According to the instructions, the agar layer was taken out onto a slide glass and dried to prepare a film specimen. Next, Kon
walinka.

The method of G. et al. (Exp, Hemat, + vol. 8, 434
Colonies were classified by triple staining of esterase triple staining and Bieblich scarlet staining according to the method (1980). Details of the method are shown below.

■Fixative: Buffered formalin autoacetone solution (pH 6゜Na
2 HPO420mg KH2PO4100mg H2030ml Acetone 45ml Total volume of Forma 1 100ml Store at 4℃ ■ Non-specific esterase staining reaction solution (prepared before use) (A) 1/15m
ol/l, phosphate buffer (pHe, 3) 9.5ml
Fast, garnet GBC salt 10-g (B) α-naphthyl butyrate 10-g ethylene glycol monomethyl ether 0.5 ml Mix (A) and (B) solutions, filter and use O ■Chloroacetate esterase staining reaction solution (Prepared before use) (A) l/15mol l/1. Phosphate buffer (
pH7,4) 9.5ml Fast Blue RR Salt 51Ig (B) Naphthol As-D
1 ml of chloroacetate, 0.5 ml of N,N-dimethylformamide Mix liquids (A) and (B), then filter and use.

■Biebrich Scarlet staining reaction solution (A) Biebrich Scarlet (manufactured by MC/B)
5g dimethyl sulfoxide 100ml (B) 0.1M Tris-HCl buffer (pH 7.4) (A
Use by mixing 2 ml of solution () with 98 ml of solution (B).

Colony staining was performed in the following order using the fixative solution and reaction solution prepared as described above.

(i) After fixing with the fixing solution (4 to 10°C) for 30 seconds, wash with distilled water three times and dry at room temperature for 10 to 30 minutes.

(11) Immerse in the reaction solution (1) and leave at room temperature for 20 to 30 minutes, then wash three times with distilled water.

(1st item) Immerse in the reaction solution (■) and leave at room temperature for 15 minutes, then wash 3 times with distilled water.

(iv) Immerse in the reaction solution (2) and leave at room temperature for 2 hours, then wash with running water.

(v) Observation after drying. Cells containing blue granules were classified as neutrophils, cells containing brown granules as monocytes-macrophages, and cells containing red granules as eosinophils.

As a result, all of the colonies formed using the CSF of the present invention were chloroacetate esterase-positive neutrophil colonies on days 7, 10, and 14 of culture, and no colonies of other strains were observed. There wasn't.

[Effects of the Invention] The CSF of the present invention is almost completely purified, as is clear from the following experimental results (1) to (2). That is, (1) it shows a single peak in high performance liquid chromatography based on a reverse phase system and a molecular sieve, and the activity peak also coincides with this. (2) Give a single band on 5DS-PAGE. (2) It is separated into three components with different isoelectric points by isoelectric focusing, but each component is a single component that exhibits CSA. Furthermore, if the terminal sialic acid is removed enzymatically or chemically, a single band will be obtained in isoelectric focusing. ■In the amino acid sequence analysis up to the N-terminal 21 residues, only one type of PTH amino acid appears at each step. ■ Previously reported effective C for humans
Compared to the purity of SF, a value about 10 times higher in specific activity is obtained.

CSF in such a purified form has never been seen before, and the CSF of the present invention is a CSF produced using genetic engineering technology.
In addition to being extremely suitable as a material for establishing mass production technology for SF, it can also be used as a reagent for clinical testing or research. Further, the CSF of the present invention has the effect of promoting proliferation of bone marrow cells during bone marrow transplantation, promoting recovery of bone marrow tissue after exposure to radiation, promoting recovery of white blood cell level after use of anticancer drugs, or promoting the differentiation and proliferation of bone marrow cells into neutrophils. Therefore, it is expected to be effective as a therapeutic agent for severe infections that cannot be treated with antibiotics.

[Brief explanation of drawings]

FIG. 1 shows the results of 5DS-PAGE of the CSF of the present invention. In the figure, * is the CSF of the present invention. FIG. 2 shows the results of isoelectric focusing of the CSF of the present invention. Figure 3 is the UV absorption spectrum of the CSF of the present invention. Figure 1 is a fir (J) Procedural amendment (method) July 3, 1985 Michibu Uga, Commissioner of the Patent Office 1, Indication of the case Showa 1981 Patent Application No. 77579 2, Title of invention: New CSF and its acquisition method 3, Relationship with the amendment case Patent applicant: 5-5-1-4 Ukima, Kita-ku, Tokyo Date of amendment order: 1988 June 3, 2016 (Shipping date: June 17, 1986) (Note) Address and contact information for sending documents 5, Full text of specification subject to amendment 6, Contents of amendment

Claims (1)

[Claims] (1) CSF having the following physical and chemical properties. i) Molecular weight: 19000 ± 1000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ii) Isoelectric point: pI=5.5±0.1, pI=5.8±
0.1, pI=6. It has at least one of three isoelectric points of 1±0.1. iii) Ultraviolet absorption: maximum absorption at 280 nm, 2
It has a minimum value at 50 nm. iv) The amino acid sequence from the N-terminus to the 21st residue is as follows. [There is an amino acid sequence] (2) The CSF according to claim 1, which has an isoelectric point pI of 5.5±0.1. (3) The CSF according to claim 1, which has an isoelectric point pI of 5.8±0.1. (4) The CSF according to claim 1, which has an isoelectric point pI of 6.1±0.1. (5) A cell line capable of producing human CSF is cultured in a serum-free culture medium, and the culture supernatant is subjected to the following treatments (1) to (3), and as necessary (
4) A method for obtaining CSF having the following physicochemical properties by subjecting it to the treatment of (5). (1) The culture supernatant is subjected to gel filtration using a gel with an effective fractionation range of 5,000 to 70,000 daltons, and a fraction having neutrophil-dominated colony formation promoting activity (hereinafter referred to as "CSA") is collected. . (2) The fraction from (1) is adsorbed onto a reversed-phase high performance liquid chromatography carrier, and eluted with a concentration gradient of a mixture of water and an organic solvent to collect a fraction having CSA dominated by neutrophils. (3) The fractions from (2) are subjected to high-performance molecular sieve chromatography to collect fractions containing CSA dominated by neutrophils. (4) The fractions in (3) are subjected to isoelectric focusing, and the fractions having CSA dominated by neutrophils are collected. (5) After performing a sialic acid removal operation on the fraction in (3), a fraction having CSA dominated by neutrophils is collected. "Physical and chemical properties" i) Molecular weight: 19,000±1,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ii) Isoelectric point: pI=5.5±0.1, pI=5.8±
0.1, pI=6.1 ±0.1. iii) Ultraviolet absorption: maximum absorption at 280 nm, 2
It has a minimum value at 50 nm. iv) The amino acid sequence from the N-terminus to the 21st residue is as follows. [There is an amino acid sequence] (6) The method according to claim 5, wherein the cell line capable of producing human CSF is CHU-1.