JPH06102035B2 - Production method of erythropoietin - Google Patents

Description

TECHNICAL FIELD The present invention relates to the production of an animal cell capable of producing erythropoietin (human erythropoietin), which is a hematopoietic factor, that is, an erythropoiesis-promoting factor, and culturing the animal cell to produce erythropoietin. On how to produce.

BACKGROUND ART Erythropoietin is an erythropoiesis-promoting factor that acts on erythroid progenitor cells (CFU-E) present in bone marrow to promote differentiation into erythroid cells. Human erythropoietin has the following physical properties. It is reported as follows.

Human erythropoietin is a glycoprotein having a molecular weight of 35,000 and is described in [Yanagawa S. et al. “J. Biol.
m. "(Journal of Biological Chemistry), 259 , 2707-2710 (1984)], the peptide portion of which is a single-chain polypeptide consisting of 166 amino acids [Jacob K. et al." Nature "(Nature). , 313 , 806-8
10 (1985)].

The structures of human erythropoietin cDNA and genomic DNA have also been clarified as seen in the report of Jacob K. et al.

Regarding the clinical efficacy of erythropoietin, it has been confirmed that erythropoietin has an effect of enhancing erythropoiesis based on animal experiments using a purified sample prepared from urine of anemia patients [Masunaga H. et al. .``Acta Hematal
Jpn in press ".

Therefore, erythropoietin is used as a clinically applicable drug for the treatment of anemia in patients with renal diseases, prevention of anemia in hemodialysis patients after nephrectomy, and promotion of recovery by enhancing red blood cell production in postoperative patients. To be

Although erythropoietin is expected to be clinically applied to the treatment of anemia as described above, it is difficult to supply a large amount of high-purity erythropoietin as a drug. It is the current situation. That is, since erythropoietin is contained in the urine of patients with aplastic anemia, conventionally, what was isolated and collected from the urine and purified was only used for the test study.

In view of such a situation, the present inventors, by using an adsorbent bound to a monoclonal anti-erythropoietin antibody obtained from a hybridoma cell fusion of spleen cells and myeloma cells of an experimental animal recently immunized with erythropoietin, A method for producing pure erythropoietin from urine from anemia with high yield was developed (JP-A-60-4161).
No. 4).

However, since the supply of the urine as a raw material by the above method is limited, it is difficult to produce erythropoietin in a large amount and constantly supply it as a medicine.

Therefore, it is considered necessary to establish a technique using a genetic engineering technique to make erythropoietin a medicine for treating anemia.

Problems to be Solved by the Invention The present invention has been made in view of the above-mentioned problems in erythropoietin production, in which erythropoietin-producing cells are produced by using a genetic engineering method, and the cells are cultured. It is an object of the present invention to provide a method for producing erythropoietin.

That is, the present invention enables the production of erythropoietin by a large-scale production method by eliminating the restriction on the raw material, which was a problem of the conventional method for producing erythropoietin from urine of anemia patient as a raw material.

The present inventor succeeded in producing a cell which constantly produces erythropoietin by introducing the erythropoietin gene into a mouse L929 cell via a specially produced vector, and completed the present invention. The present invention will be described in detail below.

Structure of the invention The feature of the method for producing erythropoietin-producing cells according to the present invention is to produce the erythropoietin transduction vector pSVhEPX by inserting the erythropoietin gene downstream of the SV40 early gene promoter of the mammalian vector shuttle vector pKSV-10. On the other hand, the selection marker Tn-derived aminoglycoside 3'-phosphotransferase gene (neo gene) was added to the shuttle vector pKSV-10 SV40.
Selectable marker ne by inserting downstream of the early gene
o The ^r gene transfer vector pKSVNeo was prepared, and the resulting erythropoietin transduction vector pSVhEPX and the selectable marker neo ^r gene transfer vector pKSVNeo were mixed and introduced into mouse L929 cells by the DNA transfer method using calcium phosphate. The purpose of this is to culture the transformed cells thus obtained to produce erythropoietin.

That is, the present invention, the erythropoietin gene transfer vector and the selectable marker neo ^r gene transfer vector were respectively prepared by the transfection method of both vectors obtained, by introducing the erythropoietin gene into L-929 cells to produce erythropoietin-producing cells. It is obtained and cultured to produce erythropoietin.

Means for Solving the Problems In the present invention, first, an erythropoietin transduction vector and a selection marker neo ^r gene transfection vector are prepared according to the following procedures.

Construction of erythropoietin transduction vector: The entire erythropoietin genomic gene obtained from the human genomic DNA library was transformed with the restriction enzyme Ec of the plasmid PUC8.
Plasmid phEP 14 inserted at the cleavage site with oRI and SmaI
25 using the plasmid phEP 1425 with the restriction enzymes BglII and
The erythropoietin gene is isolated by cutting with BamHI, and the erythropoietin gene is inserted into the cleavage site of the shuttle vector pKSV-10 for mammalian cells by the restriction enzyme BglII, and then the early gene promoter of SV40 of the shuttle vector pKSV-10. An erythropoietin transduction vector is obtained by selecting a vector in which the erythropoietin gene is correctly inserted in the downstream thereof by restriction enzyme map analysis. The expression of the erythropoietin gene in animal cells of this vector can be confirmed by transient expression in COS cells.

Creating the gene transfer vector of the selectable marker neo ^r: after cutting the plasmid pNE0 containing a selectable marker neo ^r gene with restriction enzyme HindIII, with Klenow enzyme (DNA polymerase I) 5 'to repair the projections, then connect the BamHI linker Then, the neo gene (1946
bp) fragment was inserted into the cleavage site of shuttle vector pKSV-10 by BglII to obtain SV of the shuttle vector pKSV-10.
A selection marker neo ^r gene transfer vector is obtained by selecting a vector in which the neo gene is correctly inserted downstream of 40 early gene promoters by restriction map analysis.

The expression of the neo gene in the animal cells of this vector was carried out by transducing the neo gene into L929 cells using G418.
This can be confirmed by the generation of resistant cells.

Introduction of erythropoietin gene into L929 cells: In the present invention, a vector obtained by mixing the erythropoietin transduction vector and the selection marker neo ^r gene introduction vector prepared as described above is then used in a DNA transfer method using calcium phosphate (cotransfection method). The erythropoietin gene is introduced into mouse L929 cells by the excision method). The above vector is preferably mixed with the erythropoietin transduction vector and the selection marker neo gene transfection vector at a ratio of 10: 1.

Generation of erythropoietin-producing cells: L9 into which the erythropoietin gene was introduced as described above
Twenty-nine cells were diluted about 6-fold 3 days after the above-mentioned transfer, inoculated into the medium and cultured, and the day after the inoculation, G418 was added to the culture medium and cultured for 2 weeks. After selecting G418 resistant cells (selective marker transduced cells), cells releasing erythropoietin in the culture medium were selected. Then, after cloning the cells expressing the erythropoietin gene by the limiting dilution method with respect to the selected cells,
A cell line L-92 which constantly produces erythropoietin by selecting a cell line with high expression and subculturing for 1 month.
9-SVhEP is established.

In addition, the erythropoietin produced by the above cell line was obtained by the radioimmunoassay method and mouse fetal liver cells. It can be confirmed by the in vitro bioassay method. Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 Preparation of erythropoietin transduction vector (pSVhEPX) 58 μl of TE-buffer (10 mM Tris-HCl, 1 mM EDTA pH7.
4) plasmid phEP 1425 (plasmid pUC 8)
Erythropoietin genomic gene inserted into the EcoRI and SmaI cleavage sites of 5 μg of BglII reaction buffer (50 mM Tris-HCl, 35 mM MgCl)
₂ , 500 mM NaCl, 35 mM mercaptoethanol) 20 μl, and then 10 units each of the restriction enzymes BglII and BamHI,
After reacting at 37 ° C for 2 hours, 3.5% polyacrylamide gel electrophoresis was performed to cut out the gel site corresponding to the 2.6 kb BglII-BamHI fragment (erythropoietin gene).
After finely crushing the gel, add 0.5 ml of elution buffer (0.5 M ammonium acetate, 1 mM EDTA, 0.1% SDS pH8.0),
DNA was extracted by incubating overnight at 37 ° C. DNA
Collects the upper aqueous layer by centrifugation, collects the upper aqueous layer, removes the phenol contained in the aqueous layer by one extraction with phenol, and three times with ether, and then adds 2 volumes of ethanol to remove the DNA fragment. Allowed to settle. Collect DNA fragments (precipitate) by centrifugation, dry the DNA, and then
It was dissolved in 1 sterilized water to obtain an erythropoietin gene solution.

On the other hand, 2 μg of shuttle vector pKSV-10 (4 μl of TE-
(Dissolved in buffer) 5 times concentration of BglII reaction buffer and BgLII
10 units were added, and the mixture was reacted at 37 ° C. for 1 hour to open the ring. The reaction solution was subjected to 1 time of phenol extraction, 3 times of ether extraction, and 1 time of ethanol precipitation to dry the DNA.
Dissolve in 10 μl of TE buffer and 10 μl of 5 times concentration CIP reaction buffer (250 mM Tris-HCl, 5 mM MgCl ₂ , 0.5 mM ZnCl ₂ , 5 mM
spermidiene pH9.0), 28 μl sterile water, 24 units CIP
(Calf intestinal alkaline phosphatase) was added, and 37
The reaction was carried out at 30 ° C for 30 minutes. After that, 24 units of CIP was added, and the mixture was reacted at 37 ° C for 30 minutes, followed by two phenol extractions, three ether extractions, and one ethanol precipitation treatment.
DNA is dried, dissolved in 10 μl of sterilized water and BglII cleaved CIP
This was a treated pKSV-10 solution.

100ng (nanogram 10 ^-9 g) of BglII cleaved CIP treated pKSV-1
0 (dissolved in 2 μl water), 100 ng erythropoietin gene (dissolved in 8 μl water), 1 μl 10 times concentration ligation
Reaction buffer (660 mM Tris-HCl, 66 mM MgCl ₂ , 100 mM DT
T, pH 7.6), 1 μl of 9 mM ATP and 1.5 μl of T4 DNA Iig
Add ase (4.2 units) (final DNA end concentration 10.9 pmol / ml)
The reaction was performed overnight at 4 ° C. After the reaction solution was subjected to 2 times of phenol extraction, 3 times of ether extraction and 1 time of ethanol precipitation, the DNA was dried and dissolved in 20 μl of TE-buffer to give a DNA solution for transformation. Using 10 μl of the DNA solution, 200 μl of E. coli DH-1 competent (competen
t) The cells were transformed. The transformation frequency of the DH-1 competent cells used was 1.0 × 10 ⁶ cells / μg of pBR322 DNA. By the above operation, 46 ampicillin-resistant transformants were obtained. Of these, 16 strains were subjected to restriction enzyme digestion map analysis of plasmids, and 3 strains having the desired erythropoietin transduction vector were selected. further,
Using one of these strains, a plasmid was prepared according to a conventional method to prepare an erythropoietin transduction vector (pSVhEPX).
The expression of the erythropoietin gene in the animal cells of this vector was confirmed by performing transient expression using COS cells.

Selection marker neo ^r Gene transfer vector preparation 2.5 μg of plasmid pNE dissolved in 10 μl of TE-buffer
0 to 10 times Hind III reaction buffer (100 mM Tris
-HCl, 100 mM MgCl ₂ , 500 mM NaCl, 10 mM DTT, pH 7.5) 1
After adding 0 μl and 76 μl of sterilized water, 50 units of Hind III was added and reacted at 37 ° C. for 2 hours, followed by phenol extraction once.
3 times ether extraction, ethanol precipitation 1 time treatment,
After drying the DNA, it was dissolved in 50 μl of sterile water. This solution was mixed with 10 times Klenow reaction buffer (500 mM Tris-HCl, 1
00 mM MgSO ₄ , 1 mM DTT, 500 μg / ml BSA), 25 μl of 2 mM d
NTP (dATP, dGTP, dCTP, dTTP mixed solution), sterile water 113μ
l and 10 units of Klenow enzyme (DNA polymerase I large
fragment) was added and the reaction was carried out at 22 ° C. for 30 minutes to repair the 5′-end projection site. DNA extracted once, extracted three times with ether, and once extracted with ethanol.
Was dried and dissolved in 10 μl of sterile water. Next, 3 μg
BamHI linker [d (pCGGATCCG) dissolved in 1 μl of water), 10 μl concentration of ligation reaction buffer 3 μl, sterile water 16
Add μl and 4 μl of T4-DNA ligase (11.2 units),
The reaction was carried out at 22 ° C. for 6 hours. After treatment with 1 time of phenol extraction, 3 times of ether extraction, and 1 time of ethanol precipitation, the DNA is dried and dissolved in 80 μl of sterilized water.
Add 20 μl of reaction buffer and 50 units of BamHI and mix at 37 ℃.
The reaction of time went. DNA fragments are separated by subjecting this reaction solution to 3.5% polyacrylamide gel electrophoresis.
Cut out the part of the gel corresponding to the 496 bp neo gene,
After finely crushing the gel, add 400 μl of elution buffer,
Extraction was performed overnight at 7 ° C. The acrylamide gel and the aqueous layer were separated by centrifugation, and the aqueous layer was purified by performing one extraction with phenol, three extractions with ether, and one precipitation with ethanol.

50 ng neo gene (dissolved in 19 μl water) and 110 μg Bg
lII-cleaved CIP-treated pKSV-10 (dissolved in 4 µl of water) was added with 10 µl concentration of ligation reaction buffer (3 µl), 9 mM ATP solution (1.2 µl) and 2 µl of T4-DNA ligase (5.6 units), and reacted at 4 ° C overnight. I went.

After the reaction solution was subjected to 2 times of phenol extraction, 3 times of ether extraction and 1 time of ethanol precipitation, the DNA was dried and dissolved in 20 μl of TE-buffer to give a DNA solution for transformation. Using 5 μl of the solution, 200 μl of E. coli DH-
Transformed into 1 competent cells.

(Transformation frequency 3 × 10 ⁶ cells / μg pBR322). By the above procedure, 21 ampicillin-kanamycin resistant transformants were obtained. Among them, 11 strains were subjected to restriction enzyme digestion map analysis of plasmids, and 4 strains having the selectable marker neo ^r gene transfer vector in which the neo gene was inserted in the correct direction were selected. Furthermore, one of them was used to prepare a plasmid according to a conventional method to prepare a selection marker neo ^r gene transfer vector (pKSV Neo). The expression of the neo gene in the animal cells of this vector was confirmed by transducing the neo gene into L-929 cells and generating G-418 resistant cells.

Introduction of erythropoietin gene into L-929 cells 2 × 10 ⁶ mouse L-929 cells were seeded in a 25 cm ² T-flask, and on the next day, DNA transfection was carried out by the calcium phosphate method. The transfer excision was performed by the cotransfection method using the erythropoietin transduction vector pSVhEPX and the selection marker neo ^r transduction vector pSV Neo. That is, 2.5 μg of cyclic p
SV Neo and 22.5 μg of cyclic pSVhEPX were dissolved in 22 μl of TE-buffer (pH 7.5) and 500 μl of isotonic HEPESSaline
(137 mM NaCl, 5 mM KCl, 0.7 mM disodium phosphate,
After adding 6 mM glucose and 21 mM HEPES), 33.3 μl
2M Calcium chloride solution of 2 is slowly added with stirring and then 20
The mixture was allowed to stand at room temperature for a minute to prepare a DNA-calcium phosphate solution.

The previously prepared L-929 cell culture medium [Eagle's MEM (Ear
le's salt) + 10% FCS], add 500 μl of DNA-calcium phosphate solution and leave at room temperature for 20 minutes, then 4 ml 150 μl
Add the culture solution containing M chloroquine, incubate for 4 hours in a CO ₂ incubator, and wash with the culture solution. Isotonic HEPES Saline containing 15% glycerol
After adding 2 ml and incubating at room temperature for 3 minutes and washing with the culture solution, 4 ml of the culture solution was newly added and cultured. 2 days later, 80c
^Two m ² T-flasks were reseeded (1/6 split).
The next day, the medium was exchanged with a medium containing 400 μg / ml G418, and the medium was exchanged at appropriate times to select G418-resistant cells.
Seven days later, G418 resistant strains were obtained. Three days later, a limiting dilution method was performed in which 50 cells were seeded in one 96-well microwell, the culture medium containing 400 μg / ml of G418 was replaced at appropriate times, and 16 days later, colonies were found in 45 wells. As a result of examining the erythropoietin activity contained in the culture supernatant in these 45 wells by the radioimmunoassay method, the activity was found in 38 wells. Among them, cells of 6 wells showing a high amount of erythropoietin were selected, grown in the presence of G418, and again subjected to limiting dilution in the same manner to establish a cell line that constantly produces erythropoietin, and L-929 SVhEP 001 I named it. SVhEP 001
Were cultured to 10 ⁶ / m1, and the erythropoietin production was examined by radioimmunoassay method and in vitro bioassay method for forming CFU-E using mouse fetal hepatocytes. Shown, 100 units / 10 ⁶ cells
It was / day.

Example 2 This example shows a mode of introduction of the erythropoietin gene into L-929 using a chain-like transduction vector.

The erythropoietin transduction vector pSVhEPX and the selection marker neo ^r gene transfection vector used were those prepared in Example 1.

Preparation of chain-like transduction vector 2.5 μg of pKSV Neo (dissolved in 2.1 μl of TE buffer) and 2
2.5 μg of pSVhEPX (dissolved in 19.8 μl of TE buffer) at 2 times concentration of EcoRI reaction buffer (100 mM Tris-HCl, 14 mM MgC)
l ₂ , 200 mM NaCl, 14 mM 2-mercaptoethanol, 0.02
% BSA) 110 μl, sterilized water 68 μl and EcoRI 5 μl (100 units) were added and reacted at 37 ° C. for 2 hours, followed by phenol extraction once, ether extraction three times, ethanol precipitation one time, and DNA was dried. Then, it was dissolved in 22 μl of TE-buffer to give a DNA solution.

Introduction of erythropoietin gene into L-929 cells A procedure similar to that described in Example 1 was performed. That is,
2 × 10 ⁶ mouse L-929 cells were seeded in a 25 cm ² T-flask on the day before DNA transfer excision, and transferred by the L-929 cell calcium helate method using the above DNA solution. As a result of selecting G418 resistant cells, resistant strains
10 strains were obtained. Using this strain, cells were cloned using the limiting dilution method twice to establish a cell line that constantly produces erythropoietin, and was named L-929 SVhEP 002. The erythropoietin production of this strain was examined by the radioimmunoassay method and the CFU-E forming in vitro bioassay method using mouse fetal hepatocytes.
It was 100 units / 10 ⁶ cell / day.

Example 3 Production of erythropoietin in serum-free medium L-929 SVEP 001 1.5 × 10 ⁷ c prepared in Example 1
ell was seeded in a 600 cm ² self-acting single tray (Nunc), and 200 ml of serum-containing medium [Eagle's ME
After culturing in M (Earle's salt) + 10% FCS for 1 day,
Replace with serum-free medium and culture for 3 days, then replace with serum-free medium and culture for 3 days.
got l.

The erythropoietin activity contained in this medium was determined by the radioimmunoassay method to be 12 units / ml.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ryuzo Sasaki 14 Tanaka Kogenmachi, Sakyo-ku, Kyoto City, Kyoto Prefecture (56) References International Publication 85/2610 (WO, A) Nature, 313, 806-810 (1985) Pharmacia Biotechnology catalog (Japanese version) "Molecular Biologicals: Chemicals and Egui equipment for Molecular biology 1985" P. 61 Journal of Molecular Biology, 150 (1), 1-14 (1981) Cell, 41 (2), 489-496 (1985)

Claims (5)

[Claims] 1. An erythropoietin transduction vector pSVhEPX is prepared by inserting the erythropoietin gene into the mammalian vector shuttle vector pKSV-10 downstream of the SV40 early gene promoter.
The aminoglycoside 3'-phosphotransferase gene (neo gene) derived from S was added to the shuttle vector pKSV-10.
A selection marker neo ^r gene transfer vector pKSVNeo was prepared by inserting it downstream of the V40 early gene, and the resulting erythropoietin transduction vector pSVhEPX and a selection marker neo ^r gene transfer vector pKSVNeo were mixed in a vector using calcium phosphate. A method for producing erythropoietin, which comprises introducing erythropoietin-producing cells by introducing the cells into mouse L929 cells by a transfection method, culturing the cells to produce erythropoietin, and obtaining erythropoietin. 2. The erythropoietin gene is the whole erythropoietin genomic gene, which is the restriction enzyme EcoR of the plasmid pUC8.
Plasmid phEP142 inserted at the cleavage site by I and SmaI
The production method according to claim (1), which is obtained by cleaving 5 with the restriction enzymes BglII and BamHI and separating. 3. An erythropoietin transduction vector pSVh.
For the production of EPX, after inserting the erythropoietin gene into the cleavage site of the shuttle vector pSV-10 with the restriction enzyme BglII, the erythropoietin gene was inserted correctly downstream of the SV40 early gene promoter of the shuttle vector pKSV10 into a restriction enzyme map. The production method according to claim (1), which is selected by analysis. 4. A selectable marker neo ^r gene transfer vector pKSV.
Neo was prepared by cutting the plasmid pNE0 containing the selectable marker neo ^r gene with the restriction enzyme HindIII, repairing the 5'projection site with Klenow enzyme, connecting the BamHI linker, and then cutting with the restriction enzyme BemHI. Gene fragment,
Inserted into the cleavage site of the shuttle vector pKSV-10 by the restriction enzyme BglII, the neo gene correctly inserted downstream of the SV40 early gene promoter of the shuttle vector pKSV-10 is selected by restriction enzyme map analysis. The production method according to claim (1). 5. Erythropoietin transduction vector pSVh
EPX and selectable marker neo ^r gene transfer vector pKSVNeo 1
The production method according to claim (1), wherein mixing is performed at a ratio of 0: 1.