JPH029388A - Production of physiologically active protein - Google Patents

Abstract

PURPOSE:To mass produce a physiologically active protein by transforming a cell of Chinese hamster overlay dihydrofolate reductase deficient strain (CHO) with a specific plasmid to give a cell and cultivating the cell. CONSTITUTION:A gene to code physiologically active protein is cloned to give a gene (A) of human differentiation-inducing factor BUF-3, etc. Then a plasmid is transduced with the gene A and dihydrofolate reductase (dhfr) gene (B) to give a plasmid (C) consisting of a gene to code pSD(x)/BUF-3 SV40 initial promoter BUF-3-SV40 splicing signal-SV40 initial promoter dfhr gene-SV40 splicing signal, etc. Then cell of (CHO) is transformed with the component C to give a cell, which is subjected to floating spinner culture at pH about 7 in the presence of desired dissolved oxygen to give a culture mixture (D). Then the component D is purified by salting out with ammonium sulfate, dialysis, chromatography, etc., to produce a physiologically active protein.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a Chinese hamster opalinohyde stomatofolate reductase-deficient strain (hereinafter abbreviated as CHOdhfr-).
The present invention relates to a method for efficiently producing large quantities of physiologically active proteins by using cells as production hosts and carrying out suspension agitation culture.

Prior Art Conventionally, CIOdhfr"" cells have been widely used as protein production hosts, but a single cell has been used for its growth (
Proliferation) Since these cells are adherent cells that require a K support, it was difficult to culture the cells in the dark at tK or to obtain a large amount of bioactive protein/4' protein. A method has been developed to grow cells on microbeads and produce substances in a floating agitation system, but! It was not put into practical use due to problems such as cells not growing uniformly on the microbeads. In addition, a high-density culture device for growing cells on a fibrous support has been developed, but it has not been applied to mass production of substances due to limitations in the amount of culture solution.

CHOdhfr cells transformed with a plasmid containing the target gene (containing the DHFR gene) are cultured in large quantities,
The biggest problem when producing large quantities of physiologically active proteins is the need for growth (proliferation) K proliferation supports. Therefore, an object of the present invention is to provide a method for producing a target physiologically active substance in large quantities by culturing transformed cells in a floating agitation system in large quantities without using a growth support.

As a result of extensive studies to solve the above problems, the present inventors have discovered that CHOdhf, which requires a growth (proliferation) support,
The inventors have completed the present invention by discovering that 〇HOdhfr- cells transformed with r''' cells and a plasmid containing a target gene can be grown under suspension agitation culture.

That is, 1 the present invention involves transforming CHOdbfr- cells with a plasmid having a gene encoding a physiologically active protein and a dhfr gene in an expressible state, suspending the obtained cells in a suspension culture, and adding the culture solution This is a method for producing a physiologically active protein, which is characterized by producing the desired physiologically active protein in the protein and obtaining the desired physiologically active protein and matrix.

The present invention will be explained in more detail.

First, CHOdhfr- cells that can be cultured in suspension with agitation (in this case, these cells have been transformed with a plasmid that has a gene encoding a physiologically active protein and a dhfr gene in an expressible state);

Gene and dhfr encoding physiologically active protein
Any cell that has not yet been transformed with a plasmid carrying the gene in an expressible state may be used.

) can be obtained as follows.

After growing cells on the support surface, C1-M containing nucleic acid
EM medium (GIBCO, catalog 4410-1900
) (contains 10% bovine serum) K as low density as possible (1-4
Suspend the cells to 10' cells/ml),
Perform suspension 1 stirring incubation.

Although there are no particular restrictions on the conditions for carrying out suspension agitation culture, it is preferable not to exceed the initial cell concentration Fi4XIo'/,d as much as possible.

Also, at this time, it is desirable to accurately control the temperature of the culture solution, but as long as a completely sealed state can be maintained, the temperature of 7. OK, there is no problem as long as you match it correctly.

By repeating this operation, cells suitable for floating agitation culture can be obtained. Further, the medium may be any medium containing a nucleic acid concentration equivalent to or higher than that of the α-MEM medium described above. However, since each cell requires proline, the medium must contain proline.

Initially, cell growth is poor and the maximum cell density is low, but by repeated culturing, cells with good growth (proliferation) and a high maximum cell density can be obtained.

The cells obtained in this manner can be used in conventionally known suspension culture strains 1, such as human acute monocytic leukemia cells (THP-
1, Int, J, Canc@r 26: 171-
176 (1980) χ H of human acute promyelocytic leukemia cells
L-60 (ATCCCCCL240), U-937 (ATCCCCRL 1593) of human acute monocytic leukemia cells
), human chronic myeloid leukemia cells -562 (AT
CCCCL 243 ), human acute myeloid leukemia cell KG-1 (ATCCCCCL 246 ), and human monocytic leukemia cell J-1) 1 (ATCCCCCL 24 ).

The obtained CHOdhfr cells suitable for suspension agitation culture are preferably cultured in a spinner flask equipped with a stirring base.

If it is unavoidable, use a flask container for microorganisms (
It is also possible to culture in a datesch flask for tissue cells (as long as it is not coated to make it easier for cells to adhere).

Further, methods other than those described above may also be used. Even if CHOdhfr cells suitable for suspension agitation culture are established in this way, if they do not contain a gene encoding the desired physiologically active protein or a plasmid containing the dhfr gene, perform the operations shown below. By doing so, the desired physiologically active protein can be produced.

In other words, cells into which a plasmid containing the dhfr gene and a gene encoding the target physiologically active protein are to be introduced, ie, CHOdbfr- cells, are separated in advance as cells suitable for suspension agitation culture). : Transformation is performed with a plasmid containing the dhfr gene and a gene encoding the physiologically active substance of interest, and then a transformed strain producing the physiologically active protein of interest is isolated.

The thus obtained transformed strain was grown in a medium containing mentrexate (hereinafter abbreviated as MTX), and rJt
Isolate sexually transformed cells. In such cells, the dhfr gene is amplified by becoming MTX resistant. Therefore, at this time, the gene encoding the biologically active protein of interest is also amplified, so that the biologically active protein of interest is produced in large quantities.

Cells that produce a large amount of this objective physiologically active protein may be used thereafter.

As mentioned again, of course, cells suitable for suspension agitation culture may be established after first transforming CHOdhfr- cells with a plasmid containing the dhfr gene and a gene encoding the physiologically active protein of interest.

That is, cells suitable for suspension agitation culture may be established after transformation, or cells suitable for suspension agitation may be established and then transformed.

In principle, the plasmid used in the present invention must have the dhfr gene and a gene encoding a physiologically active protein, but preferably a promoter that can be transcribed in eukaryotic cells and an SV40 splicing signal (in addition, in the present invention, The SV40 splicing signal refers to a signal consisting of both the SV40 splicing signal and the polyA addition signal.

) is preferable.

The promoter is usually the SV40 early promoter (
5V4Q early), bovine papilloma virus (abbreviated as BPV), etc., are used, and a gene encoding a biologically active protein of interest is inserted between the promoter and the splicing signal.

It is preferable that the plasmid contains a gene encoding the physiologically active protein of interest and the dhfr gene in the same plasmid.

However, even if the 74 lasmid does not have the Kdhfr gene, it may have the dhfr gene on another plasmid.

At this time, both are combined to perform transformation. In addition, enhancer sequences that increase transcriptional activity in the promoter (e.g. SV40 'yobp repeats,
Retrovirus Long Terminal Libby) (LTR)
It is also preferable to have an ennocer sequence present in the protein.

Now, a gene encoding a physiologically active protein of interest is introduced downstream of the transcription unit (promoter) of such a plasmid.

Considering the above, the preferred plasmid form is the SV40 early promoter (SV40 early promoter).
).

The gene encoding the target physiologically active protein, SV40 splicing signal (SV40 spHclng &
polyA).

A plasmid containing a gene such as dhfr is preferable.

In addition, SV40 splicing signals are R, C, Mul
Reported by llgan and P, B@rg K (Proc.
, Natl, Aead, Scl.

USA, vol 78.2072-7076 (19
81) K is listed.

Moreover, it is available.

Now, specific examples of preferred plasmids are as follows: plasmid ΔSV40 early promoter - gene encoding the target physiologically active protein - SV40 splicing signal - 8v4o early f-mouth motor - dhf r gene - S
V40 splicing signal or plasmid ΔSV4
0 early promoter - dhfr gene - SV40 splicing signal - SV40 early promoter - gene encoding the target physiologically active protein - SV40 splicing signal.

Now, in the present invention, the target physiologically active protein is human and mouse interleukin. Human and mouse interleukin 2, human and mouse interleukin 3. Human and mouse α-interferon, human and mouse β-interferon, human and mouse r-inter7epn, erythropoietin, human differentiation factor BUF-3, human B#1 cell differentiation factor BSF-2, etc. Good too.

Among these physiologically active proteins, human differentiation factor BU
Particularly preferred are F-3 (BUF-3), human intera-2, and human B cell differentiation factor (BSF-2), hereinafter referred to as IL-2, BUF-3, BSF-2.
Unless otherwise specified, human IL-2, human BUF-3, and human BSF-2 are indicated.

BSF-2 is also called BCDF or IL-6, but in the present invention, the name BSF-2, which is most commonly used in Rt, is used in the present invention.

The transformation method used in the present invention involves co-precipitation of DNA with calcium phosphate.

F, L van dsr Eb, A, J: V
irology, 52.456 (1973)) or the DEAg-text run method (Stow, N.D.
atal: J, G@n Virol 33.4
47 (1976), microinjection method for introducing genes into the nucleus using a micromanipulator (Gra@mamah
nsA, +et ml M@thod ln Enzy
mol.

65, 816 (1980)), the lysome fusion method (Grsgoliadis*
G, at a1283, 814 (1980))
, the protograst method (Scbmffne
rm W, Proe, Natl.

Acad, Scl IJ, s, a 77, 2163
(1980)).

Incidentally, as a method for purifying the target physiologically active mennoplasma produced in the culture solution, a general method that has been used less frequently may be used.

The present invention will be explained below based on examples.

Example! About human differentiation factor BUF-3 BUF-3 is a protein differentiation factor produced by human acute monocytic leukemia TIP-1 cells.

BUF'-3 is a protein having differentiation-inducing activity that acts on mouse 7rent9 leukemia cells and induces differentiation of these cells into normal erythroblast cells.

The BUF-3 gene has been fJK cloned.

Its nucleotide sequence and amino acid sequence have also been determined (first
(see figure). Regarding BUF-3, JP-A-62-2340
It is described in detail in Publication No. 97.

Now, an animal cell expression vector pSDcx)/BUF-3 containing the gene encoding this BUF-3 was constructed as shown in FIG. Plasmid vector pSV
The mentrexate resistance gene (dhfr gene) was transferred from 2dhfr and pBR322 to the SV40 early promoter (
SV40 @arly ) and SV40 splicing signal (SV40 spHclng & polyA
) plasmid pBR322-
I got dhfr.

In addition, as shown in Figure 2, plasmid psV2-dhfr
After cutting with restriction enzyme It Hlnd ml Bgl II, one fragment was converted into T4 DNA/! After treatment with J merase to make blunt ends.

By agarose gel electrophoresis, 2-
After separating the di7 fragment, this f'L K XhoI
A linker was added.

This frangand was treated with restriction enzyme Xbol again and then treated with T4ONAI7 gauze to close the ring. this!
It was named lasmid psV(x).

Next, after treating the aforementioned Gerasumi)' pBR322-dhfr with restriction enzyme BamHI, a7. / By performing low flow electrophoresis, SV40% -1--dhfr
A fragment of the gene-SV40 splicing signal was isolated (Figure 2).

This SV40 early promoter-dhfr gene-SV
A BamHI fragment containing 40 splicing signals was introduced into the BamH1 site of plasmid pSV(3C) to construct plasmid PSD(x) (FIG. 2).

Next, the DNA of BUF-3 cloned by Kanade 1 Yuchuba was treated with T4 DNA4 lymase, and then an Xhol linker was ligated, and the fragment obtained by Xhol treatment was inserted into the XhoI site of plasmid pSD(x). (Figure 2).

In this way, plasmid p containing the BUF-3 gene
SD(x)/ntyp-3 was constructed.

That is, the expression vector pSD(x)/BUF-3 is
The gene encoding SV40 first M2 first born 20 motor UF-3 - 5V40 tin rising signal - SV40 early promoter - dhfr gene - has SV40 splicing signal.

In addition, in this Example 1, pSD(x)/BUF −
The 3 plasmids were previously transformed into a Chinese hamster opaline dihydrofolate reductase-deficient strain.

A method for isolating a transformed strain that produces BUF-3, which was then subjected to suspension agitation culture after death, will be described.

Plasmid p8Dcx)/BUF-310tti-+
Transfection was carried out into mouse CHO cells using the phosphorescent method. After the introduction, a predetermined selective medium containing 1O% calf serum (α part M medium CAT4 manufactured by GIBCO) was used.
Selection was carried out at 37°C in the presence of 5% CO2 (10-2000, without nucleic acids), and the medium was replaced every 3 days.

A cell population that grew after about 2 weeks was obtained (IF
O50125). Next, in order to increase the copy number of the introduced plasmid, resistant cells were obtained by culturing in a selective medium containing 0.1 μM of dhfr antagonist Mentorex. and sequentially increase the mentrexate concentration to 0.2μ
M, 0.4 μM, 1 μM, 2 μM, and 4 μM to obtain resistant colonies at various stages.

The amount of BUF-3 protein produced by transformed cells showing resistance at each concentration of mentrexate was measured by the amount of hemoglobin synthesized using Friend cells.

Transformed cells that were resistant to 4 μMa of mentrexate accumulated BUF-3 at a concentration of 2000 U/G over 3 days. This is 1 μ, 9 when converted to protein amount.
BUF-3 was produced at a concentration of /mj.

Next, transformed cells growing in an adherent state (4 μM mentrexate resistant 2000 U/1) Lt/3 da
ys ) cells more suitable for suspension (CHO-5USP
) (IFO50146) was separated as follows.

Cells resistant to 4 μM mentrexate were placed in a plastic flask F-75 for cell culture (A156502 manufactured by Nuwa).
301) 1) Selective medium containing 7 io% calf serum (
(containing 4 μM mentrexate), 1×10 6 cells were added, and cultured at 37° C. in the presence of 5% CO 2 . Collect the harvested cells after they have grown and place them in a 400D spinner flask containing 100-medium (GI) containing 10% serum.
BCO α-MEM medium CAT410-1900,
Nucleic acid added to the above selection medium. ). Next 4
The cells were suspended in a medium at a concentration of X10' cells/mjPc, and after death, cultured with agitation at 37°C. After culturing for 8 days.

Maximum m cell 'llj degree 8.8X1 o'4R/ml,
tft epoch M 192 fI or more (see Table 1). Then, by repeating the culture for another 8 days, 4
After a week, cells with a maximum cell density of 2.6×10 5 cells in 7 ml and a generation time of 87 hrg were obtained (see Table 1). By repeating the culture at an initial cell density of 1 x 10 cells/ml, we obtained a strain that grows well with a generation time of 48 hr and a maximum cell density of 7.8 x 10 cells/M, namely CHO.
-8USP (IFO-50146) was obtained (Table-
1).

Table-1 Cslls (4v+e*ks) 2.6 X
10587Cel1m (10weeks) 7.
8 X 10548 CHO-8 thus obtained
USP strain (IFO50146) has a stable BUF -3
8000 U/6 pieces of debris were accumulated in the medium. Na then CH
The O-5USP strain was cultured in the same manner for 20 cycles, but there was no change in the maximum cell density and BUF-3 accumulation after one generation, and the growth was stable.

BUF-3 production amount is shown (Figure 3).

Furthermore, this CHO-8USP strain (rpo 50146)
-1 is 7.0, dissolved oxygen is 0.05, 0.015
Atm K precisely controlled culture system has a generation time of 24hrs and a maximum cell density of 1.0X1.
It reached 06f hard/lnl iC.

Mass production of BUF-3 became easy by separating the cells passed through the suspension agitation culture thus obtained.

The isolation and purification of BUF-3 was described in JP-A No. 62-23049.
It was carried out according to Publication No. 7. That is, 100 m of the culture solution containing BUF-3 was salted out with 65% saturated ammonium sulfate at 5°C for 12 hours. The salted-out precipitate obtained by centrifugation was
M) Lis-hydrochloric acid buffer (PH7,8) 2.5ml 1K dissolved in dialysis tube (manufactured by Swiktrobore).

cyrw6. ooo-s, ooo) and was thoroughly dialyzed at 5C.

The mixture was stirred at 23° C. for 30 minutes. Next, this suspension was centrifuged, and the centrifuged supernatant was collected from room temperature of 23°C to -5°C.
It was left to stand for 3 hours or more in a cooled state. This is K91
This liquid caused two-phase separation.

At that time, BUF-3 selectively lowers the lower fraction (acetate) =
It was concentrated and purified to about 45% tolyl. This is 0.1%
It was diluted 2 times with TFA and purified by reverse phase high performance liquid chromatography. As a reversed phase column, Hi-A RP-304 (manufactured by Bio-Rad, C4, 300X, 5 μm) was used.

The solvents were 0.1% TFA, 80% acetonitrile, 0.1% TFA, and 1 part cloud vinegar, and eluted with a linear gradient program. Ta.

Next, this eluted nine BUF-3 fraction was again subjected to reverse phase high performance liquid chromatography. The column was Hi〆A RP-304 (Bio-Rad ff1c4゜3001
, 5 pm) was used.

Soluble Ktd, o, 13% hebutafluorobutyric acid (HFBA
) and 80% acetonitrile-0.13% butafluorobutyric acid, and elution was performed using a linear gradient gelogram.

Next, 8D8 polyacrylamide gel electrophoresis (
(concentration 15%) was carried out. As a result, a single band (silver staining method) was observed at 25 kd in the absence of mercaptoethanol and at 16 kd in the presence of mercaptoethanol, and no other protein bands were detected. The specific activity of the sample purified in this way is approximately 2×10 U.
/ It was a protein.

Furthermore, this 1 m product was subjected to reverse phase high performance liquid chromatography. At this time, the column used was eluted with MMC (1% acetonitrile).

As a result, a completely single peak was obtained, and BUF-3 could be completely isolated and purified.

As a precaution, the nine-BUF-3 purified temple was subjected to amino acid analysis, and it was confirmed that it had the same amino acid sequence as shown in Figure 1.

Example 2 Human interleukin-2 b, human interleukin-2 (IL-2) l'i,
It is a protein factor that promotes the proliferation of T cells.

The IL-2 gene was described by Nobokuchi et al. (Nature, 302.
305 (1983)) has already been cloned by K.

Moreover, as shown in FIG. 4, its nucleotide sequence has already been determined (Japanese Patent Application Laid-Open No. 140882/1982). Ko (7) IL
-2c DNA is contained in pcEIL-2 (
Figure 5). The following operations were performed as shown in FIG. This IL-2eDNA has several dozen bases of GC on both ends.
It has a tail. If these identical bases, G or C, line up, it is thought that it will interfere with the expression of cDNA.
(Takara Shuzo) treatment to separate 7 IL-2eDNA fragments, which were treated with Ba, an exonuclease.
131 (NIB) processing was performed to remove GC tails. And * T4 DNA polym@ras
* After making both ends blunt with (Takara Shuzo), pCEI
L-2, the fragment from which IL-2 cDNA was removed by 9Pstl treatment is also T4 DNApolym@r
blunt-ended KL with aa*, both with T4 DNllllas
- Combined with (Takara Shuzo). After introducing the exploded plasmid into the colon IH8101 strain, a plasmid was prepared from the resulting transformed strain.

Among the obtained plasmids, one with the GC tail removed and the expression efficiency increased was selected, and this plasmid was transformed into pCE.
It was named IL-2 (Bat 7).

The pIL-2e DNA fragment was isolated from pCE-2 (Bal 7 ) by restriction enzyme treatment with B (Takara Shuzo), and the fragments were made blunt-ended with T4 DNA polymrase.

Furthermore, T4 DNA llgaa treatment was performed with each linker (Takara Shuzo) of K Barr + H (, H4ndm), and then BamHl, Hlndll (Takara Shuzo)
processed. The 7 fragments thus obtained were then transformed into pSV2-dhfr (S, Subraman
l, at at, : Mo1. Cs1). Bi
ol.

1.854 (1981)) Hlnd m, Bgl
The larger fragment obtained by M treatment was combined and transformed into Escherichia coli DHI strain, from which the psV shown in Figure 5 was
2-obtain one with IL2. That is, psV2
-dhfr derived SV40 early glomotor (SV40
@early promoter) and SV40 Sugerai'/7 Gusigna k (SV40 spliting
& polyA) in which K IL-2 cDNA is inserted. next? knee (DpSV2-LL-2
Pvull the plasmid.

A BamHI linker was added to the fragment containing IL-2a DNA obtained by BamHI treatment, and llamH
After 1 treatment, it was inserted into the BamHl cleavage site of psV2 dhfr.

gramxmid pSD(1)/n, -zΔSV4o early glomotor-d h fr gene SV40 splicing signal-SV40 early promoter In,
Gene-SV40 splicing signal encoding gene-2 was obtained. This plasmid 1) SD(1)/IL-2
has mouse dhfr cDNA derived from psV2-dhfr upstream of IL-2 cDNA.

CHOdhfr-plasmid pSDI/IL-2
The cells were transfected with the calcium phosphate method, and transformed cells into which the pSDI/IL-2 plasmid had been introduced were isolated. Immediately after introduction, 22.9 U/TL
T's human! It was producing L-2. Next, MTX the cells
MTX-resistant cells were isolated by growing in a medium containing 1 μM.

1 pMMTX resistant transformed cells are 512 U/m
1 of human IL-2. Next, cells suitable for suspension agitation culture were isolated from these cells. 1 μM MTX
After growing the resistant transformed cells on the support.

A total of 400 rnt in nine spinner flasks equipped with a stirring base was suspended in a medium (medium containing the nucleic acid shown in Example 1) so that the initial cell density was 4 x 10 cells/MK.

Then, agitation culture was carried out at 37°C, and culture was continued until the cell density reached the maximum. By repeating this agitation culture for 8 cycles, the maximum cell density was 2.9 x 10548/Ill, and the generation time was 80 hours. By repeating 6 cycles, the maximum cell density is 6.8 x 105 cells/cell, generation time 3
Cells were separated by suspension agitation for 0 hours. The cells thus obtained, which were suitable for floating agitation, had the ability to stably produce human IL-2. Furthermore, as shown in Example 1, even if the obtained cells suitable for floating agitation are repeatedly passaged, the generation time will be shortened.

Maximum cell density and human IL-2 production ability were stably maintained.

Example 3 (1) BSF-2 is a factor that differentiates human mature B cells into antibody-producing cells, and its D
The NA sequence and amino acid sequence have also been determined (Japanese Patent Application Laid-open Nos. 63-42688 and 63-56291).

The present inventors have also discovered that BSF-2 is useful for the treatment of infectious diseases and cancer (Japanese Patent Application No. 62-2890
07).

Now, in order to express such a useful gene encoding 5F-2 in hamster CIO cells, plasmid pSD(x)/BSF-2 was constructed as follows.

1) First, pCDa was cut with the restriction enzyme Hpto1, and large NA fragments were collected by agarose gel electrophoresis (
Figure 7).

ij) Plasmid p carrying BSF-Z eDNA
BSF2-38 (T, Hlrano atml, N
ature 324e 73 (1986)) After cutting with restriction enzymes BamHI and B&%■, treating with DNA polymerase, BS was analyzed by agarose gel electrophoresis.
A fragment containing F-2 cDNA was recovered (Figure 7).

1ii) The two types of DNA fragments obtained in i) and 1)) were combined using total T4 DNA ligase (7th
figure).

The obtained recombinant DNA was then introduced into Escherichia coli strain H8101, and strains resistant to ampicillin were selected. Plasmid DNA was obtained from the resulting strain and subjected to a restriction enzyme 51C cleavage test to select bacteria that harbor pcD BSF-2.

IV) 77-DNAλBSF 2.5 (T, HIr
ano at alNature 324.73. (
(1986)) f EcoRi, and the smallest fragment containing BSF-Z eDNA was recovered by agarose gel electrophoresis (Fig. 7).

(2) Plasmid pSP 65 (manufactured by Amajam) was cut with the restriction enzyme HeoRI, and then ligated to the DNA fragment purified with I using T4 DNA ligase (Figure 7). The obtained recombinant DNA is used in the large intestine! ¥I (81
01 strain, and a strain having ampicillin resistance was selected. Plasmid DNA was obtained from the resulting strain, and pBSF'2-108 was obtained by performing a restriction enzyme cleavage test.
6 was selected.

vi) Plasmid pCDB obtained in 1ii) above
SF-2 was cut with restriction enzymes Xhol and Xba[, and small fragments were collected by agarose gel electrophoresis (No. 8
figure).

Cilasmid pBsF2-1086 obtained from vIO above
After cleaving with the restriction enzyme SmaI, an Xhol linker was ligated using T4 DNA ligase, and further cleavage with the restriction enzymes XhoI and Xbal was performed, and a fragment containing the latter half of BSF-2e DNA was recovered by agarose gel electrophoresis. (Figure 8).

vin) Plasmid PAD (restriction enzyme Xbol
This fragment and the fragment obtained in vl) and vi
i) T4 DNA with 3 of 9 fragments! They were bound using J gauze (Fig. 8). That is,! Lasmid pSD (
x) / BSF-2ΔSV40 early promoter d
hfr gene-SV40 splicing signal-SV
The gene encoding the 40 early 7'a-v-ter-BSF-2-SV40 splicing signal was constructed.

This constructed plasmid was introduced into Escherichia coli strain H8101, and a strain having ampicillin resistance was selected. Cilasmid DNA was prepared from the obtained strain, and a restriction enzyme cleavage test was performed to determine the plasmid pSD(x)/BS.
A strain carrying F-2 was selected.

(2) Plasmid pSD(x)/BSF-2 was introduced into No. Muster CHO cells according to Example IK by the VNumlin method, and a nucleic acid non-requiring strain was selected. The culture supernatant was measured by ELISA method, and it was found that 10
It showed a BSF-2 activity of 0 U/IILl.

(3) CHO cell line obtained in (2) at 1X10 M
When cultured in a medium containing mentrexate (MTX), BSF-2 activity in the culture supernatant increased to 200 U/d. Furthermore, when cultured in a medium containing I x 10M MTX, the BSF-2 activity was 800U/
1) It rose to 7.

Cells with BSF-2 activity of 800 U/yil were obtained, but since one cell was an adherent cell, cells suitable for floating agitation culture were separated.

Cells with BSF-2 activity of 800 U/17 were placed at an initial cell density of 4.
Culture was carried out for 8 days at a culture temperature of 37° C. and a rotation speed of 100 rpm in a spinner flask with a capacity of 500 M using XIO'/-. When this culture was performed for 9 cycles, the maximum cell density was 7XIO'/I1) at the initial stage of culture, but it increased to 3x105/m2, and the 1 generation time was 1XIO'/I1) at the initial stage.
It was more than 30 hours, but it increased to 56 hours.

Further, the initial cell density was set to I x 105/-, and the 4-day culture was repeated for 6 cycles. As a result, the final generation time was 30 hours, the maximum cell density was 6X105/mj, and cells with good growth were CHO-BSF2 (FEBM P-997
0) was obtained. In this way, cells suitable for suspension agitation culture (
FERMP-9970) was obtained, but the BSF-2 production ability was stably maintained.

B19F-2 unit is I
K%IgM production in V-a14 cells to 50% of maximum
The activity that induces only IU is defined as IU.

Effect Conventional CHOdhfr'' cells have BUF-3, IL-2,
Although it is suitable for producing various bioactive tannins such as BSF'-2, it has not been possible to produce large quantities of the desired bioactive tannins because floating agitation culture is not possible. However, using the method of the present invention, CHOdhfr
'It becomes possible to carry out suspension agitation culture of cells, and therefore it is possible to produce extremely large quantities of the target bioactive tanno-matrix.

[Brief explanation of the drawing]

FIG. 1 shows the amino acid sequence of human differentiation-inducing factor BUF-3 and the nucleotide sequence encoding it. FIG. 2 is a construction diagram of plasmid pSD(x)/BUF-3. Figure 3 shows the EDF production ability and growth stability of the obtained 〇HO-5USP strain. FIG. 4 shows the amino acid sequence of human interleukin/2 and the nucleotide sequence encoding it. FIG. 5 is a diagram of the construction of plasmid LPSDI/IL-2. FIG. 6 shows the amino acid sequence of human B cell differentiation factor and the nucleotide sequence encoding it. FIG. 7 shows construction diagrams of plasmid pcD BSF-2 and plasmid pBsF2-1086. FIG. 8 shows a construction diagram of plasmid pSD(x)/BSF-2.

Claims (5)

[Claims] (1) A plasmid containing a gene encoding a physiologically active protein and a dihydrofolate reductase (hereinafter referred to as dhfr) gene in an expressible state is introduced into a Chinese hamster overly dihydrofolate reductase-deficient strain (CHOd).
Production of a physiologically active protein, which is characterized in that the cells obtained by transforming the hfr^-) cells are subjected to floating agitation culture, the desired physiologically active protein is produced in the culture solution, and the desired physiologically active protein is obtained. Law. (2) The physiologically active protein is human differentiation factor BUF-
3 (hereinafter referred to as BUF-3), human interleukin 2
(hereinafter referred to as IL-2), and human B cell differentiation factor (hereinafter referred to as BSF-2). (3) The plasmid is pSD(x)/BUF-3ΔSV4
0 early promoter - gene encoding BUF-3 -
SV40 splicing signal - SV40 early promoter - dihydrofolate reductase (hereinafter referred to as dhfr)
The production method according to claim (1), wherein the gene-SV40 splicing signal is used. (4) The plasmid is pSD(x)/BSF-2ΔSV4
0 early promoter - dhf gene - SV40 splicing signal - SV40 early promoter - BSF
The production method according to claim (1), wherein the gene encoding -2 is a splicing signal of SV40. (5) The plasmid is pSD(I)/IL-2ΔSV40
Early promoter-dhf gene-SV40 splicing signal-SV40 early promoter-IL-2
The production method according to claim (1), wherein the gene encoding the SV40 splicing signal.