JPH09163983A - Efficient production of useful substance by apoptosis inhibition and cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To extremely improve productivity of a useful substance such as an antibody, a vector for gene therapy, cytokinin, an antigen for vaccine, etc., by transducing an apoptosis inhibitory gene to an animal cell capable of producing a useful substance. SOLUTION: An apoptosis inhibitory gene such as bcl-2, BAG-1, Bcl-XL, Ad.Elb or CrmA is transduced to bcl-2 transduced COS-1 cell (FERM P-15,808) as an animal cell capable of producing a useful substance by an electroporation method. A selective pressure is applied to the system to select a gene-transduced cell, which is cultured in a medium to mass produce a useful substance such as various antibodies produced by a hybridoma, a virus vector for treating a gene, various recombinant protein such as a cytokine, e.g. interferon, an antigen substance for a vaccine, etc., in improved productivity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel method for producing useful substances and cells. More specifically, for example, various antibodies produced by hybridomas, viral vectors for gene therapy, various recombinant proteins, for example, cytokines such as interferon, methods for improving productivity of useful substances such as antigenic substances for vaccines, and One of the cells obtained by the method and used for that purpose
Regarding the species.

[0002]

2. Description of the Related Art Due to the rapid progress of genetic engineering in recent years,
Various molecular biology techniques have been developed. As a result, remarkable progress has been made in the analysis of genetic information or the elucidation of gene functions, and various findings have been obtained for many useful substances. As one of the attempts to apply the results obtained from these findings to actual industrial fields, many production of useful substances by animal cells has been started. For example, an antibody is a protein produced in vivo by an immune reaction as a result of antigen stimulation, has an activity of specifically binding to an immunogen, and plays a role of a defense mechanism in the body. Utilizing the specific binding activity of antibodies to such immunogens,
Various analytical agents, diagnostic agents, or therapeutic agents have been developed. The antibodies used in these industries are mainly produced by culturing hybridomas that produce the target antibody.

Further, other useful substances such as cytokines such as interferon, vaccines, enzymes, peptide hormones, viral vectors for gene therapy, etc. are also produced by various animal cells.

Examples of useful substances produced by animal cells are shown below.

(1) Monoclonal antibody In general, the method for producing a monoclonal antibody is as follows.
A mammal is immunized with the immunogen, and B cells are collected. next,
These B cells and myeloma cells, specifically mouse-derived X63-Ag8 (X63), NSI-Ag4 / 1
(NS1), P3X63-Ag8.654 (X63 / 6
54), SP2 / 0-Ag14 (SP2 / 0), MPC
11-45.6TG 1.7 (45.6TG), FO, S
149 / 5XXO, BU. 1, etc .; 2 from rat
10. RSY3. Ag1.2.3 (Y3), etc .; in human origin, U-226AR (SKO-007), GM15
00 · GTG-A12 (GM1500), UC729-
6, LICR-LOW-HMy2 (HMy2), 822
6AR / NIP4-1 (NIP41) etc. (however, the symbols in parentheses are shown) are mixed with a fusion promoter such as polyethylene glycol (PEG) or Sendai virus (HVJ) to cause cell fusion, and the antibody producing ability is obtained. And a hybridoma having both proliferation ability are produced. In addition, the search for the antibody-producing strain from the prepared hybridoma is performed by EL
ISA method (Method in Enzymology)
vol. 70. pp419-439), agglutination reaction method,
It is performed by the RIA method, the double immunodiffusion method, or the like. Cloning is performed by the limiting dilution method. To obtain an antibody from these hybridomas, there is a method of culturing the hybridoma in a medium and separating it from the culture supernatant, or a method of administering the hybridoma intraperitoneally to a mouse and collecting it from the ascites.

(2) Vaccine against virus In order to obtain a vaccine against virus,
It is introduced into an appropriate cell line to produce the target recombinant protein. As specific examples, cultured monkey kidney cells, vaccine production against polio (dead virus, live virus) by human diploid cells, Hela cells, vaccine production against adenovirus by human diploid cells, vaccine against measles by cultured chicken embryo cells. Production, vaccine production against mumps by cultured chicken embryo cells, vaccine production against rubella by cultured chicken embryo cells, cultured monkey kidney cells, human diploid cells, dog kidney cells or rabbit kidney cells, rabies caused by human diploid cells , Vaccine production against hepatitis B with hepatitis B antibody-positive human plasma, vaccine production against foot-and-mouth disease with cultured monkey kidney cells, cultured monkey kidney cells, vaccine production against Newcastle disease with hamster kidney cells, cultured monkey kidney cells, Hamster kidney cells There is a vaccine production, etc. for by distemper.

(3) Factors Acting on Immune System (Cytokines) Examples of producing cytokines using animal cells include IL-2 production by helper T cells and M-CSF production by mouse L cells. G- by CHU-2 cells
CSF production, GM-CSF production by Mo cells, endotoxin-induced embryos and placenta, IL-1 production by mononuclear cells and macrophages, IL-by T lymphocytes.
3 production, IL-4 by mouse T cell hybridoma
, IL-5 production by T lymphocytes, IL-6 production by T lymphocytes, erythropoietin production by kidneys, human myeloid leukemia cells, TN by macrophages
F production, lymphotoxin production by human B cell lines, B lymphocytes, interferon-α production by Burkitt lymphoma-derived lymphoblast cells, interferon-β production by diploid fibroblasts, T-lymph There is interferon-γ production by spheres.

(4) Cell Growth Factors Examples of cell growth factor production by animal cells include EGF production by submandibular gland and glial cells, endothelium cells, TGF-α production by placenta, platelets and vascular endothelial cells. P
DGF production, NGF production by submandibular gland, platelets,
Production of TGF-β by kidney, placenta and many cultured cells, production of IGF-I by smooth muscle cells and hepatocytes, production of IGF-II by fetal hepatocytes and placenta, production of FGF by cerebrum and chondrosarcoma cells, etc. Is mentioned.

(5) Enzymes Examples of enzyme production in animal cells include production of tissue plasminogen activator (TPA) by normal human diploid fibroblasts, Chinese hamster ovary cells and cancer cells, Chinese hamster ovary. Examples include production of renin by cells.

(6) Peptide hormone To obtain a peptide hormone from an animal cell, the gene of interest is introduced into an appropriate cell line to produce the recombinant peptide hormone of interest. Specifically, Vero, C
There are growth hormone production by 127 cells.

(7) Viral vector Currently, viral vectors are produced by recombinant virus producing cells called packaging cell lines. A packaging cell line is a cell line in which a foreign gene necessary for producing a recombinant virus has been inserted into a host cell genomic sequence.

[0012] In fact, the Moloney leukemia virus vector (AD Miller et al., Sowat. Cell Mol. Gene) was used as a viral vector for which a packaging cell line was established.
t., 12: 175, 1986).

However, the production of useful substances by these animal cells described above is (1) expensive because a large amount of medium is used for the culture; (2) animal cells are useful substances produced by prokaryotes. The production rate of useful substances per volume is lower than that in production; and (3) For example, useful substance-producing cells such as antibody-producing hybridomas rapidly undergo apoptosis when they lack nutrients (Biotechnolo).
gy and Bioengineering, vo
l. 44, pp. 1140-1154, 1994).

Therefore, the development of a useful substance production capacity enhancing method, that is, a method for producing a useful substance more than the useful substance producing capacity during ordinary culture has been a major problem.

Currently, as a method for collecting a large amount of useful substances from animal cells, there is a method of increasing the amount of culture used for one-time culture, that is, a method of using a large-scale culture device. Animal cells are CHO, CV-1, COS, BHK,
The cells can be roughly classified into adhesion-dependent cells such as mouse L cells, C127, Rat2, NIH3T3, or Hela cells, and floating cells such as Namalwa cells, and various methods have been devised as large-scale culture methods for each. That is, in adhesion-dependent cells, in order to expand the adhesion area, a method using multiplate, enamel fiber, ultrafiltration membrane, porous ceramics, microcarriers, etc., in floating cells, microcarriers, spinner flasks, stirring culture There is a method using a tank, a perfusion culture tank, an air lift culture tank, or the like. However, none of the culture methods has reached the target level of high-density culture (1 kL-10 kL in the jar fermenter).
Equivalent number of cells 10 ⁴ -10 ⁷ cells / ml).

For culturing animal cells, it is usually necessary to add about 10% serum in addition to the basal medium. However, the addition of serum has a great influence on the production cost, the cell growth activity is different between lots of each serum, and thus stable production of useful substances is not possible. However, there is a problem that it is difficult to purify the product. For this reason, a serum-free medium that does not require the addition of serum is used in industrial scale production, but the production efficiency of useful substances is low compared to the case of culturing in a medium containing serum. The points still remain.

Several methods have been devised to improve the production efficiency of these useful substances. Specifically, when transplanting an antibody-producing hybridoma into the abdominal cavity of a mouse,
Mitogen, Freund's complete adjuvant and the like are intraperitoneally administered to increase the amount of ascites to increase the absolute amount of the antibody (JP-A-02-53736).
By culturing the isolated animal cells as a three-dimensional aggregate using a serum-free culture medium on a porous substrate, the animal cells can be stably produced at high density over a long period of time, A method for enabling efficient preparation of a product (Japanese Patent Laid-Open No. 03-160988), and a method for adding a lymphocyte growth factor such as egg yolk lipoprotein, royal jelly or lactoferrin to a medium (Japanese Patent Laid-Open No. 02-2).
57892).

However, in either method,
It is unavoidable that eutrophication associated with cell proliferation and cell apoptosis associated therewith are limited, and the amount of useful substances produced is limited.

[0019]

The object of the present invention is to produce useful substances such as antibodies, cytokines such as interferons, antigenic substances for vaccines, viral vectors for gene therapy, etc. from animal cells for a long period of time. It is intended to enable cells to survive and thereby improve the production efficiency of useful substances per unit culture amount.

[0020]

[Means for Solving the Problems] In order to achieve the above object, as a result of intensive studies, the present inventors have found that cytokines such as antibodies and interferons can be suppressed by suppressing apoptosis of animal cells that produce useful substances. It was discovered that the killing of animal cells that produce useful substances such as vaccine antigenic substances and viral vectors for gene therapy can be suppressed, thereby significantly improving the ability of animal cells to produce useful substances per unit culture amount. Then, the present invention was completed.

That is, the present invention provides a method for improving the productivity of a desired useful substance by introducing an apoptosis-suppressing gene into an animal cell that produces the useful substance, and a method obtained by the method. One of the cells used for this purpose is to provide BCMGSneo-bcl-2-transfected COS-1 cells. Use of the method or cells of the present invention can not only improve the survival rate of useful substance-producing cells, but also increase the amount of useful substance produced per unit culture amount and improve the rate of useful substance production. it can.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION Useful substances whose productivity can be improved by using the method of the present invention include antibodies, cytokines such as interferon, vaccines, enzymes, peptide hormones, viral vectors for gene therapy and the like. The kind does not matter as long as it is produced by animal cells.

The term "apoptosis suppressor gene" as used herein means
For example, removal of cell growth factor, removal of hormone, receptor stimulation by cytotoxic lymphocytes, anticancer drug, radiation, toxin, virus infection, lack of zinc ion, heat shock, tumor necrosis of TNF-α, β, etc. Factor, anti-Fas antibody,
It means a gene encoding a protein that has a function of prolonging the life of cells by suppressing apoptosis induced by various factors such as. Apoptosis-suppressing genes that can be used in the present invention include, for example, bcl-2, BAG-1,
Bcl-XL, Ad. E1b, CrmA, etc. are included. One example thereof, bcl-2, is prominently expressed in a wide range of tissues in vivo, particularly in the immune system and nervous system, and is also observed in epithelial cells such as skin and small intestine where long-term surviving stem cells are present. Ori (Negrini, M. et al., Ce
ll, 49: 455-463, 1987; Eguchi, Y. et al., Nuc. Acid.
Res., 20: 4187-4192, 1992; Hockenbery, DM et a
L., Proc. Natl. Acad. Sci. USA, 88: 6961-6965, 199
1) It is thought to be involved in cell suicide prevention and life prolongation in many aspects of the body (Vaux, DL et al.,
Nature, 335: 440-442, 1988). The bcl-2 gene is a chromosomal translocation t (14; 18) in human non-Hodgkin's lymphoma
More cloned (Science vol.
226. (pp1097 (1984)) It is known to be a gene encoding bcl-2α of 26 kD protein and bcl-2β of 22 kD protein.

The method for enhancing the useful substance-producing ability of the present invention is carried out by introducing an apoptosis-suppressing gene into useful substance-producing cells by the following method, but is not limited to this method. That is, for example, bcl-2 incorporated into an expression vector plasmid containing a drug resistance gene sequence is introduced into a useful substance-producing cell that produces a useful substance. As the gene transfer method into cells, in addition to the known physical transfer methods such as the electroporation method, the calcium phosphate method and the liposome method, the biological transfer method using a viral vector such as mouse leukemia virus vector is used. After the introduction, drug selection is performed with a drug corresponding to the drug resistance gene, and only cells into which bcl-2 has been introduced are selected. As for the cloning method, known methods such as methyl cellulose method, soft agarose method, and limiting dilution method can be used without particular limitation. The cell line obtained by drug selection has an excellent ability to produce useful substances, especially in long-term culture, as compared with a cell line into which an apoptosis suppressor gene has not been introduced.

Regarding the production of antibodies, it has been known that hybridomas have a long culture period and lack nutrients in the medium, and at the same time, have a low survival rate. However, an apoptosis-suppressing gene such as bcl-2 has been introduced. By doing so, it is possible to prolong the life of the cells and significantly increase the production amount and production rate of the antibody.

The present invention can also be applied to the production of recombinant viral vectors used for gene therapy. Generally, a recombinant virus vector is produced by a recombinant virus vector producing cell called a packaging cell. It is known that packaging cells die due to oligotrophy when they are cultured for a long period of time. However, if an apoptosis suppressor gene is introduced into this packaging cell, the life of the packaging cell can be prolonged and the titer can be improved. like this,
Although cell death due to eutrophication also occurs in other useful recombinant protein-producing cells as described above, by introducing an apoptosis-suppressing gene, the cell death of cells can be performed in the same manner as in packaging cells that produce recombinant viral vectors. The life can be extended and the production amount can be increased.

The BCMGSneo-bcl-2-introduced COS-1 cells of the present invention (hereinafter, COS1 / bcl2) are used as animal cells capable of significantly increasing the production amount and production rate of a protein by prolonging the life of the cells. The present invention has been found, and the above (1) to
It can be applied to mass production of the useful substance of (7).
The effect of COS1 / bcl2 can be confirmed by using a λ chain protein with high detection sensitivity as a substitute for the protein.

COS1 / bcl2 was sent to the Institute of Biotechnology, Institute of Biotechnology, FERM P- on August 26, 1996.
Deposited as 15808.

[0029]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1: Construction of plasmid In this example, the following plasmids were used.

The plasmid BCMGSneo-bcl-2 (FIG. 1) containing the human bcl-2 gene sequence was used as an expression vector BCMGNSneo (Karasuyama, H.,
Kudo, A .; Melchers, J .; Exp.
Med. , 172: 969-972, 1990) Xh.
It was constructed by inserting the bcl-2 gene into the oI-NotI site. In addition, bcl-2α was used as the bcl-2 gene.

In addition, as a comparative example, BCMGSneo (FIG. 2) was used as a plasmid containing no bcl-2 gene.
Was used in subsequent tests.

Example 2: Introduction of human bcl-2 gene into hybridoma cells BCMGSneo-bcl-2 or BCMGSneo-bcl-2 prepared in Example 1 was added to mouse hybridoma 2E3 strain producing an antibody that recognizes trinitrophenyl group as a hapten. As a comparative example, BCMGSneo was introduced by the electroporation method (Biomanual Series 4 Gene introduction and expression analysis method p23-27). After introduction, G41
8 (GIBCO) selection pressure was applied to select only cells into which the plasmid had been introduced.

Example 3: Detection of Bcl-2 protein production in human bcl-2 gene-introduced hybridoma cells (analysis by Western blotting) BCMGSneo-bcl established in Example 2
The Bcl-2 protein production of the -2 introduced hybridoma cells was detected. As a negative control, the same study was performed on the hybridoma cells introduced with BCMGSneo. (1) Preparation of cell lysate BCMGSneo-bcl-2-introduced hybridoma cells and BCMGSneo-introduced hybridoma cell suspension were diluted with 1% Triton X-100, 0.15 mM N.
aCl, 10 mM Tris (pH 7.4), 50 μg
/ ML phenyl methanesulfony
l fluoride and 2 μg / mL apro
A cell lysate containing tinin was used to react at 4 ° C. for 30 minutes to lyse the cells. This cell lysate was used as a sample for Western blotting.

(2) Analysis by Western blotting Analysis by Western blotting was carried out according to a known method (Biomanual Series 4 Gene introduction and expression analysis method p123-127). A cell lysate corresponding to 1 × 10 ⁵ cells was added to Tris-SDS-mer.
After mixing with the captoethanol buffer and boiling for 5 minutes, the sodium dodecyl sulf
ate (SDS) -polyacrylamide gel (bcl-
The concentration of polyacrylamide gel when detecting 2 is 1
3% was most preferred) and the sample was loaded. After electrophoresis, remove the gel and wait 30 minutes or longer Tris-g
Immersed in lycine buffer. A nitrocellulose filter was attached to the gel, the protein was transferred to the nitrocellulose filter, the nitrocellulose filter was taken out, and Tris buffered saline-0.
Soak for 15 minutes in 05% Tween 20 (TBS-T),
After soaking in TBS-T containing 5% skim milk for 2 hours, washing with TBS-T was performed 3 times.

Using the nitrocellulose filter thus prepared, Bcl-2 antibody (Boehring)
er Manneheim), the nitrocellulose filter was dipped in a TBS-T solution, gently shaken for 1 hour, washed three times with the TBS-T solution, and then the secondary antibody peroxidase-conjugated anti-mouse Ig polyclonal antibody (B
io Source International, I
TBS-T including nc-Tago Products)
A nitrocellulose filter was immersed in the solution. The mixture was gently shaken for 1 hour, washed three times with a TBS-T solution, immersed in an ECl detection reagent (Amersham), and then exposed to a film.

FIG. 3 shows the results. BCMGSneo-b
In the cells into which cl-2 was introduced (shown as Example 3 in FIG. 3), a band showing the expression of Bcl-2 protein was detected, but cells into which the negative control group BCMGSneo was introduced (compared in FIG. 3). (Shown as Example 3) from Bcl-
No band indicating the expression of 2 proteins was detected.

Example 4: Improvement of survival rate of hybridoma cells by introduction of human bcl-2 gene BCMGSneo-bcl established in Example 2
The survival rates of the -2 introduced hybridoma cells and the negative control group of BCMGSneo introduced hybridoma cells were compared.

Dulbecco's modified eagle medium (Dulbecc) containing 9% fetal bovine serum was used for each cell.
o's Modified Eagle Media
m) (hereinafter, DMEM medium) (GIBCO) and suspended in a CO ₂ incubator. The cells were collected every 2 days after the start of the culture, and the survival rate of each hybridoma was calculated by the trypan blue staining method.

The results are shown in FIG. In the negative control group (shown as Comparative Example 4 in FIG. 4), about 5 days after the 6th culture.
The survival rate was 0%, while the survival rate was 10 days in the bcl-2-introduced cell group (shown as Example 4 in FIG. 4). This prolongation of cell viability was due to the introduction of bcl into cells.
-2 It is considered that this is due to the effect of gene.

Example 5: Improvement of antibody production efficiency by introduction of human bcl-2 gene BCMGSneo-bcl established in Example 2
The antibody-producing ability of the -2 introduced hybridoma cells and the negative control group BCMGSneo introduced hybridoma cells were compared.

DME containing 9% fetal calf serum in each cell
The cells were suspended in the medium and left in a CO ₂ incubator. The culture supernatant was collected every 2 days after the start of the culture, and the concentration of the antibody contained in the supernatant was measured by the ELISA method. E
LISA is an anti-mouse IgG polyclonal antibody (ZYM
ED Laboratories) and peroxidase-conjugated anti-mouse Ig polyclonal antibody (Bio S
source International, Inc-T
It was performed by a sandwich method using ago Products).

The results are shown in FIG. Compared with the negative control group (shown as Comparative Example 5 in FIG. 5), in the bcl-2 introduced cell group (shown as Example 5 in FIG. 5), the antibody concentration remarkably increased with the increase of culture time. On the 10th day, there was a difference of more than 4 times.

Example 6: Improvement of antibody production rate by introduction of human bcl-2 gene Further, the antibody production rate of each hybridoma cell whose antibody production efficiency was measured in Example 5 was also compared and examined. As shown in FIG. 6, the antibody production rate of the bcl-2-introduced cell group (shown as Example 6 in FIG. 6) was 2 times that of the negative control group (shown as Comparative example 6 in FIG. 6) at any period.
It was more than double.

Example 7: Construction of plasmid In this example, the following plasmids were used. All operations are performed by known methods (J. Sambrook, et a.
l. , Molecular Cloning, Cold
Spring Harbor Laboratory
Press, 1989).

The plasmid BCMGSneo-bc1-2 (FIG. 1) containing the human bc1-2 gene sequence was used to express the expression vector BCMGNSneo (Karasuyama, H.,
Kudo, A .; Melchers, J .; Exd. , M
ed. , 172: 969-972, 1990).
It was constructed by inserting the bcl-2 gene into the I-NotI site.

As a comparative example, a plasmid not containing the bcl-2 gene, BCMGSneo (FIG. 2), was used in the subsequent tests.

The plasmid pcDNA-λ (FIG. 7) containing the gene sequence (hereinafter mouse λ1) encoding the protein λ chain constituting the L chain of mouse immunoglobulin 1 gM is p
CMV-λl (Toyoshima, H. et a.
l. , Ce 11, 8, 67-74, 1994) PC
The mouse λ1 chain portion was amplified by the R method to synthesize cDNA, and the plasmid vector pcDNA3 (ln vitr
Ogen).

Example 8: COS of human bcl-2 gene
-1 Introduction into cells BCMGSneo-bcl-2 or BCMGSn
eo by electroporation (J. Sambrook, et a
l. , Molecular Cloning, Cold
Spring Harbor Laboratory
COS-1 cells according to Press, 1989).

10 ⁷ COS-1 cells were prepared and suspended in 450 μl of phosphate buffer (PBS). Plasmid D
10 μg of NA was dissolved in 50 μl of PBS, mixed with the above cell suspension, and placed in a cuvette. 6 pulse devices
The cuvette containing the cell suspension was pulsed 20 times at a setting of 50 V and 250 μsec. After leaving on ice for 10 minutes, 400 μg / ml G418 (GIBCO
And 10% fetal bovine serum (Fetal Bovin)
e Serum) (FBS) (GIBCO) containing Dulbecco Modified Eagle Medium (Dulbe)
cco's Modified Eagle Medi
um) (hereinafter referred to as DMEM medium) (GIBCO) and left in a CO ₂ incubator for 30 days to select only the cells into which the gene has been introduced, and then BCMGS
neo-bcl-2-introduced COS-1 cells (hereinafter referred to as COS
1 / bcl2) and BCMGSneo-introduced COS-1 cells (hereinafter, COS1 / vec) were obtained. BCMGSne
o-bcl-2 introduced COS-1 cells (hereinafter, COS1 /
bcl2) was deposited as FERM P-15808 at the Institute of Biotechnology, Institute of Biotechnology, August 26, 1996.

Test Example 7: Apoptosis of bcl-2 introduced cells
Evaluation of resistance to cis It is known that when cells are cultured in a medium having a low serum concentration, this triggers apoptosis to cause cell death. Therefore, the following studies were conducted to investigate the resistance of cells into which bcl-2 was introduced to apoptosis.

(COS introduced with human bcl-2 gene)
-1 cell culture in low serum concentration medium and measurement of viable cell number) COS1 / bcl2 (Example 8) and COS1 / v
ec (Comparative Example 7), 10 ⁵ each, 0.2% FBS
It culture | cultivated in the DMEM culture medium containing the fixed period. On the 2nd and 9th days after the start of the culture, the culture dish was washed with PBS and treated with trypsin to remove it from the culture dish to prepare a cell suspension. After staining the cells with trypan blue, the number of viable cells was counted by observing with a microscope. The results are shown in Fig. 8. The cells not introduced with the bcl-2 gene shown in Comparative Example 7 are the number of cells at the start of culture (10 ⁵ cells).
Although the number of upstream traffic is also reduced, b shown in Example 8 is used.
On the 9th day, the cells into which the cl-2 gene had been introduced proliferated to more than 4 times the initial number of cells. From this result, Example 8
It is clear that the COS1 / bcl2 cells established in 1. showed strong apoptosis resistance.

Test Example 8 Protein by introducing bcl-2 gene
White Productivity Improvement African Green Monkey-derived COS-1 cells are widely used for the expression and production of recombinant proteins. Therefore,
The protein productivity when the apoptosis inhibitor gene was introduced was evaluated.

(PcDNA-λ COS1 / bcl2,
Transfection of COS1 / vec and COS-1 cells) On the day before transfection, 5 × 10 ⁵ cells of each were seeded on a culture dish having a diameter of 10 cm. 1
0 μg of pcDNA-μ was added to the DEAE-dextran method (J. Sambrook, et al., Molec.
ullar Cloning, Cold Spring
Harbor Laboratory Press, 1
989), and allowed to stand in a carbon dioxide incubator for 4 hours. Aspirate the culture supernatant and add 10% DM
Immediately after adding 5 ml of SO aqueous solution for 2 minutes, PBS
It was washed twice with (1). DMEM containing 10% bovine serum
10 ml of the medium was added and cultured for a certain period.

(Collection of culture supernatant and λ in the culture supernatant)
Quantification of chain protein) 350 μl after 4, 5, 6, 7 and 14 days from the day of transfection of the culture supernatant of each cell into which pcDNA-1λ was introduced
Collected one by one. The amount of the λ chain protein contained in the culture supernatant is horseradish peroxidase (Horse R
It was quantified by an ELISA method using an add peroxidase-labeled goat anti-mouse λ chain antibody, and the amount on the 4th day was defined as 1 and expressed as the ratio. The accumulated amount of λ chain protein was measured by the absorbance at 410 nm. As shown in FIG. 9, in the cell lines into which the apoptosis-suppressing gene bcl-2 was not introduced (Comparative Example 7 and Comparative Example 8), the accumulated amount of λ chain protein did not increase after 7 days. On the other hand, in the cell line into which bcl-2 was introduced (Example 8), 7
It was more than double the number of days.

[0056]

As described above, the method for enhancing the ability to produce useful substances and cells according to the present invention not only significantly improve the survival rate of cells producing useful substances, but also increase the production amount and production rate of useful substances. Significantly improved.

Therefore, using the method and cells provided by the present invention, for example, cytokines such as hybridoma and interferon producing various antibodies,
It can be applied to cells producing other useful substances such as antigenic substances for vaccines and viral vectors for gene therapy.

The antibody-producing ability-enhancing method or cell of the present invention is not limited to bcl-2 exemplified in this Example, but other apoptosis-suppressing genes (Mad / Max, BAG-).
1, Bcl-XL, Ad. (E1b, CrmA, etc.) can be implemented or introduced, and its industrial application range is extremely large.

[Brief description of the drawings]

FIG. 1 is a diagram showing a structure of BCMGsneo-bcl-2.

FIG. 2 is a diagram showing a structure of BCMGSneo.

FIG. 3 is a diagram showing the results of Western blotting.

FIG. 4 is a graph showing the relationship between culture time and the survival rate of hybridoma cells.

FIG. 5 is a graph showing the relationship between culture time and the concentration of antibody contained in the culture supernatant.

FIG. 6 is a graph showing the measurement of antibody production rate at regular intervals during the culture period.

FIG. 7 is a diagram showing the structure of pcDNA-λ.

FIG. 8 is a graph showing the relationship between the culture time and the survival number of gene-transferred COS-1 cells.

FIG. 9 is a graph showing the amount of protein produced and accumulated for each fixed period during the culture period.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 39/00 A61K 39/395 A 39/395 D C12N 5/00 B (C12N 5/10 C12R 1 : 91) (C12P 21/02 C12R 1:91) (72) Inventor Takeshi Goto 1-25-11 Kannondai, Tsukuba City, Ibaraki Prefecture Hisamitsu Pharmaceutical Co., Ltd. Tsukuba Research Institute (72) Hideji Sato Tsukuba City, Ibaraki Prefecture Kannondai 1-25-11 Hisamitsu Pharmaceutical Co., Ltd. Tsukuba Research Institute

Claims (4)

[Claims] 1. A method for improving the productivity of a desired useful substance by introducing an apoptosis suppressor gene into an animal cell producing the useful substance. 2. The apoptosis-suppressing gene is bcl-
2, BAG-1, Bcl-XL, Ad. E1b or C
It is rmA, The manufacturing method of Claim 1 characterized by the above-mentioned. 3. The animal cell is bcl-2 introduced COS-1.
The method of claim 1, wherein the cell is a cell deposited as FERM P-15808. 4. A bcl-2-transfected COS-1 cell deposited as FERM P-15808.