JP5811321B2 - Novel protein expression method - Google Patents

Description

  The present invention relates to a technique for producing proteins in large quantities using mammalian cells as hosts.

  Conventionally, techniques for producing useful substances using Escherichia coli, yeast, animal cells and the like as hosts have been developed, and the produced substances are used in many fields such as pharmaceuticals and diagnostic reagents. The substances produced are mainly proteins, but not only the wild-type protein originally possessed by each organism, but also by introducing mutations into the gene encoding the wild-type protein using genetic recombination technology, Various recombinant proteins with improved activity over the wild type and recombinant proteins with functions different from the wild type have been produced (see Patent Document 1 and Patent Document 2).

  A method for producing a protein in a host by introducing a gene encoding various proteins into the host and expressing the gene is generally used in industrial production of the protein (hereinafter, the protein to be produced is referred to as “ The target protein "and the gene encoding it are sometimes called" target protein expression gene "). The host used in the production method is appropriately selected from bacteria such as Escherichia coli and Bacillus bacteria, yeasts, molds, insect cells, animal cells, and the like. Escherichia coli is used because there are many. However, production of a target protein derived from animals using E. coli as a host is not recognized, and even if production is confirmed, the target protein accumulates in the cell as an insolubilized protein or is soluble. Although it is produced as a protein, it may be produced as a protein having no physiological activity (Patent Document 3).

  On the other hand, when an animal-derived target protein is produced using yeast, insect cells, or animal cells as hosts, it can be produced as a protein that retains physiological activity with high probability (see Patent Document 2). However, when yeast is used as a host, the added sugar chain may be different from that derived from mammalian cells, which may affect the activity of the produced target protein (Patent Document 4). ). In addition, when an insect cell is used as a host, a virus must be used when the target protein gene is introduced into the host, and much effort is required for removing the virus during purification of the target protein. On the other hand, when producing an animal-derived target protein using an animal cell as a host, the added sugar chain is substantially the same sugar chain as that originally modified the target protein. This has the advantage that the bioactivity is unlikely to be affected by the difference. Moreover, since it is not necessary to use a virus for introduction of the target protein gene, it is not necessary to spend much effort for its removal, which is advantageous in terms of time and cost.

  When using animal cells as a host, while having the advantages as described above, there is a problem that the production amount is low only by introducing a target protein expression gene into the host. Therefore, in order to increase the production amount of the target protein, a Chinese hamster ovary (CHO) cell or the like is used as a host, and a gene encoding the target protein gene and dihydrofolate reductase (DHFR) is added thereto. A method is known in which methotrexate (MTX), which is an inhibitor of DHFR, is introduced at the same time and added to a culture solution (Non-patent Document 1). In this method, by gradually increasing the MTX concentration in the medium, cells expressing the DHFR gene (producing DHFR) are selected, and the target protein gene is amplified together with the DHRF gene. As a result, the production amount of the target protein is increased, but it takes 6 months or more to complete the operation.

  Also known is a method for amplifying a target protein gene by introducing a mammalian replication initiation region (IR) and a nuclear matrix binding region (MAR) into a host (Patent Document 5). .

Japanese Patent Laid-Open No. 7-284394 (Japanese Patent No. 2694440) Japanese Patent Laid-Open No. 7-67665 (Japanese Patent No. 2511251) JP-T-2002-531086 JP-A-6-277086 (Patent No. 3091851) JP 2003-245083 A (Patent No. 3755028)

R J Kaufman, L C Wasley, A J Spiliotes, S D Gossels, S A Latt, G R Larsen and R M Kay, "Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells" Mol Cell Biol. 1985 July; 5 (7): 1750-1759. Shimizu et al. , "Amplification of Plasmids Containing a Mammalian Replication Initiation Region Is Mediated by Controllable Conflict Replication and Transcrib. 63, p5281-5290.

  As described above, when an animal-derived target protein is produced using an animal cell as a host, the produced target protein retains physiological activity and does not require virus removal during purification. However, when animal cells are used as the host, the production amount of the target protein is lower than when E. coli is used as the host. Therefore, in order to produce the target protein in animal cells for the purpose of use in medicines and diagnostics, it is necessary to increase the production amount. In addition, from the viewpoint of supply of medicines and diagnostic agents, it is also necessary to produce a necessary target protein in large quantities in a short period of time.

  Accordingly, an object of the present invention is to provide a technique for producing mammalian cells that produce a target protein in a large amount in a short period of time.

  The present inventors introduce four elements of mammalian replication initiation region (IR), nuclear matrix binding region (MAR), dihydrofolate reductase (DHFR) expression gene group and target protein expression gene group into mammalian cells. Thus, not only the level of the conventional gene amplification method using DHFR and MTX, and the gene amplification method using IR and MAR, but also significantly faster than the level normally expected when these are used in combination, The inventors have found that the target protein can be produced and have completed the present invention.

  That is, the present invention made to solve the above problems includes a mammalian replication initiation region (IR), a nuclear matrix binding region (MAR), a dihydrofolate reductase (DHFR) expression gene group, and a target protein expression gene group. There is provided a method for producing a mammalian cell, comprising a step of treating a mammalian cell with methotrexate (MTX).

Typically, the step of treating methotrexate includes a plurality of steps of culturing the mammalian cell in a medium containing methotrexate while increasing the concentration stepwise.
For example, the step of treating methotrexate includes at least a first step of culturing the mammalian cell in a medium containing methotrexate at a concentration of 5 to 25 nM, and methotrexate at a concentration 5 to 10 times that of the first step. A second step of culturing the mammalian cells in a culture medium.

  In order to produce a mammalian cell comprising the mammalian replication initiation region, the nuclear matrix binding region, the dihydrofolate reductase expression gene group and the target protein expression gene group usually in advance, A step of introducing a single or a plurality of types of vectors, including a mammalian replication initiation region, a nuclear matrix binding region, a dihydrofolate reductase expression gene group, and a target protein expression gene group, into a mammalian cell, and then transforming it.

  Examples of the mammalian replication origin include those derived from the c-myc locus replication origin, the dihydrofolate reductase locus replication origin, or the β-globin locus replication origin.

  Examples of the matrix binding region include those derived from an Igκ locus matrix binding region, an SV40 initial region matrix binding region, or a dihydrofolate reductase matrix binding region.

Examples of the target protein include a receptor, an antibody, and a fluorescent protein.
More specifically, the above-described method for producing mammalian cells includes the steps (1) to (4) performed in the following order, for example.
(1) introducing a mammalian replication initiation region, a nuclear matrix binding region, a dihydrofolate reductase expression gene group and a target protein expression gene group into animal cells;
(2) culturing mammalian cells that have undergone the introduction step, and selecting and isolating cells expressing the target protein;
(3) transferring the cells that have undergone the isolation step to a medium containing 5 to 25 nM methotrexate, and selecting viable cells; and
(4) A step of transferring the cells that have undergone the selection step to a medium containing 125 to 500 nM methotrexate, and selecting the cells in which the gene has been amplified.

And this invention provides the method of manufacturing a target protein using the mammalian cell obtained by said manufacturing method.
In another aspect, the present invention relates to a mammalian replication initiation region (IR), a nuclear matrix binding region (MAR), a dihydrofolate reductase (DHFR) -expressing gene group, which are used in the method for producing a mammal as described above. A mammalian cell containing (introduced with) a target protein expression gene group is provided. The present invention also provides a mammalian cell obtained by subjecting this mammalian cell to methotrexate (MTX) treatment, that is, a mammalian cell obtained by the above-described method for producing a mammalian cell. Furthermore, the present invention provides a single vector comprising a mammalian replication origin region (IR), a nuclear matrix binding region (MAR), a dihydrofolate reductase (DHFR) expression gene group and a target protein expression gene group.

Hereinafter, the present invention will be described in detail.
IR and MAR may be any one as long as the target protein expression gene group is amplified in mammalian cells by the combination of both and separated and distributed to daughter cells. Preferred IRs include those derived from replication origins such as EB virus latent origin of origin (EBV lattice origin), c-myc locus replication origin, DHFR locus replication origin, β-globin locus replication origin and the like. It is done. Preferred MARs include those derived from MARs typified by Igκ locus MAR (AR1), SV40 early region MAR, DHFR locus MAR, and the like.

  The “DHFR expression gene group” or “target protein expression gene group” in the present invention is a base sequence for producing DHFR or a target protein by expressing a gene encoding DHFR or the target protein, and functions at least in the host. It contains a promoter and a gene encoding DHFR or target protein connected downstream thereof, and a polyadenylation signal may be further connected downstream thereof. The promoters constituting the DHFR expression gene group and the target protein expression gene group are not particularly limited as long as these genes can be expressed in the host. For example, when a host is a cell derived from a mammal such as a CHO cell, an African green monkey kidney fibroblast-derived cell (COS7 cell), a human cervical cancer-derived cell (Hela cell), or a mouse fetal skin-derived cell (NIH3T3), Examples of the promoter include SV40 early and late promoters, CMV promoter and the like.

  Host cells are cells derived from mammals such as CHO cells, COS7 cells, Hela cells, and NIH3T3 cells, and are DHFR-deficient strains that significantly lack or lack the ability to produce DHFR. For example, DHFR-deficient strains of CHO cells are established and can be obtained very easily, and other cells can be selected by culturing in a medium containing MTX, for example. .

  Mammalian cells containing the IR, MAR, DHFR expression gene group and the target protein expression gene group (hereinafter each of these four sequences or gene groups may be referred to as “elements”) according to the present invention include, for example, Various forms of cells, such as mammalian cells that have taken up and retained a single or multiple types of vectors containing four elements, and mammalian cells that have these four elements incorporated into chromosomes Is included. This mammalian cell can be prepared by introducing four elements of IR, MAR, DHFR expression gene group and target protein expression gene group into the mammalian cell. IR, MAR, and target protein expression gene group are simultaneously introduced into cells (Patent Document 5), and DHFR expression gene group is simultaneously or prior to introduction of IR, MAR, and target protein expression gene group, IR, MAR, and target protein. After the expression gene group is introduced, it is introduced into the cell.

  IR, MAR, and target protein expression gene group may be in the form of the same vector or in different vectors, and there is no particular limitation other than that as long as they are simultaneously introduced into the cell. When IR, MAR, and target protein expression genes are loaded on the same vector, it does not matter which element is upstream. As a form mounted on different vectors, any two or more of IR, MAR and target protein expression genes are loaded on the same vector, the remaining genes are loaded on another vector, or these three elements are loaded on three different vectors. Can be illustrated. When two elements are placed on the same vector, it does not matter which element is upstream.

  When introducing a DHFR expression gene group simultaneously with IR, for example, all four elements are placed on the same vector, or two or three of the four elements are placed on the same vector, and the remaining elements are placed on different vectors. The introduction at the same time can be exemplified. When not introduced simultaneously with IR or the like, a vector carrying only the DHFR expression gene group may be prepared and used. In addition, this invention provides the cell which produces a target protein in large quantities by carrying out MTX process of said mammalian cell for amplification of the target protein expression gene group. Therefore, when the DHFR expression gene group is not introduced at the same time as IR or the like in the above, the DHFR expression gene group is introduced prior to the MTX treatment. When four elements are introduced and a mammalian cell holds them, the target protein is produced in large quantities by MTX treatment.

  When a single vector including the four elements exemplified above is used, the probability that the four elements are connected to the host increases, and as a result, the target protein is amplified together with the amplification of DHFR-expressing genes by MTX treatment. Since it is thought that the probability that an expressed gene group is amplified can also be improved, it is preferable as an aspect of the present invention. However, in the case of using a plurality of types of vectors including one or more of the four elements, for example, a first vector including IR, MAR and a target protein expression gene group and a second vector including a DHFR expression gene group Even if there is a possibility that each element is kept in close proximity in mammalian cells when using the type and the target protein expression gene group is amplified together with the amplification of the DHFR expression gene group by MTX treatment, In consideration of cell growth, the effect of the present invention of producing a large amount of the target protein can be achieved without any particular inferiority. Further, a single vector including four elements will be further described. The positional relationship between the four elements is not limited, any element may be located upstream, and the direction of transcription is not limited. It is preferable that the four genes are close to each other. For example, as shown in the examples, a gene containing each element separated by several tens to several hundred bases can be exemplified.

  Mammalian cells that have been transfected (transfected) with the vector containing the above four elements are preferably cultured first, and then the cells expressing the target protein are selected and isolated, and then treated with MTX. A cell into which a DHFR expression gene group has been introduced can be selected. Then, by gradually increasing the MTX concentration in the medium, DHFR produced by the DHFR-expressing gene group is inhibited, but at that time, the DHFR-expressing gene group is amplified. When the DHFR expression gene group is amplified, the target protein expression gene group held close to the DHFR expression gene group is also amplified simultaneously. In general, the MTX concentration is gradually increased by adding 5 to 25 nM in the medium first, and at least one more step, and further one or more steps as necessary. A method of increasing the concentration by 10 times may be used. What is necessary is just to raise MTX density | concentration in the stage which the cell grew and made the colony with the culture medium which added MTX of each density | concentration. Conditions such as the number of steps to increase the concentration of methotrexate, the range of increase in concentration, the number of days of culture in each step, and the final concentration for selecting mammalian cells used for the production of the target protein are the conditions of the mammalian cell or the target protein. Although the production efficiency can be appropriately adjusted so as to achieve a desired one, if the method of the present invention is used, the overall process period can be shortened compared to the conventional gene amplification method. As a more specific procedure, for example, the isolated cells are first transferred to a medium containing 5 to 25 nM MTX to select viable cells, and if necessary, transferred to a medium containing 25 to 125 nM MTX. After the culturing step, the cells can be selected by transferring to a medium containing 125 to 500 nM MTX and amplifying the gene. In addition, after the cells grown in the medium supplemented with each concentration of MTX were once cloned, cells with a high gene amplification number, that is, cells with a large production amount of the target protein were isolated, and MTX was further isolated from the cells. The concentration may be increased, or the MTX concentration may be increased while keeping the polyclone without monocloning.

  The DHFR expression gene group and target protein expression gene group amplified by the method of the present invention are localized in the structure of either DM (Double Minutes) which is an extrachromosomal genetic factor or HSR (Homogeneously Staining Resion) on the chromosome. And can be observed using a technique such as FISH. HSR is further classified into homogeneous HSR, linear ladder, and point ladder (see FIG. 5). It is known that DM and HSR related to such gene amplification are also generated in a gene amplification method using only IR and MAR, and a huge HSR is formed by BFB (Breakage-Fusion-Bridge) cycle. (See, for example, Non-Patent Document 2). In the present invention, a longer point-like ladder HSR (see FIG. 5) is formed earlier than in the conventional gene amplification method. Correspondingly, the production of the target protein at a higher level is faster. Achieved.

  The method for producing a target protein of the present invention may be further combined with known means for improving the production amount of the target protein. For example, a method for improving the production amount of a target protein by inhibiting epigenetic methylation of a target protein expression gene using a histone deacetylation inhibitor (Sodium Butyrate or the like) is known. In the method for producing a target protein of the present invention, when culturing animal cells into which IR, MAR, DHFR, and a target protein expression gene group have been cultured, a histone deacetylation inhibitor is added to reduce the production amount of the target protein. This can be further improved.

  A method for introducing a gene into a host mammalian cell and transforming it can be appropriately selected according to the vector composition and the host. Examples include transfection by electroporation, lipofection, calcium phosphate, etc. can do. For example, when a plurality of types of vectors are used, the lipofection method can be exemplified as a preferable method. The selection of whether or not mammalian cells have been transformed with the vector prior to initiating MTX treatment is, for example, aminoglycoside 3′-phosphotransferase (kanamycin resistance gene), neomycin phosphotransferase gene (neomycin resistance gene) A drug resistance gene such as a hygromycin B phosphotransferase gene (hygromycin resistance gene), a blasticidin S deaminase gene (blasticidin resistance gene), a glutamine synthetase gene, a green fluorescent protein gene, or the like as a selection marker, What is necessary is just to follow a well-known general method. Alternatively, instead of using these selection markers, mammalian cells (originally DHFR-deficient strains) are cultured in a medium that does not contain ribonucleotides and deoxyribonucleotides, so that the mammal into which the dihydrofolate reductase gene (DHFR) has been introduced. Animal cells can also be selected.

  Culture of cells obtained by transformation may be performed under conditions suitable for the cells. The target protein produced by the expression of the target protein expression gene can be recovered from the culture supernatant by centrifugation or the like and purified by an appropriate method when the protein is secreted outside the cell. When the produced target protein accumulates in the cell, it can be extracted and purified by an appropriate method such as disrupting the cell.

  The target protein that can be expressed using the method for producing the target protein in large quantities of the present invention is not particularly limited as long as the polynucleotide sequence encoding the target protein to be expressed is known. For example, an antibody (H chain) And L chain), cytokines such as IL-6 (interleukin 6), erythropoietin, interferon, fluorescent protein and the like. In particular, antibodies that target antigens (for example, cell surface receptors) involved in specific diseases (for example, cancer and rheumatism) that are required in large quantities for the production of antibody drugs that have attracted attention in recent years are It is a target protein expected to improve production efficiency according to the invention.

  In the present invention, an IR, MAR, DHFR expression gene group and a target protein expression gene group are introduced into animal cells, and the cells are subjected to gene amplification treatment with MTX, so that mammalian cells producing a large amount of the target protein can be produced in a short period of time. Can be made between.

  And when cells that produce a large amount of useful target proteins such as pharmaceuticals are produced according to the present invention, as shown in the Examples, only IR and MAR are used, or only gene amplification treatment with MTX is performed. Compared with the production method of the target protein, the production amount of the target protein can be dramatically increased (for example, 10 times or more as shown in Examples), and a large amount can be produced in a short time. Therefore, the target protein can be produced in a short time and at a low cost as compared with the conventional production method of the target protein.

FIG. 1 is a graph showing the expression level of FcR expressed in the culture supernatant in steps 1 to 5 in Example 1 and Comparative Example 1-1. FIG. 2 shows the vector pΔBM. It is the schematic of AR1dhfr-FcR. FIG. 3 shows plasmids A (pΔBN AR1 P _sv40 -dhfr), B (pSFV-V P _sv40 -dhfr) used in Example 2, Comparative Example 2, Example 3, Comparative Examples 3-1 to 3-3, It is a map of C (pCMV-d2EGFR) and D (pMycLH). FIG. 4 is a diagram showing the steps of the selection method of Example 2 and Comparative Example 2 (top), and Example 3 and Comparative Examples 3-1 to 3-3 (bottom) (days after transformation and drugs used). It is. FIG. 5 is a photograph of a representative example of the gene amplification structure observed in Example 2 by FISH. FIG. 6 is a graph showing the results of formation frequency (right) and antibody protein expression level (left) measured by ELISA in Example 2 and Comparative Example 2 in cell populations by gene amplification structure observed by FISH. . In FIG. 6 (left), But (-) and But (+) are the case where Sodium Butyrate was not added and the case where it was added, respectively. FIG. 7 is a graph showing the results of d2EGFP protein expression distribution measured by FACS in Example 3 and Comparative Examples 3-1 to 3-3.

Hereinafter, examples will be shown to describe the present invention in more detail, but the present invention is not limited to the examples.
[Example 1]
Step 1: Preparation of a vector containing a polynucleotide encoding IR, MAR, DHFR expression genes and FcR (soluble human Fc receptor FcγRI) expression genes

  (1) pΔB. A multicloning site consisting of KpnI, AscI, SalI, SwaI, AsiSI, SbfI, BamHI, MluI was introduced between BamH1-MluI of AR1 (Non-patent Document 2), and pΔBM. AR1. It was set as MCS. The pΔBM. AR1. MCS was cut with restriction enzymes BamHI and NruI. Both cut ends were blunted with T4 DNA polymerase, further dephosphorylated with alkaline phosphatase, and pΔBM. It was set as AR1 / NBb *. The obtained pΔBM. In AR1 / NBb *, IR and MAR are both derived from the DHFR locus.

  (2) pECEFcRdhfr (patent document: Japanese Patent Application Laid-Open No. 2009-278948) was cut with a restriction enzyme BamHI, and a band containing a DHFR-FcR expression gene group was cut out by electrophoresis. The nucleotide was smoothed and phosphorylated with a BKL kit (manufactured by Takara Bio Inc.) to obtain dhfr-FcR.

  (3) pΔBM. The dhfr-FcR prepared in AR1 / NBb * and (2) was ligated using DNA Ligation Kit <Mighty Mix> (manufactured by Takara Bio Inc.) to prepare an FcR expression vector (pΔBM.AR1dhfr-FcR). .

  In the above examples, four elements of DHFR (dhfr) expression gene group, target protein (FcR) expression gene group, IR, and MAR are mounted on the same vector, but the produced vector pΔBM. The details of AR1dhfr-FcR are as shown in FIG. In FIG. 2, MAR (AR1 in the figure) is about 0.5 kb, IR derived from the DHFR locus (DHFR IR in the figure) is about 4.6 kb, dhfr expression gene group is about 1 kb, and FcR expression gene group is about kb. Although it is 1.4 kb, MAR and IR are about 100 b, and IR and dhfr-expressing genes are separated by about 50 b, so these four genes are about 7.5 kb in total length.

Step 2: pΔBM. Introduction of AR1dhfr-FcR into CHO cells (1) CHO cells cultured until 10% FBS (Fetal Bovine Serum: manufactured by JRH Biosciences) in MEM Alpha medium (Invitrogen) was grown to the logarithmic growth phase. The FcR expression vector (pΔBM.AR1dhfr-FcR) prepared in 1 was transfected using the electroporation system GenePulserXcell (manufactured by Bio-Rad) according to the attached document.

  (2) 10 mL of CHO cells transfected by the method of (1) with 10% dialyzed FBS (Fetal Bovine Serum: bioest) added to MEMAlpha medium (Invitrogen) without ribonucleoside and deoxyribonucleotide For 10 days.

Step 3: Isolation of FcR-expressing CHO cells (1) The CHO cells transfected in Step 2 are diluted to 1 cell / well with the medium used in Step 2- (2), and 96-well culture plate ( 200 microliters were dispensed into Falcon).

  (2) After culturing for 10 days, the expression level of FcR in the culture supernatant was measured by ELISA (Enzyme-Linked Immunosorbent Assay) using a microtiter plate in which human FcR was immobilized at 1 μg / well. Among them, the cell with the highest expression level and a single colony was selected and isolated.

Step 4: Gene amplification treatment of CHO cells with methotrexate and cell isolation The CHO cells isolated in Step 3 were cultured in the medium used in Step 2- (2) to the logarithmic growth phase, and then 5 × 10 ⁴ cells were placed in a 10 cm plate ( I went to Falcon). After 24 hours, the cells were cultured for 15 days in 10 mL of the medium used in Step 2- (2) containing 25 nM methotrexate (manufactured by Sigma). Surviving cells were isolated by the same method as in Step 3. Furthermore, the isolated CHO cells were subjected to gene amplification treatment and cell isolation using 125 nM methotrexate in the same manner as described above.

Step 5: Measurement of FcR expression level (1) The CHO cells isolated in Step 4 were cultured in the medium used in Step 2- (2) until the late logarithmic growth phase, and the medium was newly replaced. After culturing for 3 days, the supernatant was collected.

  (2) The amount of FcR secreted in the supernatant was measured by ELISA (Enzyme-Linked Immunosorbent Assay) using a microtiter plate in which human FcR was solid-phased at 1 μg / well.

[Comparative Example 1-1]
In Step 2- (1) above, as a negative control, the FcR expression vector (pECEFcRdhfr) not containing IR (DHFR) and MAR (AR1) used in Step 1- (2) was transferred in the same manner as in (1). The amount of FcR expression was measured in the same manner as in Example 1 except that the control was performed.

[Comparative Example 1-2]
In the above Step 4, FcR expression level was measured in the same manner as in Example 1 except that the culture was performed in 10 mL of the medium used in Step 2- (2) not containing 25 nM methotrexate for 15 days.

Results The results of Example 1 and Comparative Examples 1-1 to 1-2 are shown in Table 1 and FIG. The values shown in Table 1 indicate the expression level of FcR expressed in the culture supernatant by steps 1 to 5. The graph of FIG. 1 shows the expression level of FcR expressed in the culture supernatant in steps 1 to 5 for Example 1 and Comparative Example 1-1.

  It can be seen that FcR was produced in large quantities in cells (Examples) in which IR, MAR and FcR-expressing genes were simultaneously introduced into animal cells and the cells were subjected to gene amplification treatment with methotrexate. On the other hand, it can be seen that the cells in which IR and MAR are not introduced simultaneously with the FcR-expressing gene (Comparative Example 1-1) have a low expression level comparable to that of the target protein expressed by animal cells by the conventional method. On the other hand, in cells (Comparative Example 1-2) in which IR and MAR were introduced at the same time as the FcR-expressing gene and not subjected to gene amplification treatment with methotrexate, the expression level of FcR was higher than that in Comparative Example 1 above. However, the expression level was significantly lower than in the above examples. From the above, it can be seen that the target protein can be produced in large quantities by simultaneously introducing IR and MAR and the target protein expression gene into animal cells and subjecting the cells to gene amplification treatment with methotrexate.

[Example 2] (Examination of antibody expression: IR / MAR-DHFR / MTX fusion method)
DHFR (dhfr) protein expression gene group shown in FIG. 3, plasmid A (pΔBN AR1 P _sv40 -dhfr) containing IR and MAR (AR1) derived from DHFR locus, and target protein (anti-c-myc antibody L chain) CHO-DG44 cells were transduced with Lipofectamine 2000 (Invitrogen) using plasmid D (pMycLH) containing the MycL and MycH) expressed genes. Subsequently, as shown in FIG. 4 (Example 2), selection was performed with 0.5 mg / ml G418 and 10 μg / ml Blasticidine (1d), and 5 nM methotrexate (11d), 50 nM methotrexate (28d) and 500 nM. Selection was performed with methotrexate (40d). Chromosome specimens were prepared from the cells of each step, FISH was performed using the introduced plasmid full-length DNA as a probe, and the formation frequency for each gene amplification structure was investigated. At the same time, the cells at each step were collected, planted in a 96-well plate at 5 × 10 ⁵ cells / mL, and when sodium butyrate was added, the amount of antibody protein expression in the culture supernatant after 3 days for each case without addition of sodium butyrate Was determined by ELISA.

  Plasmid A is a plasmid prepared by removing the BamHI / NruI fragment (including the hygromycin resistance gene) of pDBN.AR1 and inserting the SV40promoter amplified by PCR and the DHFRcoding sequence at that position. A blastcidin resistance gene (Bsr) and its promoter (SR αPromoter) for selection with saidin are also included. On the other hand, plasmid D also contains a kanamycin resistance gene (Kan) for selection using G418. These base sequences are known.

[Comparative Example 2] (Examination of antibody expression: DHFR / MTX single method)
Instead of the plasmid A, shown in FIG. 3, and change DHFR (dhfr) expression gene plasmid containing no IR and MAR although containing group _{B (pSFV-V P sv40 -dhfr} ) to use, also the selection, FIG. As shown in 4 (Comparative Example 2), the same as Example 2 except that 0.5 mg / ml G418 and 10 μg / ml Blasticidine (1d), 5 nM methotrexate (17d) and 50 nM methotrexate (50d) were used. According to the procedure described above, the frequency of formation for each gene amplification structure by FISH and the amount of antibody protein expression by ELISA were examined.

  Plasmid B is a plasmid prepared by removing the BamHI / NruI fragment (including the hygromycin resistance gene) of pSFV-V and inserting the SV40 promoter and DHFRcoding sequence amplified by PCR at that position. A saidine resistance gene (Bsr) and its promoter (SR αPromoter) are also included. These base sequences are known.

Results A photograph of a representative example of the gene amplification structure observed by FISH in Example 2 and Comparative Example 2 above is shown in FIG. 5, and the results of formation frequency (FISH) and antibody protein expression level (ELISA) by gene amplification structure are shown. Shown on the right and left of FIG. Compared with the conventional method (DHFR / MTX single method), the method of the present invention (IR / MAR-DHFR / MTX fusion method) formed a faster and longer point ladder HSR. Correspondingly, higher and higher levels of antibody production were detected. The antibody production level could be further improved by adding Sodium Butyrate.

[Example 3] (Examination of d2EGFP expression: IR / MAR-DHFR / MTX fusion method)
As shown in FIG. 3, DHFR (dhfr) expression gene group, plasmid A (pΔBN AR1 P _sv40 -dhfr) containing IR (DHFR) and MAR (AR1), and plasmid C (pCMV-) containing the target protein (d2EGFP) expression gene group CHO-DG44 cells were transduced with Lipofectamine 2000 using d2EGFR, N. Shimizu, et al. (2007) J. Cell. Biochem., vol. 102, p515-529). Subsequently, as shown in FIG. 4 (Example 3), 10 μg / ml Blasticidine was selected (1d), and 5 nM methotrexate and 100 μg / ml Blasticidine (11d), 50 nM methotrexate (28d) and 500 nM Selection was performed with methotrexate (56d). Thereafter, the d2EGFP protein expression distribution of the cultured cell population was measured using a FACS (fluorescent-activated cell sorter).

[Comparative Example 3-1] (Examination of d2EGFP expression: IR / MAR single method)
Examples except that the selection of transduced CHO-DG44 cells was changed to 10 μg / ml Blasticidine (1d) and 100 μg / ml Blasticidine (11d) as shown in FIG. 4 (Comparative Example 3-1). According to the procedure similar to 2, d2EGFP protein expression distribution was measured using FACS.

[Comparative Example 3-2] (Examination of d2EGFP expression: DHFR / MTX single method)
Instead of the plasmid A, shown in FIG. 3, but including DHFR (dhfr) expression genes were modified to use a plasmid B containing no IR and _{MAR (pSFV-V P sv40 -dhfr} ), also transduced CHO -Selection of DG44 cells, as shown in FIG. 4 (Comparative Example 3-2), 10 μg / ml Blasticidine (1d), 5 nM methotrexate and 100 μg / ml Blasticidine (17d), 50 nM methotrexate (39d) and Except for changing to 500 nM methotrexate (53d), the d2EGFP protein expression distribution was measured using FACS by the same procedure as in Example 3.

[Comparative Example 3-3] (Examination of d2EGFP expression: normal method)
Instead of the plasmid A, shown in FIG. 3, but including DHFR (dhfr) expression genes were modified to use a plasmid B containing no IR and _{MAR (pSFV-V P sv40 -dhfr} ), also transduced CHO -Procedure similar to Example 3 except that the selection of DG44 cells was changed to 10 μg / ml Blasticidine (1d) and 100 μg / ml Blasticidine (15d) as shown in FIG. 4 (Comparative Example 3-3). The d2EGFP protein expression distribution was measured using FACS.

Results The results of d2EGFP protein expression distribution measured by FACS in Example 3 and Comparative Examples 3-1 to 3-3 are shown in FIG. In the method of the present invention (IR / MAR-DHFR / MTX fusion method) compared to the conventional method (IR / MAR single method, DHFR / MTX single method, normal method), cells with a high expression level of d2EGFP protein It has been shown to appear more frequently in the middle.

Claims (9)

Introducing a single vector containing a mammalian replication initiation region, a nuclear matrix-binding region, a dihydrofolate reductase expression gene group and a target protein expression gene group into a mammalian cell in the medium, and transforming the medium; and A method for producing a mammalian cell, comprising a step of treating the transformed mammalian cell with methotrexate.   The method for producing a mammalian cell according to claim 1, wherein the step of treating with methotrexate includes a plurality of steps of culturing the mammalian cell in a medium containing methotrexate while increasing the concentration stepwise.   The step of treating with methotrexate comprises at least a first step of culturing the mammalian cells in a medium containing methotrexate at a concentration of 5 to 25 nM, and a medium containing methotrexate at a concentration 5 to 10 times that of the first step. The method for producing a mammalian cell according to claim 1 or 2, comprising a second step of culturing the mammalian cell therein. The mammalian replication initiation region is derived from a c-myc locus origin of replication, a dihydrofolate reductase locus origin of replication, or a β-globin locus origin of replication. Of producing mammalian cells. The mammalian cell according to any one of claims 1 to 4, wherein the nuclear matrix binding region is derived from an Igκ locus matrix binding region, an SV40 initial region matrix binding region, or a dihydrofolate reductase matrix binding region. Manufacturing method.   The method for producing a mammalian cell according to any one of claims 1 to 5, wherein the target protein is a receptor, an antibody or a fluorescent protein. The method for producing a mammalian cell according to any one of claims 1 to 6, comprising the steps (1) to (4) performed in the following order.
(1) introducing a mammalian replication initiation region, a nuclear matrix binding region, a dihydrofolate reductase expression gene group and a target protein expression gene group into animal cells in a medium;
(2) culturing mammalian cells that have undergone the introduction step, and selecting and isolating cells expressing the target protein;
(3) transferring the cells that have undergone the isolation step to a medium containing 5 to 25 nM methotrexate, and selecting viable cells; and
(4) A step of transferring the cells that have undergone the selection step to a medium containing 125 to 500 nM methotrexate, and selecting the cells in which the gene has been amplified.   A method for producing a target protein in a medium using mammalian cells obtained by the production method according to claim 1.   A single vector comprising a mammalian replication initiation region, a nuclear matrix binding region, a dihydrofolate reductase expression gene group and a target protein expression gene group.