JP6005421B2 - Expression vector for the establishment of highly productive cells containing drug-selective fusion genes - Google Patents

Description

  The present invention relates to an expression vector for efficiently establishing a cell that expresses a gene of a target protein at a high level from among transformed cells, and a transformed highly productive cell. The expression vector of the present invention is useful for producing useful proteins such as pharmaceuticals in animal cells, particularly mammalian animal cells, by genetic engineering techniques.

  As a method for establishing a cell that produces a recombinant protein, generally, a gene construct that expresses a gene of a target protein is introduced into a host cell, and the gene construct is converted into the genome of the host cell from the obtained transformed cells. The cells stably introduced above are selected. At this time, a drug resistance gene serving as a drug selection marker is inserted into the gene construct so as to be expressed under the same promoter as the target protein gene or under a separate promoter, and the cells that survived the drug selection are inserted into the target protein. The cells are selected as stably transfected cells. The present inventors invented an mRNA destabilizing sequence that attenuates the expression of a drug selection marker, and succeeded in dramatically improving the drug selection effect (Patent Document 1).

  Furthermore, in order to reduce the decrease in expression due to the position effect of the genome introduction site, the present inventors have invented a gene expression stabilizing element and were able to maintain high expression for a long time. Furthermore, by combining the mRNA destabilizing sequence and the gene expression stabilizing element, the synergistic effect enables efficient selection of cells that highly express the target gene and stable maintenance of those highly expressing cells. (Patent Document 2).

Japanese Patent No. 4491808 Japanese Patent No. 4525863 International Publication No. 2010/023787

Bakheet T, Frevel M, Williams B. et al. R. G, Greer W, Khabar K .; S. A. (2001) Nucleic Acids Research 29: 246-254. Lagnado C.I. A, Brown C.I. L, Goodall G.G. J. et al. (1994) Molecular and Cellular Biology 14: 7984-7995. Zubiaga A.I. M, Belasco J.M. G, Greenberg M.M. E. (1995) Molecular and Cellular Biology 15: 2219-2230

  An object of the present invention is to provide an expression vector and a highly productive cell that are effective for rapidly and efficiently establishing a recombinant protein-producing cell in an animal cell, particularly a mammalian animal cell. is there.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors combined a drug selection marker gene containing an mRNA destabilizing sequence and an expression vector having a gene expression stabilizing element with a system for inducing gene amplification. Thus, it has been found that the production amount of the target protein is dramatically improved by these synergistic effects, and the present invention has been achieved.

  The feature of the present invention is that the drug selection marker gene and the dihydrofolate reductase gene expression cassette are not independently present, but the drug selection marker gene and the dihydrofolate reductase gene are expressed in one expression cassette. That is.

  That is, the following invention is provided.

[1] Expression vector [1-1]
An expression vector having at least the following (1) to (3).
(1) an expression cassette comprising a drug selection marker gene and a dihydrofolate reductase gene present adjacent to the drug selection marker gene,
(2) Gene expression stabilizing element, and (3) Target protein gene expression cassette [1-2]
An expression vector comprising at least the following (1) to (3) (1) a drug selection marker gene, an expression cassette comprising a dihydrofolate reductase gene and an mRNA destabilizing sequence present adjacent to the drug selection marker gene,
(2) gene expression stabilizing element, and (3) target protein gene expression cassette [1-3]
The expression vector according to [1-2], wherein the mRNA destabilizing sequence is derived from an AT-rich sequence present in the 3 ′ untranslated region of a cytokine, interleukin, or proto-oncogene.
[1-4]
The expression vector according to [1-2], wherein the mRNA destabilizing sequence has a motif sequence of TTATTTA (A / T) (A / T).
[1-5]
The expression vector according to [1-4], wherein the motif sequence is repeated twice or more.
[1-6]
[1-5] The expression vector according to [1-5], comprising a spacer sequence of one or more bases between repeated motif sequences.
[1-7]
The expression vector according to any one of [1-3] to [1-6], wherein the mRNA destabilizing sequence contains substitution, insertion or deletion of 1 to several bases.
[1-8]
The expression vector according to any one of [1-1] to [1-7], wherein the gene expression stabilizing element is any of the following.
(I) DNA consisting of the sequence from 41601 to 46746 in the sequence shown in SEQ ID NO: 15 or a partial sequence of the sequence from 41601 to 46746, comprising at least the sequence from 41601 to 42700 DNA
(II) a DNA comprising a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the sequence (I) and has a function of stabilizing gene expression
(III) DNA comprising a base sequence complementary to the sequence (I) or (II)
[1-9]
The expression vector according to any of [1-1] to [1-8], comprising two or more gene expression stabilizing elements.
[1-10]
The expression vector according to any of [1-1] to [1-9], wherein the drug selection marker gene is a protein synthesis inhibitory antibiotic resistance gene.
[1-11]
The expression vector according to [1-10], wherein the drug selection marker gene is selected from the group consisting of puromycin-N-acetyltransferase, hygromycin-B-phosphotransferase, and neomycin phosphotransferase.
[1-12]
The expression vector according to any one of [1-1] to [1-11], wherein the gene expression cassette of the target protein has a multicloning site for inserting a desired target protein gene.
[1-13]
The expression vector according to any one of [1-1] to [1-12], wherein the target protein gene is an antibody heavy chain and / or light chain polypeptide gene.

[2] Expression cassette [2-1]
An expression cassette comprising a drug selection marker gene, a dihydrofolate reductase gene present adjacent to the drug selection marker gene and an mRNA destabilizing sequence.

[3] Method for producing high-producing cells [3-1]
A method for producing a high-producing cell, comprising the following steps.
(A) a step of transforming a host cell using the expression vector according to any of [1-1] to [1-13] (b) a step of culturing the transformed cell in the presence of a drug (c ) A step of culturing the transformed cells in the presence of methotrexate (MTX) (d) a step of isolating a cell line showing high expression.
[3-2]
A method for producing a high-producing cell, comprising the following steps.
(A) a step of transforming a host cell using the expression vector according to any of [1-1] to [1-13] (b) culturing the transformed cell in a hypoxanthine- and thymidine-free medium (C) culturing the transformed cells in the presence of methotrexate (MTX) (d) isolating a cell line showing high expression.
[3-3]
A method for producing a high-producing cell, comprising the following steps.
(A) a step of transforming a host cell with the expression vector according to any one of [1-1] to [1-13] (b) the transformed cell does not contain hypoxanthine and thymidine, And (c) culturing the transformed cells in the presence of methotrexate (MTX) (d) isolating a cell line exhibiting high expression.
[3-4]
[3-1] In the method according to any one of [3-3], (c) a step of culturing the transformed cell in the presence of methotrexate (MTX), and (d) a cell exhibiting high expression A method for producing high-producing cells, wherein the step of isolating the strain is repeated a plurality of times.
[3-5]
The method for producing a high-producing cell according to any one of [3-1] to [3-4], wherein the host cell is a cell derived from a mammalian cell.
[3-6]
The method for producing a high-producing cell according to any one of [3-1] to [3-4], wherein the host cell is a cell derived from Chinese hamster ovary (CHO).
[3-7]
The method for producing a high-producing cell according to any one of [3-1] to [3-4], wherein the host cell is a dihydrofolate reductase gene-deficient cell.
[3-8]
The method for producing a high-producing cell according to any one of [3-1] to [3-4], wherein the host cell is a serum-free acclimatized cell.

[4] High-producing cells [4-1]
High-producing cells obtained by the method according to any one of [3-1] to [3-7].

[5] Method for producing using high-producing cells [5-1]
A method for producing a polypeptide using the high-producing cells obtained by the method according to any one of [3-1] to [3-7].
[5-2]
[3-1] A method for producing an antibody using a high-producing cell obtained by the method according to any one of [3-7].
[5-3]
[3-1] A method for producing a vaccine using high-producing cells obtained by the method according to any one of [3-7].

  In the present invention, a drug is selected from transformed cells by drug selection, and a drug typified by methotrexate (MTX) is present in the cell culturing process to cause gene amplification, thereby producing a large number of gene copies in the host cell. Cell lines can be generated. However, when a plurality of expression cassettes are present in the expression vector, transcription interference occurs, the expression level of each expression cassette is reduced, and the function of each gene is not fully exhibited. This transcription interference also affects the expression of the target protein, leading to a decrease in protein productivity as a whole. Thus, in the present invention, the drug selection marker gene and the dihydrofolate reductase gene are placed adjacent to each other and expressed as a so-called fusion gene in one expression cassette, thereby reducing transcription interference and improving productivity.

  In another aspect of the present invention, since an mRNA destabilizing sequence is inserted into an expression cassette containing a drug selection marker gene and a dihydrofolate reductase gene, the conventional drug selection marker having no mRNA destabilizing sequence The expression of the drug selection marker is attenuated as compared with the gene expression cassette, and unless it is integrated into the high expression region in the host cell genome, it becomes difficult to survive the transformed cells in the presence of the drug. For this reason, only transformed cells in which the drug selection marker gene and the target protein gene are integrated in the high expression region in the host cell genome can survive by drug selection. In addition, with regard to gene amplification, the maximum effect can be obtained with a small amount of a drug such as MTX. As a result, high-expressing cells that express the target protein gene at a high level can be efficiently selected.

  By stabilizing the gene expression of the recombinant protein gene and maintaining high expression for a long time by reducing the influence of adjacent chromosomes and regulatory elements on the recombinant protein gene in the cell genome by the gene expression stabilizing element of the present invention Can do. Therefore, the present invention can quickly, easily and efficiently establish a high-producing cell line exhibiting a very high level of protein productivity by these synergistic effects.

That is, according to the present invention, the target protein gene is introduced into the high expression region of the host cell, the target protein gene introduced into the high expression region has a large number of gene copies, and the gene expression Can be established very quickly, easily, and efficiently.

It is a figure which shows construction of pPUR * N4. Hereinafter, in the figure, Pur is a puromycin resistance gene. In the figure, iPCR means Inverse PCR. It is a figure which shows construction | assembly of pEF1 (alpha) -KOD3G8HC-RE2. Hereinafter, Neo in the figures refers to a neomycin resistance gene. It is a figure which shows construction | assembly of pEF1 (alpha) -SNAP26m-Pur * N4 and pEF1 (alpha) -SNAP26m-Pur-RE2. It is a figure which shows construction | assembly of pEF1 (alpha) -KOD3G8HC-1.1k / 1.1k. It is a figure which shows construction | assembly of pEF1 (alpha) -SNAP26m-Pur * N4-1.1k / 1.1k, pEF1 (alpha) -SNAP26m-Pur-1.1k / 1.1k. It is a figure which shows the vector map of pEHX. It is a figure which shows the vector map of pELX. It is a figure which shows the vector map of pELX2. It is a figure which shows the vector map of pEHX-dhfr * N4. It is a figure which shows construction of pEHX-Puro * dhfr and pEHX-Puro * dhfr * N4. It is a figure which shows construction | assembly of pELH2-dhfr * N4 (KOD3G8). It is a figure which shows construction | assembly of pELH2-Puro * dhfr (KOD3G8) and pELH2-Puro * dhfr * N4 (KOD3G8). Each of the puromycin resistance gene and the dhfr gene includes an expression cassette to which an mRNA destabilizing sequence (N4 sequence) is added downstream (Pur · N4 + dhfr · N4), the puromycin resistance gene (upstream) and the dhfr gene (downstream) ) Containing a fusion protein gene expression cassette (Puro · dhfr), an expression in which an mRNA destabilizing sequence (N4 sequence) is added downstream of the fusion protein gene of puromycin resistance gene (upstream) and dhfr gene (downstream) It is the graph which plotted the antibody production amount computed by performing ELISA using the culture supernatant of a polyclonal about what contains a cassette (Puro * dhfr * N4). It is the graph which plotted the antibody production amount of each clone computed by performing ELISA using the culture supernatant of a 6-well plate. Each of the puromycin resistance gene and the dhfr gene includes an expression cassette to which an mRNA destabilizing sequence (N4 sequence) is added downstream (Pur · N4 + dhfr · N4), the puromycin resistance gene (upstream) and the dhfr gene (downstream) ) Containing a fusion protein gene expression cassette (Puro · dhfr), an expression in which an mRNA destabilizing sequence (N4 sequence) is added downstream of the fusion protein gene of puromycin resistance gene (upstream) and dhfr gene (downstream) The top 5 clones of production amount are plotted for those containing the cassette (Puro · dhfr · N4). It is the graph which plotted the antibody production amount in the polyclonal after the selective culture | cultivation by MTX computed by performing ELISA using the culture supernatant of a 6 well plate. Each of the puromycin resistance gene and the dhfr gene includes an expression cassette to which an mRNA destabilizing sequence (N4 sequence) is added downstream (Pur · N4 + dhfr · N4), the puromycin resistance gene (upstream) and the dhfr gene (downstream) ) Containing a fusion protein gene expression cassette (Puro · dhfr), an expression in which an mRNA destabilizing sequence (N4 sequence) is added downstream of the fusion protein gene of puromycin resistance gene (upstream) and dhfr gene (downstream) Plot the results of the top 5 production clones of the cell population obtained after selective culture with MTX for the top 10 production clones obtained by selection with puromycin for those containing cassettes (Puro, dhfr, N4) doing. It is a figure which shows construction | assembly of pEHX-dhfr * Puro * N4. It is a figure which shows construction | assembly of pELH2-dhfr * Puro * N4 (KOD3G8). Containing an expression cassette in which an mRNA destabilizing sequence (N4 sequence) is added downstream of a fusion protein gene of a puromycin resistance gene (upstream) and a dhfr gene (downstream) (Puro / dhfr / N4), dhfr gene (upstream) And a puromycin resistance gene (downstream) fusion protein gene containing an expression cassette with an mRNA destabilizing sequence (N4 sequence) added (dhfr, Puro, N4) It is the graph which plotted the antibody production amount computed by performing ELISA. About the one containing an expression cassette (dhfr · Puro · N4) containing an mRNA destabilizing sequence (N4 sequence) downstream of a fusion protein gene of dhfr gene (upstream) and puromycin resistance gene (downstream) It is the graph which performed ELISA using a culture supernatant, calculated the antibody production amount of each clone, and plotted the top 5 production amount clones. It is the graph which plotted the antibody production amount of the cell population of the polyclone after selection by MTX calculated by performing ELISA using the culture supernatant of a 6-well plate. Select with puromycin for an expression cassette (dhfr / Puro / N4) containing an mRNA destabilizing sequence (N4 sequence) downstream of a fusion protein gene of dhfr gene (upstream) and puromycin resistance gene (downstream) The results of the top 5 production yield clones of the cell population obtained after selective culture of the top 10 production yield clones by MTX are plotted. It is the graph which plotted the amount of antibody production per day of 1 cell of the high expression strain after selection with puromycin, and calculated by performing ELISA using the culture supernatant of a 6-well plate. Serum-free acclimation of an expression cassette (dhfr, Puro, N4) containing an mRNA destabilizing sequence (N4 sequence) added downstream of a fusion protein gene of dhfr gene (upstream) and puromycin resistance gene (downstream) The results of the top 5 production clones obtained by gene transfer into CHO / dhfr-cells and selection with puromycin are plotted. It is the graph which plotted the amount of antibody production per day of 1 cell of the cell population of the polyclonal cell after selection by MTX calculated by performing ELISA using the culture supernatant of a 6-well plate. Serum-free acclimation of an expression cassette (dhfr, Puro, N4) containing an mRNA destabilizing sequence (N4 sequence) added downstream of a fusion protein gene of dhfr gene (upstream) and puromycin resistance gene (downstream) The results of the top 5 production clones of the cell population obtained after selective culturing of the top 10 production yield clones obtained by gene transfer into CHO / dhfr-cells and selection with puromycin are plotted.

  The present invention is described in detail below.

  According to the first aspect of the present invention, (1) an expression cassette comprising a drug selection marker gene and a dihydrofolate reductase gene present adjacent to the drug selection marker gene, (2) a gene expression stabilizing element, and ( 3) An expression vector characterized by having at least a target protein gene expression cassette is provided.

  According to the second aspect of the present invention, (1) an expression cassette comprising a drug selection marker gene, a dihydrofolate reductase gene and an mRNA destabilizing sequence present adjacent to the drug selection marker gene, (2) gene expression An expression vector comprising at least a stabilizing element and (3) a target protein gene expression cassette is provided.

  In the present invention, the expression cassette refers to a unit of gene expression from a promoter to a gene coding sequence and a terminator sequence (polyadenylation signal). In addition, introns, spacer sequences, translation enhancing regions, and the like may be included. Therefore, for example, when referring to “target protein gene expression cassette”, it means that the gene coding sequence of the expression cassette is the coding sequence of the gene of the target protein.

(An expression cassette containing a drug selection marker gene and a dihydrofolate reductase gene present adjacent to the drug selection marker gene)

The expression cassette containing a drug selection marker gene and a dihydrofolate reductase gene present adjacent to the drug selection marker gene refers to an expression cassette containing the drug selection marker gene and the dihydrofolate reductase gene as gene coding sequences.

  Examples of the drug used for drug selection using the drug selection marker gene of the present invention include blastcidin, geneticin (G418), hygromycin (Hygromycin; Hyg), Puromycin (Pur) is exemplified. In addition, as the drug selection marker gene of the present invention, a gene exhibiting resistance to drugs generally used for drug selection is preferably used. When a protein synthesis inhibitor antibiotic is used as a drug, it is preferable to use a protein synthesis inhibitor antibiotic resistance gene. Although not particularly limited, as a neomycin resistance gene (also having a function as a geneticin resistance gene), a Tn5-derived neomycin phosphotransferase (Aminoglycide 3'-phosphotransferase) (Neo), a hygromycin resistance gene as E. coli. E. coli-derived hygromycin-B-phosphotransferase (Hph, described as Hyg in the examples and drawings), and puromycin resistance gene, Streptomyces-derived puromycin-N-acetyltransferase gene (pac, described as Pur in the examples and drawings) Examples of the blasticidin resistance gene include a Bacillus cereus-derived blasticidin resistance gene (bsr). Among these, puromycin-N-acetyltransferase, hygromycin-B-phosphotransferase, and neomycin phosphotransferase are preferable, more preferably puromycin-N-acetyltransferase, and hygromycin-B-phosphotransferase, and more preferably. Puromycin-N-acetyltransferase.

  Dihydrofolate reductase (DHFR / dhfr) is an essential enzyme for nucleic acid biosynthesis, and cells lacking the dhfr gene cannot grow in a medium containing no nucleic acid. When cells deficient in the dhfr gene are transformed with an expression vector having the dhfr gene, a transformant with a restored nucleic acid requirement can be obtained.

  It is known that duplication (amplification) of specific genes occurs selectively and stabilizes in the cell genome when an appropriate pressure is applied from the outside. When methotrexate (MTX), a DHFR competitive inhibitor, is added to cultured cells transformed with an expression vector having the dhfr gene, most cells die but some rarely survive. Such cells have an increased copy number of the dhfr gene and can synthesize large amounts of DHFR, so that DHFR, which is not inhibited by MTX, can create the tetrahydrofolate necessary for the cells to survive and grow. If selection is continued step by step, a mutant cell in which the dhfr gene, which usually has only one or several copies, is finally amplified 100 to 1000 times can be obtained. When gene amplification occurs in animal cells, not only the dhfr gene itself subjected to selective pressure but also other genes present in the vicinity of the gene are amplified simultaneously, and as a result, the target protein gene is also amplified simultaneously.

  A known dhfr gene in the present invention can be used, and its origin is not limited, but a mouse-derived dhfr gene is preferred. The dhfr gene can be obtained from a commercially available vector, such as pSV2-dhfr (ATCC 37146) and pOptiVEC-TOPO (registered trademark) (Invitrogen), but can also be obtained by artificial synthesis.

The phrase “the drug selection marker gene and the dhfr gene are adjacent to each other” means that the drug selection marker gene and the dhfr gene are expressed as a fusion protein, and can be achieved by deleting the stop codon of the upstream gene. . Between the drug selection marker gene and the dhfr gene, for example, a sequence having substantially no function such as a glycine / serine linker may be present, or may be directly connected without the sequence. It is also preferable to remove the ATG sequence encoding the initiation codon methionine from the downstream gene.

The arrangement order of the drug selection marker gene and the dfhr gene may be any.

  The promoter in the expression cassette containing the drug selection marker gene of the present invention and the dihydrofolate reductase gene present adjacent to the drug selection marker gene is particularly limited as long as it is a promoter that can be expressed in animal cells, particularly mammalian cells. Not derived from viruses such as human and mouse cytomegalovirus (CMV) promoter, simian virus 40 (SV40) promoter, human herpes simplex virus thymidine kinase gene (HSV-tk) promoter, mouse Examples include promoters derived from non-viral cellular genes such as phosphoglycerate-kinase 1 gene (PGK) promoter, and hybrids of different promoters. Here, the promoter is not limited to the core region of the promoter, and may include an enhancer region. In order to attenuate the expression of the drug selection marker, those having low transcription activity are more preferred. A mutant promoter or the like may be used as a promoter with low transcription activity, or a Kozak sequence may be substituted. Furthermore, it is also a possible embodiment to reduce the number of repetitions of the motif sequence of the mRNA destabilizing sequence and to use a promoter with a weak transcription activity.

  The polyadenylation signal (terminator sequence) in the expression cassette containing the drug selection marker gene of the present invention and the dihydrofolate reductase gene present adjacent to the drug selection marker gene includes SV40 virus-derived late polyA signal, early polyA signal, HSV-tk-derived polyA signal, bovine growth factor gene-derived polyA signal, rabbit β-globin gene-derived polyA signal and the like are exemplified, but are not particularly limited.

  The arrangement order of the promoter, the drug selection marker gene and the dhfr gene, and the polyadenylation signal in the expression cassette containing the drug selection marker gene of the present invention and the dihydrofolate reductase gene present adjacent to the drug selection marker gene is as follows: The expression is not particularly limited as long as the drug selection marker gene and the dhfr gene can be expressed. Generally, the promoter, the drug selection marker gene and the dfhr gene, and the polyadenylation signal are arranged in this order from upstream to downstream. These three elements do not need to be directly linked to each other, but may optionally have an intron, a spacer sequence, a translation enhancing region, etc. between them.

(Expression cassette containing drug selection marker gene, dihydrofolate reductase gene and mRNA destabilizing sequence present adjacent to drug selection marker gene)
The expression cassette used in the present invention is the same as the expression cassette containing the drug selection marker gene and the dihydrofolate reductase gene existing adjacent to the drug selection marker gene except that it has an mRNA destabilizing sequence. It has a configuration. Therefore, the mRNA destabilizing sequence will be described.

  An “mRNA destabilizing sequence” is a nucleotide sequence that has a function of reducing the intracellular half-life of mRNA transcribed from DNA having this sequence, and is found to exist in early response genes in nature. Has been issued.

  The mRNA destabilizing sequence is present in the coding sequence of some genes or in the 5'UTR (untranslated region), but most is present in the 3'UTR. Examples of mRNA destabilizing sequences present in the 3 ′ UTR include AU-rich element (ARE), histone mRNA 3′-terminal stem-loop, iron-responsible element (IRE), and insulin-like growth factor II (IGF-II). ), Long stem-loop, and the like are known. Among these, ARE is preferable as an mRNA destabilizing sequence used in the present invention because ARE can destabilize mRNA constantly.

  ARE means a sequence or region containing a high percentage of “AU-rich elements” in mRNA, ie, adenine (A) and uracil (U). ARE is also used to indicate an “AT-rich element” in the DNA encoding the element, that is, a sequence or region containing a high percentage of adenine (A) and thymine (T).

  ARE is found in hematopoietic cell growth factor genes, growth factor genes, interleukin genes, cytokine genes such as interferon, and some proto-oncogenes (Non-patent Document 1). In the expression cassette of the present invention, the nucleic acid sequence corresponding to ARE in these genes may be used as it is, but TATTTTAT (Non-patent Document 2) and TTATTTA (T / A) (known as motif sequences in ARE) ( T / A) (Non-patent Document 3) and the like can avoid inserting unnecessary restriction enzyme recognition sequences into an expression vector, and is more convenient.

  Examples of genes having an ARE include: Granulocyte-monocyte colony simplification factor (GM-CSF) for hematopoietic cell growth factor, Interleukin-1β, 2, 3, 4, 6, 8, 10, 11, interferon for interleukin Is exemplified by Interferon-α and c-fos, c-myc, c-jun, c-myb, Pim-1, etc. as proto-oncogenes. In addition to this, many genes such as Tumor necrosis factor, Cyclin D1, Cyclooxygenase, Plasminogen activator inhibitor type 2 are known to have ARE, and are not limited to the exemplified genes.

Examples of mRNA destabilizing sequences that can be used in the present invention include the following.
One or more repeats of the ATTT motif sequence.
One or more repetitions of the ATTTA motif sequence.
One or more repetitions of the TATTTTAT motif sequence.
TTATTTA (T / A) (T / A) motif sequence repeated 1 to 2 times or more.
(T / A) is either T or A.

  These sequences may contain substitution / insertion / deletion of 1 to several bases, and those due to natural mutation such as DNA replication error and mutation, and those due to artificial mutation introduction are assumed. In addition, a spacer sequence or linker sequence of about 1 to 100 bases may be included between repeated motif sequences. In addition, an inversion of the motif sequence may be included.

  The number of repeats of these motif sequences may be one, but by making it two or more, more preferably four or more, the effect of destabilizing mRNA can be further enhanced, and the selection efficiency is remarkably increased. be able to.

  The upper limit of the number of repetitions is not particularly limited, but it is desirable that the expression vector has a maximum length of 10 kbp to 25 kbp from the viewpoint of vector incorporation into cells. However, as the number of repetitive sequences increases, the effect of destabilization of mRNA tends to be saturated, and when the number of repetitive numbers exceeds a certain number of repeats, no more remarkable effect can be expected. Although an upper limit is not defined, a repeated arrangement of 10 times or more is not practically meaningful.

  Promoter, drug selection marker gene, dhfr gene, mRNA destabilization in expression cassette containing drug selection marker gene of the present invention, dihydrofolate reductase gene and mRNA destabilizing sequence present adjacent to drug selection marker gene The sequence and the arrangement order of the polyadenylation signals are not particularly limited as long as the drug selection marker gene and the dhfr gene can be expressed. Generally, the promoter, the drug selection marker gene and the dfhr gene, an mRNA destabilizing sequence, In addition, the polyadenylation signals are arranged from upstream to downstream in this order. These four elements do not need to be directly linked to each other, but may optionally have an intron, a spacer sequence, a translation enhancing region, etc. between them.

(Gene expression stabilizing element)
The “gene expression stabilizing element” in the present invention eliminates the positional effect that the target protein gene expression cassette is affected by the transcriptional activation status in the vicinity of the site where the target protein gene expression cassette is inserted into the host genome, It means a nucleic acid region having a function of stabilizing gene expression.

  Nucleic acid sequences that have the function of stabilizing gene expression include insulators, scaffold / matrix binding region (S / MAR), locus control region (LCR), ubiquitous chromatin opening element (UCOE), etc. it can.

  Examples of the insulator include 1.2 kb DNaseI Hypersensitive site (cHS4) derived from chicken β-globin LCR, UR1 derived from Bafun sea urchin. Examples of S / MAR include chicken lysozyme 5 'MAR element, human β-globin MAR element and human interferon βSAR element.

  As a gene construct for introducing a target gene into a host cell genome, a plasmid vector is generally used. Since the maximum length of a plasmid vector is 10 to 25 kbp, the chain length of a gene expression stabilizing element is as short as possible. Is preferred. The preferred length is 5 kbp or less, more preferably about 1 kbp.

  In the embodiment of the present invention, any of the following (a) to (e) or a combination thereof can be used as the gene expression stabilizing element.

(A) DNA consisting of the sequence represented by SEQ ID NO: 15;
(B) a DNA comprising a partial sequence of the sequence represented by SEQ ID NO: 15 and comprising at least the sequence of the region represented by the 41820th to 41839th bases of the sequence represented by SEQ ID NO: 15;
(C) DNA comprising a partial sequence of the sequence represented by SEQ ID NO: 15 and comprising at least the sequence of the region represented by the bases from 41821 to 41840 in the sequence represented by SEQ ID NO: 15;
(D) a DNA comprising a partial sequence of the sequence represented by SEQ ID NO: 15, wherein the DNA comprises at least the sequence of the region represented by the 45182nd to 45200th bases of the sequence represented by SEQ ID NO: 15; and (e) the sequence A DNA comprising a partial sequence of the sequence represented by No. 15 and comprising at least the sequence of the region represented by the 91094th to 91113th bases of the sequence represented by SEQ ID No. 15.

  In a preferred embodiment of the gene expression stabilizing element of the present invention, the DNA of (b) or (c) is a region represented by the base sequence from the 41601st position to the 46746th of the sequence represented by SEQ ID NO: 15, or SEQ ID NO: 15 Is a base sequence region that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the 41601st to 46746th sequences and has a function of stabilizing gene expression. Also included are base sequences complementary to DNA consisting of these regions. In a further preferred embodiment of the gene expression stabilizing element of the present invention, the DNA of (b) or (c) is (1) DNA consisting of sequences from 41601 to 46746 in the sequence shown in SEQ ID NO: 15, or 41601 A DNA comprising a base sequence containing at least the positions 41601 to 42700, or a partial sequence of the sequence up to the 46746th, or a DNA comprising a base sequence complementary to the DNA shown in (2) (1) and a stringent DNA consisting of a base sequence that hybridizes under various conditions and has a function of stabilizing gene expression, or (3) DNA consisting of a base sequence complementary to DNA consisting of these regions.

  These elements were isolated and identified by the inventors from the CHO genome (CHO DR1000L-4N strain) (Patent Document 3), but elements homologous to these elements also exist in other mammalian cell genomes. Is expected to. Accordingly, (f) DNA consisting of a partial sequence of the sequence shown in SEQ ID NO: 15, and when placed in the host cell so as to be close to the foreign gene expression cassette, from the foreign gene contained in the foreign gene expression cassette DNA capable of increasing or stabilizing the expression of the target recombinant protein can also be used as a gene expression stabilizing element. Such homologous elements can be readily isolated and identified by well-known techniques in the art, such as interspecies hybridization or PCR.

  In addition, (g) DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to any of the DNAs of (a) to (f) and has a gene expression stabilizing function is naturally also a gene. It can be used as an expression stabilizing element. The gene expression stabilizing element of the present invention consists of any one of these (a) to (g) or any combination thereof. Furthermore, (h) DNA having a base sequence complementary to any of the DNAs of (a) to (g) also has a gene expression stabilizing function and can be used as a gene expression stabilizing element. Are included in the present invention.

  The stringent conditions differ depending on the probe and labeling method used, but, for example, 40-50 ° C. in 0.2 × SSC containing 0.1% SDS or 55 in 2 × SSC containing 0.1% SDS. It is a base sequence that hybridizes under a condition of ˜65 ° C. Furthermore, stringent conditions can be determined based on the melting temperature (Tm value) of the nucleic acid to be bound. Moreover, as washing conditions after hybridization, conditions such as “6 × SSC, 0.1% SDS, a temperature about 15 to 30 ° C. lower than the Tm value” can be given. Higher stringent conditions include “2 × SSC, 0.1% SDS, a temperature about 5 to 15 ° C. lower than the Tm value”, and higher stringent conditions include “1 × SSC, 0.1%. A cleaning condition of about “temperature lower by about 5 to 15 ° C. than SDS and Tm values” can be given. As a more stringent condition, a cleaning condition of about “0.1 × SSC, 0.1% SDS” is exemplified.

  In order for the gene expression stabilizing element of the present invention to exert its expression stabilizing effect, it is necessary that the element is arranged in the host cell genome at a position close to the introduction site of the target protein gene expression cassette. In the present invention, the term “adjacent” is not particularly limited, but preferably the distance between the gene expression stabilizing element of the present invention and the target protein gene expression cassette is 5000 bp or less, more preferably 500 bp. It means the following.

  Such close arrangement is achieved by introducing a mixture of a nucleic acid sequence fragment containing the element of the present invention and a nucleic acid sequence fragment containing an expression cassette of a foreign gene into a host cell by electroporation or transfection, etc. Although it can be realized by selecting a host cell clone with a high expression level, in order to reliably achieve close placement, the gene expression stabilizing element of the present invention, and expression of a foreign gene placed close thereto It is desirable to prepare a foreign gene expression vector containing a cassette in advance and transform host cells with this expression vector.

(Target protein gene expression cassette)
The target protein gene expression cassette in the present invention comprises a promoter, a gene coding sequence, and a terminator sequence (polyadenylation signal). In addition, introns, spacer sequences, and translation enhancing regions may be included. The expression promoter for the target protein gene is preferably one having as high a transcriptional activity as possible. Those derived from viruses such as human and mouse cytomegalovirus (CMV) promoter, simian virus 40 (SV40) promoter, human and mouse, Includes CHO-derived Elongation Factor 1 (EF1α) gene, ubiquitin gene, β-actin gene and other promoters derived from non-viral cellular genes, and different promoter / enhancer hybrids such as CAG promoter.

  In the target protein gene expression cassette, a multicloning site comprising a plurality of restriction enzyme recognition sequences can be arranged instead of the coding sequence of the protein gene. When introducing cDNA or coding sequence of a foreign gene, cloning work is simple and preferable.

  Another embodiment is a target protein gene expression cassette, wherein the target protein is an antibody heavy and / or light chain polypeptide. In this case, a plurality of expression cassettes of the target protein gene may be arranged, or a plurality of polypeptide genes may be connected by IRES to form a single polycistronic expression cassette.

(Expression vector)
In the present invention, the expression vector is a vector used for genetic engineering. The expression vector can be a plasmid vector. However, the present invention is not limited thereto, and for example, viral vectors, cosmid vectors, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), and other non-plasmid vectors can be used.

  One embodiment of the expression vector in the present invention is an expression cassette comprising a target protein gene expression cassette, a drug selection marker gene and a dihydrofolate reductase gene existing adjacent to the drug selection marker gene, An expression vector in which a gene expression stabilizing element is arranged upstream or downstream. Preferably, it is an expression vector in which a gene expression stabilizing element is arranged upstream of an expression cassette containing a target protein gene expression cassette, a drug selection marker gene and a dihydrofolate reductase gene present adjacent to the drug selection marker gene. . In this case, either the target protein gene expression cassette and the drug selection marker gene and the expression cassette containing the dihydrofolate reductase gene existing adjacent to the drug selection marker gene may be upstream, but preferably the target protein gene An expression cassette is located upstream.

  In a preferred embodiment of the expression vector in the present invention, an expression cassette comprising a target protein gene expression cassette, a drug selection marker gene and a dihydrofolate reductase gene existing adjacent to the drug selection marker gene is arranged, It is a vector in which gene expression stabilizing elements are arranged downstream of these expression cassettes. The target protein gene expression cassette, the drug selection marker gene, and the expression cassette containing the dihydrofolate reductase gene existing adjacent to the drug selection marker gene may be in any order, but the target protein gene expression cassette is located upstream. Is preferred.

  The gene expression stabilizing element may be arranged between the target protein gene expression cassette and an expression cassette containing a drug selection marker gene and a dihydrofolate reductase gene existing adjacent to the drug selection marker gene.

  The gene expression stabilizing element, the target protein gene expression cassette, and the drug selection marker gene and the expression cassette containing the dihydrofolate reductase gene present adjacent to the drug selection marker gene may be adjacent, A spacer sequence or the like may be inserted.

  Two or more expression cassettes containing a drug selection marker gene and a dihydrofolate reductase gene present adjacent to the drug selection marker gene may be included in one vector. In this case, the same drug selection marker may be selected, or different drug selection markers may be selected. Moreover, each expression cassette may be arrange | positioned adjacently and may be arrange | positioned separately.

  When the target protein is composed of multiple polypeptides such as antibody heavy chain and light chain, multiple expression cassettes of the target protein gene may be arranged, or multiple polypeptide genes are connected by IRES, and polycistronic It may be in the form of one expression cassette.

  Further, a target protein can be produced by inserting a plurality of polypeptide expression cassettes into different vectors and introducing each vector into one host cell to produce a transformed cell. In this case, an expression cassette containing a gene expression stabilizing element and / or a drug selection marker gene and a dihydrofolate reductase gene existing adjacent to the drug selection marker gene can be arranged in each vector, and each drug selection is the same. A marker can be used, but using a different drug selection marker is advantageous and preferable from the viewpoint of efficiently obtaining cells in which each polypeptide is highly expressed.

    When producing an antibody, the heavy chain polypeptide gene and the light chain polypeptide gene of the antibody may be inserted into different vectors for expression, but the antibody heavy chain polypeptide gene and the light chain polypeptide gene are the same. When inserted into one vector and expressed, one type of drug resistance gene on the same vector is used, which reduces the difference in the number of transgenes and balances the production of antibody heavy and light chain polypeptides. This is preferable because the productivity as an antibody consisting of two heavy chain / light chain polypeptides is stabilized.

  In the third aspect of the present invention, only an expression cassette comprising a drug selection marker gene, a dihydrofolate reductase gene present adjacent to the drug selection marker gene and an mRNA destabilizing sequence is provided. This is not limited to the expression vector of the present invention and can be applied to any expression vector.

According to the 4th side surface of this invention, the method of producing a high production cell including the following processes is provided.
(A) a host cell using an expression vector comprising a drug selection marker gene and an expression cassette comprising a dihydrofolate reductase gene present adjacent to the drug selection marker gene, a gene expression stabilizing element, and a target protein gene expression cassette; A step of transforming (b) a step of culturing the transformed cell in the presence of a drug (c) a step of culturing the transformed cell in the presence of methotrexate (MTX) (d) a cell line exhibiting high expression Isolating.

Another embodiment of the aspect of the present invention is a method for producing a high-producing cell, comprising the following steps.
(A) a host cell using an expression vector comprising a drug selection marker gene and an expression cassette comprising a dihydrofolate reductase gene present adjacent to the drug selection marker gene, a gene expression stabilizing element, and a target protein gene expression cassette; Step of transforming (b) Step of culturing the transformed cell in hypoxanthine and thymidine-free medium (c) Step of culturing the transformed cell in the presence of methotrexate (MTX) (d) High expression Isolating the cell line shown.

Yet another embodiment of an aspect of the present invention is a method for producing a highly producing cell comprising the following steps.
(A) a host cell using an expression vector comprising a drug selection marker gene and an expression cassette comprising a dihydrofolate reductase gene present adjacent to the drug selection marker gene, a gene expression stabilizing element, and a target protein gene expression cassette; Transforming step (b) culturing the transformed cell without hypoxanthine and thymidine in the presence of a drug (d) culturing the transformed cell in the presence of methotrexate (MTX) (E) A step of isolating a cell line showing high expression.

Similar to the above embodiment, a method for producing a high-producing cell comprising the following steps is also provided.
(A) an expression vector comprising a drug selection marker gene, an expression cassette comprising a dihydrofolate reductase gene and an mRNA destabilizing sequence present adjacent to the drug selection marker gene, a gene expression stabilizing element, and a target protein gene expression cassette (B) a step of culturing the transformed cell in the presence of a drug (c) a step of culturing the transformed cell in the presence of methotrexate (MTX) (d) high Isolating cell lines exhibiting expression.

(A) an expression vector comprising a drug selection marker gene, an expression cassette comprising a dihydrofolate reductase gene and an mRNA destabilizing sequence present adjacent to the drug selection marker gene, a gene expression stabilizing element, and a target protein gene expression cassette (B) culturing the transformed cell in a hypoxanthine and thymidine-free medium (c) culturing the transformed cell in the presence of methotrexate (MTX) (D) A step of isolating a cell line showing high expression.

(A) an expression vector comprising a drug selection marker gene, an expression cassette comprising a dihydrofolate reductase gene and an mRNA destabilizing sequence present adjacent to the drug selection marker gene, a gene expression stabilizing element, and a target protein gene expression cassette (B) a step of culturing the transformed cell without hypoxanthine and thymidine and in the presence of a drug (d) transforming the transformed cell with methotrexate (MTX) Culturing in the presence (e) isolating a cell line exhibiting high expression.

  In the above embodiment, (c) the step of culturing transformed cells in the presence of methotrexate (MTX) and (d) the step of isolating a cell line exhibiting high expression can be repeated a plurality of times.

  As host cells, in addition to Chinese hamster ovary cells (CHO) generally used for the production of recombinant proteins, mouse myeloma cells (NSO), baby hamster kidney cells (BHK), human fibrosarcoma cells (HT1080), COS Although cells derived from mammals such as cells are exemplified, Chinese hamster ovary cells (CHO) are preferably used.

Even if the host cell is not a cell deficient in the dihydrofolate reductase gene, the effects of the present invention including gene amplification can be obtained, but only cells transformed with an expression vector having the dhfr gene can survive. The host cell is preferably a dhfr gene-deficient cell. Specific examples of dhfr gene-deficient cells include CHO DG44 cells, CHO DUKX-B11 cells, and CHO / dhfr- cells.

  In general, for industrial production of biopharmaceuticals, in order to eliminate contamination by harmful components such as viruses, the production cells are cultured in a medium that does not contain animal components, more preferably a chemically defined medium. It is required to do. For this reason, after screening high-producing cells with a medium containing fetal bovine serum or the like that is generally used, the cells are adapted to a medium that does not contain animal components or a medium that is chemically defined over about 1 to 2 months. (Serum-free acclimation) is required. However, there are cases where the highly productive cells obtained with great effort cannot be well adapted. Thus, introducing a gene into a cell that has been conditioned in advance without serum can be very effective because it eliminates the step of acclimation to serum and shortens the establishment time of production cells. In order to achieve such problems, the host cells used in the present invention are further exemplified by using serum-free conditioned cells such as CHO cells.

  A host cell transformation method can be appropriately selected by those skilled in the art, and examples thereof include a lipofection method, a calcium phosphate method, an electroporation method, a DEAE dextran method, and a microinjection method.

  In the step of culturing transformed cells in the presence of a drug, the drug used for drug selection is determined according to the type of drug selection marker gene. Moreover, as the concentration of the drug to be used, it is possible to concentrate highly expressing cells within the range of concentrations generally used, but a slightly higher concentration is preferable. The optimum concentration varies depending on the host cell and the type of medium used. These concentration setting methods are known to those skilled in the art and can be set as appropriate. For example, the cells were transformed with an expression vector having an expression cassette containing a puromycin resistance gene, a dihydrofolate reductase gene and an mRNA destabilizing sequence in this order under culture conditions in IMDM medium containing 10% fetal bovine serum. The concentration of puromycin for CHO / dhfr − cells is preferably 5 μg / ml or more, more preferably 7.5 μg / ml or more, and the upper limit is preferably 15 μg / ml or less. Similarly, the concentration of puromycin for CHO / dhfr-cells transformed with an expression vector having an expression cassette containing in this order a dihydrofolate reductase gene, a puromycin resistance gene and an mRNA destabilizing sequence is 5 μg / ml or more is preferable, 7.5 μg / ml or more is more preferable, and the upper limit is preferably 10 μg / ml or less.

In the selection method using this method, a cell transformed with an expression vector having an expression cassette containing a drug selection marker gene, a dihydrofolate reductase gene adjacent to the drug selection marker gene and an mRNA destabilizing sequence is used. Is a drug selection marker gene, and a drug selected by transforming an expression vector having an expression cassette containing the dihydrofolate reductase gene present adjacent to the drug selection marker gene and not containing the mRNA destabilizing sequence. Compared to the group, the proportion of cells expressing the target protein gene at a high level is significantly higher. According to the present invention, the number of samples that are candidates for selection can be greatly reduced only by drug selection including mRNA destabilization of the drug selection marker. By using the selection method of the present invention, the target protein production amount is significantly increased in the state of cell groups (polyclone) that survive even after drug selection, and by isolating and culturing cells from these cell groups A cell line (monoclone) showing high expression can be easily obtained. As an example of an embodiment of the present invention, 4.0 × 10 ⁵ cells contain the drug selection marker gene of the present invention, the dihydrofolate reductase gene and mRNA destabilizing sequence present adjacent to the drug selection marker gene After introducing a vector into which an expression cassette has been inserted, 100 cell lines are randomly selected from a cell group generated by drug selection, and the expression level of each cell line is examined to highly express the target protein. Stocks can be acquired easily.

  In some embodiments of the invention, culturing the transformed cells in a hypoxanthine and thymidine (HT) free medium is included. The dhfr gene-deficient cells require hypoxanthine and thymidine for cell proliferation, and cannot grow unless hypoxanthine and thymidine are added to the medium. Therefore, by successfully culturing in a hypoxanthine and thymidine-free medium, cells that have been successfully transformed with the expression vector containing the dhfr gene can be selected. The concentration of hypoxanthine and thymidine can be appropriately set by those skilled in the art.

  The present invention includes the step of culturing transformed cells in the presence of methotrexate (MTX). MTX is a competitive inhibitor of DHFR, and cells cannot grow unless MTX resistance is acquired. In general, animal cells acquire MTX resistance by gene amplification of the dhfr gene, and the applicants utilize that other genes existing in the vicinity of the dhfr gene are simultaneously amplified. The concentration of MTX is desirably used between 1 nM and 1000 nM. If the concentration is less than 1 nM, the effect of gene amplification is small, and if the concentration is higher than 1000 nM, cells are killed. In the step of culturing in the presence of MTX, culturing may be performed at a constant MTX concentration, or culturing may be first performed at a low MTX concentration, and then the concentration may be increased stepwise. Since gene amplification does not occur in all cells at the same time but in some cells at various timings, it is preferable to increase the concentration of MTX stepwise from a low concentration. When transformed cells are cultured at multi-stage MTX concentrations, if the host cells are suddenly exposed to high concentrations of MTX, more cells will die before gene amplification occurs. In addition, if the MTX concentration is rapidly increased, the frequency of appearance of cells that acquire MTX resistance by a mechanism other than gene amplification increases.

  The optimal MTX concentration and the method for increasing the optimal MTX concentration differ depending on the cell type, the drug selection marker gene type, the arrangement of the drug selection marker gene and the dihydrofolate reductase gene, and the presence or absence of an mRNA destabilizing sequence. When a host cell is transformed with an expression vector having a drug selection marker gene, a dihydrofolate reductase gene adjacent to the drug selection marker gene and an mRNA destabilizing sequence, the expression level of DHFR is not mRNA. It is attenuated by the stabilizing sequence. Since MTX resistance is also attenuated, it is transformed with an expression vector having an expression cassette comprising a drug selection marker gene having no mRNA destabilizing sequence and a dihydrofolate reductase gene present adjacent to the drug selection marker gene. The effect of gene amplification can be obtained with a low concentration of MTX compared to the cells. As an example, when a cell transformed with an expression vector having an expression cassette containing a puromycin resistance gene, a dihydrofolate reductase gene and an mRNA destabilizing sequence in this order is cultured, the MTX concentration is preferably 20 nM or less. When culturing at multi-stage MTX concentrations, for example, the first stage can be cultured at 4 nM, the second stage at 10 nM, and the third stage at 20 nM.

  The step of culturing in the presence of MTX is preferably performed after the step of culturing transformed cells in the presence of a drug. By selecting a drug, it is possible to obtain a group of cells that have been introduced into a high-expressing region of the cell. Therefore, if this step is performed after the step of culturing in the presence of a drug, gene amplification occurs in the high-expressing region. This is because gene expression can be expected more efficiently.

  The step of isolating a cell line exhibiting high production is performed by first obtaining a cell line from a cell group (polyclone), then examining the expression level of the cell line, and selecting a cell line exhibiting high production. As a method for obtaining a cell line from a cell group (polyclone), there is a method of limiting dilution of a polyclone. The limiting dilution is performed by, for example, performing trypsinization on a cell population in a polyclonal state to disperse the cells, and seeding and culturing the cells in a 96-well plate so that the number of cells becomes 1.0 / well or less. be able to. If necessary, those skilled in the art can change the dilution concentration (for example, a concentration of 3.0 cells / well or less), or can be cultured in a mixed medium of Conditioned Medium and a normal medium.

The expression level of the cells can be examined by the expression level of the reporter gene. As an example, an expression cassette containing a fluorescent protein gene such as a SNAP26m gene (Covalents) or a GFP (Green Fluorescent Protein) gene is introduced into the expression vector of the present invention, and a flow cytometer such as FACS (fluorescence activated cell sorting) is used. Thus, it can be examined by analyzing the fluorescence intensity. As another example, it can be examined by introducing an expression cassette containing a luciferase gene into the expression vector according to the present invention, adding D-luciferin to the cell lysate, and measuring the amount of luminescence with a luminometer. In addition to the reporter gene, expression of a target protein such as an antibody can also be examined by using ELISA (Enzyme-Linked Immunosorbent Assay), enzyme immunoassay (EIA), or the like.

  This step may be performed after the step of culturing the transformed cell in the presence of a drug, or after the step of culturing the transformed cell in the presence of methotrexate (MTX), or at any stage after both steps. You may go.

  The method for producing a high-producing cell in the fourth aspect of the present invention will be illustrated more specifically.

  The first example is performed according to the following procedure. (1) A dhfr gene-deficient host cell is transfected with an expression vector according to the present invention into which a target protein gene has been inserted. (2) The transformed cells are cultured in the presence of a drug (for example, geneticin). (3) Polyclone (cell group) showing high expression is selected and cultured in the presence of MTX. (4) Polyclone showing higher expression after culture in the presence of MTX is selected and isolated into a monoclone (cell line). (5) The expression level of each cell line is examined, and a cell line showing high expression is selected.

  The second example is performed according to the following procedure. (1) A dhfr gene-deficient host cell is transfected with an expression vector according to the present invention into which a target protein gene has been inserted. (2) The transformed cells are cultured in the presence of a drug (for example, geneticin). (3) Polyclone showing high expression level is selected and isolated to monoclone. (4) The expression level of each cell line is examined, a cell line showing high expression is selected, and cultured in the presence of MTX. (5) The cell population after selective culture with MTX is again limit diluted to isolate a cell line and cultured for a certain period. (6) The expression level of the cell line isolated in (5) is examined, and a cell line showing high expression is selected.

  The third example is performed according to the following procedure. (1) A dhfr gene-deficient host cell is transfected with an expression vector according to the present invention into which a target protein gene has been inserted. (2) The transformed cells are cultured in a hypoxanthine and thymidine (HT) -free medium. (3) A polyclone showing high expression under HT-free conditions is selected and cultured in the presence of MTX. (4) Polyclone showing higher expression after culture in the presence of MTX is selected and isolated to a monoclone. (5) The expression level of each monoclone is examined, and a cell line showing high expression is selected.

  The fourth example is performed according to the following procedure. (1) A dhfr gene-deficient host cell is transfected with an expression vector according to the present invention into which a target protein gene has been inserted. (2) The transformed cells are cultured in a medium not containing hypoxanthine and thymidine (HT) in the presence of a drug (for example, geneticin). (3) Polyclone showing high expression is selected and cultured in the presence of MTX. (4) Polyclone showing higher expression after culture in the presence of MTX is selected and isolated to a monoclone. (5) The expression level of each cell line is examined, and a cell line showing high expression is selected.

  The fifth example is performed according to the following procedure. (1) A dhfr gene-deficient host cell is transfected with an expression vector according to the present invention into which a target protein gene has been inserted. (2) The transformed cells are cultured in a medium not containing hypoxanthine and thymidine (HT) in the presence of a drug (for example, geneticin). (3) Polyclone showing high expression is selected and isolated to a monoclone. (4) A cell line showing high expression is selected and cultured in the presence of MTX. (5) The cell population after selective culture with MTX is again limit diluted to isolate a cell line and cultured for a certain period. (6) The expression level of the cell line isolated in (5) is examined, and a cell line showing high expression is selected.

  The sixth example is performed according to the following procedure. (1) A dhfr gene-deficient host cell is transfected with an expression vector according to the present invention into which a target protein gene has been inserted. (2) The transformed cells are cultured in a medium not containing hypoxanthine and thymidine (HT) in the presence of a drug (for example, geneticin). (3) Polyclone showing high expression is selected and isolated to a monoclone. (4) A cell line showing high expression is selected and cultured in the presence of MTX. (5) The cell population after selective culture with MTX is again limit diluted to isolate a cell line and cultured for a certain period. (6) The expression level of the cell line isolated in (5) is examined, and a cell line showing high expression is selected. (7) Incubate again in the presence of MTX for a certain period. (8) The expression level of the cell line isolated in (7) is examined, and a cell line showing high expression is selected.

  According to the present invention, the cell selection process and time can be greatly shortened, and the ratio of cells exhibiting high expression can be increased so that cells expressing a target protein gene at a high level can be efficiently expressed in a short time. It can be obtained and shows a very remarkable effect compared with the prior art.

  The fifth aspect of the present invention is a transformed cell obtained by the method according to the fourth aspect of the present invention.

  According to a sixth aspect of the present invention, there is provided a method for producing a protein, characterized by using a transformed cell obtained by the method according to the fourth aspect of the present invention.

  In one embodiment, the protein produced is an antibody. In this case, the heavy chain polypeptide gene and the light chain polypeptide gene of the antibody may be inserted into the same vector (single vector), or may be inserted into different vectors to form two constructs.

  There are five classes of mammalian antibodies, IgM, IgD, IgG, IgA, and IgE, but they have a long blood half-life for the diagnosis, prevention, and treatment of various human diseases. From the viewpoint of functional properties such as having various effector functions, antibodies of human IgG class are mainly used. The antibody can be purified from the culture supernatant of the transformed cell using a protein A column. In addition, a purification method usually used in protein purification can be used. For example, it can be purified by a combination of gel filtration, ion exchange chromatography, ultrafiltration and the like. The molecular weight of the purified humanized antibody H chain, L chain, or whole antibody molecule can be measured by two-dimensional electrophoresis or the like.

  The antibody produced by the method of the present invention can be used as a medicament. Such medicaments can be administered alone as therapeutic agents, but are usually mixed together with one or more pharmacologically acceptable carriers, and any known in the pharmaceutical arts. It is preferable to be provided as a pharmaceutical preparation produced by the method.

  It is desirable to use the most effective route for treatment, and oral administration or parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous can be used. In the case of a preparation, intravenous administration is preferable. Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

  Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as emulsions and syrups include saccharides such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoic acid Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used as additives. Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin and the like can be used as additives.

  Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. The injection is prepared using a carrier made of a salt solution, a glucose solution, or a mixture of both. Alternatively, a powdered injection can be prepared by lyophilizing the humanized antibody according to a conventional method and adding sodium chloride thereto. Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid. The spray is prepared using a carrier that does not irritate the compound itself or the recipient's oral cavity and airway mucosa, and that disperses the compound as fine particles to facilitate absorption.

  Specific examples of the carrier include lactose and glycerin. Depending on the properties of the compound and the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.

  In another embodiment, the protein produced is a vaccine.

  As a vaccine, a protein comprising an amino acid sequence of an epitope of a pathogen can be used as a vaccine. In addition to producing all the constituent proteins of the virus body, envelope proteins and the like can be produced and used as vaccines.

  The vaccine can be formulated for administration by any route. Examples include vaccine preparations for mucosal administration, such as oral, nasal, respiratory, vaginal and rectal routes, as well as non-mucosal routes such as subcutaneous, intravenous or intramuscular administration. .

  Hereinafter, the effect of the present invention will be further clarified by illustrating examples.

Example 1
Construction of various plasmid vectors Example 1-1
1. Vector having mRNA destabilizing sequence (1) Construction of pPUR · N4 According to the scheme shown in FIG. 1, the primers of SEQ ID NOs: 1 and 2 were added to the 3 ′ end of the coding sequence of the puromycin resistant gene of pPUR (Clontech) PPUR · N4 was constructed by inserting the ARE sequence motif TATTTTATT four times (hereinafter referred to as “N4”) by an inverse PCR method using KOD-Plus-Mutageness Kit (manufactured by Toyobo Co., Ltd.).

(2) Construction of pEF1α-SNAP26m-Pur · N4 and pEF1α-SNAP26m-Pur-RE2 Next, plasmids were constructed in which the promoter of the target gene expression cassette was replaced with the EF1α promoter. In this experiment, the plasmid pEF1α-KOD3G8HC-RE2 constructed according to the scheme shown in FIG. 2 was used. That is, first, the plasmid pEF1α-KOD3G8HC in which the heavy chain of the anti-KOD antibody was inserted into the restriction enzyme XbaI-NotI site of the pCI-neo plasmid (Promega) substituted for the CMV promoter with the EF-1α promoter was used as a template. -PEF1α-KOD3G8HC-RE was constructed by adding the restriction enzymes SalI and NheI sites upstream of the expression cassette using the primers of SEQ ID NOs: 3 and 4 by the Inverse PCR method using Plus-Mutageness Kit. Furthermore, a plasmid constructed by adding restriction enzymes BsiWI and XhoI sites downstream of the expression cassette using the primers of SEQ ID NOs: 5 and 6 by the inverse PCR method using KOD-Plus-Mutageness Kit using this plasmid as a template. pEF1α-KOD3G8HC-RE2.

  Using this pEF1α-KOD3G8HC-RE2, plasmids pEF1α-SNAP26m-Pur · N4 and pEF1α-SNAP26m-Pur-RE2 were constructed according to the scheme shown in FIG. That is, the heavy chain gene of the anti-KOD antibody was excised from the plasmid pEF1α-KOD3G8HC-RE2 with restriction enzymes EcoRI and NotI. On the other hand, the SNAP26m gene from the SNAPm expression plasmid pSNAPm was amplified by PCR using the primers of SEQ ID NOs: 7 and 8, and introduced into the restriction enzymes EcoRI and NotI sites of the pEF1α-KOD3G8HC-RE2 plasmid to introduce pEF1α-SNAP26m-Neo-RE2. It was constructed. A neomycin resistance gene was excised from this plasmid with restriction enzymes AflII and BstBI. On the other hand, the Puromycin resistance gene added with N4 sequence downstream from pPUR · N4 constructed in (1) above was amplified by PCR using the primers of SEQ ID NOs: 9 and 10, and the restriction enzyme AflII of pEF1α-SNAP26m-RE2 plasmid, It was introduced into the BstBI site to construct pEF1α-SNAP26m-Pur · N4. Subsequently, the plasmid pEF1α-SNAP26m-Pur-, in which the N4 sequence downstream of the Puromycin resistance gene was deleted using the primers of SEQ ID NOs: 11 and 12, by the inverse PCR method using KOD-Plus-Mutageness Kit, using this plasmid as a template. RE2 was constructed.

Example 1-2
Construction of vector (1) pEF1α-SNAP26m-Pur · N4-1.1k / 1.1k having mRNA destabilizing sequence and gene expression stabilizing element In this experiment, pEF1α-KOD3G8HC-1.1k described in FIG. /1.1k was used. That is, first, a test amplified by PCR using the primer set of SEQ ID NOs: 13 and 14 on the restriction enzyme NheI and BglII sites upstream of the expression cassette of pEF1α-KOD3G8HC-RE2 constructed in the middle of Example 1-1. The sequence pCHO1Δ-KOD3G8HC-1.1k was constructed by inserting the sequence CHO5Δ3-3 (base sequence consisting of the positions 41601 to 42700 of SEQ ID NO: 15; hereinafter referred to as 1.1k in the construct). Further, a plasmid constructed by inserting the test sequence CHO5Δ3-3 amplified by PCR using the primer set of SEQ ID NOs: 16 and 17 into the restriction enzyme BsiWI and XhoI sites downstream of the expression cassette of the plasmid pEF1α-KOD3G8HC-1.1k Is pEF1α-KOD3G8HC-1.1k / 1.1k.

  On the other hand, by the inverse PCR method using the plasmids pEF1α-SNAP26m-Pur · N4 and pEF1α-SNAP26m-Pur-RE2 constructed in Example 1-1 and the KOD-Plus-Mutageness Kit, SEQ ID NOS: 18, 19 Plasmids pEF1α-SNAP26m-Pur2 · N4 and pEF1α-SNAP26m-Pur2-RE2 from which the restriction enzyme BsiWI site present in the Puromycin resistance gene was deleted were constructed using primers.

  Subsequently, pEF1α-SNAP26m-Pur · N4-1.1k / 1.1k was constructed according to the scheme shown in FIG. That is, SNAPm-pA-SV40Promoter-Pur2 · N4-pA was excised from pEF1α-SNAP26m-Pur2 · N4 with restriction enzymes EcoRI and BsiWI, and restriction enzymes EcoRI and BsiWI sites of pEF1α-KOD3G8HC-1.1k / 1.1k PEF1α-SNAP26m-Pur · N4-1.1k / 1.1k was constructed.

Example 1-3
Vector in which restriction enzyme recognition sequence is deleted from each sequence element in expression vector (1) Deletion of restriction enzyme recognition sequence from gene expression stabilization element CHO5Δ3-3 Subsequently, restriction enzyme recognition sequence from gene expression stabilization element CHO5Δ3-3 Deleted. First, by using the PCR of pEF1α-KOD3G8HC-1.1k described in Example 1-2 as a template and the inverse PCR method using KOD-Plus-Mutageness Kit, using the primers of SEQ ID NOs: 22 and 23, CHO5Δ3-3 PEF1α-KOD3G8HC-1.1k-ΔRE was constructed by deleting the existing restriction enzyme SacI site. Next, the restriction enzyme SmaI present in CHO5Δ3-3 by using the PCR of pEF1α-KOD3G8HC-1.1k-ΔRE as a template and the PCR of KOD-Plus-Mutageness Kit using the primers of SEQ ID NOs: 24 and 25. PEF1α-KOD3G8HC-1.1k-ΔRE2 from which the site was deleted was constructed. Furthermore, the restriction enzyme BamHI site present in CHO5Δ3-3 by the inverse PCR method using pEF1α-KOD3G8HC-1.1k-ΔRE2 as a template and the KOD-Plus-Mutageness Kit using the primers of SEQ ID NOs: 26 and 27 PEF1α-KOD3G8HC-1.1k-ΔRE3 was constructed in which was deleted. Finally, the restriction enzyme HindIII present in CHO5Δ3-3 using the primers of SEQ ID NOs: 28 and 29 by the inverse PCR method using pEF1α-KOD3G8HC-1.1k-ΔRE3 as a template and KOD-Plus-Mutageness Kit. PEF1α-KOD3G8HC-1.1k-ΔRE4 from which the site was deleted was constructed.

(2) Construction of pEHX The gene expression stabilizing element CHO5Δ3-3 from which the restriction enzyme recognition sequence prepared in Example 1-3 (1) above was deleted is arranged upstream and downstream of the expression cassette. The pEHX described in FIG. 6 was constructed. Specifically, SV40 promoter using primers of SEQ ID NOS: 30 and 31 by the inverse PCR method using pEF1α-SNAP26m-Pur2 · N4 described in Example 1-2 as a template and KOD-Plus-Mutageness Kit. PEF1α-SNAP26m-Pur2 · N4-ΔH was constructed by deleting the restriction enzyme HindIII site existing downstream. Subsequently, the restriction enzyme SalI present in the Puromycin-resistant gene using the primers of SEQ ID NOs: 32 and 33 by the inverse PCR method using KEF-Plus-Mutageness Kit using pEF1α-SNAP26m-Pur2 · N4-ΔH as a template. PEF1α-SNAP26m-Pur3 · N4-ΔH from which the site was deleted was constructed. Furthermore, the restriction enzyme SmaI site existing in the Puromycin-resistant gene using the primers of SEQ ID NOs: 34 and 35 by the inverse PCR method using KOD-Plus-Mutageness Kit using pEF1α-SNAP26m-Pur3 · N4-ΔH as a template. PEF1α-SNAP26m-Pur4 · N4-ΔH was deleted. After that, pEF1α-SNAP26m-Pur4 · N4-ΔH was used as a template, and pEF1α-SNAP26m-Pur4 · from which f1 ori was deleted using the primers of SEQ ID NOs: 36 and 37 by the inverse PCR method using KOD-Plus-Mutageness Kit. N4-ΔH-Δf1 was constructed. Next, pEF1α-SNAP26m-Pur4 · N4-ΔH-Δf1 was used as a template by the inverse PCR method using KOD-Plus-Mutageness Kit, using the primers of SEQ ID NOs: 38 and 39, restriction enzymes NheI, EcoRI and SpeI sites. PEF1α-SNAP26m-Pur4 · N4-ΔH-Δf1-RE was inserted. Subsequently, by using the PCR of pEF1α-SNAP26m-Pur4 · N4-ΔH-Δf1-RE as a template and the Inverse PCR method using KOD-Plus-Mutageness Kit, using the primers of SEQ ID NOs: 40 and 41, restriction enzymes HindIII, BsiWI, PEF1α-MCSpre-Pur4 · N4-ΔH-Δf1-RE in which XbaI and BclI sites were inserted was constructed. Then, after mixing the primers of SEQ ID NOs: 42 and 43 to the HindIII and XbaI sites of pEF1α-MCSpre-Pur4 · N4-ΔH-Δf1-RE, the DNA was prepared by gradually lowering the temperature from 95 degrees to 60 degrees and annealing. The fragment was inserted to construct pEF1α-MCS-Pur4 · N4-ΔH-Δf1-RE. Next, PCR amplification was performed using pEF1α-KOD3G8HC-1.1k-ΔRE4 as a template at the NheI and EcoRI sites of pEF1α-MCS-Pur4 · N4-ΔH-Δf1-RE using the primer sets of SEQ ID NOS: 44 and 45. CHO5Δ3-3 from which 4 restriction enzyme recognition sequences were deleted was inserted to construct pEF1α-MCS-Pur4 · N4-1.1k. Furthermore, 4 sites were amplified by PCR using pEF1α-KOD3G8HC-1.1k-ΔRE4 as a template at the BamHI and XhoI sites of pEF1α-MCS-Pur4 · N4-1.1k using the primer sets of SEQ ID NOS: 16 and 46. CHO5Δ3-3 from which the restriction enzyme recognition sequence was deleted was inserted to construct pEHX.

Example 1-4
A vector with the restriction enzyme recognition sequence deleted from each sequence element, with the antibody gene inserted

(1) Construction of pELX The pELX described in FIG. 7 including a multicloning site was constructed.

Specifically, the plasmid pEF1α-KOD3G8LC in which the light chain of the anti-KOD antibody was inserted into the restriction enzyme XbaI-NotI site of the pCI-neo plasmid (Promega) changed from the CMV promoter to the EF1α promoter was used as a template. PEF1α-KOD3G8LC-RE was constructed by adding the restriction enzymes SalI and NheI sites upstream of the expression cassette using the primers of SEQ ID NOs: 3 and 4 by the Inverse PCR method using Plus-Mutageness Kit. Furthermore, restriction enzyme BsiWI and XhoI sites were added downstream of the expression cassette using the primers of SEQ ID NOS: 5 and 6 by the inverse PCR method using KOD-Plus-Mutageness Kit using this plasmid as a template, and pEF1α-KOD3G8LC- The RE2 plasmid was constructed. This pEF1α-KOD3G8LC-RE2 was treated with AflII and BstBI to excise a neomycin resistance gene. On the other hand, a hygromycin resistance gene was amplified from pTK-Hyg (Clontech) using the primers of SEQ ID NOs: 47 and 48, introduced into the restriction enzyme AflII and BstBI sites of pEF1α-KOD3G8LC-RE2, and pEF1α-KOD3G8LC. -Hyg-RE2 was constructed.

Using pEF1α-KOD3G8LC-Hyg-RE2 as a template, pEF1α-KOD3G8LC-Hyg- in which restriction enzymes BglII and SalI sites were inserted by the Inverse PCR method using KOD-Plus-Mutageness Kit using the primers of SEQ ID NOs: 49 and 50 RE3 was constructed. Subsequently, using pEF1α-KOD3G8LC-Hyg-RE3 as a template, an inverse PCR method using KOD-Plus-Mutageness Kit, and using primers of SEQ ID NOs: 51 and 52, an expression cassette for f1 ori and hygromycin resistance gene was prepared. The pEF1α-KOD3G8LC-RE4 was constructed by deleting and inserting the restriction enzymes EcoRI and SpeI sites. After that, pEF1α-KOD3G8LC-RE4 was used as a template, and by the Inverse PCR method using KOD-Plus-Mutageness Kit, restriction enzyme HindIII, BsiWI, XbaI, and BclI sites were inserted using the primers of SEQ ID NOs: 40 and 41. MSpre-RE4 was constructed. Then, after mixing the primers of SEQ ID NOs: 42 and 43 into the restriction enzyme HindIII and XbaI sites of pEF1α-MCSpre-RE4, the DNA fragment prepared by gradually lowering the temperature from 95 degrees to 60 degrees and annealing is inserted, and pELX Built.

(2) Construction of pELX2 FIG. 8 shows a gene expression stabilizing element CHO5Δ3-3 in which four restriction enzyme recognition sequences are deleted downstream from the expression cassette of pELX constructed in Example 1-4 (1). PELX2 was constructed. Specifically, the pELX constructed in Example 1-4 (1) was used as a template, and the PCR was restricted downstream of the expression cassette using the primers of SEQ ID NOS: 51 and 53 by the inverse PCR method using KOD-Plus-Mutageness Kit. PELX-N to which an enzyme NheI site was added was constructed. Then, pEFXα-KOD3G8HC-1.1k-ΔRE4 constructed in Example 1-3 was amplified by PCR using the primer set of SEQ ID NOS: 13 and 54 at the NheI and EcoRI sites of pELX-N. 4 CHO5Δ3-3 from which the restriction enzyme recognition sequence was deleted was inserted to construct pELX2.

(3) Construction of pEL2 (KOD3G8) The light chain gene of the anti-KOD antibody was inserted into the restriction enzyme MluI and NotI sites of pELX2 to construct pEL2 (KOD3G8). Specifically, an anti-KOD antibody prepared by PCR amplification from pEF1α-KOD3G8LC described in Example 1-4 (1) using primers of SEQ ID NOs: 20 and 21, and treatment with restriction enzymes MluI and NotI. The light chain gene was introduced into the restriction enzyme MluI and NotI sites of pELX2 to construct pEL2 (KOD3G8).

Example 2
Examination of combined effect of puromycin resistance gene and dhfr gene fusion protein gene and mRNA destabilizing sequence in anti-KOD antibody expression system, followed by dhfr gene, puromycin resistance gene and dhfr gene fusion protein gene In addition, an mRNA destabilizing sequence added to the downstream of the fusion protein gene of the puromycin resistance gene and the dhfr gene was obtained by selecting with puromycin in an anti-KOD antibody expression system in order to compare the respective effects. Monoclonal cells were further selected by MTX, and the cell productivity of the polyclonal cells obtained was evaluated.
Example 2-1
Construction of expression vector having dhfr gene (1) Construction of pEHX-dhfr · N4 pEHX-dhfr · N4 shown in FIG. 9 was constructed by inserting the expression cassette of dhfr gene into pEHX constructed in Example 1-3. . Specifically, at the BamHI and XhoI sites of pEHX, SV40 promoter prepared by PCR amplification using pEHX as a template using the primer set of SEQ ID NOs: 55 and 56, and treatment with restriction enzymes BglII and XhoI was inserted. pEHX-p was constructed. Subsequently, pEF1α-KOD3G8HC-1.1k-ΔRE4 described in Example 1-3 was amplified by PCR using the primer set of SEQ ID NOS: 57 and 58 at the BamHI and XhoI sites of pEHX-p, followed by restriction. CHO5Δ3-3, which was prepared by treating with the enzymes BamHI and SalI and without the four restriction enzyme recognition sequences, was inserted to construct pEHX-p2. Next, at the BamHI and XhoI sites of pEHX-p2, using the primer set of SEQ ID NOs: 59 and 60, PCR amplification was performed using pEHX as a template, and SV40 pA prepared by treatment with restriction enzymes BamHI and SalI was inserted. pEHX-p3 was constructed. On the other hand, the DNA of SEQ ID NO: 61 in which the PEST sequence of the proteolysis promoting sequence was added to the 3 ′ end of the dhfr gene was artificially synthesized and TOPO TA cloned into the plasmid vector pCR2.1-TOPO (Invitrogen), pCR2. 1-dhfr · PEST was constructed. Subsequently, the PEST sequence was deleted from the 3 ′ end of the dhfr gene using the primers of SEQ ID NOs: 62 and 63 by the inverse PCR method using pCR2.1-dhfr · PEST as a template and KOD-Plus-Mutageness Kit. PCR2.1-dhfr · N4 added with the N4 sequence was constructed. Then, DNA amplified by PCR using pCR2.1-dhfr · N4 as a template at the BamHI and XhoI sites of pEHX-p3 using the primer set of SEQ ID NOS: 64 and 65, and treated with restriction enzymes BglII and SalI. The fragment was inserted to construct pEHX-dhfr · N4.

(2) Construction of expression vectors having fusion protein gene of puromycin resistance gene (upstream) and dhfr gene (downstream): pEHX-Puro · dhfr, pEHX-Puro · dhfr · N4 According to the scheme shown in FIG. In the pEHX constructed in -3, instead of the puromycin resistance gene with the N4 sequence added downstream, a fusion protein gene of the puromycin resistance gene and the dhfr gene, or the puromycin resistance gene with the N4 sequence added downstream pEHX-Puro · dhfr and pEHX-Puro · dhfr · N4 into which a fusion protein gene of dhfr gene was inserted were constructed. Specifically, first, a DNA fragment composed of SV40 promoter, puromycin resistance gene amplified by PCR using pEHX as a template using the primer set of SEQ ID NOs: 36 and 66, and SEQ ID NOs: 67 and 68, or SEQ ID NO: 67 , A DNA fragment consisting of dhfr gene or dhfr gene, N4 sequence amplified by PCR using pCR2.1-dhfr · N4 as a template using the primer set of 69, was assembled by overlap-extension PCR, SEQ ID NO: 36, 68, or a DNA fragment consisting of SV40 promoter, puromycin resistance gene, dhfr gene, or SV40 promoter, puromycin resistance gene, dhfr residue, using the primer set of SEQ ID NOs: 36 and 69 A DNA fragment consisting of a gene, N4 sequence was prepared. These DNA fragments were digested with KpnI and Csp45I and inserted into the KpnI and Csp45I sites of pEHX to construct pEHX-Puro · dhfr and pEHX-Puro · dhfr · N4.

(3) Construction of anti-KOD antibody expression vectors: pELH2-dhfr · N4 (KOD3G8), pELH2-Puro · dhfr (KOD3G8), pELH2-Puro · dhfr · N4 (KOD3G8) Subsequently, gene amplification in an anti-KOD antibody expression system PELH2-dhfr · N4 (KOD3G8), pELH2-Puro · dhfr (KOD3G8), and pELH2-Puro · dhfr · N4 (KOD3G8) were constructed.

  First, the heavy chain genes of anti-KOD antibodies were inserted into the restriction enzyme MluI and NotI sites of pEHX-dhfr · N4, pEHX-Puro · dhfr, and pEHX-Puro · dhfr · N4, respectively, and pEH-dhfr · N4 ( KOD3G8), pEH-Puro · dhfr (KOD3G8), and pEH-Puro · dhfr · N4 (KOD3G8) were constructed. Specifically, the heavy chain gene of the anti-KOD antibody prepared by PCR amplification from pEF1α-KOD3G8HC described in Example 1-1 using the primers of SEQ ID NOs: 20 and 21 and treatment with restriction enzymes MluI and NotI Are introduced into the restriction enzyme MluI and NotI sites of pEHX-dhfr · N4, pEHX-Puro · dhfr, and pEHX-Puro · dhfr · N4, and pEH-dhfr · N4 (KOD3G8), pEH-Puro · dhfr ( KOD3G8) and pEH-Puro · dhfr · N4 (KOD3G8) were constructed.

  Then, according to the scheme shown in FIG. 11, the anti-KOD antibody light chain expression cassette prepared by treating pEL2 (KOD3G8) described in Example 1-4 with BglII, EcoRI, and ScaI was transformed into pEH-dhfr · N4 (KOD3G8). PELH2-dhfr · N4 (KOD3G8) was constructed by inserting into restriction enzyme BglII and EcoRI sites. Similarly, according to the scheme shown in FIG. 12, a light chain expression cassette of an anti-KOD antibody prepared by treating pEL2 (KOD3G8) with BglII, EcoRI, and ScaI was transformed into pEH-Puro dhfr (KOD3G8) and pEH- It was inserted into restriction sites BglII and EcoRI of Puro.dhfr.N4 (KOD3G8) to construct pELH2-Puro.dhfr (KOD3G8) and pELH2-Puro.dhfr.N4 (KOD3G8).

Example 2-2
Evaluation of cell productivity of polyclonal cells obtained by selection with puromycin in an anti-KOD antibody expression system

The dhfr-deficient CHO / dhfr-cells (ATCC, CRL-9096) were adjusted to 1 × 10 ⁵ cells / ml, seeded in a 12-well plate 2 ml the day before, cultured overnight, and transfected CHO. / Dhfr- cells were prepared. At this time, Iscove's Modified Dulbecco's Medium (manufactured by Sigma) supplemented with 10% fetal calf serum, 4 mM Glutamine (manufactured by Nacalai), and 1xHT supplement (manufactured by Invitrogen) was used.

On the other hand, the anti-KOD antibody expression vector constructed in Example 2-1 was linearized with the restriction enzyme AhdI. For transfection, 1.6 μg of the linearized plasmid was diluted with 200 μl Opti-MEM I Reduced-Serum Medium, 4 μl of Lipofectamine LTX (manufactured by Invitrogen) was added, and the mixture was allowed to stand for 30 minutes. In addition to dhfr-cells, incubation was carried out for 24 hours. One day after transfection, the medium was removed, cells were dispersed by treatment with 2.5 g / l-Trypsin · 1 mmol / l-EDTA Solution, transferred to a 90 mm Petri dish, 10 μg / ml Puromycin, 10% fetal calf serum Selective culture was performed in Iscove's Modified Dulbecco's Medium supplemented with 4 mM Glutamine for 2 weeks. During selective culture, the medium was changed every 3-4 days. After completion of selective culture, cells were dispersed by treatment with 2.5 g / l-Trypsin · 1 mmol / l-EDTA Solution, and 1.84 × 10 ⁵ cells per well were seeded in a 6-well plate.

After culturing for 3 days, the culture supernatant was collected, and the production amount of KOD3G8 antibody in the polyclonal cells obtained by selection with puromycin was measured by ELISA. The antibody concentration was calculated based on a calibration curve prepared from a standard product.

  Specifically, the culture supernatant was added to an ELISA plate on which a DNA polymerase antigen derived from KOD1 at a concentration of 30 μg / ml was immobilized, and incubated at 35 ° C. for 2 hours. Next, 50 μl of anti-mouse antibody-HRP (manufactured by DAKO), which was washed three times with PBS-T (phosphate buffered saline containing 0.1% Tween 20) and diluted 10,000 times, was added at 35 ° C. for 1 hour. After the incubation, the plate was further washed three times with PBS-T and reacted with TMB + (3,3 ′, 5,5′-tetramethylbenzidine; manufactured by DAKO) for 5 minutes. After stopping the reaction by adding 50 μl of 1N sulfuric acid, the absorbance was measured with a plate reader (product name: SPECTRA CLASSIC, manufactured by TECAN Australia) (main wavelength: 450 nm, subwavelength: 620 nm). In addition, as a standard product, from mouse hybridoma cell line 3G8 that produces an antibody specific to DNA polymerase derived from KOD1 (obtained from: National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Deposit Number: FERM BP-6056) The obtained antibody was used.

The results are shown in FIG. As a result, in pELH2-dhfr · N4 (KOD3G8) containing the expression cassettes for both the puromycin resistance gene and the dhfr gene, the expression level was reduced despite the addition of the mRNA destabilizing sequence to the puromycin resistance gene. It was low. As a cause of the low expression level, transcription interference due to coding of four expression cassettes in one construct was considered. On the other hand, in pELH2-Puro · dhfr (KOD3G8) employing a fusion protein gene of a puromycin resistance gene and a dhfr gene, the expression cassette can be reduced to three, and the expression level compared to pELH2-dhfr · N4 (KOD3G8) Became high. Furthermore, in pELH2-Puro · dhfr · N4 (KOD3G8), which is weakened by adding an mRNA destabilizing sequence to the fusion protein gene of the puromycin resistance gene and the dhfr gene, the expression level in the polyclone is extremely high, and pELH2- It was found that the production amount was about 1.9 times that of dhfr · N4 (KOD3G8) and about 1.5 times that of pELH2-Puro · dhfr (KOD3G8).

Example 2-3
Evaluation of cell productivity of monoclonal cells obtained by selection with puromycin in an anti-KOD antibody expression system

Subsequently, limiting dilution was performed in order to compare the productivity of the monoclone. Specifically, the polyclonal cells stably transfected with the KOD3G8 antibody obtained in Example 2-2 were treated with 2.5 g / l-Trypsin · 1 mmol / l-EDTA Solution to disperse the cells. . Seed 96 well plates at a concentration of 75 cells / well. After culturing for 2 weeks, the culture supernatant was collected, and the production amount of KOD3G8 antibody was measured by ELISA in the same manner as in Example 2-2. The antibody production amount of each clone was calculated based on a calibration curve prepared from a standard product. Then, the selected clone with the highest expression level was expanded and cultured, and 1.84 × 10 ⁵ cells per well were seeded in a 6-well plate. After culturing for 3 days, the culture supernatant was collected, and the production amount of KOD3G8 antibody in the monoclonal cells obtained by selection with puromycin was measured by ELISA in the same manner as in Example 2-2.

The antibody production amount of each clone was calculated based on a calibration curve prepared from a standard product. The results are shown in FIG. As a result, as in the case of the polyclone, the maximum was 6 mg / L in pELH2-dhfr · N4 (KOD3G8) containing the expression cassettes of both the puromycin resistance gene and the dhfr gene, but the puromycin resistance gene and the dhfr gene In pELH2-Puro · dhfr (KOD3G8) employing the fusion protein gene, the maximum was 10 mg / L. On the other hand, in pELH2-Puro · dhfr · N4 (KOD3G8), which was weakened by adding an mRNA destabilizing sequence to the fusion protein gene of the puromycin resistance gene and the dhfr gene, the expression level was significantly higher, and the value was maximum. Reached 18 mg / L.

Example 2-4
Monoclonal cells obtained by selecting with puromycin in the anti-KOD antibody expression system and further evaluating with polyclonal cells obtained by selecting with MTX

Next, 10 μg / ml Puromycin and 10% dialyzed for each of the top 10 expression clones obtained by introducing each anti-KOD antibody expression vector obtained in Example 2-3 and selecting with puromycin. It was subcultured for 2 weeks in Iscove's Modified Dulbecco's Medium supplemented with fetal calf serum, 4 mM Glutamine. Thereafter, selective culture was further performed in Iscove's Modified Dulbecco's Medium supplemented with 10 nM MTX (manufactured by Sigma), 10 μg / ml Puromycin, dialyzed 10% fetal bovine serum, and 4 mM Glutamine for 6 weeks.

After completion of the selective culture, the obtained polyclonal cells were treated with 2.5 g / l-Trypsin · 1 mmol / l-EDTA Solution to disperse the cells, and 1.84 × 10 ⁵ cells per well were added to a 6-well plate. Sowing. After culturing for 3 days, the culture supernatant was collected, and the production amount of KOD3G8 antibody in the cells of the polyclonal cells obtained by selection with MTX was measured by ELISA in the same manner as in Example 2-2.

The antibody concentration was calculated based on a calibration curve prepared from a standard product. The results are shown in FIG. As a result, in pELH2-dhfr · N4 (KOD3G8), the expression level was increased by about 2 times by selection with MTX, but the highest productivity was 11 mg / L. On the other hand, in pELH2-Puro · dhfr (KOD3G8), the highest productivity was 12 mg / L. As a cause, since the dhfr gene is not weakened, it is thought that the increase in the expression level by MTX selection was not so much seen. On the other hand, in the case of pELH2-Puro · dhfr · N4 (KOD3G8), which had been able to obtain a clone with a high expression level at the time of selection with puromycin, the increase in the expression level by about 2 times was observed due to the selection with MTX. The highly probable product was 45 mg / L. By using an expression cassette in which the puromycin resistance gene and the dhfr gene are fused to form a selection gene and an mRNA destabilizing sequence is added to the 3 ′ end to weaken the gene, A rise was possible.

Example 3
Examination of the arrangement order of the dhfr gene and the puromycin resistance gene Subsequently, in order to examine the arrangement order of the dhfr gene and the puromycin resistance gene, the dhfr gene is arranged upstream and the puromycin resistance gene is arranged downstream. Construction of an expression vector having the arranged fusion protein gene, and the productivity of the polyclonal cell obtained by further selecting the monoclonal cells obtained by selecting with puromycin in the anti-KOD antibody expression system and further with MTX evaluated.
Example 3-1
Construction of expression vector having dhfr gene (1) Construction of expression vector having fusion protein gene of dhfr gene (upstream) and puromycin resistance gene (downstream): Construction of pEHX-dhfr · Puro · N4 According to the scheme shown in FIG. In pEHX constructed in Example 1-3, pEHX-dhfr in which a fusion protein gene of a dhfr gene added with N4 sequence downstream and a puromycin resistance gene was inserted instead of the puromycin resistance gene added downstream with N4 sequence・ Puro N4 was constructed. Specifically, first, a DNA fragment comprising the SV40 promoter and dhfr genes amplified by PCR using pEHX-dhfr · N4 as a template using the primer sets of SEQ ID NOS: 11 and 70, and the primer sets of SEQ ID NOS: 69 and 71 were used. The puromycin resistance gene to which N4 sequence amplified by PCR using pEHX as a template was added downstream was assembled by overlap-extension PCR, and using the primer set of SEQ ID NOs: 11 and 69, SV40 promoter, dhfr gene, puromycin A DNA fragment consisting of the resistance gene, N4 sequence was prepared. This DNA fragment was digested with KpnI and Csp45I and inserted into the KpnI and Csp45I sites of pEHX to construct pEHX-dhfr · Puro · N4.

(2) Construction of anti-KOD antibody expression vector: pELH2-dhfr · Puro · N4 (KOD3G8) Subsequently, in order to confirm the effect of gene amplification in the anti-KOD antibody expression system, pELH2-dhfr · Puro · N4 (KOD3G8) was used. It was constructed.

  First, the heavy chain gene of the anti-KOD antibody was inserted into the restriction enzyme MluI and NotI sites of pEHX-dhfr · Puro · N4 to construct pEH-dhfr · Puro · N4 (KOD3G8). Specifically, the heavy chain gene of the anti-KOD antibody prepared by PCR amplification from pEF1α-KOD3G8HC described in Example 1-1 using the primers of SEQ ID NOs: 20 and 21 and treatment with restriction enzymes MluI and NotI Was introduced into the restriction enzyme MluI and NotI sites of pEHX-dhfr · Puro · N4 to construct pEH-dhfr · Puro · N4 (KOD3G8).

  Then, according to the scheme shown in FIG. 17, the anti-KOD antibody light chain expression cassette prepared by treating pEL2 (KOD3G8) described in Example 1-4 with BglII, EcoRI, and ScaI was used as pEH-dhfr · Puro · N4. It was inserted into the restriction enzyme BglII and EcoRI sites of (KOD3G8) to construct pELH2-dhfr · Puro · N4 (KOD3G8).

Example 3-2
Cell productivity evaluation of polyclonal cells obtained by selection with puromycin in the anti-KOD antibody expression system The effect of placing the dhfr gene upstream of the puromycin resistance gene was examined in the anti-KOD antibody expression system. As a single vector for expressing an anti-KOD antibody in which the dhfr gene is placed upstream of the puromycin resistance gene, pELH2-dhfr · Puro · N4 (KOD3G8) constructed in Example 3-1 is used, and the dhfr gene is downstream of the puromycin resistance gene. The pELH2-Puro · dhfr · N4 (KOD3G8) constructed in Example 2-1 was used as a single vector for expressing the anti-KOD antibody arranged in 1. 1.6 μg of the plasmid linearized with the restriction enzyme AhdI was transfected into CHO / dhfr− cells prepared by the method described in Example 2-2 and incubated for 24 hours. The next day, the medium was removed, cells were dispersed by treatment with 2.5 g / l-Trypsin · 1 mmol / l-EDTA Solution, transferred to a 90 mm Petri dish, 7.5 μg / ml Puromycin, 10% fetal bovine serum, 4 mM Selective culture was performed in Iscove's Modified Dulbecco's Medium supplemented with Glutamine for 2 weeks. During selective culture, the medium was changed every 3-4 days. After completion of selective culture, cells were dispersed by treatment with 2.5 g / l-Trypsin · 1 mmol / l-EDTA Solution, and 1.84 × 10 ⁵ cells per well were seeded in a 6-well plate. After culturing for 3 days, the culture supernatant was collected, and the production amount of KOD3G8 antibody in the cells of the polyclonal cells obtained by selection with puromycin was measured by ELISA in the same manner as in Example 2-2.

Antibody production was calculated based on a calibration curve prepared from standard products. The results are shown in FIG. Antibody production of pELH2-Puro · dhfr · N4 (KOD3G8) in which the dhfr gene is arranged downstream of the puromycin resistance gene and pELH2-dhfr · Puro · N4 (KOD3G8) in which the dhfr gene is arranged upstream of the puromycin resistance gene Both amounts were as high as 7 mg / L. In addition, no difference was observed between the two, and it was found that a high-expressing cell population could be obtained after selection with puromycin, regardless of which of the dhfr gene and the puromycin resistance gene was placed upstream.

Example 3-3
Evaluation of cell productivity of monoclonal cells obtained by selection with puromycin in an anti-KOD antibody expression system

Subsequently, limiting dilution was performed in order to compare the productivity of the monoclone. Specifically, a polyclone cell obtained by gene transfer of pELH2-dhfr · Puro · N4 (KOD3G8) obtained in Example 3-2 and selecting with puromycin was used as 2.5 g / l-Trypsin. • Cells were dispersed by treatment with 1 mmol / l-EDTA Solution and seeded in 96-well plates at a concentration of 0.75 cells / well. After culturing for 2 weeks, the culture supernatant was collected, and the production amount of KOD3G8 antibody was measured by ELISA in the same manner as in Example 2-2. The antibody production amount of each clone was calculated based on a calibration curve prepared from a standard product. Then, the selected clone with the highest expression level was expanded and cultured, and 1.84 × 10 ⁵ cells per well were seeded in a 6-well plate. After culturing for 3 days, the culture supernatant was collected, and the production amount of KOD3G8 antibody in the monoclonal cells obtained by selection with puromycin was measured by ELISA in the same manner as in Example 2-2.

The antibody production amount of each clone was calculated based on a calibration curve prepared from a standard product. The results are shown in FIG. As a result, as in the case of the polyclone, in pELH2-dhfr · Puro · N4 (KOD3G8) in which the dhfr gene is arranged upstream of the puromycin resistance gene, a clone with a high expression level can be obtained, and the maximum value is 22 mg. / L reached.

Example 3-4
Monoclonal cells obtained by selecting with puromycin in the anti-KOD antibody expression system and further evaluating with polyclonal cells obtained by selecting with MTX

Next, with respect to the 10 expression clones obtained by gene transfer of pELH2-dhfr · Puro · N4 (KOD3G8) obtained in Example 3-3 and selected with puromycin, 7.5 μg / ml Puromycin, Subcultured for 2 weeks in Iscove's Modified Dulbecco's Medium supplemented with dialyzed 10% fetal bovine serum and 4 mM Glutamine. Thereafter, selective culture was further performed in Iscove's Modified Dulbecco's Medium supplemented with 10 nM MTX (manufactured by Sigma), 7.5 μg / ml Puromycin, dialyzed 10% fetal bovine serum, 4 mM Glutamine, for 6 weeks.

After completion of the selective culture, the obtained polyclonal cells were treated with 2.5 g / l-Trypsin · 1 mmol / l-EDTA Solution to disperse the cells, and 1.84 × 10 ⁵ cells per well were added to a 6-well plate. Sowing. After culturing for 3 days, the culture supernatant was collected, and the production amount of KOD3G8 antibody in the cells of the polyclonal cells obtained by selection with MTX was measured by ELISA in the same manner as in Example 2-2.

The antibody concentration was calculated based on a calibration curve prepared from a standard product. The results are shown in FIG. As a result, as in the case of pELH2-Puro · dhfr · N4 (KOD3G8), the expression level of pELH2-dhfr · Puro · N4 (KOD3G8) in which the dhfr gene is arranged upstream of the puromycin resistance gene is increased by MTX selection. The highest productivity was 35 mg / L. From the above, regardless of the arrangement order of the puromycin resistance gene and the dhfr gene, the puromycin resistance gene and the dhfr gene were fused to form a selection gene, and an mRNA destabilizing sequence was added to the 3 ′ end to weaken the gene. By using an expression cassette, it was possible to obtain a high-expression cell line after selection with puromycin, and it was possible to further increase the expression level by selectively culturing this cell line with MTX.

Example 4
Examination of combined effect of fusion protein gene of dhfr gene and puromycin resistance gene and mRNA destabilizing sequence in anti-KOD antibody expression system using serum-free CHO / dhfr-cells Using serum-conditioned CHO / dhfr-cells, the effect of a fusion protein gene comprising a dhfr gene (upstream) with an mRNA destabilizing sequence added downstream and a puromycin resistance gene (downstream) was examined.

Example 4-1
Evaluation of cell productivity of monoclonal cells obtained by selection with puromycin in an anti-KOD antibody expression system

1 x Yeastolate UF solution (SAFC Biosciences), IS CHO-CD supplemented with 1 x HT supplement, 4 mM Glutamine, Chemically Defined Media for CHO cells (Irvine Scientific CH) and sera without using Irvine Scientific CH cells. Serum-free CHO / dhfr-cells were suspended in Opti-MEM I Reduced-Serum Medium supplemented with 1xHT supplement so as to be 2.5 × 10 ⁵ cells / ml, and an ultra-low adhesion surface 24-well plate (Corning Life Science). 1 ml each was inoculated into 2 wells.

Meanwhile, 3 μg of pELH2-dhfr · Puro · N4 (KOD3G8) linearized with the restriction enzyme AhdI prepared in Example 3-2 was diluted with 400 μl Opti-MEM I Reduced-Serum Medium, and then 10 μl of Lipofectamine LTX was added. Left for a minute. Thereafter, half of the aliquots were added to the 2 wells of serum-free CHO / dhfr- cells and incubated for 24 hours. One day after transfection, the cells were collected, centrifuged at 800 g × 3 minutes to remove the supernatant, and suspended in IS CHO-CD, Chemically Defined Media for CHO cells medium supplemented with 4 ml of 1x Yeastolate UF solution, 1xHT supplement, 4 mM Glutamine. 7.5 μg / ml Puromycin was added and selective culture was performed.

After completion of the selective culture, the polyclonal cells were seeded on a 12-well plate using Iscove's Modified Dulbecco's Medium supplemented with 10% fetal bovine serum, 4 mM Glutamine, and 1 × HT supplement, and cultured in an adherent state. After culturing for 2 days, cells were dispersed by treatment with 2.5 g / l-Trypsin · 1 mmol / l-EDTA Solution, seeded in a 96-well plate at a concentration of 0.75 cells / well, and limiting dilution was performed in an adherent state. went. After culturing for 2 weeks, the culture supernatant was collected, and the production amount of KOD3G8 antibody was measured by ELISA in the same manner as in Example 2-2. The antibody production amount of each clone was calculated based on a calibration curve prepared from a standard product. Then, for the selected clone with the highest expression level, cells were dispersed by treatment with 2.5 g / l-Trypsin · 1 mmol / l-EDTA Solution, IS CHO-CD supplemented with 1x Yeastolate UF solution, 1xHT supplement, 4 mM Glutamine, Using Chemically Defined Media for CHO cells medium, expansion culture was performed in a floating state, and then 4.0 × 10 ⁵ cells per well were seeded on an ultra-low adhesion surface 6-well plate. After culturing for 3 days, the culture supernatant was collected, and the production amount of KOD3G8 antibody in the monoclonal cells obtained by selection with puromycin was measured by ELISA in the same manner as in Example 2-2. Furthermore, the productivity per day (pcd: pg / cell / day) was calculated from the amount of antibody production calculated based on a calibration curve prepared from a standard product and the number of cells counted after 3 days of culture. . The results are shown in FIG. As a result, even when serum-free CHO / dhfr-cells that had been conditioned in advance were used as the host, a high expression strain could be obtained after selection with puromycin.

Example 4-2
Monoclonal cells obtained by selecting with puromycin in the anti-KOD antibody expression system and further evaluating with polyclonal cells obtained by selecting with MTX

Next, 7.5 μg / ml Puromycin, about 10 expression clones obtained by gene transfer of pELH2-dhfr · Puro · N4 (KOD3G8) obtained in Example 4-1 and selecting with puromycin, The cells were subcultured for 2 weeks in IS CHO-CD, Chemically Defined Media for CHO cells medium supplemented with 1 × Yeastolate UF solution, 4 mM Glutamine. Thereafter, 7.5 μg / ml Puromycin, 3 nM MTX, 1 × Yeastolate UF solution, 4 mM Glutamine added IS CHO-CD, Chemically Defined Media for CHO cells, followed by 7.5 μg / ml PurMin, 10 μM Purincin, 1 x Yeastolate UF solution, IS CHO-CD, Chemically Defined Media for CHO cells medium supplemented with 4 mM Glutamine, and 7.5 μg / ml Puromycin, 30 nM MTF, 1 x Yeastolate UF solution Defend Media Selective culture was performed in for CHO cells medium for a total of 6 weeks.

After completion of the selective culture, 4.0 × 10 ⁵ cells per well of the obtained polyclonal cells were seeded in a 6-well plate with an ultra-low adhesion surface. After culturing for 3 days, the culture supernatant was collected, and the production amount of KOD3G8 antibody in the cells of the polyclonal cells selected by MTX was measured by ELISA in the same manner as in Example 2-2. Furthermore, the productivity per day (pcd: pg / cell / day) was calculated from the amount of antibody production calculated based on a calibration curve prepared from a standard product and the number of cells counted after 3 days of culture. . The results are shown in FIG. Based on the above, even when CHO / dhfr-cells previously serum-free is used as a host, the puromycin resistance gene and the dhfr gene are fused to form a selection gene, and an mRNA destabilizing sequence is added to the 3 'end. By using a weakened expression cassette, it is possible to obtain a high-expression cell line after selection with puromycin, and it is possible to further increase the expression level by selectively culturing this cell line with MTX. It was.

  Expression vector having drug selection marker gene of the present invention, expression cassette containing dihydrofolate reductase gene and mRNA destabilizing sequence adjacent to drug selection marker gene, gene expression stabilizing element, and target protein gene expression cassette Is very effective for performing rapidly and efficiently from cloning of the target protein gene to establishment of cells with high productivity of the recombinant protein. Therefore, the cell expression system using the expression vector of the present invention contributes not only to the functional analysis of proteins in various animal cells, particularly mammalian cells, but also to the drug discovery and medical industries as biopharmaceuticals. is there.

Claims (22)

An expression vector comprising at least the following (1) to (3): (1) a drug selection marker gene, a dihydrofolate reductase gene present adjacent to the drug selection marker gene, and an mRNA destabilizing sequence, An expression cassette configured so that the drug selection marker gene and the dihydrofolate reductase gene are expressed as a fusion gene in one expression cassette;
(2) gene expression stabilizing element, and (3) target protein gene expression cassette   The expression vector according to claim 1, wherein the mRNA destabilizing sequence is derived from an AT-rich sequence present in a 3 'untranslated region of a cytokine, interleukin, or proto-oncogene.   The expression vector according to claim 2, wherein the mRNA destabilizing sequence has a motif sequence of TTATTTA (A / T) (A / T).   The expression vector according to claim 3, wherein the motif sequence is repeated twice or more.   The expression vector according to claim 4, comprising a spacer sequence of 1 base or more between repeating motif sequences.   The expression vector according to any one of claims 2 to 5, wherein the mRNA destabilizing sequence contains one to several base substitutions, insertions or deletions. The expression vector according to any one of claims 1 to 6, wherein the gene expression stabilizing element is any of the following.
(I) DNA consisting of the sequence from 41601 to 46746 in the sequence shown in SEQ ID NO: 15 or a partial sequence of the sequence from 41601 to 46746, comprising at least the sequence from 41601 to 42700 DNA
(II) a DNA comprising a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the sequence (I) and has a function of stabilizing gene expression
(III) DNA comprising a base sequence complementary to the sequence (I) or (II)   The expression vector according to any one of claims 1 to 7, comprising two or more gene expression stabilizing elements.   The expression vector according to any one of claims 1 to 8, wherein the drug selection marker gene is a protein synthesis inhibition antibiotic resistance gene.   The expression vector according to claim 9, wherein the drug selection marker gene is selected from the group consisting of puromycin-N-acetyltransferase, hygromycin-B-phosphotransferase and neomycin phosphotransferase.   The expression vector according to any one of claims 1 to 10, wherein the gene expression cassette of the target protein comprises a multicloning site for inserting a desired target protein gene.   The expression vector according to any one of claims 1 to 11, wherein the target protein gene is an antibody heavy chain and / or light chain polypeptide gene. An expression cassette comprising a drug selection marker gene, a dihydrofolate reductase gene and an mRNA destabilizing sequence present adjacent to the drug selection marker gene, wherein the drug selection marker gene and the dihydrofolate reductase gene are fusion genes An expression cassette that is configured to express as one expression cassette. A method for producing a high-producing cell, comprising the following steps.
(A) a step of transforming a host cell using the expression vector according to any one of claims 1 to 12 (b) a step of culturing the transformed cell in the presence of a drug (c) the transformed cell (D) a step of isolating a cell line exhibiting high expression, in the presence of methotrexate (MTX). A method for producing a high-producing cell, comprising the following steps.
(A) a step of transforming a host cell using the expression vector according to any one of claims 1 to 12 (b) a step of culturing the transformed cell in a hypoxanthine- and thymidine-free medium (c) Culturing the transformed cells in the presence of methotrexate (MTX) (d) isolating a cell line exhibiting high expression. A method for producing a high-producing cell, comprising the following steps.
(A) a step of transforming a host cell using the expression vector according to any one of claims 1 to 12 (b) culturing the transformed cell in the presence of a drug without hypoxanthine and thymidine (C) culturing the transformed cells in the presence of methotrexate (MTX) (d) isolating a cell line showing high expression.   The method according to any one of claims 14 to 16, wherein (c) a step of culturing transformed cells in the presence of methotrexate (MTX), and (d) a step of isolating a cell line exhibiting high expression. A method for producing a high-producing cell by repeating a plurality of times. The method for producing a high-producing cell according to any one of claims 14 to 17, wherein the host cell is a cell derived from a mammalian cell. The method for producing a high-producing cell according to any one of claims 14 to 17, wherein the host cell is a cell derived from Chinese hamster ovary (CHO). The method for producing a high-producing cell according to any one of claims 14 to 17, wherein the host cell is a cell deficient in dihydrofolate reductase gene.   The method for producing a high-producing cell according to any one of claims 14 to 17, wherein the host cell is a serum-free conditioned cell. A high-production cell obtained by the method according to any one of claims 14 to 21, wherein the high-production cell is transformed with an expression vector containing an antibody heavy chain and / or light chain polypeptide gene as a target protein gene. A method of producing an antibody by culturing cells .