WO2022030094A1 - Method and kit for promoting expression of target protein in mammalian cells, and utilization thereof - Google Patents

Abstract

According to the present invention, the expression of a target protein is promoted in mammalian cells by co-introducing a polynucleotide containing an expression cassette of the target protein, a polynucleotide containing IR/MAR and a polynucleotide promoting the expression of the target protein, each as a separate polynucleotide, into the mammalian cells.

Description

Methods and kits for promoting expression of the protein of interest in mammalian cells, and their use.

The present invention relates to a method and a kit for promoting the expression of a target protein in mammalian cells, and its use.

A production method for producing a protein by introducing and expressing genes encoding various proteins into a host is generally used when producing a protein industrially (hereinafter, the protein to be produced is "purpose". "Protein", the gene that encodes it is sometimes called the "target gene"). The host used in the above production method is appropriately selected from bacteria such as Escherichia coli and Bacillus spp., Yeast, mold, animal cells and the like. When an animal cell is used as a host, the sugar chain added to the produced target protein is almost the same sugar chain as that originally modified the target protein. Therefore, it has the advantage that the physiological activity of the target protein is unlikely to be affected by the different sugar chains added. Moreover, since it is not necessary to use a virus to introduce the target gene, it is not necessary to put a lot of effort into removing the virus. Therefore, the use of animal cells as a host is advantageous both in terms of time and cost.

When an animal cell is used as a host, it has the above-mentioned advantages, but there is a problem that the production amount is low only by introducing the target gene into the host. Therefore, in order to increase the production of the target protein, Chinese hamster ovary (CHO: Chinese Hamster Ovary) cells deficient in dihydrofolate reductase (DHFR) are used as hosts, and the target protein gene and DHFR are used as hosts. A method is known in which a gene encoding a gene is simultaneously introduced and a DHFR inhibitor, Methotrexate (MTX), is added to a culture solution (see Patent Document 1). The method described in Patent Document 1 selects cells expressing the DHFR gene (producing DHFR) by gradually increasing the MTX concentration in the medium, and the target protein together with the DHFR gene. Amplifies the gene. As a result, the production amount of the target protein is increased by this method. On the other hand, the method described in Patent Document 1 requires a time of 6 months or more to complete the operation.

The present inventor refers to a plasmid vector (“IR / MAR vector” or “IR / MAR plasmid”) having a mammalian replication initiation region (IR: Initiative Region) and a nuclear matrix binding region (MAR: Matrix Attachment Region). ) Was introduced into human-derived cancer cells (COLO320 colon cancer cell line and HeLa cell line) by a plasmid method. Then, they found that the intracellular copy number of the target gene can be amplified to about 10,000 copies simply by selecting using the drug resistance gene (Blasticidin: Blasticidin or Neomycin: Neomycin) existing on the plasmid. rice field. In addition, the number of copies of the target gene can be determined regardless of whether the target gene is introduced into the IR / MAR vector as the same gene construct (cis) or as a different gene construct (trans). We also found that it can be highly amplified. Then, a system for highly amplifying a target gene (referred to as "advanced gene amplification system" or "IR / MAR gene amplification system") using the above IR / MAR vector has been completed (for example, Patent Document 2 and Patent Document 2 and). 3. See Non-Patent Documents 1 and 2). Here, a gene amplification method using an advanced gene amplification system (IR / MAR gene amplification system) is referred to as an "IR / MAR gene amplification method".

When gene amplification is performed by the IR / MAR gene amplification method, a simple repetitive sequence with the target gene is formed in the amplified region. In general, it is known that repetitive sequences are heterochromatinized and transcriptionally repressed by a phenomenon called Repeat-Induced Gene Silencing (RIGS). It is also known that repetitive sequences activate the RNAi system with high efficiency and induce DNA methylation. Therefore, in the gene amplified by the IR / MAR gene amplification method, the amplification of the copy number and the expression level of the target gene may not always correlate (see Non-Patent Document 3). Development of a method for releasing such transcriptional repression is underway.

As a method for releasing the transcriptional repression caused by the repetitive sequence of a gene, a method of treating cells with a histone deacetylase inhibitor such as trichostatin A is known (see Non-Patent Document 4).

In order to solve the problem that gene expression is suppressed due to repetitive sequences even if the number of copies increases in the IR / MAR gene amplification method, the present inventor of the present invention is B-3-31 (human No. 2). We have found expression-promoting polynucleotides such as (human genome fragment) derived from a region with low gene density of chromosome short arm 16.1.

The present inventor further studied the IR / MAR plasmid, the target gene, and the expression-promoting polynucleotide. It was found that when the IR / MAR plasmid and the target gene were placed on the same vector, the gene cluster expressing the target protein was effectively amplified, and the production amount of the target protein was increased (see Patent Document 6). ..

Japan Special Table No. 57-50410 Japanese Patent Application Laid-Open No. 2003-245083 Japanese Patent Laid-Open No. 2004-337066 International Publication No. 2006/054561 Pamphlet Japanese Patent Application Laid-Open No. 2016-127817 Japanese Patent Application Laid-Open No. 2012-143225

Noriaki Shimizu, et al. (2001) Plasmaids with a Mammalian Replication Origin and a Matrix Attachment Region Initiate the Event Similar to Gene Amplification. Cancer Research vol.61, no.19, p6987-6990. Noriaki Shimizu, et al (2003) Amplification of plasmids containing a mammal replication initiation region is mediated by controllable conflict between replication and transcription. Cancer Research, vol.63, no.17, p5281-5290. Shimizu, N., Hanada, N., Utani, K., and Sekiguchi, N. (2007). Interconversion of intra-and extra-chromosomal sites of gene expression by modulation of gene expression and DNA methylation 102 , 515-529. McBurney, M. W. et al, Exp Cell Res (2002), vol 274, p1-8

On the other hand, even if the above-mentioned technique was used, the expression level of the target protein finally produced by transcription / translation of the target gene was not sufficient, and there was room for further improvement.

The present invention has been made in view of the above-mentioned conventional problems, and an object of the present invention is to provide a method and a kit for promoting the expression of a target protein in mammalian cells, and the use thereof.

Normally, when introducing multiple elements into cells, by arranging them on the same vector, it is predicted that the probability that each element will be retained in the host in a connected state will increase. However, contrary to the above prediction, the present inventor does not place the IR / MAR plasmid, the target gene, and the expression-promoting polynucleotide on the same vector, but rather promotes the expression of the target protein by introducing the IR / MAR plasmid, the target gene, and the expression-promoting polynucleotide into cells separately. We found that and came to complete the present invention.

In order to solve the above-mentioned problems, in the method for promoting the expression of the target protein in the mammalian cells according to one aspect of the present invention, the above-mentioned mammalian cells are cultured cells derived from mammals, and the following (i) Including the step of co-introducing the polynucleotide according to (iii) into a mammalian cell as a separate polynucleotide: (i) a polynucleotide containing an expression cassette of the protein of interest, (ii) functioning in the mammalian cell. Polynucleotides containing a mammalian replication initiation region and a nuclear matrix binding region, (iii) a polynucleotide that promotes the expression of the above-mentioned target protein.

In addition, the kit for promoting the expression of the target gene in the mammalian cell according to one aspect of the present invention contains the polynucleotides described in (ii) to (iii) below as separate polynucleotides: ( ii) A polynucleotide containing a mammalian replication initiation region and a nuclear matrix binding region that function in mammalian cells, and (iii) a polynucleotide that promotes the expression of a target protein.

In addition, the mammalian cell according to one aspect of the present invention is a poly containing (i) a polynucleotide containing an expression cassette of a protein of interest, (ii) a mammalian replication initiation region and a nuclear matrix binding region that function in the mammalian cell. Nucleotides (iii) and polynucleotides that promote the expression of the above-mentioned target protein are co-introduced into cultured cells derived from mammals as separate polynucleotides.

According to the present invention, it has the effect of promoting and maintaining the expression of the target gene in mammalian cells. Therefore, according to the present invention, it is possible to efficiently establish a system for producing a large amount of a target protein such as a useful protein.

In Example 1, it is a figure which shows the result of agarose gel electrophoresis of the product which amplified each expression-promoting polynucleotide by PCR, the product digested by the type I restriction enzyme RsrII, and the product which made them into a series repeat sequence by ligation. .. It is a schematic diagram which shows the antibody expression plasmid used in an Example. It is a schematic diagram which shows the IR / MAR plasmid used in an Example. It is a schematic diagram which shows the outline of the schedule of Step 3 of Example 1. FIG. It is a schematic diagram which shows the outline of the Fed-Batch test of Step 4 of Example 1. FIG. It is a schematic diagram which shows the drug treatment schedule to the cell carried out in Example 3. It is a graph which shows the antibody production amount in each test group of Example 3. FIG. It is a schematic diagram which shows the drug treatment schedule to the cell carried out in Example 4. It is a graph which shows the antibody production amount in each test group of Example 4. It is a graph which shows the value which divided the antibody production amount in each test group of Example 4 by Confluency.

An embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments and examples can be used. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention. In addition, all of the academic and patent documents described in this specification are incorporated herein by reference. Further, unless otherwise specified in the present specification, "AB" representing a numerical range is intended to be "A or more and B or less".

[1. How to promote the expression of the target protein in mammalian cells]
The method for promoting the expression of the target protein in the mammalian cell of the present embodiment (hereinafter, may be abbreviated as "method of the present embodiment") is the polynucleotide described in the following (i) to (iii). Is a method comprising the step of co-introducing into mammalian cells as separate polynucleotides. In the method of this embodiment, the mammalian cell is a cultured cell derived from a mammal.
(I) A polynucleotide containing an expression cassette of the protein of interest (in other words, polynucleotide (i)),
(Ii) A polynucleotide containing a mammalian replication initiation region and a nuclear matrix binding region that function within a mammalian cell (in other words, a polynucleotide (ii)),
(Iii) A polynucleotide that promotes the expression of the target protein (in other words, a polynucleotide (iii)).

(1-1. Polynucleotide (i))
The polynucleotide (i) contains an expression cassette of the protein of interest. As used herein, the term "protein of interest" indicates a protein to be expressed. The "target gene" indicates a polynucleotide encoding a target protein. The target gene is not particularly limited, and a polynucleotide encoding a desired protein may be appropriately selected and adopted. The polynucleotide may be obtained by using a known technique (for example, PCR method, chemical synthesis method) based on the base sequence information.

The target protein is not particularly limited as long as it is a polynucleotide sequence encoding the target protein to be expressed. Examples of the target protein include antibodies (H chain and L chain), cytokines such as IL-6 (interleukin 6), erythropoietin, interferon, fluorescent protein and the like. Antibodies that have attracted particular attention in recent years Antibodies that target antigens (eg, cell surface receptors) involved in specific diseases (eg, cancer and rheumatism) that are required in large quantities for the production of pharmaceutical products. Is a target protein that is expected to improve production efficiency by the method of this embodiment.

The expression cassette of the target protein is not particularly limited as long as it contains the polynucleotide necessary for the expression of the target gene, and may be at least one containing the target gene controlledly linked to the promoter. The gene expression cassette may contain a terminator, an IRES sequence, and the like, as well as a target gene that is ligated to a promoter in a controllable manner.

The target gene is preferably linked to a promoter so that its expression can be controlled. The promoter is not particularly limited as long as it functions in the introduced mammalian cell. For example, it may be a promoter in which the transcriptional activity of the promoter is controlled and activated or inactivated by a predetermined operation with a transcription factor or the like (in other words, a transcriptional activity-regulating promoter). Further, it may be a constitutive promoter in which the transcriptional activity is constitutively activated.

The transcriptional activity-regulating promoter is not particularly limited, and for example, commercially available products such as the TRE promoter (manufactured by Clontech) and the T-REX promoter (manufactured by Invitrogen) can be used. As the constitutive promoter, a CMV promoter, an SV40 early region-derived promoter (SV40 promoter), an SRalpha promoter (SRα promoter), an LTR promoter, an MMTV promoter and the like can be used.

The expression cassette of the target protein may also contain a polynucleotide necessary for expression of the target gene such as a terminator, a restriction enzyme recognition site, a polynucleotide necessary for cloning a drug resistance gene, and the like.

Examples of the polynucleotide (i) include an expression vector (for example, a plasmid vector) comprising a polynucleotide encoding a target protein.

(1-2. Polynucleotide (ii))
The polynucleotide (ii) comprises a mammalian replication initiation region and a nuclear matrix binding region that function within mammalian cells.

The mammalian replication initiation region (hereinafter, may be abbreviated as “IR”) that functions in the mammalian cell is not particularly limited, and is limited to the c-myc locus, the dihydrofolate reductase locus, and β-globin. The replication initiation region of the locus can be mentioned. The replication initiation region of the c-myc locus is described in, for example, "McWhinney, C. et al., Nucleic Acids Res. Vol. 18, p1233-1242 (1990)". The replication initiation region of the dihydrofolate reductase locus is described in, for example, "Dijkwel, P.A. et al., Mol. Cell. Biol. Vol.8, p5398-5409 (1988)". The replication initiation region of the β-globin locus is described in, for example, "Aladjem, M. et al., Science vol. 281, p1005-1009 (1998)".

The nuclear matrix binding region (hereinafter, may be abbreviated as "MAR") that functions in mammalian cells is not particularly limited, and the nuclear matrix binding of the Igκ locus, the SV40 initial region, and the dihydrofolate reductase locus is not particularly limited. Areas can be mentioned. The nuclear matrix binding region of the Igκ locus is described in, for example, "Tsutsui, K. et. Al., J. Biol. Chem. Vol. 268, p12886-12894 (1993)". The nuclear matrix binding region of the SV40 initial region is described in, for example, "Pommier, Y. et. Al., J. Virol., Vol. 64, p419-423 (1990)". The nuclear matrix binding region of the dihydrofolate reductase locus is described in, for example, "Shimizu N. et al., Cancer Res. Vol. 61, p6987-6990 (2001)".

Examples of the polynucleotide (ii) include an expression vector (for example, a plasmid vector) including a polynucleotide encoding IR and a polynucleotide encoding MAR.

(1-3. Polynucleotide (iii))
The polynucleotide (iii) promotes the expression of the protein of interest. The polynucleotide (iii) can artificially create an environment suitable for the expression of the target gene in the cell. The specific composition of the polynucleotide (iii) is not particularly limited, and a desired polynucleotide can be used as appropriate (eg, (i) Haiqing Fu et. Al., Preventing gene silencing with human replicators, NATURE BIOTECHNOLOGY, Vol.24, No.5, p572-576, (May 2006), (ii) Carl L Schildkraut et. Al., Replicators lessen transcriptional silencing, NATURE BIOTECHNOLOGY, Vol.24, No.5, p523-524, (May) 2006), (iii) Liang Huang et. Al., Prevention of Transcriptional Silencing by a Replicator-Binding Complex Consisting of SWI / SNF, MeCP1, and hnRNP C1 / C2, MOLECULAR AND CELLULAR BIOLOGY, Vol.31, No.16, See p3472-3484, (Aug. 2011)).

As an example of the polynucleotide (iii), the following polynucleotide (A) or a polynucleotide containing the polynucleotide (B) can be mentioned:
(A) A polynucleotide encoding at least a portion of IR or MAR,
(B) A human genome fragment of 3,271 (bp) derived from a region of low gene density of human chromosome 2 short arm 16.1 (hereinafter, also abbreviated as "B-3-31").

(1-3-1. Polynucleotide (A))
The polynucleotide (A) is a polynucleotide encoding at least a portion of IR or MAR. Further, the polynucleotide (A) may be a polynucleotide encoding IR and / or MAR.

The IR is not particularly limited, and examples thereof include a c-myc locus, a dihydrofolate reductase locus, and a replication initiation region of the β-globin locus. The replication initiation region of the c-myc locus is described in, for example, "McWhinney, C. et al., Nucleic Acids Res. Vol. 18, p1233-1242 (1990)". The replication initiation region of the dihydrofolate reductase locus is described in, for example, "Dijkwel, P.A. et al., Mol. Cell. Biol. Vol.8, p5398-5409 (1988)". The replication initiation region of the β-globin locus is described in, for example, "Aladjem, M. et al., Science vol. 281, p1005-1009 (1998)".

More specifically, the polynucleotide (A) is a polynucleotide containing any of the following polynucleotides (f) to (i), or a polynucleotide consisting of any of the following polynucleotides (f) to (i). May be nucleotides:
(F) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 3,
(G) A polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 2 or 3, and an activity that promotes the expression of the target protein. Polynucleotide with.
(H) A poly having an activity of hybridizing under stringent conditions with a DNA having a base sequence complementary to the polynucleotide having the base sequence shown in SEQ ID NO: 2 or 3 and promoting the expression of the target protein. nucleotide.
(I) Activity having 90% or more, 95% or more, 97% or more or 99% or more homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 or 3 and promoting the expression of the target protein. Polynucleotide with.

Hereinafter, the polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 may be abbreviated as "G5", and the polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 may be abbreviated as "G5 / AR1".

(1-3-1. Polynucleotide (B))
The polynucleotide (B) is a human genome fragment (B-3-31) of 3,271 (bp) derived from the low gene density region of human chromosome 2 short arm 16.1.

The polynucleotide (B) may be a polynucleotide containing any of the following polynucleotides (a) to (e), or a polynucleotide composed of any of the following polynucleotides (a) to (e). good:
(A) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 1.
(B) A polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 1 and has an activity of promoting the expression of the target protein. Polynucleotide,
(C) A polynucleotide that hybridizes under stringent conditions with a DNA having a base sequence complementary to the polynucleotide having the base sequence shown in SEQ ID NO: 1 and has an activity of promoting the expression of the target protein.
(D) It has 90% or more, 95% or more, 97% or more or 99% or more homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 and has an activity of promoting the expression of the target protein. Polynucleotide.
(E) A polynucleotide that is a part of the polynucleotides (a) to (d) and has an activity of promoting the expression of a target protein.

In the above-mentioned polynucleotides (A) and (B), "a polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted, or added" (in other words, a mutant polynucleotide) is, for example, 30 or less, preferably 25 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 Hereinafter, a polynucleotide consisting of a base sequence in which two or less, more preferably one or less, is deleted, substituted, or added is intended.

In the above-mentioned polynucleotides (A) and (B), "stringent conditions" are hybridization solutions (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate). After incubation overnight at 42 ° C. in (pH 7.6) containing 5 × Denhart's solution, 10% dextran sulfate, and 20 μg / ml denatured shear salmon sperm DNA, 0.1 × at about 65 ° C. Although it is intended to wash the filter in SSC, the polynucleotides to be hybridized will appropriately change the washing conditions at high stringency. For example, washing at 65 ° C. (preferably 15 minutes x 2 times) in 0.5 x SSC containing 0.1% SDS is preferable. E. When using colli-derived DNA, washing at 68 ° C. (preferably 15 minutes × 2 times) in 0.1 × SSC containing 0.1% SDS is preferable. When RNA is used, washing at 68 ° C. (preferably 15 minutes x 2 times) in 0.1 x SSC containing 0.1% SDS is preferable. When an oligonucleotide is used, washing at a hybridization temperature (preferably 15 minutes x 2 times) in 0.1 x SSC containing 0.1% SDS is preferable. Further, the above hybridization can be performed by a well-known method described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989).

In the above-mentioned polynucleotides (A) and (B), "homology" can be confirmed based on a well-known method. Specifically, the base sequence shown in SEQ ID NO: 1, 2 or 3 is used as a query to execute a homology search program such as BLASTN 2.2.1, and a homology search is performed on databases such as GenBank, EMBL, and DDBJ. The polynucleotide consisting of the base sequence obtained by performing the above can be used as the polynucleotide (A) or (B).

Examples of the polynucleotide (iii) include linear polynucleotides and the like. The polynucleotide (iii) may be any as long as it contains the polynucleotide (B) in terms of improving the production amount of the target protein, etc., but the sequence other than the polynucleotide (B) is 5 of the entire polynucleotide (iii). % Or less is preferable. Further, from the viewpoint of improving the production amount of the target protein, the polynucleotide (iii) preferably contains a repetitive sequence (repeat sequence), and preferably contains a repetitive sequence of the polynucleotide (A) or the polynucleotide (B). Is more preferable, and it is further preferable to include a repeating sequence of the polynucleotide (B). Hereinafter, the repetitive sequence of polynucleotide (iii) will be described.

(1-4. Repeated sequence of polynucleotide (iii))
The repetitive sequence of the polynucleotide (iii) includes a unit sequence in the polynucleotide (iii) a plurality of times, and examples of the unit sequence include the above-mentioned polynucleotides (A) and (B). This makes it possible to artificially create an environment suitable for the expression of the target gene in the cell.

The size of the repetitive sequence is not particularly limited, but is preferably 500 to 500,000 bp, more preferably 1,000 to 500,000 bp, more preferably 2,000 to 200,000 bp, and most preferably. Is 4,000 to 100,000 bp.

The number of unit sequences included in the repeated sequence may be two or more, and is not particularly limited. For example, the number of unit sequences contained in the repetitive sequence is preferably 2 to 50, more preferably 2 to 25, and most preferably 2 to 25, from the viewpoint of creating an expression promoting environment independently of the surrounding chromosomal environment. There are 3 to 20 pieces. Here, the number of unit sequences contained in the repetitive sequence is at least 10%, more preferably 30%, and most preferably 50% or more of the polynucleotide (iii) molecules introduced as the expression-promoting polynucleotide. It means that the number of repeated sequences is within the above range.

The plurality of unit sequences contained in the repetitive sequence may be directly bound to each other, or may be indirectly bound to each other via other configurations (for example, nucleotides or polynucleotides). .. For example, the unit sequences may be bound to each other via a restriction enzyme recognition site.

The repeated sequence may include a serial repeat sequence (direct repeat) of the unit array, or may include an inverted repeat sequence (inverted repeat) of the unit sequence. That is, the repetitive sequence may include a polynucleotide (series repetitive sequence) in which a plurality of consecutively linked unit sequences are linked so as to be in the same direction. Further, the repeated sequence may include a polynucleotide (inverted repeated sequence) in which a plurality of consecutively arranged unit sequences are linked in the opposite direction.

As an example, consider two unit sequences each having a base sequence of "ATG".

When the "G" of the first unit sequence is bound to the "A" of the second unit sequence, the first polynucleotide (iii) and the second unit sequence form a serial repetitive sequence. Will be there. That is, in this case, the first unit array and the second unit array are concatenated so as to be in the same direction.

On the other hand, when the "G" of the first unit array is bound to the "G" of the second unit array, or the "A" of the first unit array is combined with the "A" of the second unit array. When combined, the first unit sequence and the second unit sequence form an inverted repeat sequence. That is, in this case, the first unit array and the second unit array are concatenated so as to be in opposite directions.

If the repetitive sequence is "contains a series repetitive sequence of unit sequences", it is not necessary that all of the unit sequences are concatenated so as to be in the same direction.

For example, two consecutive unit arrays are defined as one repeating unit. Of all the repetitive units forming the repetitive sequence, 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more. Most preferably, in a 100% repetitive unit, when the first unit sequence and the second unit sequence forming the repetitive unit form a serial repetitive sequence, the repeat sequence is referred to as "series repeat of the unit sequence". It may be defined as "including an array".

Also, of all the repeating units forming the repeat sequence, less than 50%, more preferably 40% or less, more preferably 30% or less, more preferably 20% or less, more preferably 10% or less, more preferably 5 % Or less, most preferably 0%, when the first unit sequence and the second unit sequence forming the repeat unit form an inverted repeat sequence, the repeat sequence is referred to as "unit sequence". It may be defined as "containing a serial repeat sequence of."

When the repeat sequence is "contains the inverted repeat sequence of the unit sequence", it is not necessary that all of the polynucleotides are concatenated so as to be in the opposite direction.

Similar to the above, two consecutive unit arrays are defined as one repeating unit. Of all the repeating units forming the repeat sequence, 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more. Most preferably, in a 100% repeat unit, when the first unit sequence and the second unit sequence forming the repeat unit form an inverted repeat sequence, the repeat sequence is referred to as "inverted unit sequence". It may be defined as "contains a repetitive sequence".

Also, of all the repeating units forming the repeat sequence, less than 50%, more preferably 40% or less, more preferably 30% or less, more preferably 20% or less, more preferably 10% or less, more preferably 5 % Or less, most preferably 0%, when the first and second unit sequences forming the repeat unit form a series repeat sequence, the repeat sequence is referred to as "unit sequence". It may be defined as "contains an inverted repeat sequence".

When the repetitive sequence contains a series repetitive sequence of unit sequences, the repetitive sequence tends to be stably integrated into the chromosome. On the other hand, when the repetitive sequence contains an inverted repeat sequence of a unit sequence, the repetitive sequence is cut out from the chromosome and tends to exist outside the chromosome.

(1-5. Step of co-introducing polynucleotides (i) to (iii))
The method of this embodiment includes a step of co-introducing polynucleotides (i) to (iii) into mammalian cells as separate polynucleotides (in other words, a co-introduction step).

As the mammalian cells, cultured cells derived from various mammals are used. Examples of cultured cells derived from various mammals include CHO derived from Chinese hamster and various tumor cells. Examples of the CHO include CHO-K1 (ATCC CCL-61, RIKEN RCB0285, RIKEN RCB0403, etc.), CHO DG44, and the like. CHO is a cell that is currently used in the actual production of useful proteins such as pharmaceuticals and whose safety has been confirmed, and is preferable as a mammalian cell to which the method of the present embodiment is applied.

In terms of high gene amplification efficiency, tumor cells having infinite proliferation ability are preferable as mammalian cells. Examples of the tumor cells include HeLa (source: ATCC CCL-2, ATCC CCL-2.2, RIKEN RCB0007, RIKEN RCB0191, etc.), human colorectal cancer COLO 320DM (source: ATCC CCL-, for example). 220), human colorectal cancer COLO 320HSR (source: ATCC CCL-220.1), NS0 (source: eg RIKEN RCB0213) and the like. For human colorectal cancer COLO 320DM, see "Shimizu, N., Kanda, T., and Wahl, G. M. Selective capture of acentric fragments by micronuclei provides a rapid method for purifying extrachromosomally amplified DNA. 65-71, 1996. ”.

The method for co-introducing the polynucleotides (i) to (iii) into mammalian cells as separate polynucleotides is not particularly limited, and known methods such as lipofection, electroporation, calcium phosphate method, and particle gun method are known. Methods are available. Further, the detailed conditions may be adopted after appropriately considering the optimum conditions according to the mammal to be introduced, each element and the like.

As shown in the examples, the expression vector and the polynucleotide (ii) comprising the polynucleotides (i) and (ii) by co-introducing the polynucleotides (i) to (iii) into mammalian cells as separate polynucleotides ( The productivity of the target protein is improved (the expression of the target protein is promoted) as compared with the simultaneous introduction of iii) into mammalian cells. In addition, mammalian cells co-introduced with polynucleotides (i) to (iii) as separate polynucleotides have high antibody production per cell and genetic stability (in other words, passage stability). expensive.

In addition, co-introduction of polynucleotides (i) to (iii) into mammalian cells as separate polynucleotides promotes the expression of selectable markers such as drug resistance genes contained in the expression vector (plasmid vector), and traits. Screening of converted cells can be performed in a short period of time. Therefore, since the transformed cells can be screened in a short period of time, mammalian cells that produce a large amount of the target protein can be produced in a short period of time and at low cost.

In particular, the amount of the polynucleotide (iii) introduced into the mammalian cells is 1 to 4 times, preferably 2 to 4 times, the amount of the polynucleotides (i) and (ii) introduced into the mammalian cells, respectively. , The productivity of the target protein is further improved. Percentage of introduction amount of each polynucleotide into mammalian cells in terms of improving the productivity of the target protein (introduction amount of polynucleotide (i): introduction amount of polynucleotide (ii): introduction of polynucleotide (iii) The amount) is more preferably 1: 1: 1 to 4, and even more preferably 1: 1: 2 to 4. If the introduction amount of the polynucleotide (iii) is too small, the production amount of the target protein may be low. If the amount of the polynucleotide (iii) introduced is too large, the amount of the polynucleotides (i) and (ii) introduced may be small, and the production amount of the target protein may also be low.

[1-6. Other processes]
In the method of the present embodiment, in addition to the co-introduction step, a step of separating mammalian cells co-introduced with polynucleotides (i) to (iii) (in other words, a selection step) and selection by the selection step. It may include a step (in other words, a culturing step) of culturing the animal cells (in other words, transformed cells). In addition, a step of purifying the target protein produced by the culture step (in other words, a purification step) may be included. That is, the method of the present embodiment also includes a method for producing a target protein using mammalian cells in which polynucleotides (i) to (iii) are co-introduced.

(1-6-1. Selection process)
The selection step is a step of separating mammalian cells co-introduced with polynucleotides (i) to (iii). More specifically, the selection step was a mixture of mammalian cells co-introduced with polynucleotides (i)-(iii) and mammalian cells co-introduced with polynucleotides (i)-(iii). This is a step of selecting mammalian cells co-introduced with polynucleotides (i) to (iii) from a cell population. By the selection step, mammalian cells capable of promoting the expression of the target protein can be selected.

The specific method of the selection step is not particularly limited, but for example, when the expression vector contains a drug resistance gene as a selection marker, desired cells may be selected by utilizing the drug resistance. .. Examples of drug resistance genes are aminoglycoside 3'-phosphotransferase (canamycin resistance gene), neomycin phosphotransferase gene (neomycin resistance gene), hyglomycin B phosphotransferase gene (hyglomycin resistance gene), and blastsaidin S deaminase gene. (Blastsaidin resistance gene) and the like. In addition to the drug resistance gene, the expression vector may contain a glutamine synthesizer gene or a green fluorescent protein gene as a selectable marker.

In addition, instead of using the above selection marker, dihydrofolate reductase gene (DHFR) is introduced into a mammal by culturing mammalian cells (originally a DHFR-deficient strain) in a medium containing no ribonucleotide and deoxyribonucleotide. You can also select cells.

The selection step can also be performed by detecting a polynucleotide such as a target gene contained in a cell by a PCR method or a Southern blotting method. The specific method of selection using drug resistance, PCR method or Southern blotting method is not particularly limited, and known methods can be appropriately used. In particular, it is preferable that the medium used for cell culture contains one or more of a histone deacetylase inhibitor and a DNA methylation inhibitor. The present inventors have found that histone deacetylase inhibitors and DNA methylation inhibitors can further promote the expression of the target protein. Histone deacetylase inhibitors release the epigenetic suppression of the target gene by increasing histone acetylation levels, and DNA methylation inhibitors reduce DNA methylation levels. , The expression of the target protein can be promoted.

The histone deacetylase inhibitor and the DNA methylation inhibitor are not particularly limited. Examples of histone deacetylase inhibitors include butyrate such as sodium butyrate, Trichostatin A (TSA), MS-275, Oxamflatin, DMSO and the like. Examples of the DNA methylation inhibitor include 5-aza-2'-deoxycytidine, 5-aza-2'-cytidine and the like. The amount of each of the above inhibitors added to the medium may be adopted after examining the amount of addition that promotes the expression of the target protein within a range that does not affect the growth of the transformed cells to be cultured.

(1-6-2. Culture step)
The culturing step is a step of culturing mammalian cells already selected by the selection step. By such a culture step, the target protein can be highly expressed in mammalian cells. The specific method of the culturing step is not particularly limited, and the optimum conditions for the mammalian cells to be cultivated may be examined and appropriately adopted.

(1-6-3. Purification process)
The purification step is a step of purifying the target protein produced by the culture step. As a specific method for purifying a protein in the purification step, for example, after suspending mammalian cells in a buffer solution such as PBS (Phosphate Buffered Saline), the cells are disrupted with a homogenizer or ultrasonic waves, and the suspension is applied. After the turbid liquid is subjected to centrifugation, the supernatant may be collected. A surfactant for promoting solubilization of the target protein, a reducing agent for stabilizing the three-dimensional structure of the target protein, and a protease inhibitor for preventing the decomposition of the target protein are appropriately added to the buffer solution. May be.

As the surfactant, CHAPS (3-[(3-cholamidopropyl) -dimethylammonio-1-propanesulfonate], Triton X-100, Nikkol, n-octyl glycoside and the like can be used, and as the reducing agent. Can use DTT (dithiothreitol), DET (dithioerythritol) and the like. As the above-mentioned protease inhibitor, aprotinin and leupeptin can be used.

From the above supernatant, the target protein can be purified by using column chromatography such as affinity chromatography, ion exchange chromatography and filtration chromatography. In addition, unnecessary salts may be removed from the solution containing the target protein by dialyzing the purified solution containing the target protein against an appropriate buffer solution. The purification step is preferably carried out under low temperature conditions in order to suppress the decomposition of the target protein. In particular, it is preferable that the purification step is performed under a low temperature condition of 4 ° C. or lower. The specific method of the purification step is not limited to this, and known methods can be appropriately used.

[2. Kit for promoting expression of target protein in mammalian cells]
The kit for promoting the expression of the target protein in the mammalian cell of the present embodiment (hereinafter, may be abbreviated as “kit of the present embodiment”) is the poly according to the above (ii) and (iii). Includes nucleotides as separate polynucleotides. The polynucleotide of (i) above may be contained.

In the kit of the present embodiment, the polynucleotide of (iii) above includes a polynucleotide containing the polynucleotide of (a) or (b) above, or a repeating sequence of the polynucleotide of (a) or (b) above. It is preferably a polynucleotide.

In the kit of the present embodiment, the amount of the polynucleotide (iii) introduced into the above-mentioned animal cell is the amount of each of the above-mentioned polynucleotide (i) and the above-mentioned polynucleotide (ii) introduced into the above-mentioned animal cell. It is preferably prepared to be 1 to 4 times, and more preferably 2 to 4 times.

In the kit of the present embodiment, it is preferable that the polynucleotides (i) and (ii) above are plasmid vectors, and the polynucleotides (iii) above are linear polynucleotides.

The kit of the present embodiment may further include equipment and reagents necessary for transformation, host mammalian cells (for example, cultured cells derived from mammals), instruction manuals, and the like.
[3. Mammalian cells]
The mammalian cell of the present embodiment is obtained by co-introducing the polynucleotides described in (i) to (iii) above into cultured animal-derived cells as separate polynucleotides.

In the mammalian cell of the present embodiment, the polynucleotide of (iii) above is a polynucleotide containing the polynucleotide of (a) or (b) above, or a repeating sequence of the polynucleotide of (a) or (b) above. It is preferable that it is a polynucleotide containing.

In the mammalian cell of the present embodiment, the amount of the polynucleotide (iii) introduced into the mammalian cell is such that the polynucleotide (i) and the polynucleotide (ii) are each introduced into the mammalian cell. It is preferably prepared to be 1 to 4 times the amount, and more preferably 2 to 4 times the amount.

In the kit of the present embodiment, it is preferable that the polynucleotides (i) and (ii) above are plasmid vectors, and the polynucleotides (iii) above are linear polynucleotides.

[Summary of Embodiment]
Embodiments of the present invention include, for example, the following aspects.
<1> The method for promoting the expression of a target protein in a mammalian cell according to one aspect of the present invention is described in the following (i) to (iii), wherein the mammalian cell is a cultured cell derived from a mammal. Contains the steps of co-introducing the polynucleotides of the above into mammalian cells as separate polynucleotides: (i) a polynucleotide containing an expression cassette of the protein of interest, (ii) a mammalian replication initiation region that functions within the mammalian cell and A polynucleotide containing a nuclear matrix binding region, (iii) a polynucleotide that promotes the expression of the above-mentioned target protein.
<2> In the method for promoting the expression of the target protein in the mammalian cell according to one aspect of the present invention, the polynucleotide of (iii) above is a polynucleotide containing the polynucleotide of (a) or (b) below. It is preferable: (a) a polynucleotide consisting of the base sequence shown in any of SEQ ID NOs: 1 to 3, and (b) one or several bases are missing in the base sequence shown in any of SEQ ID NOs: 1 to 3. A polynucleotide consisting of a lost, substituted, or added base sequence and having an activity of promoting the expression of a target protein.
<3> In the method for promoting the expression of the target protein in the mammalian cell according to one aspect of the present invention, the polynucleotide of (iii) above is a poly containing a repeating sequence of the polynucleotide of (a) or (b) below. It is preferably a nucleotide: (a) a polynucleotide consisting of the base sequence shown in any of SEQ ID NOs: 1 to 3, and (b) one or several in the base sequence shown in any of SEQ ID NOs: 1 to 3. A polynucleotide consisting of a base sequence in which a base has been deleted, substituted, or added, and which has an activity of promoting the expression of a target protein.
<4> In the method for promoting the expression of a target protein in a mammalian cell according to one aspect of the present invention, the amount of the polynucleotide of (iii) introduced into the mammalian cell is the same as that of the polynucleotide of (i). The amount of each of the polynucleotides (ii) introduced into the above-mentioned mammalian cells is preferably 1 to 4 times.
<5> In the method for promoting the expression of a target protein in a mammalian cell according to one aspect of the present invention, the amount of the polynucleotide of (iii) introduced into the mammalian cell is the same as that of the polynucleotide of (i). It is preferable that the amount of each of the polynucleotides (ii) introduced into the above-mentioned mammalian cells is 2 to 4 times.
<6> In the method for promoting the expression of a target protein in a mammalian cell according to one aspect of the present invention, the polynucleotides (i) and (ii) above are plasmid vectors, and the polynucleotide (iii) above is direct. It is preferably a chain polynucleotide.
<7> The kit for promoting the expression of the target gene in the mammalian cell according to one aspect of the present invention contains the polynucleotides described in (ii) to (iii) below as separate polynucleotides: (Ii) A polynucleotide containing a mammalian replication initiation region and a nuclear matrix binding region that function in mammalian cells, and (iii) a polynucleotide that promotes the expression of a target protein.
<8> The mammalian cell according to one aspect of the present invention contains (i) a polynucleotide containing an expression cassette of a protein of interest, (ii) a mammalian replication initiation region and a nuclear matrix binding region that function within the mammalian cell. The polynucleotide, (iii) the polynucleotide that promotes the expression of the above-mentioned target protein, is co-introduced into cultured cells derived from mammals as separate polynucleotides.
<9> The above mammalian cells are used as a method for producing the target protein according to one aspect of the present invention.

[Example 1]
<Step 1. Fabrication of serial repeats>
Serial repeats of B-3-31, G5 and G5 / AR1 were made. The production method will be described below.

PG5 and pTV-Rep-P were constructed according to the description of Okada N, Shimizu N. PLoS ONE, 2013. In addition, pB-3-31 in which B-3-31 was inserted into pΔBM-d2EGFP-AscI was constructed according to the description of M Fukuma, Y Ganmyo, O Miura, T Ohyama, N Shimizu. PloS ONE, 2016.

Next, as shown in Tables 1 and 2, by PCR using pB-3-31, pG5, pTV-Rep-P as a template and PrimeSTARMax DNA Polymerase (Takara) as an enzyme, B-3-1 31, G5 or G5 / AR1 was amplified. B-3-31 has a DNA fragment length of 3,271 bp and consists of the base sequence represented by SEQ ID NO: 1. G5 has a DNA fragment length of 972 bp and consists of the sequence represented by SEQ ID NO: 2. G5 / AR1 has a DNA fragment length of 2,030 bp and consists of the sequence represented by SEQ ID NO: 3. The nucleotide sequence of Forward Primer of B-3-31 is CATGCTTCGGACCGAAATTACCTACACTTCTGCA (SEQ ID NO: 4), and the nucleotide sequence of Reverse Primer is ATGCCAACGGTCCGCAGAATCTGAGAAGGCTTAA (SEQ ID NO: 5). The nucleotide sequence of Forward Primer of G5 is CATGCTTCGGACCGGCATTTTCTTTGACCCAGGA (SEQ ID NO: 6), and the nucleotide sequence of Reverse Primer is ATGCCAACGGTCCGCCCATCCCCCTGTACTTTTT (SEQ ID NO: 7). The nucleotide sequence of Forward Primer of G5 / AR1 is CATGCTTCGGACCGTTGGTTATGCCGGTACTGCC (SEQ ID NO: 8), and the nucleotide sequence of Reverse Primer is ATGCCAACGGTCCGCCCATCCCCCTGTACTTTTT (SEQ ID NO: 9). In order to prepare a serial repeat sequence, a recognition sequence of RsrII (CGG (A / T) CCG), which is a type I restriction enzyme, was added in advance to the 5'end of the primer used.

The PCR amplification product has RsrII recognition sequences at the 5'end and 3'end. The PCR amplification product was completely digested with RsrII and the short fragments produced by the digestion were removed using Nucleo Spin Gel and PCR Clean-up (Takara). The PCR amplification product after RsrII treatment was ligated using DNA Ligation Kit <Mighty Mix> (Takara). The results of electrophoresis on a 0.8% agarose gel after ligation are shown in FIG.

As shown in FIG. 1, amplification of G5 (972bp), G5 / AR1 (2030bp), and B-3-31 (3271bp) was confirmed by PCR (lanes No. 2 to 4). In addition, as a result of ligation, a repetitive sequence (repeat sequence) in which 2 to 6 unit structures were linked was also confirmed (lanes No. 10 to 12).

<Step 2. Preparation of expression vector>
The artificially synthesized DHFR, BGHpA, and TKpA sequences were incorporated into pHSG299 (Takara), and the EF1α promoter sequence amplified by the PCR method using pBApo-EF1α Pur (Takara) as a template was inserted into the plasmid used in the examples. A vector was prepared. FIG. 2 shows the configurations of the antibody expression plasmids pB7-HL and pC7-HL in which the genes of antibody B and antibody C, which are the target genes, are inserted into the plasmid vector, respectively. The left figure of FIG. 2 shows the plasmid before inserting Blasticidin S (BS), and the right figure shows the plasmid after inserting BS. A heavy chain (HC) and a light chain (LC) of antibody B are inserted into pB7-HL and pB7-HL-BS, respectively. A heavy chain (HC) and a light chain (LC) of antibody C are inserted into pC7-HL and pC7-CL-BS, respectively. The amino acid sequence of the heavy chain (HC) of antibody B is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain (LC) is shown in SEQ ID NO: 11. The amino acid sequence of HC of antibody C is shown in SEQ ID NO: 12, and the amino acid sequence of LC is shown in SEQ ID NO: 13. The IR / MAR plasmid (pBN-BS) is a plasmid in which IR (specifically, the replication initiation region derived from the human beta-globin locus) and MAR (specifically, AR1) are inserted. Shown in 3.

<Step 3. Introduction of each polynucleotide into CHO cells>
According to the procedure (1) to (5) shown below, the repeat sequence and expression vector (IR / MAR plasmid and antibody expression plasmid) of B-3-31, G5 or G5 / AR1 are transferred to CHO cells (Chinese hamster ovary). Co-introduced. FIG. 4 is a schematic diagram showing an outline of the procedures (1) to (5) shown below. The number of days in FIG. 4 indicates the number of days elapsed after transfection.
(1) Repeat sequences of B-3-31, G5 or G5 / AR1 prepared in steps 1 and 2 and IR / MAR plasmid (pBN) were added to CHO cells cultured in F-12 medium supplemented with 5% FBS until the logarithmic growth phase. -BS) and antibody expression plasmid (pB7-HL) were transfected by lipofection method. In the lipofection method, Thermo Fisher's Lipofectamine® 3000 Reagent was used and the protocol followed the product manual.
(2) The day after transfection, the CHO cells transfected by the method of (1) were peeled off with TrypLE and collected. Next, Blasticidin S (BS) was added to the medium of (1) so as to be 5 μg / mL. Then, the collected CHO cells were dispersed in the medium.
(3) 10 days after transfection, the concentration of Blasticidin S was increased to 10 μg / mL, and the cells were further cultured for 14 days.
(4) Twenty-three days after transfection, the medium was replaced with 10 mL of α-MEM medium (without ribonucleoside and deoxyribonucleotide) containing 5% dialysate FBS, and cultured for 7 days.
(5) Thirty days after transfection, 5 nM methotrexate (MTX) was added to the medium, and the culture was continued for 13 days. 50 nM or 100 nM methotrexate was added to the culture medium containing the surviving cells, and gene amplification treatment and cell isolation were performed.

<Step 4: Acclimation to serum-free medium and measurement of antibody B expression level>
The CHO cells that survived the methotrexate treatment of step 3 (5) were peeled off with TrypLE and collected. After exchanging the medium in EX-CELL Fed Batch Medium (Merck) supplemented with 8 mM Gln, only the collected cells were placed in a 125 mL Erlenmeyer flask, and swirling culture was started. When the habituation was completed, the Fed-Batch test was conducted. The outline of the Fed-Batch test is shown in FIG. As shown in FIG. 5, 5-10% of EX-CELL® Advanced CHO Feed 1 (Merck) was added on the 4th, 7th, and 11th days after the start of the test.

Then, the amount of antibody on the 14th day after the start of the test was measured by Cedex-Bio (Roche Diagnostics).

Table 3 shows the results of comparison of the expression levels of the antibody by co-introduction of the antibody expression plasmid and the IR / MAR plasmid and the expression-promoting polynucleotide. In contrast to the test group (No. 1) in which only the plasmid expressing antibody B was transfected, the test group (No. 2 to 5) in which the plasmid expressing antibody B and the IR / MAR plasmid were co-introduced showed the antibody. The amount was increasing. In particular, a significant increase in the expression level was observed in the test group (No. 3) in which the plasmid expressing antibody B, the IR / MAR plasmid, and the serial repeat sequence of B-3-31 were co-introduced.

Table 4 shows the results of examining the quantitative ratio of the serially repeated sequences of B-3-31 to be co-introduced. The amount ratio of the antibody expression plasmid (pB7-HL) to the IR / MAR plasmid (pBN-BS) was fixed at 1: 1 and the amount of the serial repeat sequence of B-3-31 was determined by the antibody expression plasmid or IR / MAR. The amount of each plasmid was examined in the range of 0.5 to 4 times. As shown in Table 4, the expression level of the antibody is remarkable when the amount of the serial repeat sequence of B-3-31 introduced is 1 to 4 times the amount of each of the antibody expression plasmid or the IR / MAR plasmid. It was found that the expression level of the antibody was maximized when the dose was doubled.

Next, a vector (pB7-BSIM) in which a plasmid expressing antibody B and an IR / MAR plasmid were fused was prepared, and the results when a serial repeat sequence of B-3-31 was added to the fusion vector are shown in Table 5. Shown in. As shown in Table 5, no productivity improving effect was observed even when the serial repeat sequence of B-3-31 was added to the fusion vector.

From the results of Example 1, a polynucleotide containing an expression cassette of the target protein, a polynucleotide containing IR and MAR, and an expression-promoting polynucleotide are co-introduced into mammalian cells as separate polynucleotides to obtain the target gene and It was found that the productivity of the target protein was improved (the expression of the target protein was promoted) as compared with the introduction of the expression vector containing the polynucleotide containing IR and MAR and the expression promoting polynucleotide.

[Example 2]
In a cell pool in which only the antibody expression plasmid expressing antibody B was introduced, and in a cell pool in which the antibody expression plasmid, IR / MAR plasmid, and B-3-31 were co-introduced at a volume ratio of 1: 1: 4. Cloning was performed using each AC-7 as a parent pool. The resulting clones were subcultured at intervals of 3-4 days. After 1 month, 2 months, and 3 months after the start of the passage, the same Fed-Batch test as in step 4 of Example 1 was carried out, and how the productivity changed compared to before the start of the passage. Tested.

Table 6 shows the results of Example 2. In this test, maintaining 70% of the productivity before the start of the passage was regarded as passing. On the other hand, if the productivity fell below 70% of the productivity before the start of passage in the middle of 3 months after the start of passage, the test for the clone was discontinued. As shown in Table 6, only 1 out of 6 clones (about 16.7%) passed in AC-4, whereas 3 out of 4 clones (75%) in AC-7 maintained high productivity. And passed. Therefore, the cells obtained as a result of co-introduction by increasing the amount of B-3-31 with respect to the amount of the antibody expression plasmid and IR / MAR plasmid introduced may have high genetic stability (passage stability). Do you get it.

[Example 3]
A plasmid expressing antibody C was prepared according to a well-known method and tested in the test group shown in Table 7. The antibody expression plasmid pC7-HL has the same composition as that of pB7-HL in FIG. 2 except that the gene of antibody B is changed to the gene of antibody C. The amino acid sequence of HC of antibody C is shown in SEQ ID NO: 12, and the amino acid sequence of LC is shown in SEQ ID NO: 13. The antibody expression plasmid pC7-HL-BS is a plasmid in which the Blasticidin S resistance gene is inserted into pC7-HL.

The outline of the drug treatment schedule for cells is shown in FIG. Specifically, the test was conducted according to the following procedures (1) to (5).
(1) CHO cells cultured in F-12 medium supplemented with 5% FBS until the logarithmic growth phase were transfected with polynucleotides according to the test group shown in Table 7 by the lipofection method.
(2) The day after transfection, the CHO cells transfected by the method of (1) were peeled off with TrypLE and collected. Next, Blasticidin S (BS) was added to the medium of (1) so as to be 5 μg / mL. Then, the collected CHO cells were dispersed in the medium.
(3) Twelve days after transfection, the concentration of Blasticidin S was increased to 10 μg / mL, and the cells were further cultured for 10 days. After culturing for 10 days, antibody productivity per cell (Qp: pg / cell / day) was evaluated.
(4) Twenty-five days after transfection, the concentration of Blasticidin S was increased to 100 μg / mL, and the cells were further cultured for 10 days. After culturing for 10 days, the expression level of antibody protein in the culture supernatant was evaluated by ELISA.
(5) 34 days after transfection, cells were detached with TrypLE. Using a flow cytometer, the collected cells were seeded on a 96-well plate to 1 cell / well (FACS sorting).
(6) The cells seeded in (5) were further cultured for 21 days, and the expression level of the antibody protein in the culture supernatant was determined by the ELISA method.

The results of Example 3 are shown in FIGS. 7 and 7. The horizontal axis of the graph in FIG. 7 indicates the test plot number in Table 7. The vertical axis shows the amount of IgG (ng / mL). As shown in FIGS. 7 and 7, in antibody C as well as in antibody B, the IR / MAR plasmid was co-introduced into the test group (No. 1) in which only the plasmid expressing antibody C was transfected. In the group (No. 2 and 3), the expression level of the antibody was increased. In particular, a further increase in the expression level was observed in the test group (No. 3) in which the antibody expression plasmid, the IR / MAR plasmid, and the serial repeat sequence of B-3-31 were co-introduced as separate polynucleotides. The antibody productivity per cell (Qp: pg / cell / day) was calculated from the calculated antibody production amount from the number of cells and the number of culture days.

[Example 4]
By performing FACS sorting and cloning at an early stage in Step 3 of Example 1, it was expected that the difference in the production effect of the target protein in each test group shown in Table 8 would be clarified. Therefore, screening was carried out according to the schedule shown in FIG. Specifically, the test was conducted according to the following procedures (1) to (7).
(1) CHO cells cultured in F-12 medium supplemented with 5% FBS up to the logarithmic growth phase were seeded on a 6-well plate, and transfection was carried out by the lipofection method in the test group shown in Table 8.
(2) The day after transfection, the CHO cells transfected by the method of (1) were peeled off with TrypLE and collected. Next, the medium (1) was replaced with a 5% FBS-added F-12 medium supplemented with Blasticidin S so that the final concentration was 5 μg / mL. Then, the collected CHO cells were dispersed in the medium.
(3) When cell proliferation was observed, the cells were peeled off with TrypLE and collected. Next, the concentration of Blasticidin S in the medium was increased to 100 μg / mL. Then, the collected CHO cells were dispersed in the medium.
(4) When the growth of the cells was sufficiently observed, the cells were peeled off by TrypLE. Using a flow cytometer, the collected cells were seeded in advance on a 96-well plate dispensed at 150 μL / well with 5% D-FBS-added α-MEM (FACS sorting and cloning). ..
(5) Nine days after seeding in a 96-well plate, the medium was replaced with 5% D-FBS-added α-MEM added so that MTX had a final concentration of 5 nM. Eight days after the medium was replaced, the amount of antibody in the well was quantified by ELISA, and Confluency was measured by Clone Select Imager (molecular device).

Table 8 shows the time required from 5 μg / mL Blasticidin S treatment to seeding cells in a 96-well plate (the number of days required from BS treatment to FACS). The test plot numbers in Table 8 correspond to the test plot numbers in Table 8. The time required from the 5 μg / mL Blasticidin S treatment to seeding the cells in the 96-well plate was 21 days in the test group (No. 1) into which only the antibody expression plasmid (pBL-HL) was introduced. In the test group (No. 2) in which only the IR / MAR plasmid was co-introduced into the antibody expression plasmid (pBL-HL), the time was 14 days. On the other hand, it took 10 to 11 days in the test group to which B-3-31 (No. 3), G5 (No. 4) or G5 / AR1 (No. 5) was further added. Therefore, it was found that co-introduction of the expression-promoting polynucleotide promotes the expression of not only the target gene but also the introduced gene such as the BS resistance gene, and the screening can be advanced at an early stage.

FIG. 9 shows a comparison of antibody expression levels by cell clones derived from each test group by the ELISA method. The vertical axis of FIG. 9 shows the antibody expression level (Titer (mg / L)), and the horizontal axis shows the test group number in Table 8. As shown in FIG. 9, particularly when B-3-31 was co-introduced (No. 3) as an expression-promoting polynucleotide, the proportion of clones having a high antibody expression level was significantly increased.

Further, FIG. 10 shows a graph plotting the value (mg / L /%) obtained by dividing the antibody expression level (Titer (mg / L)) by Confluency (%). The horizontal axis of FIG. 10 indicates the test plot number in Table 8. The short horizontal bar in FIG. 10 shows the average value in each test group. As shown in FIG. 10, it was suggested that the increase in the amount of antibody was not due to the increase in the number of cells but due to the increase in the amount of antibody produced per cell.

The present invention can be used in a wide range of industries such as pharmaceuticals, chemistry, foods, cosmetics, textiles, etc., which produce a large amount of desired proteins (for example, useful proteins).

Claims (9)

A method for promoting the expression of a target protein in mammalian cells.
The above-mentioned mammalian cells are cultured cells derived from mammals, and are
A method comprising the step of co-introducing the polynucleotides according to (i) to (iii) below into mammalian cells as separate polynucleotides:
(I) A polynucleotide containing an expression cassette of the protein of interest,
(Ii) A polynucleotide containing a mammalian replication initiation region and a nuclear matrix binding region that function in mammalian cells,
(Iii) A polynucleotide that promotes the expression of the target protein. The method according to claim 1, wherein the polynucleotide of (iii) above is a polynucleotide containing the polynucleotide of (a) or (b) below.
(A) A polynucleotide consisting of the base sequence shown in any of SEQ ID NOs: 1 to 3.
(B) A polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted, or added in the base sequence shown in any of SEQ ID NOs: 1 to 3, and the expression of the target protein is expressed. A polynucleotide having an activity that promotes it. The method according to claim 1, wherein the polynucleotide of (iii) above is a polynucleotide containing a repeating sequence of the polynucleotide of (a) or (b) below.
(A) A polynucleotide consisting of the base sequence shown in any of SEQ ID NOs: 1 to 3.
(B) A polynucleotide consisting of a base sequence in which one or several bases are deleted, substituted, or added in the base sequence shown in any of SEQ ID NOs: 1 to 3, and the expression of the target protein is expressed. A polynucleotide having an activity that promotes it. The amount of the polynucleotide of (iii) introduced into the mammalian cell is 1 to 4 times the amount of each of the polynucleotide of (i) and the polynucleotide of (ii) introduced into the mammalian cell. The method according to any one of claims 1 to 3. The amount of the polynucleotide of (iii) introduced into the mammalian cell is 2 to 4 times the amount of each of the polynucleotide of (i) and the polynucleotide of (ii) introduced into the mammalian cell. The method according to any one of claims 1 to 3. The polynucleotides (i) and (ii) above are plasmid vectors.
The method according to any one of claims 1 to 5, wherein the polynucleotide of (iii) is a linear polynucleotide. A kit for promoting the expression of the target protein in mammalian cells.
A kit containing the polynucleotides described in (ii) to (iii) below as separate polynucleotides:
(Ii) A polynucleotide containing a mammalian replication initiation region and a nuclear matrix binding region that function in mammalian cells,
(Iii) A polynucleotide that promotes the expression of a target protein. (I) A polynucleotide containing an expression cassette of the protein of interest,
(Ii) A polynucleotide containing a mammalian replication initiation region and a nuclear matrix binding region that function in mammalian cells,
(Iii) A mammalian cell obtained by co-introducing a polynucleotide that promotes the expression of the target protein and a polynucleotide that promotes the expression of the target protein into cultured animal-derived cells as separate polynucleotides. A method for producing a target protein using the mammalian cell according to claim 8.

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