JP4568378B2 - Gene expression stabilizing element - Google Patents

Description

  The present invention relates to a gene expression stabilizing element that can be used to efficiently and stably produce a recombinant protein in a mammalian cell into which a foreign gene has been introduced by genetic recombination. The gene expression stabilizing element of the present invention is useful for producing useful proteins such as pharmaceuticals in mammalian cells by genetic engineering techniques.

  The development of expression systems that produce recombinant proteins is important in providing a source of protein for research or therapy. As expression systems, both prokaryotes such as Escherichia coli, and eukaryotic cells including yeast (Saccharomyces, Pichia and Kluyveromyces species) and mammalian cells are used. Of these, expression systems in mammalian cells are particularly preferred for the production of therapeutic proteins. Because the post-translational modifications of proteins that occur in mammals including humans sometimes contribute deeply to their biological activity, it is better to use mammalian cell expression systems that allow post-translational modifications similar to those to which proteins are administered. This is because the effectiveness of the therapeutic protein can be enhanced.

  As a mammalian cell expression system, Chinese hamster ovary cells (CHO) are widely used. Various approaches have been performed to highly express recombinant proteins using CHO cells. A classical approach is to select a high expression clone called transgene amplification with an increased copy number of the transgene expressing the target recombinant protein. According to this method, a selective gene such as dihydrofolate reductase and a gene that expresses a recombinant protein are simultaneously introduced into a mutant cell that requires the expression of the selective gene for growth under selective conditions, and gradually a selection pressure is applied. A high expression clone is acquired by raising (Non-patent Documents 1 and 2). Further, as a recent approach, there is a method of isolating an endogenous promoter or a nucleic acid sequence of a preferable integration site from nucleic acid sequences of CHO cells and introducing it into a vector. As an endogenous promoter, Chinese hamster EF-1alpha (CHEF1) is used (Non-patent Document 3). On the other hand, in the method using an integration site, a rare site (hot spot) on a chromosome having high transcription activity is identified, and a transgene is introduced into this site (Patent Document 1). In addition, selection of high-expressing cells, improvement of screening methods, and metabolic engineering approaches are also being conducted. While these approaches enable a certain level of high expression, there are cases in which obtaining a high expression strain requires enormous amounts of time or does not maintain stable synthesis of the recombinant protein during the manufacturing phase. is there.

  Recombinant protein expression varies greatly depending on which site in the host cell genome the gene encoding the recombinant protein is introduced into, but control of the introduced site is usually not possible. Therefore, even if the gene is introduced, most cells do not express the recombinant protein or the expression level is low, so that enormous labor is required for screening. In addition, even cells that had been sufficiently expressed at the time of screening may gradually decrease in expression as culture continues. To overcome these positional effects and the need for extensive screening, chromosomal elements (cis-acting DNA elements) that mitigate the effects of adjacent chromosomes and regulatory elements on the transgene are used in the production of recombinant proteins. It's getting on. One such element is a sequence called an insulator, and the 1.2 kb DNaseI Hypersensitive site (cHS4) of chicken β-globin LCR has been well analyzed and used for the expression of recombinant proteins. (Non-patent document 4), it is said that the increase in protein expression in CHO-K1 cells is not so great (Non-patent document 5).

WO00 / 17337 JP 2001-37478 A

Kaufman R, Sharp P. et al. (1982) J Mol Biol 159: 601-621. Schimke R, Brown P, Kaufman R. (1982) Natl Cancer Inst Monogr 60: 79-86. Running Deer J, Allison DS (2004) Biotechnol Prog 20: 880-889 Pikaart MJ, Recilas-Targa F, Felsenfeld G. et al. (1998) Genes Dev 12: 2852-62. Izumi M, Gilbert DM. (1999) J Cell Biochem 76: 280-289 Blasco MA (2007) Nat Rev Genet 8: 299-309 Gonzalo S, Jaco I, Fraga MF, Chen T, Li E, Esteller M, Blasco MA (2006) Nat Cell Biol 8: 416-424. Perrod S, Gasser SM (2003) Cell Mol Life Sci 60: 2303-2318 Wakimoto BT (1998) Cell 93: 321-324 McBratney S, Chen CY, Sarnow P.M. (1993) Current Opinion in Cell Biology 5: 961-965 Kaufman RJ. (1991) Nucleic Acids Res. 19: 4485-4490 Bao L, Zhou M, Cui Y (2008) Nucleic Acids Research, 36, D83-D87

  The present invention was created in view of the current state of the prior art, and its object is to provide an element capable of stabilizing the gene expression of recombinant proteins and increasing the expression efficiency in mammalian cells, particularly CHO cells. There is.

  In order to solve the above problems, the present inventors have analyzed in detail the sequence in the vicinity of the foreign gene introduction site in a CHO cell clone in which the introduced foreign gene is highly amplified and highly expressed. The inventors have succeeded in identifying a specific region that contributes to stabilization of expression and increase in expression efficiency, and have completed the present invention.

That is, according to the present invention, there is provided a gene expression stabilizing element comprising any one of the following (a) to (g) or any combination thereof:
(A) DNA consisting of the sequence represented by SEQ ID NO: 1;
(B) a DNA comprising a partial sequence of the sequence represented by SEQ ID NO: 1 and comprising at least the sequence of the region represented by the 41820th to 41839th bases of the sequence represented by SEQ ID NO: 1;
(C) a DNA comprising a partial sequence of the sequence represented by SEQ ID NO: 1 and comprising at least the sequence of the region represented by the 41821st to 41840th bases of the sequence represented by SEQ ID NO: 1;
(D) a DNA comprising a partial sequence of the sequence represented by SEQ ID NO: 1 and comprising at least the sequence of the region represented by the 45182nd to 45200th bases of the sequence represented by SEQ ID NO: 1;
(E) a DNA comprising a partial sequence of the sequence represented by SEQ ID NO: 1 and comprising at least the sequence of the region represented by the bases from 91094 to 91113 in the sequence represented by SEQ ID NO: 1;
(F) DNA consisting of a partial sequence of the sequence shown in SEQ ID NO: 1, and when placed in the host cell so as to be close to the foreign gene expression cassette, the object from the foreign gene contained in the foreign gene expression cassette DNA capable of increasing or stabilizing expression of the recombinant protein; and (g) a complement of the DNA of any one of (a) to (f) above.
According to a preferred embodiment of the gene expression stabilizing element of the present invention, the DNA of (b) or (c) is a nucleotide sequence from positions 41601 to 46746, preferably from positions 41601 to 42700, of the sequence represented by SEQ ID NO: 1. Or a part thereof.

  In addition, according to the present invention, such a gene expression stabilizing element, a foreign gene expression vector comprising a foreign gene expression cassette arranged in the vicinity thereof, and such a gene expression stabilizing element and a foreign gene expression Also provided is a transformed host cell characterized in that the cassette is positioned in close proximity to the genome.

  The gene expression stabilizing element of the present invention can stabilize the gene expression of a foreign gene and increase the expression efficiency by reducing the influence of adjacent chromosomes and regulatory elements on the adjacently placed foreign gene. It is possible to reduce and shorten the work of establishing a high expression cell line in a cell expression system, particularly a CHO cell expression system.

It is a figure which shows the prediction site | part of the binding sequence motif of CTCF in the arrangement | sequence of sequence number 1. It is a figure which shows construction | assembly of the basic construct pBS-CMV-SNAP26m used for confirmation of the gene expression stabilization effect of a nucleic acid sequence fragment. It is a figure which shows the binding sequence motif of CTCF in the sequence of sequence number 1, and the position of the nucleic acid fragment of a test sequence. It is a graph which shows the distribution of SNAP26m expression intensity | strength and cell number obtained by FACS analysis of the CHO cell stably introduce | transduced the SNAP26m expression construct containing the fragment | piece of each nucleic acid sequence. It is the graph which plotted the ratio (%) of the cell which exhibited the fluorescence signal of 10 or more SNAP26m as a result of the FACS analysis of the CHO cell stably introduce | transduced the SNAP26m expression construct containing the fragment of each nucleic acid sequence. It is a figure which shows the position of the test sequence in the deletion construction construction of CHO5, and the sequence of SEQ ID NO: 1. It is the graph which plotted the ratio (%) of the cell which exhibited the fluorescence signal of 10 or more SNAP26m as a result of the FACS analysis of the CHO cell obtained by stably introduce | transducing CHO5 and a CHO5 deletion construction. It is a figure which shows the test insertion sequence obtained by the position of the primer used for Inverse PCR method for 3 'deletion construction construction | assembly of CHO5 (DELTA) 3, and deletion. It is the graph which plotted the ratio (%) of the cell which exhibited the fluorescence signal of 10 or more SNAP26m as a result of the FACS analysis of the CHO cell obtained by stably introduce | transducing CHO5 (DELTA) 3 and the 3 'deletion construct of CHO5 (DELTA) 3. It is a figure which shows construction | assembly of the basic construct pEF1 (alpha) -KOD3G8HC-RE2 used for confirmation of the gene expression stabilization effect of the nucleic acid sequence fragment in a KOD3G8 antibody production system. It is a figure which shows construction | assembly of basic construct pEF1 (alpha) -KOD3G8LC-Hyg-RE2 used for confirmation of the gene expression stabilization effect of the nucleic acid sequence fragment in a KOD3G8 antibody production system. It is a figure which shows the H chain or L chain expression vector which inserted each test sequence | arrangement used for confirmation of the gene expression stabilization effect of the nucleic acid sequence fragment in a KOD3G8 antibody production system. Those not containing the test sequence (ctrl), those having the test sequence CHO5Δ3 inserted upstream of the expression cassette (2.6k), those having the test sequence CHO5Δ3-3 inserted upstream of the expression cassette (1.1k), test sequence It is the graph which plotted the amount of antibody production computed by performing ELISA using the culture supernatant of a polyclonal about what inserted CHO5delta3-3 in both the upstream and the downstream of an expression cassette (1.1k / 1.1k). is there. It is the graph which plotted the antibody production amount of each clone computed by performing ELISA using the culture supernatant of a 96 well plate. Those without the test sequence (ctrl), those with the test sequence CHO5Δ3 inserted upstream of the expression cassette (2.6k), those without the test sequence (ctrl) and test sequence CHO5Δ3-3 with the upstream of the expression cassette and Those inserted downstream (1.1 k / 1.1 k) are plotted on the same graph. 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. Those without the test sequence (ctrl), those with the test sequence CHO5Δ3 inserted upstream of the expression cassette (2.6k), those without the test sequence (ctrl) and test sequence CHO5Δ3-3 with the upstream of the expression cassette and Those inserted downstream (1.1 k / 1.1 k) are plotted on the same graph.

The present invention is described in detail below.
The gene expression stabilizing element of the present invention is a nucleic acid molecule identified by the present inventors from the CHO cell DR1000L-4N strain (CHO cell-4N strain) described in Patent Document 2. This strain introduces a human granulocyte macrophage colony stimulating factor (hGM-CSF) gene as an exogenous gene into an expression vector having a DHFR gene, and introduces this vector into a CHO cell DG44 strain lacking the DHFR gene. The strain was cultured in a medium supplemented with 10% fetal bovine serum in IMDM medium (containing nucleic acid) to obtain a transformant. This transformant was then added to IMDM medium (containing no nucleic acid), 10% dialyzed fetal bovine serum. It was obtained by selecting with the added medium, further culturing to 10 to 100 times using IMDM medium containing 50 nM and 100 nM methotrexate (MTX) and not containing nucleic acid, and increasing the MTX concentration. It is a telomere type clonal cell.

  The genome of eukaryotic cells is divided into two classes of chromatin, genuine chromatin and heterochromatin. Centromere and telomere sequences are a major part of constitutive heterochromatin. In particular, the telomere is a gene-poor region composed of a repetitive sequence of TTAGGG and a subtelomeric region and having a distinct heterochromatin three-dimensional structure (Non-patent Documents 6 and 7). Telomeres affect the expression of genes introduced nearby by a phenomenon known as telomere position effects, in addition to their contribution to the stability of the genome and a protective role (Non-Patent Documents 8 and 9).

  However, although the DR1000L-4N strain is a telomere type clonal cell, the introduced foreign gene is not affected by inactivation from foreign chromosomes in the vicinity of the introduction site. High production of stable hGM-CSF was maintained. This suggests that a boundary element that strongly blocks the progress of inactivation exists in the vicinity of the site where the foreign gene has been introduced, stabilizing the gene expression.

  Therefore, as a result of analyzing the sequence in the vicinity of the foreign gene introduction site in the DR1000L-4N strain, the present inventors have found that a DNA comprising an approximately 91 kbp sequence shown in SEQ ID NO: 1 in the sequence listing is involved in the stabilization of gene expression. I found out. And when this sequence was analyzed in further detail, among these sequences, (i) a region represented by bases from 41820 to 41839th, (ii) a region represented by bases from 41821st to 41840th, (iii) It was found that the four regions of the region indicated by the 45182nd to 45200th bases and (iv) the region indicated by the 91094th to 91113th bases correspond to the binding sequence motif of the insulator binding protein CTCF. Therefore, it was considered that the vicinity of these regions including at least one of these four regions is particularly involved in the stabilization of gene expression.

Based on these findings, the present inventors considered that the following five types of DNA can be used as gene expression stabilizing elements.
(A) DNA consisting of the sequence represented by SEQ ID NO: 1;
(B) a DNA comprising a partial sequence of the sequence represented by SEQ ID NO: 1 and comprising at least the sequence of the region represented by the 41820th to 41839th bases of the sequence represented by SEQ ID NO: 1;
(C) a DNA comprising a partial sequence of the sequence represented by SEQ ID NO: 1 and comprising at least the sequence of the region represented by the 41821st to 41840th bases of the sequence represented by SEQ ID NO: 1;
(D) DNA consisting of a partial sequence of the sequence represented by SEQ ID NO: 1, and comprising at least the sequence of the region represented by the 45182st to 45200th bases of the sequence represented by SEQ ID NO: 1; and (e) the sequence A DNA comprising a partial sequence of the sequence represented by No. 1 and comprising at least the sequence of the region represented by the 91094th to 91113th bases of the sequence represented by SEQ ID No. 1.

  Although these elements have been isolated and identified from the CHO genome, elements homologous to these elements are expected to be present in other mammalian cell genomes. Therefore, (f) DNA consisting of a partial sequence of the sequence shown in SEQ ID NO: 1, and when placed close to the foreign gene expression cassette in the host cell, from the foreign gene contained in the foreign gene expression cassette DNA that can increase or stabilize 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) the DNA complement of any one of (a) to (f) can be used as a gene 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.

  In a preferred embodiment of the gene expression stabilizing element of the present invention, the DNA of (b) or (c) is from the region represented by the bases from the 41601st position to the 46746th position of the sequence represented by SEQ ID NO: 1, or a part thereof. Become. In a further preferred embodiment of the gene expression stabilizing element of the present invention, the DNA of (b) or (c) is a region represented by bases from 41601 to 42700 of the sequence represented by SEQ ID NO: 1, or a part thereof. Consists of.

  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 the plasmid vector is 10 to 20 kbp, the chain length of the gene expression stabilizing element is preferably as short as possible, and the preferred length is 5 kbp or less, more preferably about 1 kb.

  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 at a position close to the introduction site of the foreign gene expression cassette in the host cell genome. 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 foreign gene expression cassette is 5000 bp or less, more preferably 500 bp or less. It means that.

  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.

  In the present invention, an exogenous gene expression cassette refers to, for example, an exogenous gene placed downstream of an enhancer and a promoter that can be expressed in a mammalian cell, and a polyadenylation signal is used to stabilize the transcribed foreign gene RNA. It is arranged downstream of. Such an expression cassette may further comprise regulatory elements such as introns. Enhancers and promoters that can be expressed in mammalian cells are not particularly limited, but those derived from viruses such as human and mouse cytomegalovirus, simian virus 40 (SV40), β-actin and ubiquitin, Enhancer / promoter derived from non-viral cellular genes such as GAPDH and EF-1α, and hybrids thereof. It is not always necessary to place foreign genes in the foreign gene expression cassette from the beginning. Instead of foreign genes, multiple cloning sites consisting of multiple restriction enzyme recognition sequences are placed, and various foreign genes can be placed there. cDNA or coding region may be introduced later.

  In the vector of the present invention, it is more preferable that the gene expression stabilizing element of the present invention is arranged so as to sandwich the foreign gene expression cassette both upstream and downstream of the foreign gene expression cassette. In addition, the vector of the present invention preferably contains a plurality of foreign genes, and these plurality of foreign genes preferably encode antibody heavy and / or light chain polypeptides.

  The vector of the present invention is more preferably a vector capable of expressing a drug resistance gene in order to select cells into which a foreign gene has been introduced. Examples of drug resistance genes include Bacillius cereus (bsr) -derived blasticidin resistance gene encoding deminase, Tn5-derived neomycin resistance gene encoding aminoglycoside 3′-phosphotransferase, hygromycin resistance gene encoding E. coli-derived hph, puromycin And a puromycin resistance gene encoding N-acetyltransferase (PAC). Furthermore, selection marker genes such as dihydrofolate reductase (DHFR) gene and glutamine synthetase (GS) gene may be used.

  The vector of the present invention may contain a plurality of expression cassettes consisting of “promoter-foreign gene-polyadenylation signal” in order to express two or more foreign proteins including the drug resistance gene. In addition, the exogenous genes 1 and 2 are placed under the transcriptional control of the same promoter, as in “promoter-foreign gene 1—exogenous gene 2—polyadenylation signal”, and the endogenous ribosome entry site is located between the exogenous genes 1 and 2. A method of enhancing the translation of the second gene product in the expression cassette by introducing (Internal ribosome entry sites, IRES) is also useful (Non-Patent Documents 10 and 11).

  Examples of cells into which the gene expression stabilizing element of the present invention is introduced include Chinese hamster ovary cells (CHO) generally used for the production of recombinant proteins, baby hamster kidney cells (BHK), human fibrosarcoma cells ( Examples include cells derived from mammals such as HT1080), but are not limited thereto, and are derived from animals such as humans, mice, rats, hamsters, guinea pigs, rabbits, dogs, cows, horses, sheep, monkeys, and pigs. These cells can also be introduced.

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

Example 1 Determination of Nucleic Acid Sequence Among the BAC library prepared from the CHO cell DR1000L-4N strain (CHO cell-4N strain), a clone Cg0031N14 containing an hGM-CSF expression cassette was isolated. A 5 ', 3' one-pass sequence of a shotgun clone prepared from BAC derived from this clone Cg0031N14 was performed, sequence information was assembled by Phred / Phrap / Consed, and CHO cells introduced with an hGM-CSF expression cassette were introduced. A nearby genomic sequence of approximately 91 kbp was determined. The sequence is shown in SEQ ID NO: 1 in the sequence listing.

Example 2 Analysis of Nucleic Acid Sequence The presence of an insulator sequence in the determined nucleic acid sequence was investigated by searching for a binding sequence motif of the insulator binding protein CTCF. As an analysis tool, In silico CTCFBS prediction tool (http://insulatordb.utmem.edu/) (Non-patent Document 12) was used. The motif sequences extracted by the tool are shown in Table 1 and FIG.

Example 3 Confirmation of Transgene Expression Stabilizing Effect Plasmid pBS-CMV-SNAP26m was constructed according to the scheme shown in FIG. That is, first, the Emerald Luc (ELuc) gene was excised with EcoRI and NotI from the pELuc-CMV plasmid in which the CMV promoter (SEQ ID NO: 6) was introduced into the restriction enzyme ClaI and EcoRI sites of the Emerald Luc vector pELuc-test (Toyobo). . On the other hand, the SNAP26m gene was amplified by PCR from the SNAPm expression plasmid pSNAPm (Covalys) using the primers of SEQ ID NOs: 7 and 8, and introduced into the EcoRI and NotI sites of the pELuc-CMV plasmid to construct pSNAP26m-CMV. Subsequently, CMV promoter / SNAP26m / SV40 polyA was amplified from pSNAP26m-CMV by PCR using the primers of SEQ ID NOs: 9 and 10, and BamHI of plasmid pBluescript II SK (-) (Stratagene) was obtained by partial digestion. The plasmid pBS-CMV-SNAP26m was constructed by introducing into the SacI site.

  Next, among the clones of the shotgun library prepared in Example 1, a clone containing a CHO genomic sequence in the vicinity of the binding sequence motif of the CTCF at positions 41820 and 45182 is extracted, and an introduced sequence derived from the CHO genome (Fig. 3) was amplified by PCR and introduced into the KpnI, XhoI or XhoI, ClaI sites of pBS-CMV-SNAP26m. Table 2 shows the correspondence between the shotgun library clone and the SNAP26m expression construct in which the introduced sequence is incorporated. In Table 2, (-) in the column of the SNAP26m expression construct is pBS-CMV-SNAP26m into which the CHO genomic sequence has not been introduced.

  These SNAP26m expression constructs and pPUR (CLONTECH, Puromycin resistance vector) were linearized with the restriction enzyme AhdI (New England Biolabs), and then mixed to prepare a mixed plasmid at a weight ratio of 9: 1. .

On the other hand, 2 ml of CHO-K1 cells adjusted to 1 × 10 ⁵ cells / ml were seeded the day before in a 12-well plate and cultured overnight to prepare CHO-K1 cells for transfection. At this time, Ham's F12 medium (Nissui Pharmaceutical) supplemented with 10% fetal bovine serum was used as the medium.

For transfection, 18 μl GeneJuice Transfection Reagent (Merck) was diluted with 600 μl Opti-MEM I Reduced-Serum Medium (GIBCO), 103 μl of this diluted solution was added to 1 μg of the mixed plasmid, and the mixture was allowed to stand for 10 minutes. Was added to the CHO-K1 cells and incubated for 24 hours. The next day, the medium is removed, cells are dispersed by treatment with 2.5 g / l-Trypsin / 1 mmol / l-EDTA Solution (Nacalai Tesque), transferred to a 90 mm Petri dish, and Ham containing 6 μg / ml Puromycin (InvivoGen). Selective culture was performed in 's F12 + 10% FBS medium for 3 weeks. During selective culture, the medium was changed every 3-4 days. After completion of selective culture, the cells were dispersed by treatment with 2.5 g / l-Trypsin / 1 mmol / l-EDTA Solution, 2 × 10 ⁵ cells were seeded in a 12-well plate, and the next day, 0.5 ml of Ham's. 2 μM SNAP-Cell-505 (Covalys) was added to the F12 medium and incubated at 37 ° C. for 60 minutes. Thereafter, rinsing was performed 3 times with Ham's F12 medium, and 10 minutes of incubation was performed 3 times while exchanging the medium to remove unreacted fluorescent dye. The cells were treated with 2.5 g / l-Trypsin / 1 mmol / l-EDTA Solution to disperse the cells, suspended in D-PBS (−) (Nacalai Tesque), and SNAP26m of flow cytometer BD FACSCalibur. The expression intensity was analyzed.

  FIG. 4 shows the results of FACS analysis of the cell population transformed with the SNAP26m expression construct into which the test sequences CHO1 to CHO6 were introduced. In addition, FIG. 5 shows that cells showing 10 or more signals (M2 in FIG. 4) based on the analysis results of untreated CHO cells (upper left of FIG. 4 (−)) are SNAP26m expressing cells, and the ratio of the cells is shown in FIG. This is a plotted graph. As is clear from FIGS. 4 and 5, a significant increase in expression was observed in the constructs into which a part of the CHO cell genome was introduced, particularly in CHO4 and 5 into which positions 41601 to 46746 of SEQ ID NO: 1 were introduced. Although the difference between CHO4 and CHO5 is considered to be within the range of variation between samples, just in case, the subsequent narrowing of the optimum region was performed based on CHO5.

Example 4 Narrowing of Sequence Regions Having Stability for Gene Expression (1)
Based on the construct CHO5 having the highest increase in expression in Example 3, an inverse PCR method using KOD-Plus-Mutageness Kit (Toyobo) was used to sequence from the test sequence using the primers of SEQ ID NOs: 21 and 22. A CHO5Δ5 was constructed in which the region corresponding to position 41601-42902 of No. 1 was deleted. Similarly, CHO5ΔM in which a region corresponding to positions 42903-44200 of SEQ ID NO: 1 was deleted from the test sequence using the primers of SEQ ID NO: 23, 24 and the sequence of the test sequence using the primers of SEQ ID NOs: 25, 26 Each of CHO5Δ3 was constructed by deleting the region corresponding to positions 44201-46746 of number 1 (FIG. 6).

  These SNAP26m expression constructs and pPUR (Puromycin resistant vector) were linearized with the restriction enzyme AhdI, and then mixed at a weight ratio of 9: 1 to prepare a mixed plasmid.

1 μg of the mixed plasmid was transfected into CHO-K1 cells that had been seeded in a 12-well plate the day before 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, and in Ham's F12 + 10% FBS medium containing 6 μg / ml Puromycin. Then, selective culture was performed for 3 weeks. During selective culture, the medium was changed every 3-4 days. After completion of selective culture, the cells were dispersed by treatment with 2.5 g / l-Trypsin / 1 mmol / l-EDTA Solution, 2 × 10 ⁵ cells were seeded in a 12-well plate, and the next day, 0.5 ml of Ham's. 2 μM SNAP-Cell-505 (Covalys) was added to the F12 medium and incubated at 37 ° C. for 60 minutes. Thereafter, rinsing was performed 3 times with Ham's F12 medium, and 10 minutes of incubation was performed 3 times while exchanging the medium to remove unreacted fluorescent dye. The cells were treated with 2.5 g / l-Trypsin / 1 mmol / l-EDTA Solution to disperse the cells, suspended in D-PBS (−) (Nacalai Tesque), and SNAP26m of flow cytometer BD FACSCalibur. The expression intensity was analyzed.

  FIG. 7 is a plot of the percentage of cells that showed a signal value of 10 or more as a result of FACS analysis of a cell population transformed with the SNAP26m expression construct into which the test sequences CHO5Δ5, CHO5ΔM, and CHO5Δ3 were introduced. As is clear from FIG. 7, the expression intensity of SNAP26m was almost the same as that before deletion for CHO5Δ3, whereas the effect of increasing the expression was clearly reduced in CHO5ΔM and CHO5Δ5. From this, it is considered that the 5 ′ side to the first half of the positions 41601 to 46746 of the sequence of SEQ ID NO: 1 mainly contribute to the stabilization of expression.

Example 5 Narrowing of sequence regions having the ability to stabilize gene expression (2)
Using the primers of SEQ ID NOs: 26 to 31, an SNAP26m expression construct in which 3 ′ of the test sequence of CHO5Δ3 was further deleted was constructed by an inverse PCR method using KOD-Plus-Mutageness Kit (Toyobo) (FIG. 8 and Table 3).

  These SNAP26m expression constructs were processed in the same manner as in Example 4, and the expression intensity of SNAP26m was analyzed.

  FIG. 9 is a plot of the percentage of cells showing a signal value of 10 or more as a result of FACS analysis of a cell population transformed with the SNAP26m expression constructs of the test sequences CHO5Δ3 and CHO5Δ3-1 to 5. As is clear from FIG. 9, SCHO26m expression intensity of CHO5Δ3-1 to CHO5Δ3-3, which includes positions 41601 to 42700 of the sequence of SEQ ID NO: 1, is almost the same as that before the deletion, although there is variation between samples. In contrast, CHO5Δ3-4 and CHO5Δ3-5 lacking the 3 ′ side of this region clearly reduced the effect of increasing expression.

Example 6 Evaluation of Gene Expression Stabilizing Capacity in Anti-KOD Antibody Expression System Plasmids pEF1α-KOD3G8HC-RE2 and pEF1α-KOD3G8LC-Hyg-RE2 were constructed according to the schemes shown in FIGS. That is, pEF1α-KOD3G8HC or pEF1α-KOD3G8LC, which is a plasmid in which the heavy chain or light chain of the anti-KOD antibody is inserted into the XbaI-NotI site of the pCI-neo plasmid (Promega) changed to the EF-1α promoter, Restriction enzymes SalI and NheI sites were added to the template for insertion of the test sequence upstream of the expression cassette by the inverse PCR method using KOD-Plus-Mutageness Kit (Toyobo) using the primers of SEQ ID NOs: 32 and 33. pEF1α-KOD3G8HC-RE and pEF1α-KOD3G8LC-RE were constructed. Furthermore, the restriction enzymes BsiWI and XhoI for insertion of the test sequence downstream of the expression cassette using the primers of SEQ ID NOs: 34 and 35 by the inverse PCR method using KOD-Plus-Mutageness Kit (Toyobo) using this plasmid as a template. PEF1α-KOD3G8HC-RE2 and pEF1α-KOD3G8LC-RE2 to which sites were added were constructed. pEF1α-KOD3G8LC-RE2 was further treated with AflII and BstBI to excise a neomycin resistance gene. On the other hand, the hygromycin resistance gene from pTK-Hyg (Clontech) was amplified by PCR using the primers of SEQ ID NOs: 36 and 37, and the above-mentioned neomycin resistance gene was excised, and the AflII and BstBI sites of pEF1α-KOD3G8LC-RE2 Into pEF1α-KOD3G8LC-Hyg-RE2.

  Next, test sequences were inserted upstream of the expression cassettes of these plasmids or both upstream and downstream of the expression cassettes to construct the plasmids shown in FIG. Specifically, the test sequence CHO5Δ3 amplified by PCR using the primer set of SEQ ID NOs: 38 and 39 was inserted into the NheI and BglII sites upstream of the expression cassettes of plasmids pEF1α-KOD3G8HC-RE2 and pEF1α-KOD3G8LC-Hyg-RE2. Then, plasmids pEF1α-KOD3G8HC-2.6k and pEF1α-KOD3G8LC-Hyg-2.6k were constructed. In addition, the test sequence CHO5Δ3-3 amplified by PCR using the primer set of SEQ ID NOs: 38 and 40 was inserted into the NheI and BglII sites upstream of the expression cassettes of plasmids pEF1α-KOD3G8HC-RE2 and pEF1α-KOD3G8LC-Hyg-RE2. Thus, plasmids pEF1α-KOD3G8HC-1.1k and pEF1α-KOD3G8LC-Hyg-1.1k were constructed. Furthermore, the test sequence CHO5Δ3- amplified by PCR using the primer set of SEQ ID NOs: 41 and 42 at the BsiWI and XhoI sites downstream of the expression cassettes of plasmids pEF1α-KOD3G8HC-1.1k and pEF1α-KOD3G8LC-Hyg-1.1k. 3 was inserted to construct plasmids pEF1α-KOD3G8HC-1.1k / 1.1k and pEF1α-KOD3G8LC-Hyg-1.1k / 1.1k.

  These KOD3G8 H chain and L chain expression constructs were linearized with the restriction enzyme AhdI, and the test sequence CHO5Δ3 was inserted upstream of the expression cassette (2.6 k), and the test sequence CHO5Δ3-3 was inserted upstream of the expression cassette. Inserted (1.1k), test sequence CHO5Δ3-3 inserted both upstream and downstream of the expression cassette (1.1k / 1.1k), and test sequence not included (ctrl) The mixed plasmid was prepared by mixing the H chain and L chain expression constructs at a weight ratio of 1: 1.

2 μg of the mixed plasmid was transfected into CHO-K1 cells seeded in a 6-well plate the day before 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, and in Ham's F12 + 10% FBS medium containing G418 and HygroGold. Selective culture was performed for 10 days. During selective culture, the medium was changed every 3 to 4 days, and the G418 concentration was gradually increased from 250 μg / ml to 700 μg / ml, and the HygroGold concentration was gradually increased from 150 μg / ml to 350 μg / ml. After completion of the selective culture, cells were dispersed by treatment with 2.5 g / l-Trypsin / 1 mmol / l-EDTA Solution, and 1.84 × 10 ⁵ cells 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 polyclone was measured by ELISA (Pyrococcus kodakaraensis KOD1-derived DNA polymerase was immobilized).
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, after washing 3 times with PBS-T and adding 50 μl of 10000-fold diluted anti-mouse antibody-HRP (manufactured by DAKO) and incubating at 35 ° C. for 1 hour, further washing 3 times with PBS-T, 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 (main wavelength: 450 nm, sub wavelength: 620 nm).

  The antibody concentration was calculated based on a calibration curve prepared from a standard product. As a standard product, an antibody obtained from mouse hybridoma cell line 3G8 that produces an antibody specific to DNA polymerase derived from KOD1 was used. The results are shown in FIG. As is clear from FIG. 13, the test sequence CHO5Δ3 or CHO5Δ3-3 was inserted upstream of the expression cassette (2.6 k or 1.1 k), and the test sequence CHO5Δ3-, compared to the test sequence not containing the test sequence (ctrl). The antibody production increased in all of the cases in which 3 was inserted both upstream and downstream of the expression cassette (1.1 k / 1.1 k).

Therefore, limiting dilution was carried out for a total of 3 series of the test sequence CHO5Δ3 inserted into the upstream of the expression cassette, the test sequence CHO5Δ3-3 inserted into both the upstream and downstream of the expression cassette, and the test sequence not containing, and the monoclonal clones were used. Productivity comparison was performed.
Specifically, the polyclonal cells stably transfected with the KOD3G8 antibody were treated with 2.5 g / l-Trypsin / 1 mmol / l-EDTA Solution to disperse the cells, and the cells were dispersed at a concentration of 0.75 cell / well. Seeded in 96 well plate. 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 described above.

  FIG. 14 shows a graph in which the antibody production amount of each clone calculated based on a calibration curve prepared from a standard product is plotted. As is clear from FIG. 14, the test sequence CHO5Δ3 was inserted upstream of the expression cassette (2.6 k), and the test sequence CHO5Δ3-3 was inserted upstream and downstream of the expression cassette, compared to the test sequence not including the test sequence (ctrl). The ratio of high-expressing clones markedly increased in both of the inserts (1.1k / 1.1k).

Next, 18 clones with the highest expression level were expanded and screened using a culture supernatant in a 96-well plate, and then 1.84 × 10 ⁵ cells were seeded on a 6-well plate. After culturing for 3 days, the culture supernatant was collected, and the production amount of KOD3G8 antibody in the monoclone was measured by ELISA in the same manner as described above.

  FIG. 15 shows a graph in which the antibody production amount of each clone calculated based on a calibration curve prepared from a standard product is plotted. As is clear from FIG. 15, the test sequence CHO5Δ3 was inserted upstream of the expression cassette (2.6 k), compared to the test sequence not containing the test sequence (ctrl), as in the 96-well plate screening, the test The ratio of high-expressing clones markedly increased both in the sequence CHO5Δ3-3 inserted both upstream and downstream of the expression cassette (1.1 k / 1.1 k).

  From the above results, it is clear that the test sequence CHO5Δ3 or CHO5Δ3-3 can stabilize the gene expression of a foreign gene arranged in close proximity and increase the expression efficiency.

  The gene expression stabilizing element of the present invention can stabilize the expression of a foreign gene in mammalian cells, particularly CHO cells, and reduce and shorten the work of establishing a high expression cell line. Therefore, the cell expression system using the gene expression element of the present invention contributes not only to the functional analysis of proteins in various mammalian cells but also to the industry such as drug discovery and medicine as a biopharmaceutical.

SEQ ID NOs: 7 to 20 are primer sequences used in Example 3.
SEQ ID NOs: 21 to 26 are primer sequences used in Example 4.
SEQ ID NOs: 27 to 31 are primer sequences used in Example 5.
SEQ ID NOs: 32 to 42 are primer sequences used in Example 6.

Claims (11)

A gene expression increasing element comprising any one of the following (a) to (c) or any combination thereof:
(A) DNA consisting of a region represented by bases 41601 to 46746 in the sequence represented by SEQ ID NO: 1;
(B) a DNA comprising a partial sequence of the sequence of (a) , comprising at least the sequence of the region represented by the 41601st to 42700th bases of the sequence represented by SEQ ID NO: 1; and
(C) The complement of the DNA of (a) or (b) . Look including gene expression increased elementary DOO and alien gene expression cassette according to claim 1, foreign gene expression, wherein a distance between the gene expression increased element and the foreign gene expression cassettes is less than 5000bp vector. 3. The foreign gene expression vector according to claim 2 , wherein the gene expression increasing element is arranged both upstream and downstream of the foreign gene expression cassette. The vector according to claim 2 or 3 , wherein the foreign gene encodes an antibody heavy or light chain polypeptide. The vector according to claim 2 or 3 , comprising a plurality of foreign genes. The vector according to claim 5 , wherein the plurality of foreign genes encode antibody heavy and / or light chain polypeptides. Transformed host cells, characterized in that the gene expression increased element of claim 1 and a foreign gene expression cassette is placed in the following distance 5000bp in the genome. 8. A transformed host cell according to claim 7 , wherein the host cell is a mammalian cell. The transformed host cell of claim 8 , wherein the mammalian cell is a CHO cell. A method for producing a protein, characterized in that the transformed host cell according to any one of claims 7 to 9 is used. The method according to claim 10 , wherein the protein is an antibody.