WO2020032153A1 - Hspa8遺伝子のプロモーター - Google Patents
Hspa8遺伝子のプロモーター Download PDFInfo
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- WO2020032153A1 WO2020032153A1 PCT/JP2019/031295 JP2019031295W WO2020032153A1 WO 2020032153 A1 WO2020032153 A1 WO 2020032153A1 JP 2019031295 W JP2019031295 W JP 2019031295W WO 2020032153 A1 WO2020032153 A1 WO 2020032153A1
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N2830/00—Vector systems having a special element relevant for transcription
Definitions
- the present invention relates to a mammalian transformed cell having enhanced transcriptional activity of a foreign protein obtained by using a foreign gene expression vector having an Hspa8 gene promoter, and a method for producing the foreign protein using the same.
- Examples of a host that produces a proteinaceous drug represented by an antibody drug include microorganisms, yeasts, insects, animal and plant cells, transgenic animals and plants, and the like. Post-translational modifications such as folding and glycosylation are essential for the biological activity and antigenicity of proteinaceous drugs, so microorganisms that cannot perform complex post-translational modifications and plants with significantly different sugar chain structures are unsuitable as hosts. It is. In consideration of safety, it has a sugar chain structure similar to that of humans and can be modified after translation, and in consideration of safety, mammalian cultured cells such as CHO cells (Chinese Hamster Ovarie: ovary of Chinese hamster) Has become.
- CHO cells Choinese Hamster Ovarie: ovary of Chinese hamster
- Non-Patent Document 1 When a cultured mammalian cell is used as a host, there are problems such as low growth rate, low productivity, and high cost as compared with microorganisms and the like (Non-Patent Document 1). In addition, since clinical use of proteinaceous drugs requires a large amount of administration, there is a problem worldwide insufficient production capacity. When manufacturing proteinaceous drugs using a mammalian cell culture expression system, production costs are higher than synthetic low-molecular-weight drugs. Increasing the production volume is also an effective method for reducing the manufacturing cost (Non-Patent Documents 2 and 3).
- CMV promoter virus-derived Human cytomegalovirus / major / immediate / early / promoter
- Non-Patent Document 7 EF-1 ⁇ which is an elongation ⁇ factor-1 ⁇ alpha
- a promoter of a heat shock protein A5 (Hspa5 / GRP78) gene which has improved productivity of a foreign protein is known (Patent Document 4).
- Patent No. 3051411 WO2006 / 123097 WO2013 / 080934 WO2018 / 066492
- An object of the present invention is to provide a promoter having a high exogenous gene expression enhancing activity in a host cell such as a cultured mammalian cell, and to enhance the production of a foreign protein to be a protein drug by using the promoter.
- the purpose is to provide a means for causing this to occur.
- the present inventors have conducted intensive studies in order to solve the above problems, and as a result, a polynucleotide of about 2.9 kbp upstream of the initiation codon of the heat shock protein A8 (Hspa8) gene has excellent promoter activity,
- the present inventors have found that it is possible to significantly improve the productivity of a foreign protein to be expressed in cultured animal cells, and have completed the present invention. That is, the present invention includes the following inventions.
- a promoter of the Chinese hamster-derived Hspa8 gene comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6, comprising the nucleotide sequence of SEQ ID NO: 1 or a partial sequence of the nucleotide sequence.
- a polynucleotide comprising (2) The polynucleotide according to (1), comprising the nucleotide sequence of SEQ ID NO: 1. (3) The polynucleotide according to (1), consisting of the nucleotide sequence of SEQ ID NO: 5. (4) The polynucleotide according to (1), comprising the nucleotide sequence of SEQ ID NO: 6. (5) A polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 2 in the Sequence Listing, which is a promoter of a human-derived Hspa8 gene.
- a polynucleotide comprising a nucleotide sequence having 99% or more identity to the nucleotide sequence according to any one of (1) to (7), wherein the polynucleotide has promoter activity.
- An exogenous gene expression unit comprising the polynucleotide according to any one of (1) to (10).
- a foreign gene expression vector comprising the foreign gene expression unit according to any one of (11) to (14).
- the transformed cell according to (17), wherein the cell is a cultured cell derived from a mammal.
- the transformed cell according to (18), wherein the cultured cell derived from a mammal is a COS-1 cell, a 293 cell, or a CHO cell.
- a method for producing a protein which comprises culturing the transformed cell according to any one of (17) to (19) and obtaining a protein derived from a foreign gene from the culture.
- (21) Use of the polynucleotide according to any one of the above (1) to (10) for expressing a foreign gene in a transformed cell.
- the promoter of the present invention can further enhance the expression of a foreign gene such as a therapeutic protein or an antibody by combining it with a DNA element.
- Schematic diagram of Y The figure which compared the production amount of the antibody expressed by the Hspa8 gene promoter with the human EF1- ⁇ gene promoter in the fed-batch culture using the stable pool expressing the humanized antibody Y.
- FIG. 2A shows the number of viable cells on each sampling day.
- FIG. 2B shows the production amount on each sampling day.
- FIG. 3A shows the number of living cells on each sampling day.
- FIG. 3B shows the production amount on each sampling day.
- FIG. 4A shows the number of viable cells on each sampling day.
- FIG. 4B shows the production amount on each sampling day.
- Hspa8 The figure which compared the antibody production amount in the fed-batch culture of the humanized antibody Y expression stable pool produced using the Hspa8 gene promoter derived from each species.
- Hspa8, hHspa8, mHspa8 and rHspa8 show the results of Hspa8 derived from Chinese hamster, human, mouse and rat, respectively.
- FIG. 5A shows the number of living cells on each sampling day.
- Hspa8, hHspa8, mHspa8 and rHspa8 show the results of Hspa8 derived from Chinese hamster, human, mouse and rat, respectively.
- 5B shows the production amount on each sampling day.
- Nucleotide sequence of polynucleotide which is promoter of Chinese hamster-derived Hspa8 gene Nucleotide sequence of a polynucleotide that is a promoter of human Hspa8 gene Nucleotide sequence of polynucleotide that is promoter of mouse-derived Hspa8 gene Nucleotide sequence of polynucleotide which is a promoter of rat-derived Hspa8 gene
- the term “gene” means a portion that is transcribed into mRNA and translated into protein, and includes not only DNA but also its mRNA, cDNA and its RNA.
- polynucleotide is used in the same meaning as nucleic acid, and also includes DNA, RNA, probe, oligonucleotide, and primer.
- polypeptide and “protein” are used interchangeably.
- gene expression means a phenomenon in which a certain gene is transcribed into mRNA and / or a phenomenon in which a protein is translated from the mRNA.
- the term “foreign gene” means a gene that is artificially introduced into a host cell.
- foreign protein means a protein encoded by a foreign gene.
- the term “gene expression unit” means a polynucleotide having at least a promoter region, a foreign gene, and a transcription terminator region (polyA addition signal) in the direction of the reading frame of transcription.
- promoter refers to a region to which a transcription factor involved in initiation of transcription from DNA to RNA is bound. In this specification, it may be referred to as a “promoter region”. Examples of the promoter include, for example, a polynucleotide from a nucleotide of about 3 kbp upstream of the start codon to a nucleotide immediately before the nucleotide sequence corresponding to the start codon, and may include a 5'UTR and an intron.
- promoter activity refers to an activity in which a transcription factor binds to a promoter, initiates transcription, and produces a protein encoded by a gene.
- a protein encoded by a reporter gene such as firefly luciferase Can be tested by using the activity of the as an index.
- “having promoter activity” means that the human EF-1 ⁇ gene is expressed under the same conditions as in the evaluation of promoter activity using an antibody expression level in fed-batch culture as an index described later (Example 2). This means that the antibody expression level is equal to or higher than that of the promoter.
- DNA element means a polynucleotide having a foreign gene expression enhancing activity when placed near a gene expression unit or on a foreign gene expression vector containing the gene expression unit.
- antigen-binding fragment of an antibody refers to a partial fragment of an antibody having an antigen-binding activity, and includes Fab, F (ab ') 2, and the like. It is not limited to these molecules as long as they have binding ability.
- identity refers to the relationship between two or more nucleotide or amino acid sequences, as known in the art, as determined by comparing the sequences. In the art, “identity” also refers to the sequence between nucleic acid molecules or between polypeptides, as the case may be, as determined by the match between two or more nucleotide sequences or two or more amino acid sequences in a row. Means the degree of relevance. "Identity” is an identity match between a small one of two or more sequences and a gap alignment, if any, addressed by a particular mathematical model or computer program (ie, "algorithm").
- ClustalW2 European ⁇ Molecular ⁇ Biology ⁇ Laboratory-European ⁇ Bioinformatics ⁇ Institute (EMBL-EBI), but it is limited to those used by those skilled in the art. Not done.
- hybridize under stringent conditions refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more of a nucleotide sequence of a nucleic acid complementary to a nucleic acid is hybridized, and the identity is higher than that. Conditions that do not allow the complementary strand of a nucleic acid having a low nucleotide sequence to hybridize can be mentioned. More specifically, hybridization is performed at 68 ° C.
- promoter used for enhancing the expression of foreign gene of the present invention is a heat shock protein A8 gene (hereinafter, “Hspa8”). It is a promoter of. There is no particular limitation as long as it is a polynucleotide having an activity as an Hspa8 promoter. Examples of the Hspa8 promoter include polynucleotides from a nucleotide at about 2.9 kbp upstream of a start codon to a nucleotide immediately before a nucleotide sequence corresponding to the start codon. preferable.
- Hspa8 promoter Although the origin of the Hspa8 promoter is not particularly limited, it may be derived from mammals, and examples thereof include Hspa8 promoters derived from Chinese hamster, human, mouse, rat and the like.
- the promoter of the present invention is preferably a rodent-derived Hspa8 promoter, more preferably a Chinese hamster, mouse or rat-derived Hspa8 promoter, and still more preferably a mouse or rat-derived Hspa8 promoter.
- Hspa8 promoter examples of the Chinese hamster-derived Hspa8 promoter include SEQ ID NO: 1 in the sequence listing and the polynucleotide described in FIG.
- the nucleotide sequence of SEQ ID NO: 1 is a sequence consisting of a nucleotide of about 2.9 kbp upstream of the initiation codon of Chinese hamster-derived Hspa8 to a nucleotide immediately before the nucleotide sequence corresponding to the initiation codon.
- nucleotide sequences of SEQ ID NOs: 2, 3, and 4 are sequences consisting of nucleotides about 2 kbp upstream of the initiation codon of human, mouse and rat-derived Hspa8 to nucleotides immediately before the nucleotide sequence corresponding to the initiation codon, respectively.
- the nucleotide sequences of SEQ ID NOs: 2, 3, and 4 are also shown in FIGS. 7, 8, and 9, respectively.
- the Chinese hamster-derived Hspa8 promoter may be a nucleotide sequence consisting of a partial sequence of the sequence set forth in SEQ ID NO: 1, corresponding to a start codon from a nucleotide of about 1.9 and 1.2 kbp upstream of the start codon of Hspa8, respectively.
- a polynucleotide comprising the sequence shown in SEQ ID NO: 5 or 6 of the sequence consisting of the nucleotide immediately before the nucleotide sequence to be described further corresponds to the start codon from nucleotides about 1.1 and 1.0 kbp upstream of the start codon of Hspa8
- a polynucleotide containing a sequence consisting of the nucleotide immediately before the nucleotide sequence to be described is also exemplified, the polynucleotide described in SEQ ID NO: 6 is preferable.
- the promoter of the present invention has 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more identity to any one of the nucleotide sequences shown in SEQ ID NOS: 1 to 6. It may be a polynucleotide comprising a nucleotide sequence having a promoter activity.
- the promoter of the present invention comprises, under stringent conditions, a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 1 to 6. It may be a polynucleotide that hybridizes and has promoter activity.
- the promoter of the present invention may have one or more, preferably 1 to 300, and more preferably 1 to 30 nucleotides in any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 1 to 6. It may be a mutant polynucleotide consisting of a nucleotide sequence in which nucleotides have been deleted, substituted, and / or added, and may have a promoter activity.
- the mutation (deletion, substitution, and / or addition) of the nucleotide sequence can be introduced by a method known in the art, such as the Kunkel method or the Gapped Duplex method, or a method analogous thereto.
- a method known in the art such as the Kunkel method or the Gapped Duplex method, or a method analogous thereto.
- Mutant-K manufactured by Takara Bio Inc.
- Mutant-G manufactured by Takara Bio Inc.
- LA PCR in vitro Mutogenesis series kit of Takara Bio Inc. can be used.
- Such a mutant polynucleotide can also be used as the promoter of the present invention.
- the activity of the promoter of the present invention to enhance the expression of a foreign gene can be assayed using the activity of a protein encoded by a reporter gene such as firefly luciferase or the amount of antibody produced in fed-batch culture as an index.
- a reporter gene such as firefly luciferase or the amount of antibody produced in fed-batch culture as an index.
- the amount of antibody production in fed-batch culture was equal to or higher, preferably 1.2 times or higher, more preferably 1.5 times.
- the enhancement is about 1.2 times or more, reduction of the culture scale of the cells, reduction of the culture time, and reduction of the purification process are expected, and as a result, it is possible to improve the yield and reduce the culture cost. If the yield is improved, it becomes possible to stably supply a foreign protein as a drug. Also, if the culturing cost is reduced, the cost of the foreign protein as a medicine is reduced.
- the foreign gene expression unit of the present invention may have at least 1. And a promoter, a foreign gene, and a transcription terminator region (poly-A addition signal) according to the present invention.
- the poly-A additional sequence may be any sequence having an activity of initiating transcription termination from transcription from the promoter, and may be of the same or different gene as the promoter gene.
- DNA element used for enhancing expression of foreign gene can be further enhanced by using the gene expression unit of the present invention described in (1) and a DNA element in combination.
- DNA elements used in combination can be obtained using the interaction with acetylated histone H3 as an index. It is generally said that histone (H3, H4) acetylation is involved in the activation of transcription, and two main theories are considered. A theory related to a nucleosome conformational change in which the histone tail is acetylated to neutralize charge and loosen the binding between DNA and histone (Mellor J. (2006) Dynamic nucleosomes and gene transcription. Trends). Genet.
- DNA A2, A7, and A18 can be mentioned as DNA elements used in combination with the promoter of the present invention and used for enhancing the expression of a foreign gene.
- A2 is a polynucleotide having a AT content of 62.2% and 8450 bp, which is located on human chromosome 15 at 80966429-80974878.
- the nucleotide sequence of A2 is described in SEQ ID NO: 7 in the sequence listing.
- ⁇ A7 is a polynucleotide having a AT content of 64.52% and 8420 bp, which is located on human chromosome 11 at 88992123 to 89000542.
- the nucleotide sequence of A7 is described in SEQ ID NO: 8 in the sequence listing.
- A18 is a polynucleotide having a AT content of 62.54% and 8475 bp, which is located on human chromosome 4 at 1112759776 to 111284450.
- the nucleotide sequence of A18 is described in SEQ ID NO: 9 in the sequence listing.
- the exogenous gene expression-enhancing activity of a DNA element used in combination with the promoter of the present invention can be assayed using the activity of a protein encoded by a reporter gene such as SEAP as an index.
- any one of the above DNA elements may be used alone, or one or more DNA elements may be used in two or more copies. Alternatively, two or more DNA elements may be used in combination.
- the DNA element used in the present invention has 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more identity to the nucleotide sequence shown in SEQ ID NOs: 7 to 9. It may be a nucleotide sequence consisting of a nucleotide sequence and having a foreign gene expression enhancing activity.
- the homology search of the nucleotide sequence can be performed using, for example, a program such as FASTA or BLAST for the Japan DNA Data Bank (DNA ⁇ Databank ⁇ of ⁇ JAPAN).
- a mutation (deletion, substitution, and / or addition) of the polynucleotide can be performed by a method known in the art such as the Kunkel method or the Gapped Duplex method, or a method analogous thereto.
- a method known in the art such as the Kunkel method or the Gapped Duplex method, or a method analogous thereto.
- Mutant-K manufactured by Takara Bio Inc.
- Mutant-G manufactured by Takara Bio Inc.
- LA PCR in vitro Mutogenesis series kit of Takara Bio Inc. can be used.
- Such a mutant polynucleotide can also be used as the DNA element of the present invention.
- a polynucleotide containing an exogenous gene encoding an exogenous protein to be described below whose production is to be enhanced can be acquired by the following general method.
- a cDNA library derived from a cell or tissue in which a foreign gene is expressed can be isolated by screening using a DNA probe synthesized based on the gene fragment.
- Preparation of mRNA can be performed by a technique commonly used in the art.
- the cells or tissues are treated with a guanidinin reagent, a phenol reagent or the like to obtain total RNA, and then subjected to an affinity column method using an oligo (dT) cellulose column or poly U-sepharose using Sepharose 2B as a carrier.
- poly (A) + RNA (mRNA) is obtained by a batch method.
- poly (A) + RNA may be further fractionated by sucrose density gradient centrifugation or the like.
- a single-stranded cDNA is synthesized using an oligo dT primer and a reverse transcriptase, and a double-stranded DNA is synthesized from the single-stranded cDNA using Synthesize cDNA.
- a double-stranded DNA is synthesized from the single-stranded cDNA using Synthesize cDNA.
- the cDNA library is obtained. Make it.
- a cDNA library can also be prepared using a plasmid vector other than the ⁇ phage. Then, a strain (positive clone) having the target DNA may be selected from the cDNA library.
- genomic DNA is extracted from the cell line of the organism to be collected, and polynucleotides are selected. Extraction of genomic DNA can be performed, for example, by the method of Cryer et al. (Methods in Cell Biology, 12, 12, 39-44 (1975)) and P.I. Philippsen et al. (Methods Enzymol., 194, 169-182 (1991)).
- the polynucleotide containing the promoter, DNA element, or foreign gene of interest can also be obtained, for example, by the PCR method (PCR, Technology, Henry, A. Erlich, Ackton, Press (1989)).
- PCR PCR, Technology, Henry, A. Erlich, Ackton, Press (1989)
- genomic DNA is used as a template.
- the amplified gene is used after confirming the polynucleotide sequence.
- a genomic DNA library such as a bacterial artificial chromosome (BAC) can be used as a template for PCR.
- a gene library is prepared by an ordinary method, (b) a desired polynucleotide is selected from the prepared gene library, and the polynucleotide is obtained.
- Amplification can be performed.
- a gene library is obtained by partially digesting chromosomal DNA obtained from a cell line of an organism to be collected by an ordinary method with an appropriate restriction enzyme to fragment the fragment, ligating the obtained fragment to an appropriate vector, and It can be prepared by introducing into a suitable host. Alternatively, it can be prepared by extracting mRNA from cells, synthesizing cDNA therefrom, ligating it to an appropriate vector, and introducing the vector into an appropriate host.
- a plasmid generally known as a known vector for preparing a gene library can be used, and a phage vector or a cosmid can also be widely used.
- a host for transformation or transduction may be used depending on the type of the vector. Selection of a polynucleotide containing a foreign gene is performed from the gene library by a colony hybridization method, a plaque hybridization method, or the like using a labeled probe containing a sequence specific to the foreign gene.
- a polynucleotide containing a foreign gene can be chemically totally synthesized.
- a method of producing a pair of complementary oligonucleotides and annealing them, a method of ligating several annealed DNAs with DNA ligase, or a method of producing several partially complementary oligonucleotides and performing PCR Genes can be synthesized by methods such as filling gaps.
- Polynucleotide sequence can be determined by a conventional method such as the dideoxy method (Sanger et al., Proc. Natl. Acad. Sci., USA, 74, 5463-5467 (1977)). Further, the polynucleotide sequence can be easily determined by using a commercially available sequence kit or the like.
- the foreign gene expression vector of the present invention includes the aforementioned 1. 2. The promoter containing the promoter described in 2. above. And a vector comprising the foreign gene expression unit described in (1).
- the foreign gene expression vector of the present invention is the same as described in 3. above. Or the combination of two or more of the DNA elements may be included.
- the DNA element When expressing a foreign gene in a host cell using the above-described foreign gene expression vector, the DNA element may be placed immediately before or immediately after the gene expression unit, or may be placed at a position away from the gene expression unit. good. Further, one foreign gene expression vector containing a plurality of DNA elements may be used. The direction of the DNA element may be either forward or backward with respect to the gene expression unit.
- the foreign gene is not particularly limited, but includes reporter genes such as secreted alkaline phosphatase (SEAP), green fluorescent protein (GFP) and luciferase; various enzyme genes such as ⁇ -amylase gene and ⁇ -galactosidase gene; Various bioactive proteins such as interferon ⁇ and interferon ⁇ ; various interleukin genes such as IL1 and IL2; erythropoietin (EPO) gene; various cytokine genes such as granulocyte colony stimulating factor (G-CSF) gene; Examples of the gene include a gene encoding a growth factor gene or a multimeric protein, such as a gene encoding a heteromultimer that is an antibody or an antigen-binding fragment thereof. These genes may be obtained by any technique.
- SEAP secreted alkaline phosphatase
- GFP green fluorescent protein
- luciferase various enzyme genes such as ⁇ -amylase gene and ⁇ -galactosidas
- the “antigen-binding fragment of an antibody” refers to a partial fragment of an antibody having an antigen-binding activity, and includes Fab, F (ab ′) 2 , Fv, scFv, diabody, linear antibody, and antibody fragment. And multispecific antibodies formed therefrom.
- Fab ' which is a monovalent fragment of the variable region of an antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the antigen-binding fragment of the antibody. However, it is not limited to these molecules as long as it has an antigen binding ability.
- These antigen-binding fragments include not only those obtained by treating the full-length molecule of an antibody protein with an appropriate enzyme, but also those produced in an appropriate host cell using an antibody gene modified by genetic engineering. included.
- the exogenous gene expression vector of the present invention may contain a selection marker for selecting a transformant.
- a selection marker for selecting a transformant for example, cerulenin, aureobasidin, zeocin, canavanine, cycloheximide, hygromycin, puromycin, blasticidin, tetracycline, kanamycin, ampicillin, by using a drug resistance marker or the like that imparts resistance to drugs such as neomycin, Transformants can be selected.
- the foreign gene expression vector of the present invention may be a vector that is not integrated into chromosomal DNA.
- a foreign gene expression vector is randomly integrated into a chromosome after gene introduction into a host cell, but components derived from a mammalian virus such as simian virus 40 (SV40), papillomavirus (BPV, HPV), and EBV.
- SV40 simian virus 40
- BPV papillomavirus
- EBV papillomavirus
- a vector having an SV40-derived replication origin and a sequence encoding SV40 large T antigen, which is a trans-acting factor and a vector having a sequence encoding EBV-derived oriP and EBNA-1 are widely used.
- EBV-derived oriP and EBNA-1 are widely used.
- Transformed cell The transformed cell of the present invention may be any one of the aforementioned 5. Transformed cells introduced using the exogenous gene expression vector.
- the host cell to be transformed is a eukaryotic cell, preferably a mammalian cell, more preferably a cell derived from human, mouse, rat, hamster, monkey, or cow.
- mammalian cells include, but are not limited to, COS-1 cells, 293 cells, CHO cells (CHO-K1, CHO-O1, CHO DG44, CHO dhfr-, CHO-S) and the like.
- any method may be used as long as the introduced gene is stably present in the host and can be appropriately expressed, and is generally used.
- Methods such as the calcium phosphate method (Ito et al., (1984) Agric. Biol. Chem., 48, 341), the electroporation method (Becker, ⁇ DM ⁇ et al. (1990) Methods. Enzymol., 194, 182-187), the spheroplast method (Creggh et al., Mol. Cell. Biol., 5, 3376 (1985)), the lithium acetate method (Itoh, H. (1983) J. Bacteriol. 153, 163). -168), Lipo Ekushon method, or the like can be mentioned.
- the method for producing a foreign protein of the present invention is described in 6. above. Can be carried out by culturing the transformed cells described in the item 3) by a known method, collecting from the culture, and purifying.
- the term “culture” refers to any of cultured cells or crushed cells in addition to the culture supernatant.
- a foreign protein that can be produced using the transformed cell described in the item not only a monomeric protein but also a multimeric protein can be selected. When a heteromultimeric protein composed of a plurality of different subunits is to be produced, a plurality of genes encoding these subunits are each transferred to 6. Need to be introduced into the host cell described in the item.
- the method of culturing the transformed cell can be performed according to a usual method used for culturing the host cell.
- the cells are cultured, for example, at 37 ° C., 5% or 8% CO 2 , and the culturing time is about 24 to 1000 hours.
- the culture is allowed to stand, shake, stir, aerate. It can be carried out by batch culture, fed-batch culture, perfusion culture, continuous culture or the like below.
- ⁇ ⁇ Confirmation of the expression product of the foreign protein gene from the culture (culture solution) can be performed by SDS-PAGE, Western analysis, ELISA or the like.
- Examples of the heteromultimeric protein produced by using the production method described in the item 3) include an antibody protein.
- Antibody proteins are tetrameric proteins consisting of two heavy chain polypeptides and two light chain polypeptides. Therefore, in order to obtain an antibody protein in a form maintaining the antigen-binding ability, the above-mentioned 6. It is necessary that both the heavy chain and the light chain genes have been introduced into the transformed cell described in the item. In this case, the heavy chain and light chain gene expression units may be present on the same expression vector, or may be present on different expression vectors.
- an antibody produced in the present invention an antibody produced by immunizing a laboratory animal such as a rabbit, a mouse or a rat with a desired antigen can be mentioned.
- chimeric antibodies and humanized antibodies using the above-mentioned antibodies as raw materials can also be mentioned as antibodies produced in the present invention.
- a human antibody obtained by a genetically modified animal or a phage display method is also an antibody produced in the present invention.
- a specific polynucleotide It is not limited to an antibody gene having a sequence.
- the antibody gene it is not necessary to encode the full-length molecule of the antibody, and a gene encoding an antigen-binding fragment of the antibody can be used. Genes encoding these antigen-binding fragments can be obtained by genetically modifying a gene encoding a full-length antibody protein molecule.
- the foreign proteins to be subjected to the production method of the present invention include various proteins derived from human or non-human animals, antigen-binding fragments thereof, variants thereof, and the like. Can be.
- Such proteins include atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), vasopressin, somatostatin, growth hormone (GH), insulin, oxytocin, ghrelin, Peptide hormones such as leptin, adiponectin, renin, calcitonin, osteoprotegerin, insulin-like growth factor (IGF), interleukins, chemokines, interferons, tumor necrosis factors (TNF ⁇ / ⁇ and other TNF superfamily, etc.), nerve growth factors (NGF ), Cell growth factors (EGF, FGF, PDGF, HGF, TGF, etc.), hematopoietic factors (CSF, G-CSF, erythropoietin, etc.), cytokines such as adipokine, receptors such as NF receptor, lysozyme , Proteases
- the plasmids, restriction enzymes, DNA modifying enzymes and the like used in the examples of the present invention are commercially available and can be used according to a conventional method.
- the procedures used for cloning DNA, determining the polynucleotide sequence, transforming host cells, culturing transformed cells, collecting and purifying proteins from the resulting culture, and the like are well known to those skilled in the art. Or can be known from the literature.
- the promoter region of Hspa8 was amplified by PCR using genomic DNA of CHO cells as a template and the following primer set and PrimeSTAR Max DNA Polymerase (Takara Bio), and purified by QIAquick PCR Purification Kit (QIAGEN).
- the nucleotide sequence of the promoter region of the cloned Chinese hamster Hspa8 is shown in SEQ ID NO: 1 in the sequence listing.
- Hspa8-XbaI-R TTCTCTAGAGGTTGCTGAAAGAAAACCAAA (SEQ ID NO: 11)
- Example 2 Evaluation by fed-batch culture of Hspa8 promoter using antibody expression level as an index 2-1
- Construction of antibody expression vector pDSLH4.1 (Okumura T et al., J Biosci Bioeng., 120 (3): 340) -346, 2015) as a vector backbone, and Hspa8 is used as a promoter for antibody H chain and L chain genes to construct a humanized antibody gene Y expression vector pDSLH3.1-Hspa8-Y containing no DNA element. did.
- Example 1 After the DNA fragment amplified and purified in Example 1 was digested with NotI-XbaI, the H chain gene expression vector pDSH1.1-hEF1 ⁇ -Y and the L chain gene expression vector pDSL2.1 described in Patent Document 4 were used. -Insertion between NotI-NheI sites of -hEF1 ⁇ -Y to construct pDSH1.1-Hspa8-Y and pDSL2.1-Hspa8-Y, respectively. Next, a DNA fragment obtained by digesting pDSL2.1-Hspa8-Y with AatII-MluI was inserted between AatII-MluI of pDSH1.1-Hspa8-Y to construct pDSLH3.1-Hspa8-Y. did. The vector outline is shown in FIG.
- FIGS. 2A and 2B Changes in the number of living cells and the amount of antibody production are shown in FIGS. 2A and 2B, respectively.
- the amount of antibody produced by the Hspa8 promoter reached a value 3.9 times that of the human EF1- ⁇ promoter, far exceeding the amount produced by the promoter frequently used at present.
- Example 3 Examination of Hspa8 promoter length using index of antibody expression in fed-batch culture 3-1) Construction of antibody expression vector Antibody H chain of humanized antibody gene Y expression vector pDSLH3.1-hEF1 ⁇ -Y
- pDSLH3.1-Hspa8-1.9-Y and pDSLH3.1-Hspa8-1.2-Y in which the promoter of the L chain gene was replaced with a partial sequence of the Hspa8 promoter were constructed.
- a sequence from a nucleotide of about 1.9, 1.2 kbp upstream of the Hspa8 start codon sequence to a nucleotide immediately before the nucleotide sequence corresponding to the start codon sequence was used as a partial sequence of the Hspa8 promoter.
- pDSLH3.1-Hspa8-1.9-Y was constructed by the following method. First, a partial sequence of the Chinese hamster Hspa8 promoter was amplified by PCR using pDSH1.1-Hspa8-Y as a template and the following primer set and PrimeSTAR Max DNA Polymerase, and purified by QIAquick PCR Purification kit. After the purified DNA fragment was digested with NotI-XbaI, the fragment was inserted between the NotI-NheI sites of the H chain gene expression vector pDSH1.1-hEF1 ⁇ -Y and the L chain gene expression vector pDSL2.1-hEF1 ⁇ -Y.
- pDSL2.1-Hspa8-1.9-Y and pDSL2.1-Hspa8-1.9-Y respectively.
- a DNA fragment obtained by digesting pDSL2.1-Hspa8-1.9-Y with AatII-MluI was inserted between AatII-MluI of pDSH1.1-Hspa8-1.9-Y, and pDSLH3 .1-Hspa8-1.9-Y was constructed.
- pDSLH3.1-Hspa8-1.2-Y was constructed.
- Hspa8 promoter 1.9 kbp primer set Hspa8-NotI-1900F: TTCGCGGCCGCAACAACCTAACTAATAGCTGTCC (SEQ ID NO: 12) Hspa8-XbaI-R: TTCTCTAGAGGTTGCTGAAAGAAAACCAAA (SEQ ID NO: 11) Hspa8 promoter 1.2 kbp primer set Hspa8-NotI-1200F: TTCGCGGCCGCAACCTTCGCGGCCATTTTGTCCC (SEQ ID NO: 13) Hspa8-XbaI-R: TTCTCTAGAGGTTGCTGAAAGAAAACCAAA (SEQ ID NO: 11)
- Example 4 Examination of combination effect of Hspa8 promoter and A7 using index of antibody expression in fed-batch culture 4-1) Construction of antibody expression vector The antibody expression vector pDSLH3.1 constructed in (3-1) The DNA element A7 described in Patent Document 3 was inserted upstream of the expression cassette of Hspa8-1.9-Y to construct pDSLHA4.1-Hspa8-1.9-Y.
- FIGS. 4A and 4B Changes in the number of living cells and the amount of antibody production are shown in FIGS. 4A and 4B, respectively.
- the amount of antibody produced by the A7-containing antibody expression vector on the 14th day of culture was 3.4 times that of the A7-free antibody expression vector. It was found that by using the DNA element A7 and the Hspa8 promoter in combination, a high production can be effectively realized by a synergistic effect.
- Example 5 Evaluation by fed-batch culture of human, mouse, and rat Hspa8 promoters using antibody expression level as an index 5-1) Construction of antibody expression vector Construction of humanized antibody gene Y expression vector pDSLH3.1-hEF1 ⁇ -Y PDSLH3.1-hHspa8-Y, pDSLH3.1-mHspa8-Y, and pDSLH3.1-rHspa8-Y, in which the promoters of the antibody H chain and L chain genes were replaced with human, mouse, and rat Hspa8 promoters, respectively.
- Hspa8 promoter a sequence from a nucleotide of about 2.0 kbp upstream of the start codon sequence of Hspa8 to a nucleotide immediately before the nucleotide sequence corresponding to the start codon sequence was used as the Hspa8 promoter.
- the nucleotide sequences of the cloned human, mouse, and rat Hspa8 promoters are shown in SEQ ID NOs: 2, 3, and 4, respectively.
- pDSLH3.1-hHspa8-Y was constructed by the following method. First, using the human genomic DNA as a template, the human Hspa8 promoter was amplified by PCR using the following primer set and PrimeSTAR Max DNA Polymerase, and purified by QIAquick PCR Purification kit. After the purified DNA fragment was digested with HindIII-EcoT14I or AatII-EcoT14I, the HindIII-NheI of the heavy chain gene expression vector pDSH1.1-hEF1 ⁇ -Y and the Lchain gene expression vector pDSL2.1-hEF1 ⁇ -Y were respectively digested.
- Hspa8-human-HindIII-F GGTGGAAGCTTATACAAACGTTCAGAAAGTCTTAA
- Hspa8-human-EcoT14I-R GGTGCCATGGGGTTTGCTGAAAAAAAGAAAAATC
- Primer set of human Hspa8 promoter for insertion of L chain gene expression vector Hspa8-human-AatII-F GGGTGACGTCCATACAAACGTTCAAAAGTCTAA
- Hspa8-human-EcoT14I-R GGTGCCATGGGGTTTGCTGAAAAAAAGAAAAATC (SEQ ID NO: 15)
- FIGS. 5A and 5B Changes in the number of living cells and the amount of antibody production are shown in FIGS. 5A and 5B, respectively.
- the production of antibodies from the rat, mouse, Chinese hamster, and human Hspa8 promoters was 7.0, 6.8, 4.5, and 2.1 times that of the human EF1- ⁇ promoter, respectively.
- the Hspa8 promoter from any species greatly exceeded the amount of antibody produced by the human EF1- ⁇ promoter.
- a further increase in antibody production was observed from the Chinese hamster Hspa8 promoter, and it was found that by selecting an appropriate species, the promoter ability could be strongly exerted.
- SEQ ID NO: 1 Chinese hamster-derived Hspa8 promoter
- SEQ ID NO: 2 Human-derived Hspa8 promoter
- SEQ ID NO: 3 Mouse-derived Hspa8 promoter
- SEQ ID NO: 4 Rat-derived Hspa8 promoter
- SEQ ID NO: 5 Chinese hamster-derived Hspa8 promoter Hspa8 Nucleotide sequence consisting of about 1.9 kbp nucleotide upstream of the codon to the nucleotide immediately preceding the nucleotide sequence corresponding to the start codon
- SEQ ID NO: 6 Promoter of Hspa8 from Chinese hamster Starting from about 1.2 kbp nucleotide upstream of the start codon of Hspa8 Nucleotide sequence consisting of up to the nucleotide immediately preceding the nucleotide sequence corresponding to the codon
- SEQ ID NO: 7 Nucleotide sequence sequence of DNA element A2 Issue 8: DNA element A7 nucleotide sequence
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Abstract
Description
(1)チャイニーズハムスター由来Hspa8遺伝子のプロモーターであって、配列番号6に記載のヌクレオチド配列からなるポリヌクレオチドを含むことを特徴とする、配列番号1に記載のヌクレオチド配列又は該ヌクレオチド配列の部分配列からなるポリヌクレオチド。
(2)配列番号1に記載のヌクレオチド配列からなる、前記(1)に記載のポリヌクレオチド。
(3)配列番号5に記載のヌクレオチド配列からなる、前記(1)に記載のポリヌクレオチド。
(4)配列番号6に記載のヌクレオチド配列からなる、前記(1)に記載のポリヌクレオチド。
(5)ヒト由来Hspa8遺伝子のプロモーターである、配列表の配列番号2に記載のヌクレオチド配列からなるポリヌクレオチド。
(6)マウス由来Hspa8遺伝子のプロモーターである、配列表の配列番号3に記載のヌクレオチド配列からなるポリヌクレオチド。
(7)ラット由来Hspa8遺伝子のプロモーターである、配列表の配列番号4に記載のヌクレオチド配列からなるポリヌクレオチド。
(8)前記(1)乃至(7)のいずれか一つに記載のヌクレオチド配列に対して95%以上同一性を有するヌクレオチド配列からなるポリヌクレオチドであって、プロモーター活性を有するポリヌクレオチド。
(9)前記(1)乃至(7)のいずれか一つに記載のヌクレオチド配列に対して99%以上同一性を有するヌクレオチド配列からなるポリヌクレオチドであって、プロモーター活性を有するポリヌクレオチド。
(10)前記(1)乃至(9)のいずれか一つに記載のヌクレオチド配列に相補的なヌクレオチド配列からなるポリヌクレオチドとストリンジェントな条件でハイブリダイズするポリヌクレオチドであって、プロモーター活性を有するポリヌクレオチド。
(12)外来遺伝子が多量体蛋白質をコードする遺伝子である、前記(11)に記載の外来遺伝子発現ユニット。
(13)外来遺伝子がヘテロ多量体蛋白質をコードする遺伝子である、前記(11)に記載の外来遺伝子発現ユニット。
(14)外来遺伝子が抗体又はその抗原結合性断片をコードする遺伝子である、前記(11)に記載の外来遺伝子発現ユニット。
(15)前記(11)乃至(14)のいずれか一つに記載の外来遺伝子発現ユニットを含む外来遺伝子発現ベクター。
(16)前記(11)乃至(14)のいずれか一つに記載の外来遺伝子発現ユニット及び下記A群の(a)乃至(e)に記載のポリヌクレオチドから選択されるいずれか一つ又は複数のポリヌクレオチドを含む外来遺伝子発現ベクター;
A群
(a)配列表の配列番号7に記載のヌクレオチド配列からなるポリヌクレオチド、
(b)配列表の配列番号8に記載のヌクレオチド配列からなるポリヌクレオチド、
(c)配列表の配列番号9に記載のヌクレオチド配列からなるポリヌクレオチド、
(d)前記(a)乃至(c)のいずれか一つに記載のヌクレオチド配列に対して95%以上同一性を有するヌクレオチド配列からなるポリヌクレオチドであって、外来遺伝子発現亢進活性を有するポリヌクレオチド、
(e)上記(a)乃至(c)のいずれか一つに記載のヌクレオチド配列に対して99%以上同一性を有するヌクレオチド配列からなるポリヌクレオチドであって、外来遺伝子発現亢進活性を有するポリヌクレオチド。
(17)前記(15)又は(16)に記載の外来遺伝子発現ベクターが導入された形質転換細胞。
(18)細胞が哺乳動物由来の培養細胞である、前記(17)に記載の形質転換細胞。
(19)哺乳動物由来の培養細胞が、COS-1細胞、293細胞、又はCHO細胞である、前記(18)に記載の形質転換細胞。
(20)前記(17)乃至(19)のいずれか一つに記載の形質転換細胞を培養し、培養物から外来遺伝子由来の蛋白質を取得することを特徴とする、該蛋白質の製造方法。
(21)形質転換細胞において外来遺伝子を発現させることを目的とする、前記(1)乃至(10)のいずれか一つに記載のポリヌクレオチドの使用。
(22)形質転換細胞において外来遺伝子を発現させることを目的とする、前記(15)又は(16)に記載の外来遺伝子発現ベクターの使用。
本発明の外来遺伝子の発現亢進に使用されるプロモーター(以下、「本発明のプロモーター」ということもある)は、ヒートショックプロテインA8遺伝子(以下、「Hspa8」という)のプロモーターである。Hspa8プロモーターとしての活性を有するポリヌクレオチドであれば特に限定されないが、Hspa8のプロモーターとしては、開始コドンの上流約2.9kbpのヌクレオチドから開始コドンに対応するヌクレオチド配列の直前のヌクレオチドまでのポリヌクレオチドが好ましい。
チャイニーズハムスター由来Hspa8のプロモーターとしては、配列表の配列番号1及び図6に記載のポリヌクレオチドが例示される。配列番号1のヌクレオチド配列は、チャイニーズハムスター由来Hspa8の開始コドンの上流約2.9kbpのヌクレオチドから開始コドンに対応するヌクレオチド配列の直前のヌクレオチドからなる配列である。
本発明の外来遺伝子発現ユニット(以下、「本発明の遺伝子発現ユニット」ということもある)は、転写の読み枠の方向に、少なくとも前記1.に記載の本発明のプロモーター、外来遺伝子、及び転写ターミネーター領域(ポリA付加シグナル)を有するものである。
前記2.に記載の本発明の遺伝子発現ユニットとDNAエレメントを組み合わせて使用することにより、外来遺伝子の発現をさらに亢進することができる。組み合わせて使用するDNAエレメントは、アセチル化ヒストンH3との相互作用を指標として取得することが可能である。一般にヒストン(H3、H4)のアセチル化は転写の活性化に関与しているといわれており、主に2つの説が考えられている。ヒストンテールがアセチル化することで電荷的に中和され、DNAとヒストンとの結合が緩くなるというヌクレオソームの立体構造変化が関係している説(Mellor J. (2006) Dynamic nucleosomes and gene transcription. Trends Genet. 22(6):320-329)と、様々な転写因子のリクルートに関与するという説(Nakatani Y. (2001) Histone acetylases-versatile players. Genes Cells. 6(2):79-86)である。いずれの説においても、ヒストンのアセチル化が転写活性化に関与している可能性は高く、抗アセチル化ヒストンH3抗体を用いたクロマチン免疫沈降(Chromatin Immunoprecipitation;ChIP)によって、アセチル化ヒストンH3と相互作用するDNAエレメントを濃縮することが可能である。
本発明において、後記の産生亢進の対象となる外来蛋白質をコードする外来遺伝子を含むポリヌクレオチドは、以下に示す一般的な方法により取得することができる。例えば、外来遺伝子が発現している細胞や組織に由来するcDNAライブラリーを、当該遺伝子断片をもとにして合成したDNAプローブを用いてスクリーニングすることにより単離することができる。mRNAの調製は、当該技術分野において通常用いられる手法により行うことができる。例えば、前記細胞又は組織を、グアニジニン試薬、フェノール試薬等で処理して全RNAを得、その後、オリゴ(dT)セルロースカラムやセファロース2Bを担体とするポリU-セファロース等を用いたアフィニティーカラム法により、あるいはバッチ法によりポリ(A)+RNA(mRNA)を得る。さらに、ショ糖密度勾配遠心法等によりポリ(A)+RNAをさらに分画してもよい。次いで、得られたmRNAを鋳型として、オリゴdTプライマー及び逆転写酵素を用いて一本鎖cDNAを合成し、該一本鎖cDNAからDNA合成酵素I、DNAリガーゼ及びRNaseH等を用いて二本鎖cDNAを合成する。合成した二本鎖cDNAをT4DNA合成酵素によって平滑化後、アダプター(例えば、EcoRIアダプター)の連結、リン酸化等を経て、λgt11等のλファージに組み込んでin vivoパッケージングすることによってcDNAライブラリーを作製する。また、λファージ以外にもプラスミドベクターを用いてcDNAライブラリーを作製することもできる。その後、cDNAライブラリーから目的のDNAを有する株(ポジティブクローン)を選択すればよい。
本発明の外来遺伝子発現ベクターとしては、前記1.に記載のプロモーターを含む前記2.に記載の外来遺伝子発現ユニットを含むベクターが提供される。本発明の外来遺伝子発現ベクターは、前記3.に記載のDNAエレメントの1種、DNAエレメントの1種を2個以上のコピー数、DNAエレメントの2種以上の組み合わせを含んでもよい。前記の外来遺伝子発現ベクターによって外来遺伝子を宿主細胞内で発現させる際には、DNAエレメントを遺伝子発現ユニットの直前又は直後に配置してもよく、又は遺伝子発現ユニットから離れた位置に配置しても良い。また、複数のDNAエレメントを含む1つの外来遺伝子発現ベクターを用いてもよい。なお、DNAエレメントの向きは、遺伝子発現ユニットに対して順方向又は逆方向のいずれであっても良い。
本発明の形質転換細胞は、前記5.の外来遺伝子発現ベクターを用いて導入した形質転換細胞である。
本発明の外来蛋白質の製造方法は、前記6.の項目に記載の形質転換細胞を公知の方法により培養し、その培養物から採取し、精製することにより行うことができる。「培養物」とは、培養上清のほか、培養細胞、又は細胞の破砕物のいずれをも意味するものである。なお、6.の項目に記載の形質転換細胞を用いて産生することのできる外来蛋白質としては、単量体蛋白質のみならず多量体蛋白質を選択することも可能である。異なる複数のサブユニットから構成されるヘテロ多量体蛋白質の生産を行う場合、これらのサブユニットをコードしている複数の遺伝子を、それぞれ6.の項目に記載の宿主細胞に導入する必要がある。
前記7.の項目に記載の製造方法を用いて製造されるヘテロ多量体蛋白質としては抗体蛋白質を挙げることができる。抗体蛋白質は、2分子の重鎖ポリペプチド及び2分子の軽鎖ポリペプチドからなる4量体蛋白質である。従って、抗原結合能を維持した形態で抗体蛋白質を取得するためには、前記6.の項目に記載の形質転換細胞において、重鎖及び軽鎖の遺伝子の双方が導入されている必要がある。この場合に、重鎖及び軽鎖の遺伝子発現ユニットは、同じ発現ベクター上に存在しても良く、あるいは異なる発現ベクター上に存在していても良い。
本発明の製造方法の対象となる外来蛋白質としては、前述の抗体に加え、ヒト又は非ヒト動物由来の各種蛋白質、その抗原結合性断片、その改変体等を挙げることができる。そのような蛋白質等としては、心房性ナトリウム利尿ペプチド(ANP)、脳性ナトリウム利尿ペプチド(BNP)、C型ナトリウム利尿ペプチド(CNP)、バソプレッシン、ソマトスタチン、成長ホルモン(GH)、インスリン、オキシトシン、グレリン、レプチン、アディポネクチン、レニン、カルシトニン、オステオプロテジェリン、インスリン様成長因子(IGF)等のペプチドホルモン、インターロイキン、ケモカイン、インターフェロン、腫瘍壊死因子(TNFα/βほかTNFスーパーファミリー等)、神経成長因子(NGF)、細胞増殖因子(EGF、FGF、PDGF、HGF、TGF等)、造血因子(CSF、G-CSF、エリスロポエチン等)、アディポカイン等のサイトカイン、ТNF受容体等の受容体、リゾチーム、プロテアーゼ、プロテイナーゼ、ペプチダーゼ等の酵素、その機能性断片(元の蛋白質の生物活性を一部又は全部保持している断片)、それらの蛋白質を含むことからなる融合蛋白質等を挙げることができるが、それらに限定されるものではない。
Hspa8のプロモーター領域としては、GenBankにNM_001246729.1で登録されているmRNAの配列およびNW_003616190.1で登録されているチャイニーズハムスターゲノムのスキャフォールド配列を参考にして、Hspa8の開始コドン配列の上流約2.9kbpのヌクレオチドから開始コドン配列に対応するヌクレオチド配列の直前のヌクレオチドまでの配列を用いた。
Hspa8プロモーターのプライマーセット
Hspa8-NotI-F:TTCGCGGCCGCCAAGGCTGAGGCAGCG(配列番号10)
Hspa8-XbaI-R:TTCTCTAGAGGTTGCTGAAAGAAAACCAAA(配列番号11)
2-1)抗体発現ベクターの構築
pDSLH4.1(Okumura T et al., J Biosci Bioeng., 120(3):340-346,2015参照)をベクター基本骨格として有し、抗体H鎖およびL鎖遺伝子のプロモーターにHspa8を使用し、DNAエレメントを含まないヒト化抗体遺伝子Y発現ベクターpDSLH3.1-Hspa8-Yを構築した。まず、実施例1で増幅、精製したDNA断片をNotI-XbaIで消化した後、特許文献4に記載のH鎖遺伝子発現ベクターpDSH1.1-hEF1α-Y、および、L鎖遺伝子発現ベクターpDSL2.1-hEF1α-YのNotI-NheIサイト間に挿入して、それぞれpDSH1.1-Hspa8-Y、pDSL2.1-Hspa8-Yを構築した。次に、pDSL2.1-Hspa8-YをAatII-MluIで消化して得られたDNA断片をpDSH1.1-Hspa8-YのAatII-MluI間に挿入して、pDSLH3.1-Hspa8-Yを構築した。ベクター概略を図1に示す。
CHO-K1細胞(ATCC)を無血清培地を用いた浮遊状態での培養が可能となるように馴化し、宿主細胞CHO-O1細胞を得た。CHO-O1細胞に、(2-1)で構築した抗体発現ベクターpDSLH3.1-Hspa8-Y、特許文献4に記載のpDSLH3.1-hEF1α-Yを、遺伝子導入装置Neon Transfection System(Invitrogen)を用いて遺伝子導入し、T-25フラスコにて5%CO2、37℃で培養した。遺伝子導入の1日後にGeneticin(Life Technologies Corporation)を終濃度800 μg/mLで添加し、1週間薬剤選択培養を行った。その後、125mL容三角フラスコにて5%CO2、37℃で培養し、ヒト化抗体Y発現ステーブルプールを作製した。
(2-2)で作製したヒト化抗体Y発現ステーブルプールを用いて、125mL容三角フラスコにて流加培養を行った。基礎培地にG13(アイエスジャパン製カスタム培地)、フィード培地にF13(アイエスジャパン製カスタム培地)を用いた。
3-1)抗体発現ベクターの構築
ヒト化抗体遺伝子Y発現ベクターpDSLH3.1-hEF1α-Yの、抗体H鎖およびL鎖遺伝子のプロモーターをHspa8プロモーターの部分配列に置換した、pDSLH3.1-Hspa8-1.9-Y、および、pDSLH3.1-Hspa8-1.2-Yを構築した。それぞれの発現ベクターで、Hspa8の開始コドン配列の上流約1.9、1.2kbpのヌクレオチドから開始コドン配列に対応するヌクレオチド配列の直前のヌクレオチドまでの配列を、Hspa8プロモーターの部分配列として用いた。
Hspa8プロモーター 1.9kbpのプライマーセット
Hspa8-NotI-1900F:TTCGCGGCCGCAACAACCTAACTAATAGCTGTCC(配列番号12)
Hspa8-XbaI-R:TTCTCTAGAGGTTGCTGAAAGAAAACCAAA(配列番号11)
Hspa8プロモーター 1.2kbpのプライマーセット
Hspa8-NotI-1200F:TTCGCGGCCGCAACCTTCGCGGCCATTTTGTCCTC(配列番号13)
Hspa8-XbaI-R:TTCTCTAGAGGTTGCTGAAAGAAAACCAAA(配列番号11)
(2-1)および(3-1)で構築した抗体発現ベクターpDSLH3.1-hEF1α-Y、pDSLH3.1-Hspa8-Y、pDSLH3.1-Hspa8-1.9-Y、あるいは、pDSLH3.1-Hspa8-1.2-Yを、(2-2)に記載の方法でCHO-O1細胞にトランスフェクション、薬剤選択培養を行い、ヒト化抗体Y発現ステーブルプールを作製した。
(3-2)で作製したヒト化抗体Y発現ステーブルプールを用いて、125mL容三角フラスコにて流加培養を行った。基礎培地にG13、フィード培地にF13を用いた。
4-1)抗体発現ベクターの構築
(3-1)で構築した抗体発現ベクターpDSLH3.1-Hspa8-1.9-Yの発現カセット上流に、特許文献3に記載のDNAエレメントA7を挿入して、pDSLHA4.1-Hspa8-1.9-Yを構築した。
(3-1)で構築したDNAエレメントA7を含まない抗体発現ベクターpDSLH3.1-Hspa8-1.9-Y、(4-1)で構築したDNAエレメントA7を含む抗体発現ベクターpDSLHA4.1-Hspa8-1.9-Yを、(2-2)に記載の方法でCHO-O1細胞にトランスフェクション、薬剤選択培養を行い、ヒト化抗体Y発現ステーブルプールを作製した。
(4-2)で作製したヒト化抗体Y発現ステーブルプールを用いて、125mL容三角フラスコにて流加培養を行った。基礎培地にG13、フィード培地にF13を用いた。
5-1)抗体発現ベクターの構築
ヒト化抗体遺伝子Y発現ベクターpDSLH3.1-hEF1α-Yの、抗体H鎖およびL鎖遺伝子のプロモーターをヒト、マウス、ラットHspa8プロモーターに置換した、pDSLH3.1-hHspa8-Y、pDSLH3.1-mHspa8-Y、pDSLH3.1-rHspa8-Yを構築した。それぞれ、Hspa8の開始コドン配列の上流約2.0kbpのヌクレオチドから開始コドン配列に対応するヌクレオチド配列の直前のヌクレオチドまでの配列を、Hspa8プロモーターとして用いた。クローニングしたヒト、マウス、ラットHspa8プロモーターのヌクレオチド配列をそれぞれ配列表の配列番号2、3、4に示す。
H鎖遺伝子発現ベクター挿入用のヒトHspa8プロモーターのプライマーセット
Hspa8-human-HindIII-F:GGTGAAGCTTATACAAACGTTCAGAAAGTCTAA(配列番号14)
Hspa8-human-EcoT14I-R:GGTGCCATGGGGTTGCTGAAAAAAAGAAAAATC(配列番号15)
L鎖遺伝子発現ベクター挿入用のヒトHspa8プロモーターのプライマーセット
Hspa8-human-AatII-F:GGGTGACGTCATACAAACGTTCAGAAAGTCTAA(配列番号16)
Hspa8-human-EcoT14I-R:GGTGCCATGGGGTTGCTGAAAAAAAGAAAAATC(配列番号15)
マウスHspa8プロモーターのプライマーセット
Hspa8-mouse-NotI-F:GGGTGCGGCCGCAGACCTTCCAATTTAAACGCCAC(配列番号17)
Hspa8-mouse-XbaI-R:GAGGTCTAGAGGTTGCTATTAGAAAAAAAAAGG(配列番号18)
ラットHspa8プロモーターのプライマーセット
Hspa8-rat-NotI-F:GGTGGCGGCCGCCTTTTGATAGCCTTCCTCACATG(配列番号19)
Hspa8-rat-NheI-R:GGTCGCTAGCGGTTGCTAGAAGGAAAAAAAAAA(配列番号20)
(2-1)、(3-1)および(5-1)で構築した抗体発現ベクターpDSLH3.1-hEF1α-Y、pDSLH3.1-Hspa8-1.9-Y、pDSLH3.1-hHspa8-Y、pDSLH3.1-mHspa8-Y、あるいは、pDSLH3.1-rHspa8-Yを、(2-2)に記載の方法でCHO-O1細胞にトランスフェクション、薬剤選択培養を行い、ヒト化抗体Y発現ステーブルプールを作製した。
5-3)ヒト化抗体Y発現ステーブルプールの流加培養による抗体生産量評価
(5-2)で作製したヒト化抗体Y発現ステーブルプールを用いて、125mL容三角フラスコにて流加培養を行った。基礎培地にG13、フィード培地にF13を用いた。
配列番号2:ヒト由来Hspa8のプロモーター
配列番号3:マウス由来Hspa8のプロモーター
配列番号4:ラット由来Hspa8のプロモーター
配列番号5:チャイニーズハムスター由来Hspa8のプロモーター Hspa8の開始コドンの上流約1.9kbpのヌクレオチドから開始コドンに対応するヌクレオチド配列の直前のヌクレオチドまでからなるヌクレオチド配列
配列番号6:チャイニーズハムスター由来Hspa8のプロモーター Hspa8の開始コドンの上流約1.2kbpのヌクレオチドから開始コドンに対応するヌクレオチド配列の直前のヌクレオチドまでからなるヌクレオチド配列
配列番号7:DNAエレメントA2のヌクレオチド配列
配列番号8:DNAエレメントA7のヌクレオチド配列
配列番号9:DNAエレメントA18のヌクレオチド配列
配列番号10:Hspa8プロモーターのプライマー Hspa8-NotI-F
配列番号11:Hspa8プロモーターのプライマー Hspa8-XbaI-R
配列番号12:Hspa8プロモーター 1.9kbpのプライマー Hspa8-NotI-1900F
配列番号13:Hspa8プロモーター 1.2kbpのプライマー Hspa8-NotI-1200F
配列番号14:ヒトHspa8プロモーターのプライマー Hspa8-human-HindIII-F
配列番号15:ヒトHspa8プロモーターのプライマー Hspa8-human-EcoT14I-R
配列番号16:ヒトHspa8プロモーターのプライマー Hspa8-human-AatII-F
配列番号17:マウスHspa8プロモーターのプライマー Hspa8-mouse-NotI-F
配列番号18:マウスHspa8プロモーターのプライマー Hspa8-mouse-XbaI-R
配列番号19:ラットHspa8プロモーターのプライマー Hspa8-rat-NotI-F
配列番号20:ラットHspa8プロモーターのプライマー Hspa8-rat-NheI-R
Claims (22)
- チャイニーズハムスター由来Hspa8遺伝子のプロモーターであって、配列番号6に記載のヌクレオチド配列からなるポリヌクレオチドを含むことを特徴とする、配列番号1に記載のヌクレオチド配列又は該ヌクレオチド配列の部分配列からなるポリヌクレオチド。
- 配列番号1に記載のヌクレオチド配列からなる、請求項1に記載のポリヌクレオチド。
- 配列番号5に記載のヌクレオチド配列からなる、請求項1に記載のポリヌクレオチド。
- 配列番号6に記載のヌクレオチド配列からなる、請求項1に記載のポリヌクレオチド。
- ヒト由来Hspa8遺伝子のプロモーターである、配列表の配列番号2に記載のヌクレオチド配列からなるポリヌクレオチド。
- マウス由来Hspa8遺伝子のプロモーターである、配列表の配列番号3に記載のヌクレオチド配列からなるポリヌクレオチド。
- ラット由来Hspa8遺伝子のプロモーターである、配列表の配列番号4に記載のヌクレオチド配列からなるポリヌクレオチド。
- 請求項1乃至7のいずれか一つに記載のヌクレオチド配列に対して95%以上同一性を有するヌクレオチド配列からなるポリヌクレオチドであって、プロモーター活性を有するポリヌクレオチド。
- 請求項1乃至7のいずれか一つに記載のヌクレオチド配列に対して99%以上同一性を有するヌクレオチド配列からなるポリヌクレオチドであって、プロモーター活性を有するポリヌクレオチド。
- 請求項1乃至9のいずれか一つに記載のヌクレオチド配列に相補的なヌクレオチド配列からなるポリヌクレオチドとストリンジェントな条件でハイブリダイズするポリヌクレオチドであって、プロモーター活性を有するポリヌクレオチド。
- 請求項1乃至10のいずれか一つに記載のポリヌクレオチドを含むことからなる、外来遺伝子発現ユニット。
- 外来遺伝子が多量体蛋白質をコードする遺伝子である、請求項11に記載の外来遺伝子発現ユニット。
- 外来遺伝子がヘテロ多量体蛋白質をコードする遺伝子である、請求項11に記載の外来遺伝子発現ユニット。
- 外来遺伝子が抗体又はその抗原結合性断片をコードする遺伝子である、請求項11に記載の外来遺伝子発現ユニット。
- 請求項11乃至14のいずれか一つに記載の外来遺伝子発現ユニットを含む外来遺伝子発現ベクター。
- 請求項11乃至14のいずれか一つに記載の外来遺伝子発現ユニット及び下記A群の(a)乃至(e)に記載のポリヌクレオチドから選択されるいずれか一つ又は複数のポリヌクレオチドを含む外来遺伝子発現ベクター;
A群
(a)配列表の配列番号7に記載のヌクレオチド配列からなるポリヌクレオチド、
(b)配列表の配列番号8に記載のヌクレオチド配列からなるポリヌクレオチド、
(c)配列表の配列番号9に記載のヌクレオチド配列からなるポリヌクレオチド、
(d)前記(a)乃至(c)のいずれか一つに記載のヌクレオチド配列に対して95%以上同一性を有するヌクレオチド配列からなるポリヌクレオチドであって、外来遺伝子発現亢進活性を有するポリヌクレオチド、
(e)上記(a)乃至(c)のいずれか一つに記載のヌクレオチド配列に対して99%以上同一性を有するヌクレオチド配列からなるポリヌクレオチドであって、外来遺伝子発現亢進活性を有するポリヌクレオチド。 - 請求項15又は16に記載の外来遺伝子発現ベクターが導入された形質転換細胞。
- 細胞が哺乳動物由来の培養細胞である、請求項17に記載の形質転換細胞。
- 哺乳動物由来の培養細胞が、COS-1細胞、293細胞、又はCHO細胞である、請求項18に記載の形質転換細胞。
- 請求項17乃至19のいずれか一つに記載の形質転換細胞を培養し、培養物から外来遺伝子由来の蛋白質を取得することを特徴とする、該蛋白質の製造方法。
- 形質転換細胞において外来遺伝子を発現させることを目的とする、請求項1乃至10のいずれか一つに記載のポリヌクレオチドの使用。
- 形質転換細胞において外来遺伝子を発現させることを目的とする、請求項15又は16に記載の外来遺伝子発現ベクターの使用。
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WO2023210607A1 (ja) * | 2022-04-26 | 2023-11-02 | 第一三共株式会社 | Eno1遺伝子のプロモーター |
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CN105018528B (zh) * | 2015-08-20 | 2018-04-03 | 重庆大学 | 热休克蛋白基因启动子和四环素基因启动子控制的多基因表达和沉默系统 |
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Cited By (2)
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WO2021201268A1 (ja) * | 2020-04-03 | 2021-10-07 | 第一三共株式会社 | 新規遺伝子発現ユニット |
WO2023210607A1 (ja) * | 2022-04-26 | 2023-11-02 | 第一三共株式会社 | Eno1遺伝子のプロモーター |
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EP3835423A1 (en) | 2021-06-16 |
JPWO2020032153A1 (ja) | 2021-08-12 |
US20210317498A1 (en) | 2021-10-14 |
AU2019318910A1 (en) | 2021-02-18 |
KR20210042084A (ko) | 2021-04-16 |
CA3109101A1 (en) | 2020-02-13 |
CN112639098A (zh) | 2021-04-09 |
SG11202100996TA (en) | 2021-03-30 |
TW202020160A (zh) | 2020-06-01 |
EP3835423A4 (en) | 2022-08-10 |
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