WO2021201268A1 - Nouvelle unité d'expression génique - Google Patents

Nouvelle unité d'expression génique Download PDF

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WO2021201268A1
WO2021201268A1 PCT/JP2021/014287 JP2021014287W WO2021201268A1 WO 2021201268 A1 WO2021201268 A1 WO 2021201268A1 JP 2021014287 W JP2021014287 W JP 2021014287W WO 2021201268 A1 WO2021201268 A1 WO 2021201268A1
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seq
gene
polynucleotide
utr
derived
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PCT/JP2021/014287
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Japanese (ja)
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雄大 酒井
真初 西尾
兼治 増田
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第一三共株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention uses a mammalian transformed cell in which the expression level of a foreign protein is enhanced by using an expression unit designed by combining a promoter having high transcriptional activity and 5'-UTR having high translational activity, and the present invention.
  • the present invention relates to a method for producing the foreign protein.
  • protein drugs such as therapeutic proteins and antibody drugs are rapidly expanding their markets.
  • antibody drugs do not cause a harmful immune reaction even when administered to the human body, and are being actively developed due to their high specificity.
  • Non-Patent Document 1 When cultured mammalian cells are used as a host, there is a problem of high production cost due to low growth rate and low productivity as compared with microorganisms (Non-Patent Document 1). In addition, due to the recent development of antibody drugs and the large doses, the production capacity of antibody drugs is insufficient worldwide. Improvements are being made to each manufacturing process of protein pharmaceuticals with the aim of improving production capacity, and improvement of the mammalian cultured cell expression system is a promising means for achieving an increase in production volume (Patent Documents 1 to 3). , Non-Patent Documents 2, 3).
  • the promoter is present on the DNA and RNA polymerase transcribes RNA from the transcription start site within the promoter region. The sequence region upstream of the transcription initiation site is mainly involved in the efficiency of transcription, and a promoter with high transcription efficiency has a binding sequence of a plurality of transcription factors in the sequence region (Non-Patent Document 4).
  • mRNA messenger RNA
  • proteins are synthesized by translation.
  • mRNA has not only gene / protein information but also 5'-Untranslated Region (5'-UTR) and 3'-UTR which are untranslated regions that do not contain gene / protein information upstream and downstream thereof.
  • the untranslated region is involved in gene expression, and in particular, 5'-UTR controls translation efficiency (Non-Patent Document 5).
  • the amount of translation and the intracellular stability of mRNA vary depending on the presence or absence of AUG sequence, RNA-binding protein, and microRNA (miRNA) binding / target sequence in 5'-UTR.
  • splicing excludes introns and matures into mRNAs composed exclusively of exons.
  • Introns are present not only in untranslated regions but also in structural gene sequences, but their numbers and positions vary from mRNA to mRNA.
  • the mRNA transcribed in the nucleus is transported to the outside of the nucleus, that is, into the cytoplasm, and translation is initiated. Introns are involved in the export of this mRNA to the nucleus and polyadenylation at the 3'end (Non-Patent Document 6).
  • a hybrid promoter consisting of a virus-derived enhancer and TATA-box, a mammalian-derived intron (Patent Document 4), and a heat that improves the productivity of foreign proteins.
  • a promoter of a shock protein A5 (Hspa5 / GRP78) gene Patent Document 5
  • a foreign intron into or downstream of a structural gene
  • An object of the present invention is to design an expression unit that combines a promoter region with high transcriptional activity, 5'-UTR, and intron having high translational activity in a host cell such as cultured mammalian cells, and use this to design a protein drug. It is an object of the present invention to provide a means for increasing the production amount of a foreign protein. By combining the promoter region with a 5'-UTR, or by substituting a different intron for the intron in the combined 5'-UTR, the introns in the CHO cells and the like are equal to or higher than those in which only the promoter region is used.
  • the present inventors have different expression units consisting of polynucleotides in which a 5'-UTR is combined with a promoter region having transcriptional activity, or further, introns in the expression unit are different.
  • the present invention includes the following inventions. (1) A polynucleotide in which 5'-UTR contained in the same or different gene as the gene containing the promoter region is linked downstream of the promoter region.
  • (21) The transformed cell according to (20) above, wherein the cell is a cultured cell derived from a mammal.
  • (22) The transformed cell according to (21) above, wherein the cultured mammalian-derived cell is a COS-1 cell, a HEK293 cell, or a CHO cell.
  • a method for producing a protein which comprises culturing the transformed cell according to any one of (20) to (22) above and obtaining a protein derived from a foreign gene from the culture.
  • (24) Use of the polynucleotide according to any one of (1) and (13) above, for the purpose of expressing a foreign gene in transformed cells.
  • (25) Use of the foreign gene expression vector according to (19) above, for the purpose of expressing a foreign gene in transformed cells.
  • the expression unit of the present invention can significantly enhance the expression of foreign genes such as therapeutic proteins and antibodies by being incorporated into a foreign gene expression vector together with a gene and introduced into a mammalian host cell.
  • FIG. 4A shows the number of viable cells on each sampling day.
  • FIG. 4B shows the production volume on each sampling day.
  • the amount of antibody produced by the expression unit containing the mouse-derived Ubc promoter region is expressed by the promoter region of the human EF1 ⁇ gene and the 5'-UTR.
  • FIG. 5A shows the number of viable cells on each sampling day.
  • FIG. 5B shows the production volume on each sampling day.
  • Transient expression was performed using an expression unit consisting of a nucleotide sequence of about 3.0 kbp upstream from the start codon of mouse-derived Ubc and a promoter region of a nucleotide sequence of about 2.1 kbp upstream from the start codon and 5'-UTR.
  • the amount of antibody produced by the expression unit containing the Hspa5 promoter region and the 5'-UTR of Hspa8 was measured by the promoter region of the human EF1 ⁇ gene and the 5'-UTR.
  • the figure which compared with the expression unit composed of. The nucleotide length after the transcription start point is described in parentheses.
  • FIG. 7A shows the number of viable cells on each sampling day.
  • the amount of antibody produced by the expression unit containing the Hspa5 promoter region and the 5'-UTR of Hspa8 was measured by the promoter region of the human EF1 ⁇ gene and the 5'-UTR.
  • FIG. 7B shows the production volume on each sampling day.
  • a stable pool prepared using a humanized antibody Y expression vector containing or not containing DNA element A7 using an expression unit containing the mUbc promoter region and 5'-UTR as a promoter for antibody H chain and L chain genes.
  • FIG. 8A shows the number of viable cells on each sampling day.
  • a stable pool prepared using a humanized antibody Y expression vector containing or not containing DNA element A7 using an expression unit containing the mUbc promoter region and 5'-UTR as a promoter for antibody H chain and L chain genes.
  • FIG. 8B shows the production volume on each sampling day.
  • promoter region means a sequence having transcriptional activity in the sequence upstream of the transcription initiation site. Also referred to herein as a “promoter.”
  • 5'-UTR is an upstream sequence of the start codon of a gene present in transcribed mRNA, and means an untranslated region that does not contain gene / protein information.
  • upstream refers to the 5'terminal side of a gene, sequence region, or base
  • downstream refers to the 3'terminal side of a gene, sequence region, or base
  • an "intron” is a non-coding portion that is excised or removed when mature messenger RNA (mRNA) is produced from DNA by splicing in gene expression, and the DNA is combined with the coding portion (exon). Means the constituent array. Introns are also included in the 5'-UTR.
  • mRNA messenger RNA
  • 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 includes DNA, RNA, probe, oligonucleotide, and primer.
  • SEQ ID NO: ⁇ is ⁇
  • [a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: ⁇ ] is abbreviated as [the polynucleotide described in SEQ ID NO: ⁇ ].
  • polypeptide and “protein” are used without distinction.
  • gene expression means a phenomenon in which a certain gene is transcribed into mRNA and a protein is translated from the mRNA.
  • the "foreign gene” means a gene artificially introduced into a host cell.
  • foreign protein means a protein encoded by a foreign gene.
  • gene expression cassette means a polynucleotide having at least a promoter region, a foreign gene, and a transcription terminator region (poly A addition signal) in the direction of the transcription reading frame.
  • transcriptional activity refers to the activity of initiating transcription from DNA by RNA polymerase and synthesizing mRNA.
  • transcriptional activity refers to the activity of synthesizing a protein from transcribed mRNA.
  • DNA element means a polynucleotide having a foreign gene expression-enhancing activity when placed in the vicinity of a gene expression cassette or on a foreign gene expression vector contained in the gene expression cassette. ..
  • the "antigen-binding fragment of an antibody” means a partial fragment of an antibody having an antigen-binding activity, including Fab, F (ab') 2, etc., but with an antigen. It is not limited to these molecules as long as it has a binding ability.
  • identity refers to the relationship between sequences of two or more nucleotide sequences or amino acid sequences, as is known in the art, determined by comparison of sequences. In the art, “identity” is also a sequence between nucleic acid molecules or polypeptides, as determined by matching between two or more nucleotide sequences in a row or between two or more amino acid sequences, as the case may be. Means the degree of relevance. “Identity” is the same match between the smaller of two or more arrays and the gap alignment (if any) addressed by a particular mathematical model or computer program (ie, "algorithm”). It can be evaluated by calculating the percentage of. Specifically, it can be evaluated by using software such as ClinicalW2 provided by European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI), but it is limited to those used by those skilled in the art. Not done.
  • EBL-EBI European Molecular Biology Laboratory-European Bioinformatics Institute
  • hybridize under stringent conditions means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
  • a complementary strand of a nucleic acid consisting of a nucleotide sequence having an identity of 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more with respect to a certain nucleic acid hybridizes, and the identity is lower than that.
  • the condition that the complementary strand of the nucleic acid consisting of the nucleotide sequence does not hybridize can be mentioned.
  • the expression unit of the present invention is a polynucleotide in which a 5'-UTR is linked downstream of a promoter region, which is a sequence region essential for transcription and exists upstream of a gene. be.
  • the 5'-UTR may be a 5'-UTR existing upstream of the gene containing the promoter region, or may be a 5'-UTR existing upstream of a gene different from the gene containing the promoter region.
  • FIG. 1 shows an outline of an expression unit in which a 5'-UTR existing upstream of a gene different from the gene containing the promoter region is linked downstream of the promoter region.
  • the 5'-UTR contains an intron
  • the intron may be replaced with an intron contained in the 5'-UTR in a gene different from the gene containing the 5'-UTR.
  • FIG. 2 shows an outline of an expression unit in which the intron contained in the expression unit is replaced with the intron contained in 5'-UTR in a gene different from the gene containing the 5'-UTR.
  • the promoter region contained in the expression unit of the present invention is a sequence region having transcriptional activity in the sequence upstream from the transcription initiation site.
  • the transcription start site, or the 5'end of the 5'-UTR is a sequence published on a database such as NCBI, or a sequence position identified by an experimental method such as the 5'-RACE method.
  • the origin of the promoter region contained in the expression unit of the present invention is not particularly limited, but is preferably derived from cultured mammalian cells, and examples of the mammal include Chinese hamsters, humans, mice, rats, and the like. ..
  • the promoter region contained in the expression unit of the present invention include a promoter region contained in the Hspa5 gene derived from Chinese hamster and a promoter region contained in the Ubc gene derived from mouse. More preferably, the promoter region of the Hspa5 gene derived from the Chinese hamster consisting of the polynucleotide shown in SEQ ID NO: 1 in the sequence listing and the promoter region of the Ubc gene derived from the mouse consisting of the polynucleotides shown in SEQ ID NOs: 2 and 3 can be mentioned.
  • the nucleotide sequence of SEQ ID NO: 1 is a sequence consisting of a nucleotide at the transcription start site from a nucleotide of about 0.6 kbp upstream of the start codon of the Chinese hamster-derived Hspa5 gene.
  • the nucleotide sequence of SEQ ID NO: 2 is a sequence consisting of nucleotides at the transcription start site from nucleotides about 3.0 kbp upstream of the start codon of the mouse-derived Ubc gene.
  • the promoter region of the mouse-derived Ubc gene may be a nucleotide sequence consisting of a partial sequence of the sequence shown in SEQ ID NO: 2, and the transcription initiation site is located at a nucleotide of about 2.1 kbp upstream of the start codon of the mouse-derived Ubc gene.
  • the polynucleotide set forth in SEQ ID NO: 3 consisting of nucleotides is preferred.
  • the nucleotide sequence of SEQ ID NO: 4 is a sequence consisting of 32 nucleotides downstream from the transcription start site of about 0.2 kbp upstream of the start codon of the Chinese hamster-derived Hspa5 gene.
  • the nucleotide sequence of SEQ ID NO: 5 is a polynucleotide consisting of a nucleotide sequence in which the nucleotide sequence of SEQ ID NO: 4 is linked to the 3'side of the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 may be contained in the promoter region in its entire length, or may be contained in a partially deleted state.
  • the origin of the 5'-UTR contained in the expression unit of the present invention is not particularly limited, but it is preferably derived from cultured mammalian cells, and examples of the mammal include Chinese hamsters, humans, mice, rats and the like. Can be mentioned.
  • the 5'-UTR derived from Chinese hamster as the 5'-UTR of the Hspa8 gene, Actb gene, Rpsa gene, and EF1 ⁇ gene is 5'-UTR of the Ubc gene.
  • UTRs include 5'-UTRs derived from Chinese hamsters and mice. More preferably, it is a 5'-UTR consisting of the polynucleotides set forth in SEQ ID NOs: 6-11.
  • the nucleotide sequence of SEQ ID NO: 6 is a sequence consisting of the nucleotide corresponding to the transcription start point at about 0.6 kbp upstream of the start codon of the Chinese hamster-derived Hspa8 gene and the nucleotide immediately preceding the nucleotide sequence corresponding to the start codon.
  • the polynucleotide set forth in SEQ ID NO: 12 is an intron contained in the 5'-UTR composed of the polynucleotide set forth in SEQ ID NO: 6.
  • the nucleotide sequence of SEQ ID NO: 7 is a sequence consisting of the nucleotide corresponding to the transcription start point at about 1.0 kbp upstream of the start codon of the Chinese hamster-derived Actb gene and the nucleotide immediately preceding the nucleotide sequence corresponding to the start codon.
  • SEQ ID NO: 13 is included in the 5'-UTR consisting of the polynucleotide shown in SEQ ID NO: 7, but is an intron.
  • the nucleotide sequence of SEQ ID NO: 8 is a sequence consisting of the nucleotide corresponding to the transcription start point at about 0.6 kbp upstream of the start codon of the Chinese hamster-derived Rpsa gene and the nucleotide immediately preceding the nucleotide sequence corresponding to the start codon.
  • SEQ ID NO: 14 is an intron contained in the 5'-UTR composed of the polynucleotide shown in SEQ ID NO: 8.
  • the nucleotide sequence of SEQ ID NO: 9 is a sequence consisting of the nucleotide corresponding to the transcription start point at about 1.7 kbp upstream of the start codon of Chinese hamster-derived Ubc and the nucleotide immediately preceding the nucleotide sequence corresponding to the start codon.
  • SEQ ID NO: 15 is an intron contained in the 5'-UTR composed of the polynucleotide shown in SEQ ID NO: 9.
  • the nucleotide sequence of SEQ ID NO: 10 is a sequence consisting of the nucleotide corresponding to the transcription start point at about 1.0 kbp upstream of the start codon of the Chinese hamster-derived EF1 ⁇ gene and the nucleotide immediately preceding the nucleotide sequence corresponding to the start codon.
  • SEQ ID NO: 16 is an intron contained in the 5'-UTR composed of the polynucleotide shown in SEQ ID NO: 10.
  • the nucleotide sequence of SEQ ID NO: 11 is a sequence consisting of the nucleotide corresponding to the transcription start point at about 1.5 kbp upstream of the start codon of the mouse-derived Ubc gene and the nucleotide immediately preceding the nucleotide sequence corresponding to the start codon.
  • SEQ ID NO: 17 is an intron contained in the 5'-UTR composed of the polynucleotide shown in SEQ ID NO: 11.
  • the intron contained in the expression unit of the present invention is a sequence published on a database such as NCBI or a sequence identified by an experimental method such as sequence analysis, and is a 5'-UTR in the expression unit of the present invention. Included in.
  • an intron in 5'-UTR contained in the expression unit of the present invention can be mentioned, and more preferably, the polynucleotide described in SEQ ID NOs: 12 to 17 described above can be mentioned.
  • the expression unit of the present invention is preferably 5'-UTR according to any one of SEQ ID NOs: 6 to 11 downstream of the promoter region set forth in any one of SEQ ID NOs: 1, 2, 3 and 5. It is a polynucleotide in which the above is linked.
  • the 5'-UTR includes an intron.
  • the intron may be replaced with an intron contained in a gene different from the gene containing the 5'-UTR.
  • the intron consisting of the polynucleotides set forth in SEQ ID NOs: 12 to 17 contained in each of the 5'-UTRs consisting of the polynucleotides set forth in SEQ ID NOs: 6 to 11 is different from the other introns set forth in SEQ ID NOs: 12 to 17. It may be replaced.
  • Preferred specific examples of the expression unit of the present invention include expression units composed of the polynucleotides set forth in SEQ ID NOs: 18 to 35 and 45 to 48.
  • more preferable specific examples include expression units consisting of the polynucleotides set forth in SEQ ID NOs: 18, 24-29, and 45-48.
  • the nucleotide sequences of SEQ ID NOs: 18 to 23 are downstream of the nucleotide sequence in which the 32 nucleotides of SEQ ID NO: 4 are linked to the promoter region of the Chinese hamster-derived Hspa5 gene consisting of the polynucleotides of SEQ ID NO: 1. It is a polynucleotide in which the 5'-UTR of the Hspa8 gene of origin, the Actb gene, the Rpsa gene, the Ubc gene, the EF1 ⁇ gene, or the Ubc gene of mouse origin is linked, respectively.
  • the nucleotide sequences of SEQ ID NOs: 24-29 are downstream of the promoter region of the mouse-derived Ubc gene consisting of the polynucleotide shown in SEQ ID NO: 2, the Chinese hamster-derived Hspa8 gene, Actb gene, Rpsa gene, Ubc gene, EF1 ⁇ gene, or , 5'-UTR of the mouse-derived Ubc gene, respectively.
  • the nucleotide sequences of SEQ ID NOs: 30 to 35 are downstream of the promoter region of the mouse-derived Ubc gene consisting of the polynucleotide shown in SEQ ID NO: 3, the Chinese hamster-derived Hspa8 gene, Actb gene, Rpsa gene, Ubc gene, EF1 ⁇ gene, or , 5'-UTR of the mouse-derived Ubc gene, respectively.
  • the nucleotide sequence of SEQ ID NO: 36 is a sequence from 33 nucleotides downstream to 48 nucleotides downstream of the transcription initiation site at about 0.2 kbp upstream of the start codon of Chinese hamster-derived Hspa5.
  • the sequence is a sequence that contributes to the maintenance or improvement of transcriptional activity or translational activity, and can be added to the nucleotide sequence of SEQ ID NO: 5.
  • the Chinese hamster-derived Hspa8 gene, Actb gene, Rpsa gene, Ubc gene, EF1 ⁇ gene, or mouse-derived Ubc gene 5'-UTR are linked downstream of this, the polynucleotides shown in SEQ ID NOs: 18 to 23 are used. Has similar transcriptional or translational activity.
  • the 3'end of the promoter region used for the expression unit and the 5'end of the 5'-UTR are preferably the nucleotides immediately before the transcription initiation site or the nucleotide corresponding to the transcription initiation site. Nucleotides can be added to the promoter region and to the ends of the 5'-UTR if the transcriptional or translational activity can be maintained or improved in the.
  • the nucleotide sequence to be added is a nucleotide sequence upstream or downstream of the transcription initiation site, and the chain length and region can be selected and identified by evaluating the transcriptional activity and translational activity of the expression unit by an experimental method. ..
  • the nucleotide sequences of SEQ ID NOs: 45 to 47 are 5'-UTRs in the polynucleotide in which the 5'-UTR of mouse-derived Ubc is linked downstream of the mouse-derived Ubc gene promoter region consisting of the polynucleotide of SEQ ID NO: 29. It is a polynucleotide sequence in which the intron contained therein is replaced with the intron contained in the 5'-UTR of the Hspa8, Actb, and Rpsa genes derived from Chinese hamster, respectively.
  • the nucleotide sequence of SEQ ID NO: 48 is a Chinese hamster-derived Hspa8 containing the sequence after the 31st base of the polynucleotide sequence of SEQ ID NO: 6 downstream of the mouse-derived Ubc gene promoter region consisting of the polynucleotide shown in SEQ ID NO: 3. It is a polynucleotide in which the gene 5'-UTR is linked.
  • the combination of the promoter region and 5'-UTR used for each expression unit of the present invention is referred to as "gene name from which the promoter region is derived-5'-the gene name from which the UTR is derived".
  • the expression unit of the present invention sequences the nucleotide sequence corresponding to the intron in any one of the nucleotide sequences set forth in SEQ ID NOs: 18 to 35 and 45 to 48, or the nucleotide sequence set forth in SEQ ID NOs: 18 to 35. 80% or more, preferably 90% or more, more preferably 95% or more, particularly more than 80%, preferably 90% or more, particularly preferably 95% or more, with respect to the nucleotide sequence corresponding to any one of the other introns described in Nos.
  • Such an expression unit of the present invention has transcriptional activity and translational activity.
  • nucleotide sequence corresponding to the intron is represented by SEQ ID NOs: 12 to 17.
  • the combination of the promoter region used for each expression unit and the substituted intron is referred to as "gene name from which the promoter region is derived-gene name from which the intron is derived”.
  • gene name from which the promoter region is derived-gene name from which the intron is derived is referred to as "gene name from which the promoter region is derived-gene name from which the intron is derived”.
  • SEQ ID NO: 29 the nucleotide sequences in which the nucleotide sequence corresponding to the intron is replaced with the nucleotide corresponding to any one of the other introns set forth in SEQ ID NOs: 12 to 17 are shown in SEQ ID NOs: 45 to 47.
  • Such an expression unit of the present invention has transcriptional activity and translational activity.
  • the nucleotide sequence corresponding to the intron is set forth in any one of SEQ ID NOs: 12 to 17.
  • One or more, preferably 1 to 300, more preferably 1 to 30 nucleotides in any one nucleotide sequence selected from the group consisting of the nucleotide sequence corresponding to one other intron and the recombinant nucleotide sequence. May be a mutant polynucleotide consisting of a nucleotide sequence that is deleted, substituted, and / or added.
  • Such an expression unit of the present invention has transcriptional activity and translational activity.
  • the introduction of the nucleotide sequence mutation can be carried out by a method known in the art such as the Kunkel method or the Gapped duplex method, or a method similar thereto, for example, site-specific.
  • Mutagenesis kits for example, Mutant-K (manufactured by Takara Bio Co., Ltd.) or Mutant-G (manufactured by Takara Bio Co., Ltd.), LA PCR in vivo Mutagenesis series kit manufactured by Takara Bio Co., Ltd., etc. can be used.
  • Such mutant polynucleotides can also be used as promoters of the present invention.
  • the foreign gene expression-enhancing activity of the expression unit of the present invention can be tested 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. Comparing the case where the expression unit consisting of the promoter region derived from human EF1 ⁇ (hEF1 ⁇ ) and 5'-UTR is used and the case where the expression unit of the present invention is used, the amount of antibody produced in fed-batch culture is preferably equal to or higher than that. When is increased 1.2 times or more, more preferably 1.5 times or more, it can be determined that the expression unit has a foreign gene expression enhancing activity.
  • the increase is about 1.2 times or more, it is expected that the cell culture scale can be reduced, the culture time, and the purification process can be shortened, and as a result, the yield can be improved and the culture cost can be reduced. If the yield is improved, it becomes possible to stably supply foreign proteins as pharmaceuticals. Moreover, if the culture cost is reduced, the cost of the foreign protein as a medicine is reduced.
  • the foreign gene expression cassette of the present invention (hereinafter, also referred to as “gene expression cassette of the present invention”) is at least 1. It has the expression unit of the present invention described in the above, a foreign gene, and a transcription terminator region (poly A addition signal).
  • poly A addition sequence may be a sequence having an activity of causing transcription termination with respect to transcription from the promoter region, and may be the same as or different from the gene from which the promoter region or 5'-UTR is derived. May be good.
  • DNA element 2 By using the expression cassette of the present invention described in the above in combination with a DNA element, the expression of a foreign gene can be further enhanced.
  • the DNA element used in combination can be obtained by using the interaction with acetylated histone H3 as an index. It is generally said that acetylation of histones (H3, H4) is involved in transcriptional activation, and two main theories are considered. The theory is related to the nucleosome conformational change that the histone tail is acetylated to neutralize the charge and loosen the bond between DNA and histone (Mellor J. (2006) Dynamic nucleosomes and gene transition. Trends). In Genet.
  • Examples of the DNA element used for enhancing the expression of a foreign gene, which is used in combination with the expression unit of the present invention, include A2, A7, and A18.
  • A2 is located on human chromosome 15 80966429-80974878 and is a polynucleotide having an AT content of 62.2% and 8450 bp.
  • the nucleotide sequence of A2 is set forth in SEQ ID NO: 42 of the Sequence Listing.
  • A7 is located on human chromosome 11 88992123 to 8900542, and is a polynucleotide having an AT content of 64.52% and 8420 bp.
  • the nucleotide sequence of A7 is set forth in SEQ ID NO: 43 of the Sequence Listing.
  • A18 is a polynucleotide located on human chromosome 4 1112757976 to 1112844450, having an AT content of 62.54% and 8475 bp.
  • the nucleotide sequence of A18 is set forth in SEQ ID NO: 44 of the Sequence Listing.
  • the foreign gene expression-enhancing activity of a DNA element used in combination with the expression unit of the present invention can be tested 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 two or more copies of one of the DNA elements may be used. Alternatively, two or more types of 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 with respect to the nucleotide sequences shown in SEQ ID NOs: 42 to 44. 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, for example, by using a program such as FASTA or BLAST for the DNA Data Bank of Japan (DNA Databank of JAPAN) or the like.
  • the introduction of a mutation (deletion, substitution, and / or addition) of the polynucleotide can be carried out by a method known in the art such as the Kunkel method or the Gapped duplex method, or a method similar thereto, for example, site-specific.
  • Mutagenesis kits for example, Mutant-K (manufactured by Takara Bio Co., Ltd.) or Mutant-G (manufactured by Takara Bio Co., Ltd.), LA PCR in vivo Mutagenesis series kit manufactured by Takara Bio Co., Ltd., etc. can be used.
  • Such mutant polynucleotides can also be used as the DNA element of the present invention.
  • a polynucleotide containing a foreign gene encoding a foreign protein to be enhanced in production can be obtained by the following general method.
  • a cDNA library derived from a cell or tissue expressing a foreign gene can be isolated by screening using a DNA probe synthesized based on the gene fragment.
  • the mRNA can be prepared by a method 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 an affinity column method using an oligo (dT) cellulose column or poly U-sepharose using Sepharose 2B as a carrier is used.
  • an affinity column method using an oligo (dT) cellulose column or poly U-sepharose using Sepharose 2B as a carrier is used.
  • poly (A) + RNA may be further fractionated by a sucrose density gradient centrifugation method or the like.
  • a single-stranded cDNA was synthesized using an oligo dT primer and a reverse transcriptase, and the single-stranded cDNA was double-stranded using DNA synthase I, DNA ligase, RNase H, or the like.
  • a cDNA library is prepared by smoothing the synthesized double-stranded cDNA with a T4 DNA synthase, ligating an adapter (for example, an EcoRI adapter), phosphorylating, etc., incorporating it into a ⁇ phage such as ⁇ gt11, and packaging it in vivo. Make.
  • a cDNA library can also be prepared using a plasmid vector. Then, a strain having the desired DNA (positive clone) 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.
  • Genomic DNA can be extracted, for example, by the method of Cryer et al. (Methods in Cell Biology, 12, 39-44 (1975)) and P. et al. It can be carried out according to the method of Philippesen et al. (Methods Enzymol., 194, 169-182 (1991)).
  • the acquisition of the polynucleotide containing the target promoter region, 5'-UTR, intron, DNA element or foreign gene can also be performed by, for example, the PCR method (PCR Technology. Henry A. Erlich, Attackton press (1989)).
  • PCR method PCR Technology. Henry A. Erlich, Attackton press (1989)
  • 20 to 30 mer synthetic single-stranded DNA is used as a primer
  • 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.
  • a gene library is prepared by a conventional method, (b) a desired polynucleotide is selected from the prepared gene library, and the polynucleotide is selected. It can be done by amplifying.
  • chromosomal DNA obtained by a conventional method from the cell line of the organism to be collected is partially digested and fragmented with an appropriate restriction enzyme, and the obtained fragment is ligated to an appropriate vector, and the vector is appropriately used. It can be prepared by introducing it into a suitable host.
  • It can also be prepared by extracting mRNA from cells, synthesizing cDNA from this, ligating it to an appropriate vector, and introducing the vector into an appropriate host.
  • a vector used at this time a plasmid known as a commonly known vector for preparing a gene library can be used, and a phage vector, cosmid, or the like can also be widely used.
  • a host for transformation or transduction a host corresponding to the type of the vector may be used.
  • a polynucleotide containing a foreign gene is selected from the gene library by a colony hybridization method, a plaque hybridization method, or the like using a labeled probe containing a sequence peculiar to the foreign gene.
  • a method of preparing two pairs of complementary oligonucleotides and annealing them a method of linking several annealed DNAs with DNA ligase, or a method of preparing several partially complementary oligonucleotides and performing PCR.
  • Genes can be synthesized by a method of filling the gap or the like.
  • the polynucleotide sequence can be determined by a usual method, for example, the dideoxy method (Sanger et al., Proc. Natl. Acad. Sci., USA, 74, 5463-5467 (1977)) or the like. 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 above 1. 2. The expression unit according to the above 2. A vector containing the foreign gene expression cassette described in the above is provided.
  • the foreign gene expression vector of the present invention is described in 3. above.
  • One of the DNA elements described in the above, one type of DNA element may include two or more copies, and a combination of two or more types of DNA elements.
  • the DNA element When the foreign gene is expressed in the host cell by the foreign gene expression vector, the DNA element may be placed immediately before or after the gene expression cassette, or may be placed at a position away from the gene expression cassette. good.
  • the orientation of the DNA element may be either forward or reverse with respect to the gene expression cassette.
  • the foreign gene is not particularly limited, but is useful for pharmaceutical purposes, such as secretory alkaline phosphatase (SEAP), green fluorescent protein (GFP), reporter genes such as luciferase, various enzyme genes such as ⁇ -amylase gene and ⁇ -galactosidase gene.
  • Various interferon genes such as interferon ⁇ and interferon ⁇ , various interleukin genes such as IL1 and IL2, various cytokine genes such as erythropoetin (EPO) gene and granulocyte colony stimulating factor (G-CSF) gene, which are physiologically active proteins. Examples thereof include a growth factor gene or a gene encoding a multimeric protein, for example, a gene encoding an antibody or a heteromultimer which is an antigen-binding fragment thereof. These genes may be obtained by any method.
  • the "antigen-binding fragment of an antibody” means a partial fragment of an antibody having an antigen-binding activity, and is a Fab, F (ab') 2, Fv, scFv, diabody, linear antibody, and antibody fragment. Includes more specific multispecific antibodies and the like.
  • Fab' which is a monovalent fragment of the variable region of the antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the antigen-binding fragment of the antibody.
  • 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 proteins produced in an appropriate host cell using a genetically modified antibody gene. included.
  • the foreign gene expression vector of the present invention can include a selection marker for selecting a transformant.
  • a selection marker for selecting a transformant for example, by using a drug resistance marker that imparts resistance to drugs such as cerulenin, aureobasidin, zeocin, canavanin, cycloheximide, hygromycin, puromycin, blastocidin, tetracycline, kanamycin, ampicillin, neomycin, etc. It is possible to select transformants. Further, it is also possible to select a transformant by using a gene that imparts solvent resistance to ethanol or the like, osmotic pressure resistance to glycerol, salts or the like, metal ion resistance to copper or the like as a marker.
  • the foreign gene expression vector of the present invention may be a vector that is not incorporated into chromosomal DNA.
  • a foreign gene expression vector is randomly integrated into a chromosome after being introduced into a host cell, but is a component derived from a mammalian virus such as simian virus 40 (SV40), papilomavirus (BPV, HPV), and EBV.
  • SV40 simian virus 40
  • BPV papilomavirus
  • EBV papilomavirus
  • a vector having a sequence encoding an SV40-derived replication origin and a trans-acting factor SV40 large T antigen a vector having a sequence encoding EBV-derived oriP and EBNA-1, and the like are widely used.
  • the effect of the DNA element can show the activity of enhancing the expression of foreign genes regardless of the type of vector or the presence or absence of integration into the chromosome.
  • Transformed cells The transformed cells of the present invention are described in 5. above. It is a transformed cell introduced by using the foreign gene expression vector of.
  • the host cell to be transformed is a eukaryotic cell, preferably a mammalian cell, more preferably a human, mouse, rat, hamster, monkey, or bovine-derived cell.
  • mammalian cells include, but are not limited to, COS-1 cells, HEK293 cells, CHO cells (CHO-K1, DG44, CHO dhfr-, CHO-S) and the like.
  • the method for introducing an expression vector into a host cell may be any method as long as the transgene is stably present in the host and can be appropriately expressed, and is generally used.
  • Methods such as, for example, calcium phosphate method (Ito et al., (1984) Vector Biol. Chem., 48, 341), electroporation method (Becker, DM et al. (1990) Methods. Enzymol., 194,182-187), spheroplast method (Cregch et al., Mol. Cell. Biol., 5,3376 (1985)), lithium acetate method (Itoh, H. (1983) J. Vector. 153, 163). -168), the lipofection method and the like can be mentioned.
  • the method for producing a foreign protein of the present invention is described in 6.
  • the transformed cells described in the above item can be cultured by a known method, collected from the culture, and purified.
  • the “culture” means not only a culture supernatant but also a cultured cell or a crushed cell product.
  • As the foreign protein that can be produced using the transformed cells described in the above item not only a monomeric protein but also a multimeric protein can be selected.
  • a plurality of genes encoding these subunits should be used for each of the genes. It is necessary to introduce into the host cell described in the item of.
  • the method for culturing transformed cells can be carried out according to the usual method used for culturing the host cells.
  • the transformed cells are mammalian cells, they are cultured under the conditions of, for example, 37 ° C., 5% or 8% CO 2 , and the culture time is about 24 to 1000 hours.
  • the culture is allowed to stand, shake, stir, and aerate. It can be carried out by the lower batch culture, fed-batch culture, perfusion culture, continuous culture and the like.
  • Confirmation of the expression product of the foreign protein gene from the above culture (culture solution) can be performed by SDS-PAGE, Western blot analysis, ELISA or the like.
  • An antibody protein can be mentioned as a heteromultimeric protein produced by using the production method described in the above item.
  • the antibody protein is a tetrameric protein consisting of two molecules of heavy chain polypeptide and two molecules of light chain polypeptide. Therefore, in order to obtain the antibody protein in a form that maintains the antigen-binding ability, the above 6.
  • both heavy chain and light chain genes need to be introduced.
  • the heavy and light chain gene expression cassettes may be present on the same expression vector or on different expression vectors.
  • Examples of the antibody produced in the present invention include an antibody produced by immunizing an experimental animal such as a rabbit, mouse, or rat with a desired antigen. Further, a chimeric antibody using the above antibody as a raw material and a humanized antibody can also be mentioned as the antibody produced in the present invention. Furthermore, the human antibody obtained by the genetically modified animal or the phage display method is also an antibody produced in the present invention.
  • a specific polynucleotide is used as long as the combination of the heavy chain polypeptide and the light chain polypeptide transcribed and translated from the antibody gene retains the activity of binding to an arbitrary antigen protein. It is not limited to the antibody gene having the sequence.
  • the antibody gene it is not always necessary to encode the full-length molecule of the antibody, and a gene encoding an antigen-binding fragment of the antibody can be used.
  • the gene encoding these antigen-binding fragments can be obtained by genetically engineering the gene encoding the full-length molecule of the antibody protein.
  • foreign protein production methods examples include various proteins derived from humans or non-human animals, antigen-binding fragments thereof, variants thereof, and the like, in addition to the above-mentioned antibodies. Can be done. Examples of such proteins include atrial natriuretic peptide (ANP), cerebral natriuretic peptide (BNP), C-type natriuretic peptide (CNP), vasopressin, somatostatin, growth hormone (GH), insulin, oxytocin, grelin, etc.
  • ADP atrial natriuretic peptide
  • BNP cerebral natriuretic peptide
  • CNP C-type natriuretic peptide
  • vasopressin vasopressin
  • somatostatin growth hormone
  • GH growth hormone
  • insulin oxytocin
  • grelin etc.
  • Peptide hormones such as leptin, adiponectin, renin, calcitonin, osteoprotegerin, insulin-like growth factor (IGF), interleukin, chemokine, interferon, tumor necrosis factor (TNF ⁇ / ⁇ and other TNF superfamily), nerve growth factor (NGF) ), Cell growth factors (EGF, FGF, PDGF, HGF, TGF, etc.), hematopoietic factors (CSF, G-CSF, erythropoetin, etc.), cytokines such as adipokine, receptors such as ⁇ NF receptor, lysoteam, protease, proteinase, Examples include, but are limited to, enzymes such as peptidases, functional fragments thereof (fragments that retain some or all of the biological activity of the original protein), fusion proteins that include those proteins, and the like. It's not something.
  • 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. Also well known to those skilled in the art are operations used for cloning DNA, determining nucleotide sequences, transforming host cells, culturing transformed cells, collecting proteins from the resulting cultures, purifying, etc. It is something that can be known from the literature.
  • PCR amplification was performed using the primer set shown in Table 1 and PrimeSTAR Max DNA Polymerase (Takara Bio) or KOD FX Neo (Toyobo), and purification was performed with QIAquick PCR Purification kit (QIAGEN) to obtain the desired nucleotide sequence.
  • the polynucleotide shown in SEQ ID NO: 39 was PCR-amplified to obtain a polynucleotide that is the 5'-UTR of the Hspa8 gene.
  • the polynucleotide set forth in SEQ ID NO: 29 was used as an expression unit to obtain a promoter region of a mouse-derived Ubc gene and a polynucleotide having a 5'-UTR sequence.
  • Example 2 Evaluation by feeding culture of an expression unit using the antibody expression level as an index (2-1) Construction of antibody expression vector
  • the humanized antibody gene Y expression described in Patent Document 5 is used for construction of the antibody expression vector.
  • the vector pDSLH3.1-Hspa5-Y was used.
  • An antibody expression vector was constructed by substituting the region of the nucleotide sequence corresponding to Hspa5 in pDSLH3.1-Hspa5-Y with the nucleotide sequence set forth in SEQ ID NOs: 18-22, 24-26, 29.
  • a schematic diagram of the vector is shown in FIG.
  • pDSLH3.1-Hspa5-Y and pDSLH3.1-hEF1 ⁇ -Y those described in Patent Document 5 were used.
  • (2-2) Preparation of humanized antibody Y expression stable pool CHO-K1 cells (ATCC) are acclimated so that they can be cultured in a suspended state using a serum-free medium, and host cell CHO-O1 cells are prepared. Obtained.
  • the antibody expression vector constructed in (2-1) was introduced into CHO-O1 cells using a gene transfer device Neon Transfection System (manufactured by Thermo Fisher Scientific), and 5% CO was introduced in a T-25 flask. 2 , 37 ° C. was cultured.
  • FIGS. 4A and 4B Changes in the number of viable cells and antibody production in the stable pool are shown in FIGS. 4A and 4B, respectively.
  • the amount of antibody produced by each expression unit on the 14th day of culture was 2.2 times higher for Hspa5-Hspa8 than for the expression unit (Hspa5-Hspa5) composed of the promoter region of the Hspa5 gene and 5'-UTR, respectively.
  • Hspa5-Actb 1.9 times for Hspa5-Actb
  • Hspa5-Rpsa 1.3 times for Hspa5-Rpsa
  • Hspa5-chUbc and Hspa5-EF1 ⁇ respectively.
  • FIGS. 5A and 5B the changes in the number of viable cells and the amount of antibody produced in the stable pool into which the expression unit consisting of the promoter of the mUbc gene and each 5'-UTR was introduced are shown in FIGS. 5A and 5B, respectively.
  • the antibody production in each expression unit on the 14th day of culture reached 1.6 times for mUbc-Hspa8, 2.1 times for mUbc-Actb, and 1.1 times for mUbc-Rpsa with respect to mUbc-mUbc. .. This reached a value of about 4.6 to 8.1 times that of hEF1 ⁇ -hEF1 ⁇ , which greatly exceeded the amount of antibody produced by the promoters currently frequently used.
  • Example 3 Examination of mouse-derived Ubc promoter region using the amount of luciferase luminescence in transient expression as an index (3-1) Construction of luciferase expression vector The luciferase expression vector is arranged in pGL4.10 (manufactured by Promega). The nucleotide sequence of No. 29 or 35 was inserted and constructed. (3-2) Evaluation of luciferase luminescence amount in transient expression Using Lipofectamine2000 CD (manufactured by Thermo Fisher Scientific Co., Ltd.) in CHO-O1 cells, the luciferase expression vector constructed in (3-1) was controlled. The gene was introduced at the same time as the vector pGL4.74.
  • luciferase luminescence was measured using a Dual-Luciferase Reporter Assay System (manufactured by Puromega).
  • the promoter activity was defined as the value obtained by dividing the luminescence amount of Firefly-derived luciferase on pGL4.10 by the luminescence amount of Renilla-derived luciferase on pGL4.74 (FIG. 6).
  • a region having a total length of about 2.1 kb upstream of TATA-box and having a total length of about 2.1 kb was obtained with a promoter activity equivalent to the total length of the promoter region of the mUbc gene, and the promoter region of the mUbc gene exhibited transcriptional activity. It was inferred that a region of 2.1 kb or more upstream of the start codon from the transcription start point was required to show.
  • Example 4 Examination by infusion culture of promoter region chain length of Hspa5 gene using antibody expression level as an index (4-1) Construction of antibody expression vector and preparation of humanized antibody Y expression stable pool
  • Example (2) -1) A human having an expression unit in which the promoter region of the Hspa5 gene contained in Hspa5-Hspa8 is contained from the transcription start site to 0, 8, 16, 24, 32, 40, 48 nucleotides downstream by the same method as described in -1). An antibody Y expression vector was constructed. Then, the gene was introduced into CHO-O1 cells by the same method as described in Example (2-2) to prepare a humanized antibody Y expression stable pool.
  • Example 5 Examination of the combined effect of the expression unit and A7 using the antibody expression level in the feed culture as an index (5-1) Construction of the antibody expression vector (2-1) The expression cassette of the antibody expression vector constructed in (2-1). An antibody expression vector in which the DNA element A7 was inserted was constructed upstream of the above. (5-2) Preparation of Humanized Antibody Y Expression Stable Pool The region of the nucleotide sequence corresponding to Hspa5 in pDSLH3.1-Hspa5-Y that does not contain the DNA element A7 constructed in (2-1) is designated by SEQ ID NO: 24.
  • An antibody expression vector containing the antibody expression vector substituted with the nucleotide sequence described in ⁇ 26, 29 and 45 to 48, and the DNA element A7 constructed in (5-1) was prepared by the method described in (2-2) for CHO-O1.
  • the cells were transfected and selectively cultured to prepare a humanized antibody Y expression stable pool.
  • (5-3) Evaluation of antibody production by fed-batch culture of humanized antibody Y-expressing stable pool Using the humanized antibody Y-expressing stable pool prepared in (5-2), an example was used in a 125 mL Erlenmeyer flask (5-3). Fed-batch culture was carried out by the same method as described in 2-3). The changes in the number of living cells and the amount of antibody produced are shown in FIGS. 8A and 8B, respectively.
  • the amount of antibody produced by the antibody expression vector containing A7 in each gene expression unit at the 14th day of culture was 1.3 to 2.1 times higher than that of the antibody expression vector containing no A7. Therefore, it was found that high production can be effectively achieved by the synergistic effect by using the expression unit consisting of the DNA element A7, the promoter of the mUbc gene, and each 5'-UTR in combination. Since it was suggested that the expression level of DNA element A7 was improved in all the expression units, it is presumed that the same synergistic effect can be realized when the promoter of the Hspa5 gene and the expression unit consisting of each 5'-UTR are combined.
  • SEQ ID NO: 1 Promoter region of Chinese hamster-derived Hspa5
  • SEQ ID NO: 2 Promoter region of mouse-derived Ubc
  • SEQ ID NO: 3 Promoter region of mouse-derived Ubc Corresponds to the transcription initiation site from about 2.1 kbp of nucleotide upstream of the Ubc start codon.
  • SEQ ID NO: 4 Nucleotide sequence that can be added to the 3'side of the promoter region of Hspa5
  • SEQ ID NO: 5 Nucleotide sequence in which 32 bases are linked to the 3'side of the promoter region of Hspa5.
  • SEQ ID NO: 6 5'-UTR of Hspa8 derived from Chinese hamster SEQ ID NO: 7: Chinese Hamster-derived Actb 5'-UTR SEQ ID NO: 8: Chinese hamster-derived Rpsa 5'-UTR SEQ ID NO: 9: Chinese hamster-derived Ubc 5'-UTR SEQ ID NO: 10: 5'-UTR of EF1 ⁇ derived from Chinese hamster SEQ ID NO: 11: Mouse-derived Ubc 5'-UTR SEQ ID NO: 12: Intron contained in 5'-UTR of Chinese hamster-derived Hspa8 SEQ ID NO: 13: Intron contained in 5'-UTR of Chinese hamster-derived Actb SEQ ID NO: 14: Included in 5'-UTR of Chinese hamster-derived Rpsa Intron SEQ ID NO: 15: Included in 5'-UTR of Chinese hamster-derived Ubc Intron SEQ ID NO: 16: Included in 5'-UTR of Chinese hamster-derived
  • Expression unit SEQ ID NO: 32 composed of 5'-UTR of Chinese hamster-derived Actb: Expression unit SEQ ID NO: 33 composed of 5'-UTR of Chinese hamster-derived Rpsa and promoter region of mouse-derived Ubc: Promoter of mouse-derived Ubc Expression unit SEQ ID NO: 34 composed of region and Chinese hamster-derived Ubc 5'-UTR: Mouse-derived Ubc promoter region and Chinese hamster-derived EF1 ⁇ 5'-UTR expression unit SEQ ID NO: 35: Mouse-derived Ubc Expression unit consisting of the promoter region of Ubc and the 5'-UTR of the mouse-derived Ubc SEQ ID NO: 36: Substance sequence that can be added to the 3'side of the promoter region of Hspa5 SEQ ID NO: 37: Primer of Hspa8 Hspa8-NotI-F SEQ ID NO: 38: Primer of Hspa8 Hspa8-XbaI-R SEQ ID NO: 39: Nucleotide sequence upstream
  • Expression unit consisting of a nucleotide sequence substituted for the intron region contained in the 5'-UTR of the hamster-derived Actb SEQ ID NO: 47: The promoter region of the mouse-derived Ubc and the intron region in the mouse-derived Ubc5'-UTR are defined as the Chinese hamster-derived Rpsa.
  • Expression unit composed of a nucleotide sequence substituted with the intron region contained in the 5'-UTR of the sequence No. 48 Expression unit composed of the mouse-derived Ubc promoter region and the Chinese hamster-derived Hspa8 5'-UTR.

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Abstract

La présente invention concerne un moyen pour renouveler la production d'une protéine étrangère qui servira de préparation médicinale protéique, dans des cellules hôtes telles que des cellules de culture dérivées d'un mammifère.　L'invention concerne une nouvelle unité d'expression qui est utilisée pour favoriser l'expression d'une protéine étrangère, des cellules transformées qui comprennent l'unité d'expression, et un procédé de production sécrétoire élevée d'une protéine étrangère à l'aide desdites cellules hôtes transformées.
PCT/JP2021/014287 2020-04-03 2021-04-02 Nouvelle unité d'expression génique WO2021201268A1 (fr)

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JP2010539946A (ja) * 2007-09-26 2010-12-24 イントレキソン コーポレーション 合成５’ｕｔｒ、発現ベクター、および導入遺伝子の発現を増加させる方法
JP2014502159A (ja) * 2010-11-30 2014-01-30 エルジー ライフ サイエンシーズ リミテッド 新たなハイブリッドプロモーター及びそれを含む組換えベクター
WO2018066492A1 (fr) * 2016-10-03 2018-04-12 第一三共株式会社 PROMOTEUR DU GÈNE Hspa5
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WO2010140387A1 (fr) * 2009-06-05 2010-12-09 東洋紡績株式会社 Vecteur d'expression pour établir des cellules hyperproductrices et cellules hyperproductrices
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