WO2021201268A1 - Novel gene expression unit - Google Patents

Abstract

The present invention provides a means for renewing the production of a foreign protein, which will serve as a protein drug, in host cells such as cultured cells from a mammal.　Provided are a novel expression unit that is used in promoting the expression of a foreign protein, transformed cells that include the expression unit, and a method for high secretory production of a foreign protein using said transformed host cells.

Description

New gene expression unit

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.

With the development of gene recombination technology, protein drugs such as therapeutic proteins and antibody drugs are rapidly expanding their markets. Among them, 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.

As a host for producing protein drugs represented by antibody drugs, it has a sugar chain structure similar to that of humans, can be modified after translation, and in consideration of safety, Chinese Hamster Ovaly (CHO: Chinese hamster). Mammalian cultured cells such as ovary) cells are currently the mainstream.

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).

In messenger RNA (mRNA) that has gene / protein information, 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. In eukaryotes, including cultured mammalian cells, 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).
As improvements to the mammalian cultured cell expression system aimed at improving production capacity, 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. Examples of inserting a promoter of a shock protein A5 (Hspa5 / GRP78) gene (Patent Document 5) and a foreign intron into or downstream of a structural gene have been reported (Non-Patent Document 7).

Patent No. 3051411 WO2006 / 123097 WO2005 / 00788 US92342111 WO2018 / 066492

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. By finding an expression unit that can obtain the protein expression level, it is possible to provide a means for the cultured mammalian cells to stably achieve high expression of the foreign gene, and to improve the production amount of the proteinaceous drug in the cultured mammalian cell expression system. That is, it is possible to provide a means that contributes to the reduction of manufacturing cost.

As a result of intensive studies to solve the above problems, 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. We have found that it is possible to significantly improve the productivity of foreign proteins to be expressed in cultured mammalian cells by using an expression unit composed of a polynucleotide substituted with an intron, and completed the present invention. That is, 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.
(2) The polynucleotide according to (1) above, wherein the intron contained in the 5'-UTR is an intron contained in the 5'-UTR contained in the same or different gene as the gene containing the 5'-UTR.
(3) The polynucleotide according to (1) or (2) above, wherein the promoter region is the polynucleotide according to any one of SEQ ID NOs: 1 to 3.
(4) The above-mentioned (1) or (2), wherein the promoter region is the polynucleotide according to SEQ ID NO: 5, wherein the polynucleotide according to SEQ ID NO: 4 is linked to the 3'end of the polynureotide set forth in SEQ ID NO: 1. The polynucleotide described in.
(5) The polynucleotide according to (4) above, wherein the polynucleotide according to SEQ ID NO: 4 is lacking in the entire length or a part thereof.
(6) The polynucleotide according to (1) or (2) above, wherein the promoter region is formed by linking all or part of the polynucleotide set forth in SEQ ID NO: 36 to the 3'end of the polynureotide set forth in SEQ ID NO: 1. ..
(7) The polynucleotide according to any one of (1) to (6) above, wherein 5'-UTR is the polynucleotide according to any one of SEQ ID NOs: 6 to 11.
(8) The polynucleotide according to any one of (2) to (7) above, wherein the intron is the polynucleotide according to any one of SEQ ID NOs: 12 to 17.
(9) The polynucleotide according to (1) above, which comprises the polynucleotide according to any one of SEQ ID NOs: 18 to 35 and 45 to 48.
(10) The polynucleotide according to (1) above, which comprises the polynucleotide according to any one of SEQ ID NOs: 18, 24 to 29, and 45 to 48.
(11) A polynucleotide having 95% or more identity with respect to the polynucleotide according to (9) or (10) above, and having transcriptional activity and translational activity.
(12) A polynucleotide having 99% or more identity with respect to the polynucleotide according to (9) or (10) above, and having transcriptional activity and translational activity.
(13) A polynucleotide that hybridizes with a polynucleotide complementary to the polynucleotide according to (9) or (10) above under stringent conditions, and has transcriptional activity and translational activity.
(14) An expression unit composed of the polynucleotide according to any one of (1) to (13) above.
(15) A foreign gene expression cassette containing the expression unit according to (14) above.
(16) The foreign gene expression cassette according to (15) above, wherein the foreign gene is a gene encoding a multimeric protein.
(17) The foreign gene expression cassette according to (15) above, wherein the foreign gene is a gene encoding a heteromultimeric protein.
(18) The foreign gene expression cassette according to (15) above, wherein the foreign gene is a gene encoding an antibody or an antigen-binding fragment thereof.
(19) A foreign gene expression vector containing the foreign gene expression cassette according to any one of (15) to (18) above.
(20) A transformed cell into which the foreign gene expression vector according to (19) has been introduced.
(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.
(23) 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.

Schematic diagram of an expression unit (Expression Unit) composed of a promoter region and 5'-UTR. In the figure, TSS indicates a transcription start site (Transcription Start Site). The schematic diagram which performed the intron substitution for the expression unit. Schematic diagram of a humanized antibody gene Y expression vector (pDSLH3.1-Expression Unit-Y) composed of an antibody H chain and an L chain gene using an expression unit. The antibody production amount expressed by the expression unit containing the Hspa5 promoter region in the feeding culture using the humanized antibody Y expression stable pool is composed of the promoter region of the human EF1α (hEF1α) gene and the 5'-UTR. The figure which compared with the expression unit. FIG. 4A shows the number of viable cells on each sampling day. In the feeding culture using the humanized antibody Y expression stable pool, the amount of antibody produced by the expression unit containing the Hspa5 promoter region was expressed by the expression unit composed of the promoter region of the human EF1α gene and the 5'-UTR. Comparison figure. FIG. 4B shows the production volume on each sampling day. In fed-batch culture using a humanized antibody Y expression stable pool, 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 EF1α gene and the 5'-UTR. Diagram compared to the unit. FIG. 5A shows the number of viable cells on each sampling day. In fed-batch culture using a humanized antibody Y expression stable pool, 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 EF1α gene and the 5'-UTR. Diagram compared to the unit. 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 figure which compared the expression level of firefly luciferase with the human EF1α gene promoter. In the feed culture using the humanized antibody Y expression stable pool, 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 EF1α 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. In the feed culture using the humanized antibody Y expression stable pool, 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 EF1α 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. 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. The figure which compared the antibody production amount by the pouring culture. 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. The figure which compared the antibody production amount by the pouring culture. FIG. 8B shows the production volume on each sampling day.

Hereinafter, the present invention will be specifically described.

As used herein, the term "promoter region" means a sequence having transcriptional activity in the sequence upstream of the transcription initiation site. Also referred to herein as a "promoter."

In the present specification, "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.

In the present specification, "upstream" refers to the 5'terminal side of a gene, sequence region, or base, and "downstream" refers to the 3'terminal side of a gene, sequence region, or base.

As used herein, 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.

In the present specification, 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.

In the present specification, "polynucleotide" is used in the same meaning as nucleic acid, and includes DNA, RNA, probe, oligonucleotide, and primer. In the present specification, for example, when the SEQ ID NO: α is α, [a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: α] is abbreviated as [the polynucleotide described in SEQ ID NO: α].

In this specification, "polypeptide" and "protein" are used without distinction.

In the present specification, "gene expression" means a phenomenon in which a certain gene is transcribed into mRNA and a protein is translated from the mRNA.

In the present specification, the "foreign gene" means a gene artificially introduced into a host cell.

In the present specification, "foreign protein" means a protein encoded by a foreign gene.

As used herein, the term "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.

As used herein, the term "transcriptional activity" refers to the activity of initiating transcription from DNA by RNA polymerase and synthesizing mRNA.

As used herein, the term "translational activity" refers to the activity of synthesizing a protein from transcribed mRNA.
As used herein, the term "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. ..

In the present specification, 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.

As used herein, "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.

In the present specification, "hybridize under stringent conditions" means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, 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. More specifically, in a commercially available hybridization solution ExpressionHyb Hybridization Solution (manufactured by Clontech), hybridizing at 68 ° C., or using a DNA-fixed filter in the presence of 0.7 to 1.0 M NaCl. After hybridization at 68 ° C., use a 0.1 to 2-fold concentration SSC solution (1-fold concentration SSC consists of 150 mM NaCl and 15 mM sodium citrate), and wash at 68 ° C. It means hybridizing under the same conditions.

1. 1. Expression unit used to enhance the expression of a foreign gene 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.

Further, the 5'-UTR contains an intron, and 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. ..

Preferred examples of 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.

As a preferable example of the 5'-UTR contained in the expression unit of the present invention, the 5'-UTR derived from Chinese hamster as the 5'-UTR of the Hspa8 gene, Actb gene, Rpsa gene, and EF1α 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. Of these, 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. Of these, 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. Of these, 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. Of these, 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 EF1α gene and the nucleotide immediately preceding the nucleotide sequence corresponding to the start codon. Of these, 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. Of these, 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. As the intron, preferably, 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. For example, 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. Among these, 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 EF1α 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, EF1α 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, EF1α 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. When the Chinese hamster-derived Hspa8 gene, Actb gene, Rpsa gene, Ubc gene, EF1α 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. 12 to 17 and the recombinant nucleotide sequence. It may be preferably a polynucleotide consisting of a nucleotide sequence having 99% or more identity. Such an expression unit of the present invention has transcriptional activity and translational activity.

In the expression unit of the present invention, among the nucleotide sequences set forth in SEQ ID NOs: 18 to 35 and 45 to 48 and the nucleotide sequences set forth in SEQ ID NOs: 18 to 35, the nucleotide sequence corresponding to the intron is represented by SEQ ID NOs: 12 to 17. Consisting with a nucleotide sequence complementary to a nucleotide consisting of any one nucleotide sequence selected from the group consisting of a nucleotide sequence corresponding to any one of the other introns described in the above and a nucleotide sequence to be recombined, and a string. It may be a polynucleotide that hybridizes under gentle conditions.

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". Among the nucleotide sequences set forth in 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.

In the expression unit of the present invention, among the nucleotide sequences set forth in SEQ ID NOs: 18 to 35 and 45 to 48, and the nucleotide sequences set forth in SEQ ID NOs: 18 to 35, 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 (deletion, substitution, and / or addition) 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 EF1α (hEF1α) 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. Even if 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.

2. Foreign gene expression cassette 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).

Further, the 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.

3. 3. 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. 22 (6): 320-329) and the theory that it is involved in the recruitment of various transcription factors (Nakatani Y. (2001) Histone acetylases-verstile players. Genes Cells. 6 (2): 79-86). be. In both theories, histone acetylation is likely to be involved in transcriptional activation and interacts with acetylated histone H3 by chromatin immunoprecipitation (ChIP) with anti-acetylated histone H3 antibodies. It is possible to concentrate the working DNA elements.

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.

When used in combination with the expression unit of the present invention, 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.

If you are a person skilled in the art, please refer to Molecular Cloning (Sambrook, J. et al., Molecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory, Plainview, etc. Such homologous genes of DNA elements can be easily obtained. In addition, the identity of the above-mentioned nucleotide sequence can be similarly determined by FASTA search or BLAST search.

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.

4. Acquisition of polynucleotide In the present invention, a polynucleotide containing a foreign gene encoding a foreign protein to be enhanced in production, which will be described later, can be obtained by the following general method. For example, 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. For example, 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. , Or the batch method to obtain poly (A) + RNA (mRNA). Further, poly (A) + RNA may be further fractionated by a sucrose density gradient centrifugation method or the like. Next, using the obtained mRNA as a template, 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. Synthesize cDNA. 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 λ phage such as λgt11, and packaging it in vivo. Make. In addition to λ phage, 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.

In addition, when isolating the expression unit used for protein production, the polynucleotide containing the terminator region, and the polynucleotide containing the DNA element or foreign gene from genomic DNA, a general method (Molecular Cloning (1989), Methods in). According to Energy 194 (1991)), 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)). For the amplification of polynucleotides using the PCR method, 20 to 30 mer synthetic single-stranded DNA is used as a primer, and genomic DNA is used as a template. The amplified gene is used after confirming the polynucleotide sequence. As a PCR template, a genomic DNA library such as a bacterial artificial chromosome (BAC) can be used.

On the other hand, in order to obtain a polynucleotide containing a foreign gene having an unknown sequence, (a) 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. In the gene library, 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. As the 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. As the 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.

It is also possible to chemically totally synthesize a polynucleotide containing a foreign gene. For example, 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.

5. Foreign gene expression vector 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. 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. Moreover, you may use one foreign gene expression vector containing a plurality of DNA elements. 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 α-amylase gene and α-galactosidase gene. Various interferon genes such as interferon α and interferon γ, 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. In addition, 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. However, it is not limited to these molecules as long as it has the ability to bind to an antigen. In addition, 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.

Further, the foreign gene expression vector of the present invention can include 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. Generally, 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. By using, it can be used as an expression vector capable of self-renewal in the introduced host cell. For example, 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.

6. 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. Examples of 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.

In the present invention, 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.

7. Method for producing foreign protein 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. In addition, 6. 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. When producing a heteromultimeric protein composed of a plurality of different subunits, 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.

When 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.

8. Method for producing antibody protein 7. 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. In the transformed cells described in the above item, both heavy chain and light chain genes need to be introduced. In this case, 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.

As an antibody gene used for antibody production, 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.

Further, as 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.

9. Other foreign protein production methods Examples of the foreign proteins to be produced by the production method of the present invention 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. Peptide hormones such as leptin, adiponectin, renin, calcitonin, osteoprotegerin, insulin-like growth factor (IGF), interleukin, chemokine, interferon, tumor necrosis factor (TNFα / β 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 ТNF 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.

Hereinafter, the present invention will be specifically described with reference to Examples. However, these examples do not limit the technical scope of the present invention in any way. 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.

(Example 1) Cloning of promoter region and 5'-UTR Cloning of the promoter region of Chinese hamster-derived Hspa5 gene, Actb gene, Rpa gene, Ubc gene, EF1α gene and 5'-UTR is described in Patent Document 5. It can be done according to the same procedure.

For the 5'-UTR of the Chinese hamster-derived Hspa8 gene and the promoter region and 5'-UTR of the mouse-derived Ubc gene, the database shown in Table 1 was referred to. 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. As pDSLH3.1-Hspa5-Y and pDSLH3.1-hEF1α-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. One day after gene transfer, Geneticin (manufactured by Thermo Fisher Scientific Co., Ltd.) was added at a final concentration of 800 μg / mL, and drug-selective culture was carried out for 1 week. Then, the cells were cultured in a 125 mL Erlenmeyer flask at 5% CO ₂ and 37 ° C. to prepare a humanized antibody Y expression stable pool.
(2-3) Evaluation of antibody production by fed-batch culture of humanized antibody Y expression stable pool Using the humanized antibody Y expression stable pool prepared in (2-2), flow in a 125 mL Erlenmeyer flask. Fed-batch was performed. G13 (custom medium manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was used as the basal medium, and F13 (custom medium manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was used as the feed medium.

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. , 1.9 times for Hspa5-Actb, 1.3 times for Hspa5-Rpsa, and 2.0 times for Hspa5-chUbc and Hspa5-EF1α, respectively. This is about 3.9 to 6.7 times higher than the expression unit (hEF1α-hEF1α) composed of the promoter region of the human EF1α gene and 5'-UTR, and is currently frequently used. It greatly exceeded the amount of antibody produced by the promoter.

In addition, 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 hEF1α-hEF1α, 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. Twenty-four hours after gene transfer, cells were centrifuged (1000 rpm, 3 min), collected in 1.5 mL tubes, and washed with PBS buffer. Then, the amount of 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). As a result of evaluating luciferase luminescence, 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.
(4-2) Evaluation of antibody production by fed-batch culture of humanized antibody Y-expressing stable pool The stable pool prepared in (4-1) was flowed by the same method as described in Example (2-3). The amount of antibody produced was evaluated by culturing. Changes in the number of viable cells and antibody production in the prepared stable pool are shown in FIGS. 7A and 7B, respectively. Regardless of the chain length of the promoter region of the Hspa5 gene contained in the expression unit, the amount of antibody produced is compared with the case of using the expression unit (Hspa5-Hspa5) composed of the promoter region of the Hspa5 gene and 5'-UTR. It reached about 1.7 to 2.3 times. This corresponds to about 3.2 to 4.4 times that when the expression unit (hEF1α-hEF1α) composed of the promoter region of the human EF1α gene and 5'-UTR is used, and is a promoter that is frequently used at present. The amount of antibody produced in Japan was significantly exceeded. In addition, from the above results, it was shown that the antibody expression level can be maintained even if a sequence from the transcription start point of the promoter region of the Hspa5 gene to +0 to +48 nucleotides downstream is linked to the expression unit, and it is constant after the transcription start point. It was suggested that nucleotide sequences could be linked.

(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.

Improving the productivity of foreign genes such as therapeutic proteins and antibodies by introducing the foreign gene expression cassette or the foreign gene expression vector of the present invention constructed using the expression unit of the present invention into mammalian host cells. Is possible.

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. Nucleotide sequence consisting up to the nucleotide sequence 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 EF1α 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 EF1α Intron SEQ ID NO: 17: Included in 5'-UTR of mouse-derived Ubc Intron SEQ ID NO: 18: Expression unit composed of Chinese hamster-derived Hspa5 promoter region and Chinese hamster-derived Hspa8 5'-UTR SEQ ID NO: 19: Chinese hamster-derived Hspa5 promoter region and Chinese hamster-derived Actb 5'-UTR Expression unit SEQ ID NO: 20: Promoted region of Chinese hamster-derived Hspa5 and 5'-UTR of Chinese hamster-derived Rpsa Expression unit SEQ ID NO: 21: Promoted region of Chinese hamster-derived Hspa5 and 5 of Chinese hamster-derived Ubc '-UTR-derived expression unit SEQ ID NO: 22: Chinese hamster-derived Hspa5 promoter region and Chinese hamster-derived EF1α 5'-UTR-based expression unit SEQ ID NO: 23: Chinese hamster-derived Hspa5 promoter region and mouse-derived Expression unit SEQ ID NO: 24 composed of Ubc 5'-UTR: mouse-derived Ubc promoter region and Chinese hamster-derived Hspa8 expression unit SEQ ID NO: 25: mouse-derived Ubc promoter region and Chinese Expression unit SEQ ID NO: 26 composed of 5'-UTR of hamster-derived Actb: expression unit SEQ ID NO: 27: promoter region of mouse-derived Ubc and 5'-UTR of Chinese hamster-derived Rpsa: promoter region of mouse-derived Ubc And Chinese hamster-derived Ubc 5'-UTR expression unit SEQ ID NO: 28: Mouse-derived Ubc promoter region and Chinese hamster-derived EF1α 5'-UTR expression unit SEQ ID NO: 29: Mouse-derived Ubc Promoter region and mouse reason The expression unit SEQ ID NO: 30 composed of the 5'-UTR of the coming Ubc: the promoter region of the mouse-derived Ubc and the expression unit SEQ ID NO: 31 composed of the 5'-UTR of the Chinese hamster-derived Hspa8: the promoter region of the mouse-derived Ubc. 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 EF1α 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 of Hspa8 gene contained in cloned DNA SEQ ID NO: 40: Mouse-derived Ubc primer mChub2-3k-NotI-f
SEQ ID NO: 41: Mouse-derived Ubc primer mChub2-2p-NheI-r
SEQ ID NO: 42: nucleotide sequence of DNA element A2 SEQ ID NO: 43: nucleotide sequence of DNA element A7 SEQ ID NO: 44: nucleotide sequence of DNA element A18 SEQ ID NO: 45: Promoter region of mouse-derived Ubc and intron in mouse-derived Ubc5'-UTR Expression unit consisting of a nucleotide sequence in which the region is replaced with the intron region contained in the 5'-UTR of Chinese hamster-derived Hspa8 Expression unit SEQ ID NO: 46: The intron region in the mouse-derived Ubc promoter region and the mouse-derived Ubc5'-UTR is Chinese. 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.

Claims (25)

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.
2. The polynucleotide according to claim 1, wherein the intron contained in the 5'-UTR is an intron contained in the 5'-UTR contained in the same or different gene as the gene containing the 5'-UTR.
3. The polynucleotide according to claim 1 or 2, wherein the promoter region is the polynucleotide according to any one of SEQ ID NOs: 1 to 3.
4. The polynucleotide according to claim 1 or 2, wherein the promoter region is the polynucleotide according to SEQ ID NO: 5, in which the polynucleotide according to SEQ ID NO: 4 is linked to the 3'end of the polynureotide set forth in SEQ ID NO: 1. ..
5. The polynucleotide according to claim 4, wherein the total length or a part of the polynucleotide according to SEQ ID NO: 4 is deleted.
6. The polynucleotide according to claim 1 or 2, wherein the promoter region is the 3'end of the polynucleotide set forth in SEQ ID NO: 1 and all or part of the polynucleotide set forth in SEQ ID NO: 36 is linked.
7. The polynucleotide according to any one of claims 1 to 6, wherein the 5'-UTR is the polynucleotide according to any one of SEQ ID NOs: 6 to 11.
8. The polynucleotide according to any one of claims 2 to 7, wherein the intron is the polynucleotide according to any one of SEQ ID NOs: 12 to 17.
9. The polynucleotide according to claim 1, which comprises the polynucleotide according to any one of SEQ ID NOs: 18 to 35 and 45 to 48.
10. The polynucleotide according to claim 1, which comprises the polynucleotide according to any one of SEQ ID NOs: 18, 24 to 29, and 45 to 48.
11. A polynucleotide having 95% or more identity with respect to the polynucleotide according to claim 9 or 10, and having transcriptional activity and translational activity.
12. A polynucleotide having 99% or more identity with respect to the polynucleotide according to claim 9 or 10, and having transcriptional activity and translational activity.
13. A polynucleotide that hybridizes with a polynucleotide complementary to the polynucleotide according to claim 9 or 10 under stringent conditions, and has transcriptional activity and translational activity.
14. An expression unit composed of the polynucleotide according to any one of claims 1 to 13.
15. A foreign gene expression cassette containing the expression unit according to claim 14.
16. The foreign gene expression cassette according to claim 15, wherein the foreign gene is a gene encoding a multimeric protein.
17. The foreign gene expression cassette according to claim 15, wherein the foreign gene is a gene encoding a heteromultimeric protein.
18. The foreign gene expression cassette according to claim 15, wherein the foreign gene is a gene encoding an antibody or an antigen-binding fragment thereof.
19. A foreign gene expression vector containing the foreign gene expression cassette according to any one of claims 15 to 18.
20. A transformed cell into which the foreign gene expression vector according to claim 19 has been introduced.
21. The transformed cell according to claim 20, wherein the cell is a cultured cell derived from a mammal.
22. The transformed cell according to claim 21, wherein the cultured mammalian cell is a COS-1 cell, a HEK293 cell, or a CHO cell.
23. A method for producing a protein, which comprises culturing the transformed cell according to any one of claims 20 to 22 and obtaining a protein derived from a foreign gene from the culture.
24. Use of the polynucleotide according to any one of claims 1 to 13 for the purpose of expressing a foreign gene in transformed cells.
25. Use of the foreign gene expression vector according to claim 19, for the purpose of expressing a foreign gene in transformed cells.

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