JP7081139B2 - New promoter and expression vector containing it - Google Patents

Description

The present invention relates to a novel promoter for producing a recombinant protein using a mammalian cell as a host and an expression vector containing the promoter. More specifically, the present invention relates to a novel promoter obtained by modifying the human growth factor 1α (EF1α) promoter and an expression vector containing the promoter.

Currently, recombinant proteins are used in a wide range of fields. The growth of biopharmacy in recent years has made it even more important. Recombinant proteins are mainly produced using Escherichia coli, yeast, and insect cells as hosts. While these hosts can be used to obtain large amounts of recombinant proteins in a short period of time, the expressed recombinant proteins do not have the correct three-dimensional structure, and their original functions are due to post-translational modifications such as glycosylation. There was a problem of not having.
In order to solve the above problems, an increasing number of cases are using a recombinant protein expression system using mammalian cells as a host. In particular, recombinant proteins produced using Chinese hamster ovary cells (hereinafter referred to as CHO cells) have been confirmed to be safe for use as pharmaceuticals, and are currently a general method. Therefore, improving the productivity of recombinant protein production using mammalian cells is extremely important from the viewpoint of reducing production costs and reducing medical costs due to the price reduction of biopharmacy. However, at present, it cannot be said that the productivity of recombinant proteins using mammalian cells as a host is sufficiently high.

In order to express a recombinant protein using a mammalian cell as a host, an expression vector containing a promoter, a polynucleotide encoding the protein, and a polyadenyl chain (hereinafter, also referred to as polyA) is introduced into the host cell. Need to be cultivated. Therefore, various factors such as the promoter, the sequence of poly A, and the culture conditions of the recombinant cells are involved in the production of the recombinant protein. Among them, the promoter is particularly important because it affects the productivity of recombinant proteins.

Expression system promoters using mammalian cells as hosts have traditionally been the SV40 promoter (hereinafter, also referred to as SV40pro), which is a promoter derived from Simian virus, and the CMV promoter (hereinafter, also referred to as CMVpro), which is a promoter derived from cytomegalovirus. Patent Document 1) and the EF1α promoter (hereinafter, also referred to as EF1apro) (Patent Document 2), which is a promoter derived from human growth factor, are used. In recent years, the CAG promoter (hereinafter also referred to as CAGpro) in which a human CMV enhancer is combined with a chicken β-actin promoter (Patent Document 3) and the CEF promoter in which a human CMV enhancer is combined with a human EF1α promoter (Non-Patent Document 1). For example, stronger expression promoters than before have also been made. However, in order to improve the productivity of recombinant protein production using mammalian cells as a host, a promoter stronger than the above-mentioned promoter is required.

US Pat. No. 5,168,062 Japanese Unexamined Patent Publication No. 2-242687 Japanese Unexamined Patent Publication No. 3-16887

Analytical Biochemistry, 247, 179-181 (1997)

An object of the present invention is to provide a novel promoter capable of highly producing a recombinant protein in mammalian cells and an expression vector containing the promoter.

As a result of diligent studies to solve the above problems, the present inventors have made a recombinant protein using an expression vector containing a novel promoter obtained by deleting a part of the sequence of the human growth factor 1α (EF1α) promoter. By constructing an expression system, we have found that recombinant protein production by mammalian cells can be performed more efficiently, and have completed the present invention.

That is, the present application includes the aspects described below.
(1) A polynucleotide lacking at least the region from the 621st guanine to the 798th guanine from the nucleotide sequence set forth in SEQ ID NO: 12, or a polynucleotide having 80% or more homology with the polynucleotide.
(2) The polynucleotide according to (1), which comprises the nucleotide sequence set forth in SEQ ID NO: 18, or which comprises a nucleotide sequence having 80% or more homology with the nucleotide sequence.
(3) A promoter consisting of the polynucleotide according to (1) or (2).
(4) A vector containing the polynucleotide encoding the promoter and protein according to (3).
(5) The vector according to (4), wherein the protein is an H chain or an L chain of an antibody.
(6) A transformant obtained by transforming a mammalian cell with the vector according to (4) or (5).
(7) The transformant according to (6), wherein the mammalian cell is a CHO cell or a COS cell.
(8) Includes a step of producing a recombinant protein by culturing the transformant according to (6) or (7), and a step of recovering the recombinant protein produced from the obtained culture. , Method for producing recombinant protein.
Hereinafter, the present invention will be described in detail.

The polynucleotide of the present invention is a polynucleotide lacking a region from at least 621st guanine to 798th guanine from the nucleotide sequence set forth in SEQ ID NO: 12, or a polynucleotide having 80% or more homology with the polynucleotide. .. At this time, it is preferable that the polynucleotide consists of the nucleotide sequence set forth in SEQ ID NO: 18, or consists of a nucleotide sequence having 80% or more homology with the nucleotide sequence. Further, it is more preferable to have 90% or more homology with those sequences.

The promoter of the present invention is a promoter that improves the productivity of recombinant proteins in mammalian cells by deleting a part of the human EF1α promoter. Specifically, among the human EF1α promoters consisting of the nucleotide sequence set forth in SEQ ID NO: 12, the promoter lacks at least the 621st guanine to the 798th guanine (hereinafter, also referred to as region A). As long as the promoter of the present invention has the characteristic of the promoter of the present invention of improving the productivity of recombinant proteins in mammalian cells, the region adjacent to the 5'end side and / or the 3'end side of region A. May be further deleted. On the other hand, according to the examples described later, in order to express the recombinant protein using the human EF1α promoter, a region 3'-terminal to the region A in the promoter is required, so that the further deletion is a region. It is not possible to delete the entire region on the 3'end side of A. Further, the promoter of the present invention may have a defect in a region that is on the 5'end side and / or 3'end side of the region A and is not adjacent to the region A. As a specific example of the promoter of the present invention, among the human EF1α promoters consisting of the nucleotide sequence shown in SEQ ID NO: 12, the poly consisting of the sequence set forth in SEQ ID NO: 18 lacking guanine at position 621 to guanine at position 798. Nucleotides can be mentioned.

In order to improve the productivity of a recombinant protein in a mammalian cell by using the promoter of the present invention, an expression vector containing the promoter of the present invention and a polynucleotide encoding the recombinant protein (hereinafter, simply the expression of the present invention). (Also referred to as a vector) is preferably produced. In addition to the promoter of the present invention and the polynucleotide encoding the recombinant protein, the expression vector of the present invention includes poly A, a secretory signal required for secret expression of the recombinant protein, a gene amplification marker gene, and host selection. It may further include an antibiotic resistance gene to be used, a replication initiation site in a host other than mammalian cells, and the like.

The poly A is not particularly limited as long as it contains a termination signal, and as an example, poly A derived from the protein to be expressed, poly A derived from the SV40 virus genome, poly A derived from herpesvirus thymidine kinase, and poly A derived from bovine growth hormone. , Poly A derived from the β-globin gene of rabbits.

Examples of the secretory signal include a secretory signal derived from a recombinant protein to be expressed, a secretory signal of human interleukin 2 (IL-2) (SEQ ID NO: 75), a secretory signal of an azlocidine precursor (SEQ ID NO: 69), and a human serum. The secretory signal of albumin (SEQ ID NO: 72) can be mentioned. For example, when expressing an antibody, it is preferable to use the secretory signal sequence of the azurosidine precursor from the examples described later because the antibody expression level is high.

As the gene amplification marker gene, a gene suitable for the method of gene amplification may be used. For example, the dhfr gene may be used when the dihydrofolate reductase (dhfr) / methotrexate (MTX) method is used, and the GS gene may be used when the glutamine synthetase (GS) / methionine sulfoxymin (MSX) method is used.

For the antibiotic resistance gene, a resistance gene corresponding to the antibiotic used for host selection may be selected. For example, when the host is Escherichia coli, an ampicillin resistance gene or a canamycin resistance gene is selected, and when the host is a mammalian cell, the resistance gene is selected. Examples thereof include G418 resistance gene, puromycin resistance gene, blastsaidin resistance gene, zeosin resistance gene, hyglomycin resistance gene, and freomycin resistance gene, respectively.

The origin of replication can be exemplified by ColE1, which has a high number of copies in Escherichia coli and a high yield of plasmid DNA when the host other than the mammalian cell is Escherichia coli.

Further, the expression vector of the present invention may further contain an enhancer for enhancing the action of the promoter. The enhancer to be used is not particularly limited, and may be appropriately selected in consideration of the recombinant protein to be expressed and the host. An example is an enhancer derived from cytomegalovirus (CMV).

Further, in order to facilitate the expression of the gene introduced into the host, the expression vector of the present invention may further contain the LoxP gene. When the expression vector is introduced into a host cell containing the LoxP gene in its genome, the host cell undergoes homologous recombination with Cre recombinase to cause homologous recombination between the host and the LoxP gene in the early expression vector. Polynucleotides encoding recombinant proteins can be introduced into the genome of the gene.

The size (base length) of the vector of the present invention should be as small as possible, preferably 4000 bases or more and 8000 bases or less, because the gene transfer efficiency into the host mammalian cell decreases as the size (base length) increases.

The recombinant protein expressed in mammalian cells using the expression vector of the present invention is not particularly limited. Examples include antibodies (immunoglobulins), various enzymes, and human-derived receptor proteins. As a specific example of the case where the recombinant protein is an antibody, a mouse, rat, rabbit, camel, or human, which is a mammal from the origin, becomes an antibody derived from a different host except for the complementarity determining region (CDR) from the structure. Examples thereof include miniaturized antibodies such as modified recombinant antibody, Fab fragment, F (ab') ² fragment, Fv fragment, scFv in which VL and VH regions are combined with a synthetic linker.

As an example of mammalian cells used for expressing recombinant protein using the expression vector of the present invention, CHO cells (K1 strain, DG44 strain, DXB11 strain), human fetal kidney-derived cells (HEK cells), human leukemia cells. Examples thereof include derived cells (HL-60 cells), human cervical cancer-derived cells (HeLa cells), and African green monkey kidney cell-derived cells (COS cells). Among them, the expression vector of the present invention is a preferred vector for a recombinant protein expression system using CHO cells or COS cells as a host.

In order to transform a mammalian cell with the expression vector of the present invention, one of the transformation methods usually used by those skilled in the art, such as electroporation and lipofection using a cationic liposome, is selected according to the mammalian cell used as a host. It may be selected as appropriate.

By culturing a transformant capable of producing a recombinant protein obtained by transforming a mammalian cell with the expression vector of the present invention, and recovering the recombinant protein produced from the obtained culture. The recombinant protein can be produced with high efficiency. When recovering the recombinant protein from the culture of the transformant, a purification operation by chromatography such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography and the like may be combined, and the operation may be used. The recombinant protein can be recovered with high efficiency and high purity.

The promoter of the present invention is a polynucleotide lacking a partial sequence of the human growth factor 1α (EF1α) promoter, and inclusion of the promoter of the present invention in an expression vector improves the productivity of recombinant proteins in mammalian cells. do. Therefore, the recombinant protein can be produced at low cost.

It is a figure which shows the outline of the plasmid pEFd prepared in Example 1. The antibody expression level in the CHO cell K1 strain was determined when a human EF1α promoter full length (EF1apro) was used as a promoter and when a promoter (EFdpro, EFS1pro, EFS2pro) in which a part of the promoter was deleted was used. It is a figure which shows the result of comparison. The antibody expression levels in COS cells were compared between when a human EF1α promoter full length (EF1apro) was used as a promoter and when a promoter (EFdpro, EFS1pro, EFS2pro) in which a part of the promoter was deleted was used. It is a figure which shows the result. The CHO cell K1 strain was used as a promoter when the human EF1α promoter full length (EF1apro) was used, when the promoter of the present invention (EFdpro) was used, and when the conventional promoters (SV40pro, CMVpro and CAGpro) were used. It is a figure which shows the result of having compared the antibody expression level in. When the human EF1α promoter full length (EF1apro) was used as the promoter, when the promoter of the present invention (EFdpro) was used, and when the conventional promoters (SV40pro, CMVpro and CAGpro) were used, in COS cells. It is a figure which shows the result of having compared the antibody expression level. It is a figure which shows the result of having compared the antibody expression level in the CHO cell K1 strain by the combination of the promoter (EFdpro) of this invention and a secretory signal. It is a figure which shows the result of having compared the antibody expression level in COS cell by the combination of the promoter (EFdpro) of this invention, and a secretory signal.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these examples.

Example 1 Preparation of a plasmid containing the human growth factor 1α (EF1α) promoter A plasmid containing the human EF1α promoter was prepared by the method shown below.

(1) The polynucleotide encoding dihydrofolate reductase (DHFR) shown in SEQ ID NO: 1 and the polynucleotide in which the restriction enzyme SacII recognition sequence (CCGCGG) was added to Poly A of SV40 were totally synthesized and cloned into a plasmid. (Consigned to Integrated DNA Technologies).

(2) Escherichia coli JM109 strain was transformed with the plasmid prepared in (1). The obtained transformant was cultured, a plasmid was extracted, and then digested with a restriction enzyme SacII to prepare a polynucleotide in which the dhfr gene and SV40 polyA were ligated (named dhfr-SV40polyAP).

(3) Described in SEQ ID NO: 2 (5'-TCC [CCGCGG] GCGGGACTCGGGGTTCGAAAATTGACCG-3') and SEQ ID NO: 3 (5'-TCC [CCGCGG] GGTGGCTAGCCTAAGTCGACTG-3') using a pIRES vector (manufactured by Clontech) as a template. Using the oligonucleotide consisting of the sequence as a primer (restriction enzyme SacII recognition sequence in square brackets), prepare a reaction solution having the composition shown in Table 1, heat the reaction solution at 98 ° C. for 30 seconds, and then heat the reaction solution at 98 ° C. for 10 seconds. PCR was performed by repeating the reaction of the first step, the second step at 55 ° C. for 5 seconds, and the third step at 72 ° C. for 5 minutes as one cycle for 25 cycles, and the region of the pIRES vector excluding the neomycin resistance gene. Was amplified (named pIRES-P).

(4) The prepared PCR product pIRES-P was purified, digested with the restriction enzyme SacII, and ligated with dhfr-SV40polyAP prepared in (2). Escherichia coli JM109 strain was transformed with the ligation product, and a plasmid was extracted from the cultured transformant to obtain a plasmid pIRES-dhfr containing the dhfr gene.

(5) Described in SEQ ID NO: 4 (5'-AGATTTAGA [CTCGAG] GAAATAACCCTGAAAAGAGGAACTTGG-3') and SEQ ID NO: 5 (5'-ATCAGATCAGACTGTTACCATTTTGTAGAGGTTAC-3') using the pIRES-dhfr prepared in (4) as a template. Using the above oligonucleotide as a primer (restriction enzyme XhoI recognition sequence in square brackets), prepare a reaction solution having the composition shown in Table 2, heat the reaction solution at 98 ° C for 5 minutes, and then heat the reaction solution at 98 ° C for 10 seconds. PCR was performed by repeating the reaction of 1 step, the second step at 55 ° C. for 5 seconds, and the third step at 72 ° C. for 2 minutes as one cycle for 30 cycles, and the polyA gene regions of the SV40 promoter, DHFR and SV40 were analyzed. Amplified. Then, the PCR product obtained by purification by agarose gel extraction was named dhfr-P.

(6) Using a commercially available pUC19 (manufactured by Takara Bio Co., Ltd.) as a template, from the sequences shown in SEQ ID NO: 6 (5'-CAGTCTGATCTGATTATTGAAAAAAGGAAGAGTAG-3') and SEQ ID NO: 7 (5'-AGA [TCTAGA] AGAATCAGGGATAAGCAGGAAAG-3'). Using the above oligonucleotide as a primer (restriction enzyme XbaI recognition sequence in square brackets), prepare a reaction solution having the composition shown in Table 2, heat the reaction solution at 98 ° C for 5 minutes, and then heat the reaction solution at 98 ° C for 10 seconds. PCR was performed by repeating the reaction of 1 step, the 2nd step at 55 ° C. for 5 seconds, and the 3rd step at 72 ° C. for 2 minutes as 1 cycle for 30 cycles, and replication with the ampicillin resistance gene ( ^Ampr ) and Escherichia coli. The origin (ColE1) gene region was amplified. Then, the PCR product obtained by purification by agarose gel extraction was named Amp + ColE1-P.

(7) Using pECE-dhfr (J. Biochem., 108, 673-676 (1990)) as a template, SEQ ID NO: 8 (5'-AGA [TCTAGA] CTGTGGAATGTGTGTCAGTGGG-3') and SEQ ID NO: 9 (5'-[ GCGGCCGC] TTT <GAATTC> AGCTTTTGCAAAAGCCCATAGGC-3') Primer (restriction enzyme XbaI recognition sequence in the square bracket of SEQ ID NO: 8 and restriction enzyme NotI recognition sequence in the square bracket of SEQ ID NO: 9) , The inside of the mountain bracket of SEQ ID NO: 9 is the recognition sequence of EcoRI), prepare a reaction solution having the composition shown in Table 2, heat the reaction solution at 98 ° C. for 5 minutes, and then perform the first step at 98 ° C. for 10 seconds. PCR was performed by repeating 30 cycles of the second step at 55 ° C. for 5 seconds and the third step at 72 ° C. for 90 seconds as one cycle to amplify the SV40 promoter (SV40pro) gene region. Then, the PCR product obtained by purification by agarose gel extraction was named SV40-P.

(8) Using the pIRES-dhfr prepared in (4) as a template, SEQ ID NO: 10 (5'-[GAATTC] AAA <GCGGCCGC> GACATGATAAGATATACTGATTG-3') and SEQ ID NO: 11 (5'-TAA [CTCGAG] TTTACCAATTTTGTTACGAGTTAC-3') Primer is an oligonucleotide consisting of the sequence shown in') (the recognition sequence of the restriction enzyme EcoRI is in the square bracket of SEQ ID NO: 10, the recognition sequence of NotI is in the mountain bracket of SEQ ID NO: 10, and the square bracket of SEQ ID NO: 11 is restricted. As a recognition sequence of the enzyme XhoI), a reaction solution having the composition shown in Table 2 was prepared, and after heat-treating the reaction solution at 98 ° C. for 5 minutes, the first step at 98 ° C. for 10 seconds and the first step at 55 ° C. for 5 seconds. PCR was performed by repeating the reaction of 2 steps, the 3rd step of 2 minutes at 72 ° C. as 1 cycle for 30 cycles, and the SV40 poly A region was amplified. Then, the PCR product obtained by purification by agarose gel extraction was named SV40polyAP.

(9) The PCR product dhfr-P prepared in (5) and the PCR product Amp + ColE1-P prepared in (6) contain overlapping regions. Therefore, using these as templates and the polynucleotides consisting of the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 7 as primers, a reaction solution having the composition shown in Table 3 was prepared, and the reaction solution was used at 98 ° C. for 5 minutes. After the heat treatment, PCR was performed by repeating 30 cycles of the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, and the third step at 72 ° C. for 2 minutes as one cycle, and PCR was performed. P and Amp + ColE1-P were synthesized and amplified as one gene. After that, purification was performed by agarose gel extraction, and the obtained PCR product was named dhfr-Amp-P.

(10) The PCR product SV40-P prepared in (7) and the PCR product SV40polyAP prepared in (8) include overlapping regions. Therefore, using these as templates and the polynucleotides consisting of the sequences shown in SEQ ID NO: 8 and SEQ ID NO: 11 as primers, a reaction solution having the composition shown in Table 3 was prepared, and the reaction solution was used at 98 ° C. for 5 minutes. After the heat treatment, PCR was performed by repeating 30 cycles of the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, and the third step at 72 ° C. for 90 seconds as one cycle, and PCR was performed. P and SV40polyA-P were synthesized and amplified as one gene. After that, purification was performed by agarose gel extraction, and the obtained PCR product was named SV40propolyAP.

(11) The dhfr-Amp-P prepared in (9) and the SV40polyA-P prepared in (10) are digested with restriction enzymes XbaI and XhoI, respectively, purified using a PCR Purification kit (manufactured by Qiagen), and then purified. Ligation. Escherichia coli JM109 strain was transformed with a ligation product, and a plasmid was extracted from the cultured transformant to obtain pSV40, which is an expression vector for mammalian cells.

(12) The polynucleotide of the promoter region of human growth factor 1α (EF1α) (hereinafter, also referred to as EF1apro) consisting of the sequence shown in SEQ ID NO: 12 was totally synthesized and cloned into a plasmid (consigned to GENEWIZ). An oligonucleotide consisting of the sequences described in SEQ ID NO: 13 (5'-AGA [TCTAGA] CGTGAGGCTCCGGTGCCCGTC-3') and SEQ ID NO: 14 (5'-[GCGGCCGC] TTT <GAATTC> TCACGAACCCTGAAATGGAAG-3') using the plasmid as a template. As a primer (restriction enzyme XbaI recognition sequence in the square bracket of SEQ ID NO: 13, restriction enzyme NotI recognition sequence in the square bracket of SEQ ID NO: 14, EcoRI recognition sequence in the mountain bracket of SEQ ID NO: 14), Table 2 A reaction solution having the composition shown in the above is prepared, and after heat-treating the reaction solution at 98 ° C. for 5 minutes, the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, and the second step at 72 ° C. for 90 seconds. PCR was performed by repeating the reaction with 3 steps as 1 cycle for 30 cycles to amplify the EF1apro gene region, and then the PCR product was purified by agarose gel extraction.

(13) The PCR products prepared in (11) and pSV40 and (12) were digested with restriction enzymes XbaI and EcoRI, respectively, purified using a PCR Purification kit (manufactured by Qiagen), and then ligated. Escherichia coli JM109 strain was transformed with a ligation product, and a plasmid was extracted from the cultured transformant to obtain pEFd, which is an expression vector for mammalian cells.

(14) The nucleotide sequence of the vector prepared in (13) was subjected to BigDye Terminator Ver. 3.1 Cycle sequence reaction was performed using a Cycle Sequencing Kit (manufactured by Thermo Fisher Scientific), and a fully automated DNA sequencer ABI Prism 3700 DNA analyzer (manufactured by Thermo Fisher Scientific) was used for cycle sequence reaction. As primers for sequencing, SEQ ID NOs: 6 to 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 (5'-CTCGGTCAGGGGGGCGGAGCC-3'), SEQ ID NO: 16 (5'-CTCTAGCTTCCCCGCAACAA-3') and SEQ ID NO: 17 ( An oligonucleotide consisting of the sequence described in 5'-ATTTTCTTGCCAAAAAGTTTG-3') was used.

As a result of sequence analysis, among the PCR products obtained in (12) (that is, EF1apro, SEQ ID NO: 12), 178 nucleotides from the 621st guanine to the 798th guanine were deleted (SEQ ID NO: 18, EFdpro thereafter). Name it). The other regions had the nucleotide sequence as designed. The outline of the plasmid pEFd prepared in this example is shown in FIG.

Example 2 Preparation of antibody expression vector (Part 1)
A polynucleotide encoding an antibody (immunoglobulin) was introduced into the plasmid pEFd prepared in Example 1 to prepare a vector for antibody expression.

(1) Preparation of plasmid containing antibody heavy chain gene (1-1) Encoding the heavy chain (H chain) variable region (VH region) of an anti-human gp130 receptor antibody consisting of the amino acid sequence set forth in SEQ ID NO: 19. A polynucleotide in which the restriction enzymes EcoRI recognition sequence (GAATTC) and NheI recognition sequence (GCTAGC) were added to the polynucleotide (SEQ ID NO: 20) was totally synthesized and cloned into a plasmid (consigned to FASMAC).

(1-2) The VH region is encoded by transforming the Escherichia coli JM109 strain with the plasmid prepared in (1-1), extracting the plasmid from the obtained transformant, and then digesting with the restriction enzymes EcoRI and NheI. A polynucleotide was prepared and named VH-P.

(1-3) pFUSEss-CHIg-hG1 (manufactured by InvivoGen) containing a constant region of human IgG1 was digested with restriction enzymes EcoRI and NheI, purified by agarose gel extraction, and VH prepared in (1-2). -Rigated with P. Escherichia coli JM109 strain was transformed with a ligation product, and a plasmid was extracted from the cultured transformant to obtain a plasmid pFU-hG1 containing a polynucleotide encoding the H chain of an anti-human gp130 receptor antibody.

(2) Preparation of plasmid containing antibody light chain gene (2-1) Encodes the light chain (L chain) variable region (VL region) of an anti-human gp130 receptor antibody consisting of the amino acid sequence set forth in SEQ ID NO: 21. A polynucleotide in which the restriction enzymes EcoRI recognition sequence (GAATTC) and BsiWI recognition sequence (CGTACG) were added to the polynucleotide (SEQ ID NO: 22) was completely synthesized and cloned into a plasmid (consigned to FASMAC).

(2-2) Escherichia coli JM109 strain is transformed with a plasmid containing a polynucleotide encoding the prepared VL region, and the obtained transformant is extracted from the plasmid and then digested with restriction enzymes EcoRI and BsiWI to VL. A polynucleotide encoding the region was prepared and named VL-P.

(2-3) pFUSE2ss-CLIg-hk (manufactured by InvivoGen) containing a constant region of human κ chain was digested with restriction enzymes EcoRI and BsiWI, purified by agarose gel extraction, and ligated with VL-P. Escherichia coli JM109 strain was transformed with a ligation product, and a plasmid was extracted from the cultured transformant to obtain a plasmid pFU-hL containing a polynucleotide encoding the L chain of an anti-human gp130 receptor antibody.

(3) The nucleotide sequences of pFU-hG1 prepared in (1) and pFU-hL prepared in (2) were confirmed by the same method as in Example 1 (14). As a primer for sequencing, an oligonucleotide having the sequence shown in SEQ ID NO: 23 (5'-CGTTACAGATCCAAAGCTGTGAC-3') was used. As a result, it was confirmed that the introduced polynucleotide sequence was as designed.

(4) Using the pFU-hG1 prepared in (1) as a template, from the sequences described in SEQ ID NO: 24 (5'-GCAGGTGTTCCTCTCATCAGGTTCAACTCCAG-3') and SEQ ID NO: 25 (5'-AAT [GCGGCCGC] TATCATTCCGGAGAGAGGGAGAG-3'). A reaction solution having the composition shown in Table 4 is prepared using the oligonucleotide (the recognition sequence of the restriction enzyme NotI in the square brackets) as a primer, and the reaction solution is heat-treated at 98 ° C. for 5 minutes and then at 98 ° C. for 10 seconds. PCR was performed by repeating the reaction of the first step, the second step at 55 ° C. for 5 seconds, and the third step at 72 ° C. for 60 seconds as one cycle for 30 cycles. Then, the PCR product encoding the full length of the H chain of the anti-human gp130 receptor obtained by purification by agarose gel extraction was named AHP.

(5) Using the pFU-hL prepared in (2) as a template, from the sequences shown in SEQ ID NO: 26 (5'-CATATATGTCCAGGGGGACATTCAGATGAC3') and SEQ ID NO: 27 (5'-AAT [GCGGCCGC] TACTAACACTCCCCCTGAAGC-3'). The oligonucleotides are used as primers (the recognition sequence of the restriction enzyme NotI in the square brackets) to prepare a reaction solution having the composition shown in Table 4, and the reaction solution is heat-treated at 98 ° C. for 5 minutes and then at 98 ° C. PCR was performed by repeating the reaction of the first step for 10 seconds, the second step for 5 seconds at 55 ° C., and the third step for 1 minute at 72 ° C. for 30 cycles. Then, the L-chain full-length PCR product of the anti-human gp130 receptor obtained by purification by agarose gel extraction was named AL-P.

(6) Described in SEQ ID NO: 29 (5'-ATGGGATGGAGCTGGATCTTTCTTTCTTCCTGTGTCAGGTACGTGTCCT), which is a polynucleotide encoding the signal sequence AH (MGWSWIFLLFFLSGTAGVLS) set forth in SEQ ID NO: 28, using the AH-P prepared in (4) as a template. The polynucleotide consisting of the sequence is used as the signal DNA, and the oligonucleotide consisting of the sequence shown in SEQ ID NO: 30 (5'-CTA [GAATTC] GCCACCATGGGGAGGAGCTGG-3') and SEQ ID NO: 25 is used as a primer (recognition of the restriction enzyme EcoRI in square brackets). As an arrangement), a reaction solution having the composition shown in Table 5 is prepared, and after heat-treating the reaction solution at 98 ° C. for 5 minutes, the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, 72. PCR was performed by repeating the reaction at ° C. for 90 seconds with the third step as one cycle for 30 cycles, and the polynucleotide encoding the H chain to which the signal sequence AH was added was amplified. Then, purification was performed by agarose gel extraction, and the obtained PCR product was named sAHP-P.

(7) The AL-P prepared in (5) is used as a template, and the polynucleotide encoding the signal sequence AL (MDFQVQIFSFLLMSASVISRG) set forth in SEQ ID NO: 31 is set forth in SEQ ID NO: 32 (5'-ATGGATTTTCAAGTGCAGATTTTTCAGCTTCCGCTAGCTAGTCAGTCAGCTAG). The polynucleotide consisting of the sequence is used as the signal DNA, and the oligonucleotide consisting of the sequence shown in SEQ ID NO: 33 (5'-CTA [GAATTC] GCACCACTGGATTTTCAAGTG-3') and SEQ ID NO: 27 is used as a primer (recognition of the restriction enzyme EcoRI in the square brackets). As an arrangement), a reaction solution having the composition shown in Table 5 is prepared, and after heat-treating the reaction solution at 98 ° C. for 5 minutes, the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, 72. PCR was performed by repeating the reaction at ° C. for 90 seconds with the third step as one cycle for 30 cycles, and the polynucleotide encoding the L chain to which the signal sequence AL was added was amplified. Then, purification was performed by agarose gel extraction, and the obtained PCR product was named sAL-P.

(8) The sAH-P prepared in (6), the sAL-P prepared in (7), and the plasmid pEFd prepared in Example 1 were digested with restriction enzymes EcoRI and NotI, respectively, and ligated after purification. By transforming Escherichia coli JM109 strain with a ligation product and extracting a plasmid from the cultured transformant, the H chain expression vector pEFd-AHsH of the anti-human gp130 receptor antibody and the L chain expression vector of the anti-human gp130 receptor antibody pEFd-ALsL was obtained.

(9) Of the expression vectors pEFd-AHsH and pEFd-ALsL obtained in (8), the cloned signal sequence and the vicinity of the anti-human gp130 receptor antibody H chain (pEFd-AHsH) or L chain (pEFd-ALsL) The nucleotide sequence was analyzed in the same manner as in Example 1 (14). As a sequence primer for pEFd-AHsH, it is described in SEQ ID NO: 34 (5'-GAGTTCCACGACACCGTCAC-3'), SEQ ID NO: 35 (5'-GCTAGCACCAAGGGCCCATC-3') and SEQ ID NO: 36 (5'-CGGGAGGAGACCAAGAAC-3'). The oligonucleotide consisting of the sequence was used as a sequence primer for pEFd-ALsL, and the oligonucleotide consisting of the sequences shown in SEQ ID NO: 33 and SEQ ID NO: 37 (5'-CACCTTCCACTGTACTTGGC-3') was used, respectively.

As a result of sequence analysis, it was confirmed that the polynucleotide sequence was as designed. The amino acid sequence of the signal sequence AH and the anti-human gp130 receptor antibody H chain is in SEQ ID NO: 38, the sequence of the polynucleotide encoding the signal sequence AH and the anti-human gp130 receptor antibody H chain is in SEQ ID NO: 39, and the signal sequence AL and The amino acid sequence of the anti-human gp130 receptor antibody L chain is shown in SEQ ID NO: 40, and the sequence of the signal sequence AL and the polynucleotide encoding the anti-human gp130 receptor antibody L chain is shown in SEQ ID NO: 41, respectively. The signal sequence AH is from the first methionine to the 19th serine of SEQ ID NO: 38, and the amino acid sequence of the anti-human gp130 receptor antibody H chain is from the 20th glutamine to the 469th lysine. The signal sequence AL is from the first methionine to the 22nd glycine of SEQ ID NO: 40, and the amino acid sequence of the anti-human gp130 receptor antibody L chain is from the 23rd aspartic acid to the 235th cysteine.

Comparative Example 1 Preparation of antibody expression vector (Part 2)
Since the promoter EFdpro inserted in the plasmid pEFd prepared in Example 1 is an embodiment in which a part of the human EF1α promoter is deleted, an expression vector containing the entire length of the human EF1α promoter (EF1apro) was prepared again.

(1) Using the plasmid containing EF1apro synthesized in Example 1 (12) as a template and the oligonucleotide consisting of the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14 as primers, a reaction solution having the composition shown in Table 6 was prepared. After heat-treating the reaction solution at 98 ° C. for 5 minutes, the reaction consists of a first step at 98 ° C. for 10 seconds, a second step at 55 ° C. for 10 seconds, and a third step at 72 ° C. for 90 seconds as one cycle. PCR was performed by repeating 30 cycles to amplify the total length of EF1apro. Then, purification was performed by agarose gel extraction, and the obtained PCR product was named EF1α-P.

(2) EF1α-P prepared in (1) and pEFd-AHsH and pEFd-ALsL prepared in Example 2 are digested with restriction enzymes XbaI and EcoRI, respectively, and after purification, EF1α-P and pEFd-AHsH, Also, EF1α-P and pEFd-ALsL were ligated.

(3) H chain expression vector pEF1α-AHsH and anti-human gp130 of anti-human gp130 receptor antibody containing full-length EF1apro by transforming Escherichia coli JM109 strain with ligation product and extracting plasmid from the cultured transformant. The L chain expression vector pEF1α-ALsL of the receptor antibody was obtained.

(4) The nucleotide sequence of the cloned region was analyzed by the same method as in Example 1 (14). As a sequencer primer, an oligonucleotide consisting of the sequences shown in SEQ ID NOs: 14, 15, 34 and 39 was used.

As a result of sequence analysis, it was confirmed that the polynucleotide sequence was as designed. The promoter, signal sequence and anti-human gp130 antibody receptor antibody L chain in pEF1α-ALsL are encoded by SEQ ID NO: 44, and the promoter, signal sequence and anti-human gp130 antibody receptor antibody L chain in pEF1α-ALsL are encoded by SEQ ID NO: 44. The sequences of the polynucleotides to be used are shown in SEQ ID NO: 45, respectively. In SEQ ID NOs: 44 and 45, the 1st to 1184th are the nucleotide sequences of the full length of the promoter EF1apro, and the 1197th and subsequent adenines are the signal sequences and the polynucleotide sequences encoding the antibody chains.

Comparative Example 2 Preparation of antibody expression vector (Part 3)
An antibody expression vector containing a polynucleotide lacking a partial sequence of the human EF1α promoter was prepared.

(1) Of the EF1apro consisting of the nucleotide sequence shown in SEQ ID NO: 12, the polynucleotides 1 to 619 (hereinafter, also referred to as EFS1) were amplified. The plasmid containing EF1apro synthesized in Example 1 (12) was used as a template, and the oligonucleotide consisting of the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 42 (5'-AAT [GAATTC] CCGTCGCCGCCCCGCGCCCC-3') was used as a primer (square bracket). A reaction solution having the composition shown in Table 6 is prepared as the restriction enzyme EcoRI recognition sequence), and the reaction solution is heat-treated at 98 ° C. for 5 minutes, then the first step at 98 ° C. for 10 seconds, and 10 at 55 ° C. PCR was performed by repeating 30 cycles of the second step of seconds and the third step of 60 seconds at 72 ° C. as one cycle, and EFS1 was amplified. Then, purification was performed by agarose gel extraction, and the obtained PCR product was named EFS1-P.

(2) The polynucleotides 1 to 366 of EF1apro shown in SEQ ID NO: 12 (hereinafter, also referred to as EFS2) were amplified. The plasmid containing EF1apro synthesized in Example 1 (12) was used as a template, and the oligonucleotide consisting of the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 43 (5'-AAT [GAATTC] CCCACCACTTCCAACCCACCGAAGC-3') was used as a primer (square bracket). A reaction solution having the composition shown in Table 6 is prepared as the restriction enzyme EcoRI recognition sequence), and the reaction solution is heat-treated at 98 ° C. for 5 minutes, then the first step at 98 ° C. for 10 seconds, and 10 at 55 ° C. PCR was performed by repeating the reaction with the second step of seconds and the third step of 60 seconds at 72 ° C. as one cycle for 30 cycles, and EFS2 was amplified. Then, purification was performed by agarose gel extraction, and the obtained PCR product was named EFS2-P.

(3) EFS1-P prepared in (1), EFS2-P prepared in (2), and pEFd-AHsH and pEFd-ALsL prepared in Example 2 are digested with restriction enzymes XbaI and EcoRI, respectively, and purified. Later, EFS1-P and pEFd-AHsH, EFS1-P and pEFd-ALsL, EFS2-P and pEFd-AHsH, and EFS2-P and pEFd-ALsL were ligated, respectively.

(4) By transforming Escherichia coli JM109 strain with a ligation product and extracting a plasmid from the cultured transformant, H chain and L chain expression vectors pEFS1-AHsH and pEFS1- of an anti-human gp130 receptor antibody containing EFS1. H-chain and L-chain expression vectors pEFS2-AHsH and pEFS2-ALsL for anti-human gp130 receptor antibody containing ALsL and EFS2 were obtained.

(5) The nucleotide sequence of the cloned region was analyzed by the same method as in Example 1 (12).

As a result of sequence analysis, it was confirmed that the polynucleotide sequence was as designed. The promoter, signal sequence and anti-human gp130 receptor antibody H chain in pEFS1-AHsH are encoded by SEQ ID NO: 46, and the promoter, signal sequence and anti-human gp130 antibody receptor antibody L chain in pEFS1-ALsL are encoded by the sequence. The sequence of the polynucleotide to be used is on SEQ ID NO: 47, the promoter, signal sequence in pEFS2-AHsH and the sequence of the polynucleotide encoding the anti-human gp130 receptor antibody H chain are in SEQ ID NO: 48, the promoter, signal sequence and in pEFS2-ALsL. The sequence of the polynucleotide encoding the anti-human gp130 antibody receptor antibody L chain is shown in SEQ ID NO: 49, respectively. In SEQ ID NOs: 46 and 47, the 1st to 619th are cloned nucleotide sequences of EFS1, and the 632th and subsequent adenines are signal sequences and polynucleotide sequences encoding antibody chains. Further, in SEQ ID NOs: 48 and 49, the 1st to 366th are cloned nucleotide sequences of EFS2, and the 379th and subsequent adenines are signal sequences and polynucleotide sequences encoding antibody chains.

Example 3 Comparison of antibody expression levels (Part 1)
The antibody expression levels were compared using the antibody expression vectors prepared in Example 2 and Comparative Examples 1 and 2.

(1) CHO cell K1 strain in RPM1640 medium (Wako) containing 10% (w / v) fetal bovine serum and 50 μg / mL kanamycin using a 24-well plate (BD) at 37 ° C. The cells were cultured under the condition of 5% CO ₂ .

(2) COS cells in D-MEM medium (manufactured by Gibco) containing 10% (w / v) fetal bovine serum and 50 μg / mL kanamycin at 37 ° C. using a 24-well plate, 5% CO ₂ Was cultured under the conditions of.

(3) After the cells became confluent, the medium of each well was replaced with 0.3 mL of Opti-MEM (manufactured by Thermo Fisher Scientific).

(4) pEFd-AHsH and pEFd-ALsL prepared in Example 2, pEF1α-AHsH and pEF1α-ALsL prepared in Comparative Example 1, pEFS1-AHsH and pEFS1-ALsL prepared in Comparative Example 2, and pEFS2-AHsH and Using pEFS2-ALsL, 250 ng each of H chain expression vector and L chain expression vector and 1 μL of P3000 Reagent were added to 50 μL of Opti-MEM, and 1 μL of Lipofectamine 3000 (manufactured by Thermo Fisher Scientific) was included. -MEM was added and allowed to stand at room temperature for 10 minutes, then the whole amount was added to the well containing the CHO cell K1 strain or COS cells whose medium was exchanged in Opti-MEM, and cultured at 37 ° C. and 5% CO ₂ for 3 days. did.

(5) The expression level of the expressed antibody was measured by the Enzyme-Linked ImmunoSorbent Assay (hereinafter referred to as ELISA) shown below.
(5-1) Anti-human IgG-Fab antibody (manufactured by Bethyl) was immobilized at 1 μg / well in a well of a 96-well microplate (manufactured by Nunc) at 1 μg / well (overnight at 4 ° C.), and 2% after completion of immobilization. Blocking was performed with (w / v) SKIM MILK (manufactured by BD) and 20 mM Tris-hydrochloric acid buffer (pH 7.4) containing 150 mM sodium chloride.
(5-2) Anti-human gp130 receptor expressed after washing with wash buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% (w / v) Tween 20, 150 mM NaCl). The antibody was reacted with solid phase anti-human IgG-Fab (1 hour at 30 ° C.).
(5-3) After completion of the reaction, the reaction was washed with the washing buffer, and an anti-mouse antibody (manufactured by Bethyl) labeled with peroxidase diluted to 100 ng / mL was added at 100 μL / well.
(5-4) After reacting at 30 ° C. for 1 hour, washing with the washing buffer, TMB Peroxidase Substrate (manufactured by KPL) was added at 50 μL / well. Color development was stopped by adding 1 M phosphoric acid at 50 μL / well, and the absorbance at 450 nm was measured using a microplate reader (manufactured by Tecan).
(5-5) Measurements similar to (5-1) to (5-4) were performed using a standard antibody (manufactured by Sigma) having a known concentration, and a calibration curve was prepared based on the obtained absorbance, and the calibration curve was prepared. The expression level of the anti-human gp130 receptor antibody was determined from the line.

The results when the CHO cell K1 strain was used as the host are shown in FIG. 2, and the results when the COS cells were used as the host are shown in FIG. 3, respectively. In any host, the antibody expression level was high when EFdpro (deficient in nucleotides from 621st guanine to 798th guanine in EF1apro (SEQ ID NO: 12)), which is the promoter of the present invention, was used as the promoter. It was the highest. Similar to EFd, most of EFS1pro (deficient in nucleotides after 620th guanine in EF1apro) and EFS2pro (deficient in nucleotides after 376th adenine in EF1apro), which are polynucleotides lacking a part of EF1apro. No antibody was expressed. From this, it can be seen that in order to express a recombinant protein using the human EF1α promoter, the 3'-terminal region of the promoter is required.

Comparative Example 3 Preparation of antibody expression vector (No. 4)
An antibody expression vector containing promoters (SV40, CMV, CAG) generally used for protein expression by mammalian cells was prepared.

(1) Preparation of antibody expression vector containing SV40 promoter (1-1) SV40-P prepared in Example 1 (7) was digested with restriction enzymes XbaI and EcoRI, and purified using agarose gel extraction. The SV40-P comprises the SV40 promoter set forth in SEQ ID NO: 50.
(1-2) The pEFd-AHsH and pEFd-ALsL prepared in Example 2 were digested with restriction enzymes XbaI and EcoRI, and purified by agarose gel extraction to prepare plasmids from which EFdpro had been removed.
(1-3) The restriction enzyme digestion products obtained in (1-1) and each plasmid prepared in (1-2) excluding EFdpro were ligated. By transforming Escherichia coli JM109 strain with a ligation product and extracting a plasmid from the cultured transformant, an anti-human gp130 receptor antibody H chain expression vector pSV40-AHsH containing SV40pro, and an anti-human gp130 receptor containing SV40pro. The antibody L chain expression vector pSV40-ALsL was obtained.

(2) Preparation of antibody expression vector containing CMV promoter (2-1) The polynucleotide of the CMV promoter region (CMVpro) consisting of the sequence shown in SEQ ID NO: 51 was totally synthesized and cloned into a plasmid (consigned to FASMAC).
(2-2) Using the plasmid synthesized in (2-1) as a template, SEQ ID NO: 52 (5'-AGA [TCTAGA] GTTGACATTGATTATTGACTAG-3') and SEQ ID NO: 53 (5'-[GCGGCCGC] TTT <GAATTC> GAGCTCTGCTATATAGACCTCCC Primer the oligonucleotide consisting of the sequence shown in -3') (the recognition sequence of the restriction enzyme XbaI is in the square bracket of SEQ ID NO: 52, the recognition sequence of the restriction enzyme NotI is in the square bracket of SEQ ID NO: 53, and the mountain of SEQ ID NO: 53. A reaction solution having the composition shown in Table 2 is prepared as a restriction enzyme EcoRI recognition sequence in parentheses), and the reaction solution is heat-treated at 98 ° C. for 5 minutes, then the first step at 98 ° C. for 10 seconds, at 55 ° C. PCR was performed and the CMV promoter was amplified by repeating the reaction with the second step for 5 seconds and the third step for 90 seconds at 72 ° C. as one cycle for 30 cycles. Then, the PCR product obtained by purification by agarose gel extraction was named CMV-P.
(2-3) CMV-P prepared in (2-2) was digested with restriction enzymes XbaI and EcoRI, and purified using agarose gel extraction.
(2-4) The restriction enzyme digestion products obtained in (2-3) and each plasmid prepared in (1-3) excluding EFdpro were ligated. By transforming Escherichia coli JM109 strain with a ligation product and extracting a plasmid from the cultured transformant, an anti-human gp130 receptor antibody H chain expression vector pCMV-AHsH containing CMVpro, and an anti-human gp130 receptor containing CMVpro. The antibody L chain expression vector pCMV-ALsL was obtained.

(3) Preparation of antibody expression vector containing CAG promoter (3-1) The polynucleotide of the CAG promoter region (CAGpro) consisting of the sequence shown in SEQ ID NO: 54 was totally synthesized and cloned into a plasmid (consigned to GENEWIZ).
(3-2) Using the plasmid synthesized in (3-1) as a template, SEQ ID NO: 55 (5'-AGA [TCTAGA] GTGAGCCCACGTTCTGCTTCAC-3') and SEQ ID NO: 56 (5'-[GCGGCCGC] TTT <GAATTC> GCCGCCGGTCACACGCCAGAGAGCC. -3') Primer (restriction enzyme XbaI recognition sequence in the square bracket of SEQ ID NO: 55, restriction enzyme NotI recognition sequence in the square bracket of SEQ ID NO: 56, and mountain bracket of SEQ ID NO: 56 A reaction solution having the composition shown in Table 6 was prepared as a restriction enzyme EcoRI recognition sequence), and after heat-treating the reaction solution at 98 ° C. for 5 minutes, the first step at 98 ° C. for 10 seconds and the first step at 55 ° C. for 5 seconds. PCR was performed by repeating the reaction of 2 steps, the 3rd step for 90 seconds at 72 ° C. as 1 cycle for 30 cycles, and the CAG promoter was amplified. Then, the PCR product obtained by purification by agarose gel extraction was named CAG-P.
(3-3) CAG-P prepared in (3-2) was digested with restriction enzymes XbaI and EcoRI, and purified using agarose gel extraction.
(3-4) The restriction enzyme digestion products obtained in (3-3) and each plasmid prepared in (1-3) excluding EFdpro were ligated. By transforming Escherichia coli JM109 strain with a ligation product and extracting a plasmid from the cultured transformant, an anti-human gp130 receptor antibody H chain expression vector pCAG-AHsH containing CAGpro, and an anti-human gp130 receptor containing CAGpro. The antibody L chain expression vector pCAG-ALsL was obtained.

(4) The antibody expression vectors (6 types in total) obtained in (1) to (3) were sequence-analyzed by the same method as in Example 1 (14). As primers for sequencing, pSV40-AHsH, pCMV-AHsH, SEQ ID NOs: 15 and 34, pCAG-AHsH, SEQ ID NOs: 15, 34 and 57 (5'-CGTACGGAGCCCCGCACCCGAAGCCGG-3'), pSV40-ALsL. , PCMV-ALsL used SEQ ID NOs: 15 and 37, and pCAG-ALsL used oligonucleotides consisting of the sequences set forth in SEQ ID NOs: 15, 37 and 57.

As a result of sequence analysis, it was confirmed that all of the antibody expression vectors contained the promoter as designed.

Example 4 Comparison of antibody expression levels (Part 2)
As antibody expression vectors, pEFd-AHsH and pEFd-ALsL prepared in Example 2, pEF1α-AHsH and pEF1α-ALsL prepared in Comparative Example 1, pSV40-AHsH and pSV40-ALsL, pCMV-AHsH prepared in Comparative Example 3 And pCMV-ALsL, and pCAG-AHsH and pCAG-ALsL were used, and the expression level of the antibody after culturing for 3 days was compared by ELISA in the same manner as in Example 3.

The results when the CHO cell K1 strain was used as the host are shown in FIG. 4, and the results when the COS cells were used as the host are shown in FIG. Regardless of which host is used, the promoter of the present invention, EFdpro (deficient in nucleotides from 621st guanine to 798th guanine in EF1apro (SEQ ID NO: 12)), is used as a promoter. It can be seen that the antibody expression level is improved as compared with (SV40pro, CMVpro, CAGpro).

Example 5 Preparation of antibody expression vector (No. 5)
The antibody expression vectors pEFd-AHsH and pEFd-ALsL prepared in Example 2 were prepared by modifying the secretory signal sequences.

(1) Using pFU-hG1 prepared in Example 2 (1) as a template, SEQ ID NO: 58 (5'-ACAGGGGTCAATTCCACAGGTTCACTACG-3'), SEQ ID NO: 59 (5'-GCCTTTCCCCCGGGCCCAGGTTCAACTCCAG-3'), SEQ ID NO: 60 (5). The oligonucleotide consisting of the sequence set forth in any of'-CTCTTCTGCCTACTCTCAGGTTCAACTCCAG-3') and SEQ ID NO: 61 (5'-CTTGTCACCAAATTTCGCAGGGTCAACTCCAG-3') is used as a forward primer, and the oligonucleotide consisting of the sequence set forth in SEQ ID NO: 25 is used as a reverse primer. The reaction solution having the composition shown in Table 4 is prepared, and after heat-treating the reaction solution at 98 ° C. for 5 minutes, the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, and 72 ° C. PCR was performed by repeating the reaction with the third step for 60 seconds as one cycle for 30 cycles, and purification was performed by agarose gel extraction. Among the obtained PCR products, the PCR product obtained when the oligonucleotide consisting of the sequence shown in SEQ ID NO: 58 was used as a forward primer was named BH-P, and the oligonucleotide consisting of the sequence shown in SEQ ID NO: 59 was named BH-P. The PCR product obtained when the above was used as the forward primer was named CH-P, and the PCR product obtained when the oligonucleotide consisting of the sequence shown in SEQ ID NO: 60 was used as the forward primer was named DH-P. , The PCR product obtained when the oligonucleotide consisting of the sequence shown in SEQ ID NO: 61 was used as a forward primer was named EHP-P.

(2) Using the pFU-hL prepared in Example 2 (2) as a template, SEQ ID NO: 62 (5'-CCAGGCTCACTGGGTGACATTCAGATGAC-3'), SEQ ID NO: 63 (5'-GCCTTTCCCGGGCCGACATTCAGATGAC-3'), SEQ ID NO: 64 (5). The oligonucleotide consisting of the sequence set forth in any of'-CTCTTCTGCCTACTCTGACATTCAGATAGAC-3') and SEQ ID NO: 65 (5'-CTTGTCACCACACTCGGACATTCAGATGAC-3') is used as a forward primer, and the oligonucleotide consisting of the sequence set forth in SEQ ID NO: 27 is used as a reverse primer. The reaction solution having the composition shown in Table 4 is prepared, and after heat-treating the reaction solution at 98 ° C. for 5 minutes, the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, and 72 ° C. PCR was performed by repeating the reaction with the third step for 60 seconds as one cycle for 30 cycles, and purification was performed by agarose gel extraction. Among the obtained PCR products, the PCR product obtained when the oligonucleotide consisting of the sequence shown in SEQ ID NO: 62 was used as a forward primer was named BL-P, and the oligonucleotide consisting of the sequence shown in SEQ ID NO: 63 was named BL-P. The PCR product obtained when the above was used as the forward primer was named CL-P, and the PCR product obtained when the oligonucleotide consisting of the sequence shown in SEQ ID NO: 64 was used as the forward primer was named DL-P. , The PCR product obtained when the oligonucleotide consisting of the sequence shown in SEQ ID NO: 65 was used as a forward primer was named EL-P.

(3) A polynucleotide encoding an anti-human gp130 receptor antibody H chain to which each signal sequence was added was prepared.

(3-1) The signal sequence set forth in SEQ ID NO: 66 using BH-P prepared with the polynucleotide (1) encoding the anti-human gp130 receptor antibody H chain to which the signal sequence BH (SEQ ID NO: 66) was added as a template. The polynucleotide having the sequence described in SEQ ID NO: 67 (5'-ATGAAATGCAGCTGGTATTTTCTTCTCTGCTGGCAGGTGGTTACAGGGGTCAATTCA-3'), which is a polynucleotide encoding BH (MKCSWVIFFLLAVVTGVNS), is used as a signal DNA, and SEQ ID NO: 68 is used as a signal DNA. Using the oligonucleotide consisting of the sequence shown in') and SEQ ID NO: 25 as a primer (the recognition sequence of the limiting enzyme EcoRI in the square brackets), prepare a reaction solution having the composition shown in Table 5, and use the reaction solution at 98 ° C. for 5 After the heat treatment for 1 minute, PCR was performed by repeating the reaction of 30 cycles including the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, and the third step at 72 ° C. for 90 seconds. The polynucleotide encoding the anti-human gp130 receptor antibody H chain to which the signal sequence BH was added was amplified. Then, the PCR product obtained by purification by agarose gel extraction was named BHs-P.

(3-2) The signal sequence set forth in SEQ ID NO: 69 using CH-P prepared with the polynucleotide (1) encoding the anti-human gp130 receptor antibody H chain to which the signal sequence C (SEQ ID NO: 69) was added as a template. The polynucleotide having the sequence described in SEQ ID NO: 70 (5'-ATGACCCGCGCTGACCGTGCTGGCCCTGCTGCGCTGGCCTGCTCGCCTCTTCCCGGGCC-3'), which is a polynucleotide encoding C (MTRLTVALLAGLLASSRA), is used as a signal DNA, and SEQ ID NO: 71 (5'CCGCCACCGATCCGCCACGCCACCGCCACGCCACGCCACGCCACGCCACGCCACGCCACGCCACGCCACGCCACGCCACGCCACGCCACGCCACGCCGCCCGCC. The oligonucleotide consisting of the sequence shown in 3') and SEQ ID NO: 25 was PCR and purified in the same manner as in (3-1) as a primer (the recognition sequence of the restriction enzyme EcoRI in the square brackets). The polynucleotide encoding the anti-human gp130 receptor antibody H chain to which the signal sequence C was added, which is the obtained PCR product, was named CHs-P.

(3-3) The signal sequence set forth in SEQ ID NO: 72 using DH-P prepared with the polynucleotide (1) encoding the anti-human gp130 receptor antibody H chain to which signal sequence D (SEQ ID NO: 72) was added as a template. The polynucleotide consisting of the sequence described in SEQ ID NO: 73 (5'-ATGAAGTGGGGTCACTTCACTCTCTTGCTGTTCGTTCTTCTTGCCTACCT-3'), which is a polynucleotide encoding D (MKWVTFISLLFLFSSAYS), is used as a signal DNA, and SEQ ID NO: 74 (5'-GTGACCAT) The oligonucleotide consisting of the sequence shown in 3') and SEQ ID NO: 25 was PCR and purified in the same manner as in (3-1) as a primer (the recognition sequence of the restriction enzyme EcoRI in the square brackets). The polynucleotide encoding the anti-human gp130 receptor antibody H chain to which the signal sequence D was added, which is the obtained PCR product, was named DHs-P.

(3-4) The signal sequence set forth in SEQ ID NO: 75 using EH-P prepared with the polynucleotide (1) encoding the anti-human gp130 receptor antibody H chain to which the signal sequence E (SEQ ID NO: 75) was added as a template. The polynucleotide having the sequence described in SEQ ID NO: 76 (5'-ATGTACAGGATGCAACTCCCTGTCTTGCATTGCACTAAGTCTGCACTTGTCACCAATTTCG-3'), which is a polynucleotide encoding E (MYRMQLSCIALSLALVTNS), is used as a signal DNA, and the polynucleotide consisting of the sequence described in E (MYRMQLSCIALSLALVTNS) is used as a signal DNA, and SEQ ID NO: 77. The oligonucleotide consisting of the sequence shown in 3') and SEQ ID NO: 25 was PCR and purified in the same manner as in (3-1) as a primer (the recognition sequence of the restriction enzyme EcoRI in the square brackets). The polynucleotide encoding the anti-human gp130 receptor antibody H chain to which the signal sequence E was added, which is the obtained PCR product, was named EHs-P.

(4) A polynucleotide encoding an anti-human gp130 receptor antibody L chain to which each signal sequence was added was prepared.

(4-1) The signal sequence set forth in SEQ ID NO: 78, using BL-P prepared with the polynucleotide (2) encoding the anti-human gp130 receptor antibody L chain to which the signal sequence BL (SEQ ID NO: 78) was added as a template. The polynucleotide having the sequence described in SEQ ID NO: 79 (5'-ATGGAGTCAGACACATCCCTGTCATGGTGTGCTGCTGCTCTGGGTTCCAGGTCCACTGGT-3'), which is a polynucleotide encoding BL (MESDTLLLWVLLLWVPGSTG), is used as a signal DNA, and SEQ ID NO: 80 is used as a signal DNA. The oligonucleotide consisting of the sequence shown in 3') and SEQ ID NO: 27 was PCR and purified in the same manner as in (3-1) as a primer (the recognition sequence of the restriction enzyme EcoRI in the square brackets). The polynucleotide encoding the anti-human gp130 receptor antibody L chain to which the signal sequence BL was added, which is the obtained PCR product, was named BLs-P.

(4-2) Using CL-P prepared from the polynucleotide (2) encoding the anti-human gp130 receptor antibody L chain to which the signal sequence C (SEQ ID NO: 69) was added as a template, from the sequence set forth in SEQ ID NO: 70. The polynucleotide encoding the signal sequence C (SEQ ID NO: 69) is used as the signal DNA, and the oligonucleotide consisting of the sequences set forth in SEQ ID NO: 71 and SEQ ID NO: 27 is used as a primer, and PCR and purification are carried out in the same manner as in (3-1). did. The polynucleotide encoding the anti-human gp130 receptor antibody L chain to which the signal sequence C was added, which is the obtained PCR product, was designated as CLs-P.

(4-3) From the sequence set forth in SEQ ID NO: 73, DL-P prepared with the polynucleotide (2) encoding the anti-human gp130 receptor antibody L chain to which the signal sequence D (SEQ ID NO: 72) was added was used as a template. The polynucleotide encoding the signal sequence D (SEQ ID NO: 72) is used as the signal DNA, and the oligonucleotide consisting of the sequences set forth in SEQ ID NO: 73 and SEQ ID NO: 27 is used as a primer, and PCR and purification are carried out in the same manner as in (3-1). did. The polynucleotide encoding the anti-human gp130 receptor antibody L chain to which the signal sequence D was added, which is the obtained PCR product, was designated as DLs-P.

(4-4) From the sequence set forth in SEQ ID NO: 76, using EL-P prepared with the polynucleotide (2) encoding the anti-human gp130 receptor antibody L chain to which the signal sequence E (SEQ ID NO: 75) was added as a template. The polynucleotide encoding the signal sequence E (SEQ ID NO: 75) is used as the signal DNA, and the oligonucleotide consisting of the sequences shown in SEQ ID NO: 77 and SEQ ID NO: 27 is used as a primer, and PCR and purification are carried out in the same manner as in (3-1). did. The polynucleotide encoding the anti-human gp130 receptor antibody L chain to which the signal sequence E was added, which is the obtained PCR product, was designated as ELs-P.

(5) BHs-P, CHs-P, DHs-P and EHs-P prepared in (3), and pEFd prepared in Example 1 were digested with restriction enzymes EcoRI and NotI, respectively, and purified. BHs-P, CHs-P, DHs-P or EHs-P treated with restriction enzymes were ligated to pEFd treated with restriction enzymes.

(6) The E. coli JM109 strain was transformed with the ligation product obtained in (5), and the plasmid was extracted from the cultured transformant to encode an anti-human gp130 receptor antibody H chain to which the signal sequence BH was added. Anti-human gp130 receptor antibody H chain-encoding antibody expression vector pEFd- CsH, an antibody expression vector pEFd-DsH containing a polynucleotide encoding an anti-human gp130 receptor antibody H chain to which signal sequence D (SEQ ID NO: 72: secretory signal of human serum albumin) is added, and signal sequence E (SEQ ID NO: 75). : An antibody expression vector pEFd-EsH containing a polynucleotide encoding an anti-human gp130 receptor antibody H chain to which a human IL-2 secretory signal) was added was obtained.

(7) BLs-P, CLs-P, DLs-P and ELs-P prepared in (4), and pEFd prepared in Example 1 were digested and purified with restriction enzymes EcoRI and NotI, respectively. BLs-P, CLs-P, DLs-P or ELs-P treated with restriction enzymes were ligated to pEFd treated with restriction enzymes.

(8) By transforming the E. coli JM109 strain with the ligation product obtained in (7) and extracting the plasmid from the cultured transformant, the anti-human gp130 receptor antibody L chain to which the signal sequence BL was added was encoded. Anti-human gp130 receptor antibody L chain-encoding antibody expression vector pEFd- CsL, an antibody expression vector pEFd-DsL containing a polynucleotide encoding an anti-human gp130 receptor antibody L chain to which signal sequence D (SEQ ID NO: 72: secretory signal of human serum albumin) is added, and signal sequence E (SEQ ID NO: 75). : An antibody expression vector pEFd-EsL containing a polynucleotide encoding an anti-human gp130 receptor antibody L chain to which a human IL-2 secretion signal) was added was obtained.

Example 6 Comparison of antibody expression levels (Part 3)
We investigated the difference in antibody expression level due to the difference in secretory signal sequence.

As antibody expression vectors, pEFd-AHsH and pEFd-ALsL prepared in Example 2, pEFd-BHsH and pEFd-BLsL prepared in Example 5, pEFd-CsH and pEFd-CsL, pEFd-DsH and pEFd-DsL, and The expression level of the antibody after culturing for 3 days was compared by ELISA in the same manner as in Example 3 except that pEFd-EsH and pEFd-EsL were used.

The results when the CHO cell K1 strain was used as the host are shown in FIG. 6, and the results when the COS cells were used as the host are shown in FIG. Regardless of which host was used, the expression level of the antibody was highest when the signal sequence C (SEQ ID NO: 69: secretory signal of azlocidine precursor) was used as the secretory signal, and hereinafter, the signal sequence B (H chain: SEQ ID NO:) was used. 66, L chain: SEQ ID NO: 78), signal sequence A (H chain: SEQ ID NO: 28, L chain: SEQ ID NO: 31), signal sequence D (SEQ ID NO: 72: secretory signal of human serum albumin), signal sequence E (sequence). The antibody expression level was higher in the order of No. 75: human IL-2 secretion signal).

Claims (7)

A polynucleotide in which the region from at least 621st guanine to 798th guanine is deleted from the nucleotide sequence set forth in SEQ ID NO: 12 and comprises the nucleotide sequence set forth in SEQ ID NO: 18 . The promoter comprising the polynucleotide according to claim 1 . A vector comprising the polynucleotide encoding the promoter and protein according to claim 2 . The vector according to claim 3 , wherein the protein is an H chain or an L chain of an antibody. A transformant obtained by transforming a mammalian cell with the vector according to claim 3 or 4 . The transformant according to claim 5 , wherein the mammalian cell is a CHO cell or a COS cell. A step of producing a recombinant protein by culturing the transformant according to claim 5 or 6 , and a step of recovering the recombinant protein produced from the obtained culture. Production method.

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