KR100493703B1 - Recombinant Vectors Useful in Animal Cells and Expression Vectors for Preparing Erythropoietin - Google Patents

Abstract

The present invention relates to a recombinant vector and a method for producing erythropoietin using the vector to achieve a vector amplification, and more particularly, a low affinity binding site in the binding sequence of the methylated DNA binding protein as a high affinity binding site. the mutated methylated DNA binding promoter of cytomegalovirus comprising a binding sequence of the protein and a replication origin which operate in operatively dhfr and eukaryotic cells associated with the said promoter dhfr comprising ^- a vector and the vector for animal cells, erythropoietin It relates to an expression vector inserted with the gene encoding the, and a method for producing erythropoietin using the same.

Description

Recombinant Vectors Useful in Animal Cells and Expression Vectors for Preparing Erythropoietin for Mass Production of Recombinant Vectors and Erythropoietin for Animal Cells

The present invention relates to a vector and a method for producing erythropoietin using the vector, and more particularly, to a recombinant vector and a method for producing erythropoietin using the vector to achieve excellent vector amplification.

Erythropoietin (EPO) is an acidic glycoprotein hormone, also known as an erythropoiesis-stimulating factor, that differentiates erythrocytes produced in the bone marrow into red blood cells. Human EPO consists of 166 amino acids and contains three N-linked sugar chains and one O-linked sugar chain, and the entire sugar chain corresponds to 40% of the molecular weight of this glycoprotein hormone.

Human EPO is a hormone that controls the final maturation and growth of erythrocyte progenitor cells. It is mainly produced in fetal liver or adult kidney, and the amount of human EPO in the blood is increased in hypoxic blood, resulting in the development of mature oxygenated red blood cells. Regulate production (Krystal et al., ExpHematol. 11: 649-660 (1983))

In healthy people, EPO is usually present in the blood of 10-20 mIU / ml, and in the event of a massive bleeding caused by an accident, the oxygen concentration in the human body is reduced, the production of EPO is stimulated and its productivity is at least 1000 times. Increases. In addition, abnormalities in the kidneys may lead to decreased production of EPO, resulting in severe anemia (Jacobson, et al., Nature , 179: 633-634 (1957)).

Therefore, EPO has been used to treat anemia that does not produce EPO due to kidney damage due to sickle cell anemia, erythrolytic anemia, hemophilia, physiological anemia, excessive bleeding before and after surgery, and chronic renal failure.

Until now, clinically used EPO was mainly extracted from sheep's plasma, human urine, and urine in patients with aplastic anemia. Therefore, in order to manufacture a large amount of human EPO, a method of recombination technology has been attempted. Specifically, cDNA of human EPO gene is cloned to reveal its DNA base sequence, and the expression vector containing the gene is derived from a mammalian cell CHO cell line. A method of producing pure EPO by transforming a dihydrofolate reductase (dhfr) mutant cell line (ATCC CRL 9096) or the like has been attempted.

On the other hand, natural human EPO is an acidic, monomeric glycoprotein with a total molecular weight of about 34-38 kDa and a molecular weight of only the protein part excluding sugar is about 18,000 Da (Wang et al., Endocrinology , 166: 2286- 2292 (1985). Since the sugar chain portion of about 40% of the total molecular weight in human EPO plays an essential role in the in vivo activity of human EPO, when the sugar content of human EPO is reduced, the biological half-life of the human EPO molecule is drastically reduced, resulting in in vivo biological activity of human EPO. This is lost. There is a growing awareness of the importance of sugar chain structures in human EPO, which leads to many problems when producing human EPO from heterogeneous host cells using genetic recombination techniques.

In the expression and production of glycoprotein hormone using genetic recombination technology, Escherichia coli, yeast, insect cells or animal cells may be generally used as host cells, but they are particularly different from human EPO because they have different glycation or glycosylation systems. Likewise, in order to prepare glycoprotein hormones in which sugar plays an important role in the in vivo activity of hormones, the glycosylation capacity or glycosylation system should be considered first when selecting host cells.

E. coli has no glycosylation capacity in host cells, and in the case of yeast or insect cells, it has glycosylation ability, but human EPO production is mainly because of low glycosylation ability to produce high-mannose-type glycoprotein or complex glycoprotein such as human EPO It is not suitable as a host cell for. On the other hand, hamster cells such as mammalian cell CHO cells are characterized by excellent ability to produce complex glycoprotein hormones such as human EPO in its glycosylation system. Moreover, CHO cells, which are Chinese hamster ovary cells, are the most used host cells for biopharmaceutical production, and have the advantage of high productivity by gene amplification using a dihydrofolate reductase-deficient cell line. Accordingly, CHO cells, which are hamster cell types, especially CHO / dhfr-, which are dehydrofolate reductase-deficient cells, can be usefully used as host cells for producing human EPO which is safe in the human body and exhibits high activity. have.

Transformed CHO cells can efficiently increase the production of EPO by amplifying the EPO gene by gradual adaptation to medium containing MTX (Malik et al., DNA and Cell Bio. , 6: 453-9 (1992). )).

Throughout this specification, a number of articles are referenced and their citations are indicated. The disclosures of cited articles are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

Accordingly, it is an object of the present invention to provide a vector for dhfr ^- animal cells.

Another object of the present invention is to provide an expression vector for dhfr ^- animal cells for mass production of erythropoietin.

Another object of the present invention to provide a dhfr ^- animal cell line transformed by the vector.

Still another object of the present invention is to provide a method for preparing erythropoietin.

According to an aspect of the present invention, the present invention relates to a promoter of cytomegalovirus and a promoter comprising a binding sequence of a methylated DNA binding protein in which a low affinity binding site in a binding sequence of a methylated DNA binding protein is transformed into a high affinity binding site. Provided are dhfr ^- animal cell vectors comprising an operatively linked dhfr and a replication origin that operates in eukaryotic cells.

According to another aspect of the present invention, the present invention provides a promoter of cytomegalovirus comprising a binding sequence of a methylated DNA binding protein in which a low affinity binding site in a binding sequence of a methylated DNA binding protein is transformed into a high affinity binding site. For mass production of erythropoietin comprising dhfr operably linked and origin of replication in eukaryotic cells, genes encoding erythropoietin and promoters in eukaryotic cells operably linked to the erythropoietin gene. dhfr ^- provides an expression vector for animal cells.

The present invention provides a large amount of vector and erythropoietin, which achieves excellent amplification of the vector at low concentrations of inhibitors of dihydrofolate reductase, particularly methotrexate (MTX), in animal cells transformed with dhfr ⁻ . It relates to an expression vector for. As used herein, the term "dhfr ^- animal cell" refers to an animal cell that is not normally expressed in dihydrofolate reductase (DHFR), so that there is little or no activity of the enzyme in the cell. The present invention utilizes the principle of gene amplification including a DHFR gene (hereinafter referred to as "dhfr") for the characterization and selection of the host cell. In other words, when dhfr ^- animal cells are transformed with dhfr-containing DNA (eg, a vector), and the transformed cells are treated with a DHFR inhibitor, the cells with amplified dhfr-containing vectors are selected. . As a result, amplification of the vector is achieved.

According to a preferred embodiment of the present invention, the vector is constructed to achieve effective vector amplification even with low concentrations of DHFR inhibitors. For this strategy, artificially reduce the transcriptional activity of a promoter operatively linked to dhfr. The promoter is a promoter of cytomegalovirus (hereinafter referred to as "CMV") commonly used in animal cells, and more preferably is an immediate early promoter of CMV. To reduce the activity of this promoter, among the various transcriptional regulatory sequences in the promoter, the binding sequence of the methylated binding protein is mutated. The methylated DNA binding protein is one of the transcriptional regulators, and the CMV promoter has three binding sequences for the methylated DNA binding protein. Two of them are high affinity binding sequences and the other one is low affinity binding sequences (Zhang, XY et al., Virology , 182: 865-869 (1991)).

The present invention mutates the low affinity binding sequence among the binding sequences. The mutation is a mutation of a low affinity binding sequence into a high affinity binding sequence, which reduces the transcriptional activity of the promoter operably linked to the dhfr gene (Xian-Yang Zhang et al., Nucl. Aci. Res. 23: 3026-3033 (1995)), eventually inhibiting the transcription of dhfr linked to the promoter in which this mutation occurred. The mutation of the low affinity binding site to the high affinity binding site is most preferably the mutation of the ATCGCCTGGAGA sequence to GTTGCCATAGTG.

Promoters operably linked to the above-mentioned dhfr gene most preferably comprise the nucleotide sequence of SEQ ID NO: 1 attached and functional equivalents thereof.

According to a preferred embodiment of the present invention, the animal cells are CHO (Chinese Hamster Ovary) cells, because CHO cells deficient in DHFR have been widely used by the FDA has been approved for safety and efficacy.

Replication origins that operate in eukaryotic cells included in the vectors of the present invention include, but are not limited to, f1 origin, SV 40 origin, pMB1 origin, Adeno origin, AAV origin and BBV origin.

Most preferably, the dhfr ^- animal cell vector of the present invention is pCBE4 with the genetic map shown in FIG. 6. In Figure 6, "mutated CMV promoter" means that the low affinity binding site of the binding sequence of the methylated DNA binding protein in the CMV promoter sequence is mutated to a high affinity binding site, "pEM" refers to the EM-7 promoter sequence do.

In the expression vector for producing a large amount of erythropoietin of the present invention, the gene for coating the inserted erythropoietin is gDNA (genomic DNA) or cDNA (complementary DNA), erythropoietin closer to the natural form GDNA is more preferable to obtain.

In the expression vector of the present invention, the promoter operating in the eukaryotic cells operably linked to the erythropoietin gene is an immediate early promoter of cytomegalovirus, a promoter of SV40 (SV 40 late promoter and SV 40 early promoter), HSV ( tk promoter of herpes simplex virus), Adeno 2 major late promoter P _Adml , adenovirus 2 early promoter (P _AdE2 ), p19 promoter of human parvo virus-associated virus (AAV), and metal of mouse Rothionine promoter (metallothionein), MT promoter and MMTV LTR promoter and the like can be used, but is not limited thereto.

As the transcription termination sequence, the expression vector of the present invention includes a polyadenylation sequence, and for example, an SV40 polA sequence and a BGH polA sequence may be used.

According to the most preferred embodiment of the present invention, the expression vector for animal cells for mass production of the erythropoietin is pCBE5M having the gene map shown in FIG. In Figure 8, "mutated CMV promoter" means that the low affinity binding site of the binding sequence of the methylated DNA binding protein in the CMV promoter sequence is mutated to a high affinity binding site.

According to another aspect of the present invention, the present invention provides a dhfr ^- animal cell line transformed with the dhfr ^- animal cell vector described above.

According to another aspect of the present invention, the present invention provides a dhfr ^- animal cell line transformed with an expression vector for animal cells for mass production of the above-mentioned erythropoietin. In the most preferred embodiment of the invention, said animal cell line is a CCHO E-2 cell line (KCTC 10023BP).

Erythropoietin expressed by such an animal cell line exhibits almost the same biochemical and physical properties as natural human erythropoietin. For example, modification by sialic acid, which determines physiological activity in human erythropoietin, also has the same pattern in erythropoietin expressed by the cell line of the present invention.

In the present invention, a method of transforming dhfr ^- animal cells with an expression vector is micro-injection (Capecchi, MR, Cell , 22: 479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology , 52 : 456 (1973)), electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982)), liposome-mediated transformation (Wong, TK et al., Gene , 10:87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol. , 5: 1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci. , 87 : 9568-9572 (1990)).

According to another aspect of the present invention, the present invention comprises the steps of (a) culturing the cell line for producing the above-mentioned erythropoietin in large quantities under the addition of an inhibitor of dihydrofolate reductase; And (b) provides a method for producing erythropoietin comprising the step of purifying the erythropoietin antigen in culture.

In a preferred embodiment of the present invention, the inhibitor of the dihydrofolate reductase, for example, aminopterin (aminopterin) and methotrexate (MTX) and the like, but is not limited thereto, most preferably methotrexate.

Since MTX used for gene amplification is expensive, it is not an important consideration in laboratory experiments conducted at the in vitro level, but it is an important consideration when carried out at a high volume level. In addition, it takes a long period of more than 6 months to adapt the cells step by step to a high concentration, such as 1 μM concentration, there is a side effect that the cell growth is reduced by high concentration culture of MTX.

Thus, the art continues to study ways to reduce the concentration of MTX added for gene amplification. On the other hand, in the art, about 0.5-1 μM of MTX is used for gene amplification. The cell line used in the production method of the present invention is transformed so that gene amplification is performed well even at low concentrations of MTX. Therefore, the concentration of methotrexate added to the present invention is preferably 5-100 nM, more preferably 30-60 nM, and most preferably 40-50 nM. As can be seen in the following examples, when the MTX is 50 nM or more in the present invention, the rate of gene amplification is greatly reduced, considering both the price and the degree of amplification of MTX, the increase in MTX added is It loses meaning.

In the production method of the present invention, the step of culturing may be carried out in a medium commonly used in the present invention, and commercially available medium may be, for example, Hams F10 (Sigma), MEM (minimal essential medium, Gibco BRL), RPMI. -1640 (Sigma), DMEM (Gibco BRL), and the like. Ham and Wallace, Meth. Enz. , 58:44 (1979) and Barnes and Sato, Anal. Biochem. , 102: 255 (1980) can be used.

In the culturing step, the erythropoietin expressed in the host cell is secreted into the medium, and purification of the erythropoietin thus secreted yields a large amount of the desired erythropoietin. In the present invention, the purification step may be carried out by employing a purification method commonly used in the art. For example, solubility fractionation using ammonium sulfate or PEG, ultrafiltration separation according to molecular weight, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Various methods may be used and are usually purified using a combination of the above methods.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example 1 Cloning of the Human EPO Genome Gene

A probe for cloning the human EPO genome gene, with reference to the EPO genome gene sequence published by Fu-Kuen Lin, et al., Proc. Natl. Acad. Sci ., 82: 7580-7584 (1985). Yong oligo nucleotides were designed and synthesized. Synthetic oligonucleotide A is derived from the antisense strand of the EPO genome gene exon 2 and is 36-mer (5'-GCA GGG ACA GGA GAA GCC ACA GCC AGG CAG GAC ATT-3 ') and synthetic oligonucleotide B is the EPO genome gene It is derived from the sense strand of exon 5 and is 30-mer (5'-CAG GGG AGG CCT GCA GGA CAG GGG ACA GAT-3 '). The synthetic oligonucleotides A and B were labeled with γ-32P using the 5'-end labling kit (Amersham pharmacia Biotech, cat # RNN1509) and then used as probes.

In addition, the search by plaque hybridization with the bacteriophage-human fetal liver genome DNA library was performed in two stages using the in situ amplification method as follows: First, the human fetal liver genomic DNA library Λ bacteriophage with ATCC, cat # 37333) was applied at a density of 20,000 phages per 150 mm NZYAM Petri dish, incubated at 37 ° C. until small plaques (approximately 0.5 mm in diameter) were seen, and then at 1 ° C. Cool for hours. Duplicate replicas of plaque patterns were then transferred to nylon filters and incubated overnight at 37 ° C. in fresh NZYAM Petri dishes. After incubation, the nylon filter was treated in 0.5 N NaOH-1.5 M NaCl solution for 10 minutes, then in 0.5 M Tris (pH 7.5) -1.5 M NaCl solution for 10 minutes to denature and neutralize and vacuum dried at 80 ° C. for 2 hours. It was. The dried filter was washed for 1 hour in 6X SSC and 0.5% SDS solution and wiped off the cell pieces on the filter surface, then the filter was washed again with water, 50% (v / v) formamide, 6X SSC, 0.5% (w / v) was added to a prehybridization solution consisting of SDS and 5X Denhardt's solution and reacted at 42 ° C. for 4-8 hours. To this was added ³² P-labeled oligonucleotide probe A at a concentration of 0.1 pmol per ml and hybridization reaction was carried out at 42 ° C. for 72 hours. After the hybridization reaction, the filter was vigorously washed with 2X SSC-0.1% SDS solution at room temperature and further washed for 1 hour at room temperature with 1X SSC-0.1% SDS solution. The filter was placed on a paper towel and dried at room temperature and then exposed to X-ray film for 16 hours at -70 ° C. Subsequently, positive plaques were collected by confirming the position where a strong radiation signal was detected. Positive plaques selected in the primary search were once again secondary searched in the same manner as described above using ³² P-labeled oligonucleotide probe B.

In the above manner, the entire 1 × 10 ⁶ bacteriophage clone was searched in two steps to obtain the final five positive phage clones. DNA was isolated from five positive phage clones by a method used by Yamamoto et al. (Yamamoto et al., Virology, 40: 734-744, (1970)), and then the DNA sequence was analyzed using an autosequencer ABI 3100. As a result, it was confirmed that one clone had the same nucleotide sequence as the human EPO genome gene published by Lin et al., And this clone was named "CBEPO-C1" genome clone. Human EPO genome gene was isolated from the CBEPO-C1 genome clone in the same manner as above and cloned into pBluescript II SK (+) (Stratagene, Cat. # 212205) plasmid to prepare pCBE1 vector.

Example 2: pCBE2 Vector Preparation and Transient Transformation in COS Cells

The pCBE1 vector obtained in Example 1 was digested with restriction enzymes Xho I and Not I, followed by electrophoresis on a 1% agarose gel to isolate an EPO genome gene of about 2.2 kb in size, and a gel extraction kit (VIOGENE, EG1001) was used. After elution, pCBE2 vector was prepared by inserting into the animal cell expression vector pCI-neo (Promega, cat. # E1841) cut with restriction enzymes Xho I and Not I using a T4 DNA linking enzyme (see Fig. 1). .

COS-7 cells (ATCC 79942) 1 × 10 ⁵ were inoculated and cultured in 6-well plates containing DMEM medium (Dulbecco's Modified Eagle Media, GiBco BRL) containing 10% fetal calf serum. After 24 hours of incubation, 3 μl of FuGENE ^™ 6 (Roche) was diluted in 100 μl of serum-free DMEM medium, mixed by adding 2 μg of pCBE2 vector prepared above, and left at room temperature for 40 minutes. Then, transformed by putting evenly on the prepared COS-7 cells. After transformed cells were cultured in DMEM medium for 72 hours, the obtained cell culture was examined for EPO production using EPO-ELISA (Medac, EPO-ELISA medac 500). As a result, it was confirmed that the EPO gene obtained in Example 1 can be normally expressed in animal cells.

Example 3: Preparation of Recombinant Plasmid pCBE4

To obtain a cell line that expresses high EPO in a low concentration of MTX-containing medium, a pCBE3 vector was first prepared as follows (see FIG. 2): First, to modify the nucleotide sequence of the transcription factor binding site in the CMV promoter, the hCMV promoter was first used. Position-directed mutagenesis was performed by PCR using pZeo-SV2 (+) vector (Invitrogen) as a template. Pfu polymerase used for the first PCR was purchased from Solgenetech, and the total temperature was 30 cycles at annealing 55 ° C., 30 seconds at 72 ° C., and 30 seconds at 94 ° C. for denaturation. It was. The primers used for the primary PCR were set to 5'-ATCGATTCCGTTACATAACTT-3 '(forward primer) and 5'-TGGATGGCGTCACTATGGCAACCTGACGGT-3' (reverse primer) and 5'-TGAACCGTCAGGTTGCCATAGTGACGCCATCCA-3 '(forward primer) ) And 5'-TGCTAGCCGGTG TCTTCTATG-3 'were used as another set. This first-order PCR results in site-directed mutations.

Then, the secondary PCR was performed using the primary PCR amplification template as a forward primer 5'-ATCGATTCCGTTACSATAACTT-3 'and the reverse primer 5'-TGCTAGCCGGTGTCTTCTATG-3'. Primers were designed to generate Cla I and Nhe I restriction enzyme sites. The temperature cycle employed in the secondary PCR is the same as the primary PCR described above. The sequence of the primer with which the mutation was induced is shown in FIG. 3. In Figure 3, the bold is the portion where the mutation occurred, the ATCGCCTGGAGA sequence was changed to GTTGCCATAGTGA, the low-affinity binding site of the methylated DNA binding protein was mutated to a high affinity binding site.

The obtained second PCR amplified product was eluted using a gel extraction kit, and then inserted into a pGEM-T Easy vector (Promega, Cat. # A1380) to prepare pCBE3. The sequencing analysis confirmed that the sequence of the low-affinity binding site of the methylation DNA binding protein, a transcription factor of the CMV promoter, was converted.

Meanwhile, pZeo-SV2 (+) vector (Invitogen) and pSV2-dhfr vector (ATCC 67110) were treated with Hin dIII and Bam HI, respectively, to insert the dhfr gene included in pSV2-dhfr into pZeo-SV2 (+). -dhfr was made (see FIG. 4). Subsequently, the vector pCBE3 was digested with restriction enzymes Cla I and Nhe I and inserted into a vector pZeo-dhfr treated with the same enzyme to finally prepare pCBE4 (see FIG. 5).

Example 5: Preparation of pCBE5M Vectors and Selection of Recombinant CHO Cell Lines

The pCBE2 vector prepared in Example 2 was digested with restriction enzymes Bgl II and Not I, followed by electrophoresis on a 1% agarose gel to isolate an EPO genome gene fragment containing a CMV promoter of about 3.3 kb in size, followed by T4 DNA. The final EPO expression vector pCBE5M was prepared by inserting into the pCBE4 vector of Example 4 digested with restriction enzymes Bam HI and Not I using a ligase. The cleavage sites of Bgl II and Bam HI form cohesive ends and are connected to each other.

Then, 3 μl of FuGENE ^™ 6 (Roche) was diluted in 100 μl of serum-free IMDM medium (Iscove's Modified Dulbecco's Media, Gibco BRL), mixed with 2 μg of the prepared pCBE5M vector, and then mixed for 40 minutes. After allowing to stand at room temperature, the prepared CHO / dhfr ⁻ (ATCC CRL 9096) cells were evenly placed on the cells. Then, incubation for 72 hours in normal medium (IMDM medium containing 10% FBS and HT (0.1 mM sodium hypoxanthine, 0.016 mM thymidine)), followed by treatment with 0.25% trypsin (Gibco BRL) and centrifugation 1 × 10 ⁴ cells per well Cells were aliquoted into 96-well plates. Then incubation (37 ° C., 5% CO) by addition of α-MEM medium (Gibco BRL) containing 10% of dialyzed serum (Gibco BRL, 26300) and 500 μg / ml of zeocine (Invitrogen, R-250) ₂ concentration) and incubated for 2 weeks while changing the medium every 4-5 days. Two weeks later, 150 surviving clones were obtained, and EPO-ELISA (Medac, EPO-ELISA medac 500) was performed to measure the ability of each clone to produce EPO. Among them, the cell line producing the most EPO protein was named "CCHOE", and the cells were further divided into 96-well plates by 1 × 10 ² cells per well, and α-MEM medium containing MTX (Sigma) 5 nM. Incubated for 2 weeks with medium exchange at 4-5 day intervals. About the colonies produced in the concentration of MTX, the expression level of EPO was measured by the same ELISA method as the above method. The highest expression cell line was named "CCHOE-1", and the final EPO high expression cell line obtained by culturing in α-MEM medium containing 50 nM MTX was named "CCHO E-2". It was designated as "CCHO E-2" and was deposited with the Korean Collection for Type Cultures on July 25, 2001, and was given accession number KCTC 10023BP.

Example 6: Purification and Analysis of EPO Produced in Recombinant Cell Line

Example 6-1. Isolation and Purification of EPO

CHO-S-SFMII (Gibco BRL Cat. # 31033-020), a serum-free medium, was cultured for 2 days in α-MEM medium containing 10% of calf serum dialyzed with the transformed CCHO E-2 cell line obtained in Example 5. ) And cultured for 5 days. The precipitate obtained by adding the same amount of saturated ammonium sulfate solution to the culture was dissolved in 10 mM Tris-HCl buffer (pH 8.6) and dialyzed in the same buffer for 2 days. Subsequently, EPO was first isolated using a buffer solution in which 0.25 M NaCl was added by salt concentration gradient in FPLC using a Mono-Q column (Pharmacia, Mono-Q HR). To further purify the sample of the primary isolated EPO, it was injected again into the Sephacryl S-200 column (Pharmacia, Sephacryl S-200 HR) and the wash solution (0.5 M NaCl, 1 mM EDTA and 0.2 mM PMSF was added to 50 mM Tris buffer). One, pH 7.5) to finally perform gel filtration to purify EPO.

Example 6-2. SDS-PAGE Analysis

The purified EPO protein was electrophoresed on a 12% SDS-polyacrylamide gel, followed by Coomassie staining (AMRESCO, Coomassi Brilliant Blue R-250, # 0472-25G). As a result of analysis using SDS-PAGE, the recombinant EPO expressed in the transformed cell line appeared to be one band spread widely due to the characteristics of glycoprotein, and the molecular weight was 34-44 kD, which is almost the same molecular weight as the natural human EPO. (See FIG. 9). In FIG. 9, the middle lane is loaded with purified EPO.

Example 6-3. Immunoblotting Assay and ELISA Assay

① Immunoblotting Analysis

Mini-Blot Electrophoretic Transfer Cell (Bio-Rad, # 170-3930) after electrophoresis of the recombinant EPO protein purified in Example 6-1 with 12% SDS-polyacrylamide gel Adsorbed onto the NC membrane by electrophoresis method. Human EPO primary antibody (Genzyme, anti-EPO mouse polyclonal antibody, cat. # 1541-01) was reacted with the NC membrane and then the secondary antibody alkaline phosphatase-conjugated anti-mouse goat antibody (Sigma) And color development substrate BCIP / NBT was added. As a result, the recombinant protein expressed in the cell line CCHO E-2 was positive for human EPO antibody, and the signal was observed at the same position as SDS-PAGE, and the difference in the degree of glycosylation by glycation, in particular sialic acid, was observed. As a result, the band was spread slightly because it is composed of isomers, which caused small differences in molecular weight (see FIG. 10). In FIG. 10, the middle lane has loaded the EPO.

② ELISA analysis

EPO expressed in each of the cell lines CCHOE, CCHOE-1 and CCHO E-2 obtained in Example 5 was quantified using Medac's EPO-ELISA kit. In the selection medium containing the initial zeocin and in the selection medium at 5 nM-MTX concentrations, 30 and 120 IU / 10 ⁶ cells / 24 hr, respectively, were obtained. E-2 showed an expression level of 420 IU / 10 ⁶ cells / 24 hr (see FIG. 11). As clearly shown in FIG. 11, CCHO E-2 exhibits superior EPO production compared to other cell lines.

Example 6-4. Isoelectric focusing analysis

After concentrating the purified EPO sample, 100 μl of the concentrate was added and mixed with 100 μl of sample buffer (Novex, cat. # LC5311), followed by electrophoresis ( Novex, X-cell II Mini-Cell, cat). Place the IEF-gel (Novex, IEF PAGE pH 3-10, cat. # EC6655A) on an electrophoretic plate of # EI9001 and load with 5 μl of isoelectric standard (Novex) in three lanes of the IEF-gel. Then, electrophoresis was performed. After electrophoresis was completed, the solution was transferred to an IEF-gel fixation solution (Novex), shaken for 30 minutes, stained in a dye solution, and the bands on the IEF-gel were read. In the electric field with the pH gradient formed, the EPO protein formed 7-8 isomer bands over pH 3.5-5.3 depending on the degree of glycation (see FIG. 12). In FIG. 12, the middle lane has loaded EPO.

Example 7: Southern blotting analysis

In order to confirm that the EPO gene was inserted into the genome DNA of CHO cells in the cell line CCHO E-2 of the present invention, Southern blotting analysis was performed. For analysis, DIG-DNA labeling and detection kit (Roche, 1 093 657) was used. ^{. CHO / dhfr - (ATCC CRL} 9096) transfected with the salting-out of the cell line genomic DNA CCHO E-2 switch (salting out) method (Steffer, et al, Proc Nat Acad Sic, 76: 4554-4558 (.... 1979)) and then quantified. 5 μg of each genome isolated was digested with restriction enzyme Eco RI and electrophoresed on 0.8% agarose gel. The DNA in the agarose gel is then transferred to the nylon membrane (Amersham Pharmacia Biotech) by capillary method, and the resulting blot is hybridized with DIG-labeled probe (probe to identify EPO gene), followed by alkaline phosphatase- It was reacted with the conjugated anti-DIG antibody and finally developed by adding NBT / BCIP, which is a chromogenic substrate (see FIG. 13).

As a result, no signal was detected in CHO / dhfr ^- cells, but the signal of EPO gene was shown in the cell lines CCHO E-1 cell line and CCHO E-2 cell line of the present invention. As a result, the dhfr gene and the EPO gene were inserted into the genome DNA of the transformed cell line, and the signal of the CCHO E-2 cell line was stronger than that of the CCHO E-1 cell line, which was adapted to the higher MTX concentration and increased the expression of EPO. In the case of cell lines, it was confirmed that increased EPO expression was caused by gene amplification.

The present invention provides dhfr ^- vector for animal cells and expression vector for erythropoietin expression. The present invention also provides a dhfr ^- animal cell line transformed with the present vector. The present invention also provides a method for preparing erythropoietin. According to the present invention, not only a large amount of erythropoietin can be obtained by a lower concentration of DHFR inhibitor than the conventional method, but the obtained erythropoietin is glycosylated in the same way as the natural form, and thus has excellent physiological activity.

1 is a schematic diagram showing the construction of a pCBE2 vector;

2 is a schematic diagram showing the construction of a pCBE3 vector;

Figure 3 is a sequence diagram comparing the sequence of the promoter induced mutations by the present invention and the sequence and the sequence of the promoter contained in pZeoSV2;

4 is a schematic diagram showing the fabrication process of pZeo-dhfr vector;

5 is a schematic diagram showing a manufacturing process of pCBE4, which is one embodiment of the vector of the present invention;

6 is a genetic map of pCBE4, which is one embodiment of the vector of the present invention;

Figure 7 is a schematic diagram showing the manufacturing process of pCBE5M an embodiment of the expression vector of the present invention;

8 is a genetic map of pCBE5M, which is one embodiment of the expression vector of the present invention;

9 is a photograph showing the results of SDS-polyacrylamide gel electrophoresis on erythropoietin obtained according to the method of the present invention;

10 is a photograph showing the results of immunoblotting confirming the production of erythropoietin obtained according to the method of the present invention;

11 is a graph showing an ELISA result showing the expression level of erythropoietin produced by the cell line of the present invention;

12 is a photograph showing isoelectric focusing analysis results confirming glycosylation of erythropoietin obtained according to the method of the present invention; And

Figure 13 is a photograph showing the Southern blotting result to confirm whether the erythropoietin gene is inserted into the genome of the host cell in the cell line of the present invention.

Claims (17)

A promoter of cytomegalovirus comprising a binding sequence for a methylated DNA binding protein mutated to a high affinity binding site by mutating an ATCGCCTGGAGA sequence at a low affinity binding site in a binding sequence for a methylated DNA binding protein to a GTTGCCATAGTGA sequence, the promoter A dhfr- animal cell vector comprising a dhfr operatively linked to and a replication origin that operates in eukaryotic cells. A promoter of cytomegalovirus comprising a binding sequence for a methylated DNA binding protein mutated to a high affinity binding site by mutating an ATCGCCTGGAGA sequence at a low affinity binding site in a binding sequence for a methylated DNA binding protein to a GTTGCCATAGTGA sequence, the promoter Mass production of erythropoietin, including dhfr operatively linked to, origin of replication in eukaryotic cells, genes encoding erythropoietin and promoters in eukaryotic cells operatively linked to said erythropoietin gene Dhfr- expression vector for animal cells. delete The vector dhfr ^- animal cell vector according to claim 1 or 2, wherein the eukaryotic cell is a CHO cell. The method of claim 1 or 2, wherein the origin of replication in the eukaryotic cell is selected from the group consisting of f1 origin, SV 40 origin, pMB1 origin, Adeno origin, AAV origin and BBV origin Characterized by dhfr ^- vector for animal cells. The dhfr ^- animal cell vector according to claim 1, wherein the animal cell vector is pCBE4 having the gene map shown in FIG. The dhfr ^- expressing vector for animal cells according to claim 2, wherein the gene for coating the erythropoietin is gDNA or cDNA. 3. The promoter of claim 2, wherein the promoter acting in the eukaryotic cell operably linked to the erythropoietin gene is an immediate early promoter of the cytomegalovirus, an SV 40 late promoter, an SV 40 early promoter, a tk promoter of HSV, or adenovirus 2 major. Dhfr ^- animal for mass production of erythropoietin, characterized in that it is selected from the group consisting of late promoter, adenovirus 2 early promoter, p19 promoter of AAV and metallothionein promoter of mouse, MT promoter and MMTV LTR promoter Expression vector for cells. The method of claim 8, wherein the erythropoietin expression for an animal cell to produce a large amount of vector is dhfr, characterized in that pCBE5M having the gene map shown in Fig. 8 ^- the expression vector for animal cells (KCTC 10023BP). A dhfr ^- CHO (Chinese Hamster Ovary) cell line transformed with the vector of claim 6. A dhfr ^- CHO (Chinese Hamster Ovary) cell line transformed with the expression vector of claim 9. 12. The transformed dhfr ^- CHO (Chinese Hamster Ovary) cell line (KCTC 10023BP) according to claim 11, wherein said animal cell line is a CCHO E-2 cell line. Method for preparing erythropoietin comprising the following steps: (a) culturing the cell line of claim 12 under the addition of an inhibitor of dihydrofolate reductase; And (b) purifying erythropoietin in culture. The method of claim 13, wherein the inhibitor of dihydrofolate reductase is aminopterin or methotrexate. 15. The method of claim 14, wherein the inhibitor of dihydrofolate reductase is methotrexate. The method of claim 15, wherein the concentration of methotrexate is 5-100 nM. The method of claim 16, wherein the concentration of methotrexate is 30-60 nM.