KR920001379B1 - Method for producing epo - Google Patents

Abstract

A erythropoietin i.e. glycoprotein hormone is prepd. by (a) cutting lamda phage DNA with restriction enzyme ECoRI and selecting erythropoietin gene-contg. fragment, (b) inserting the fragment into the pBR322 and transforming into the E. coli HB101, (C) fermenting the transformed E. coli HB101 in the LB medium and collecting plasmid by EtBr-CsCl density gradient ultracentrifugation, (d) treating the obtd. plasmid with BSSH II and Bgl II to obtain the fragment (2374 base pair), (e) treating the obtd. framgment with T4 DNA polymerase, sticking to the BamHI linker, and inserting into the pDW-M to obtain the recombinant DNA vector pDW-MO931 (I), (f) transforming the (I) into the mammal cell to obtain the recombinant (KFCC 10670), and (g) culturing the BHK to obtain the human erythropoietin.

Description

Method for producing glycoprotein hormone using genetic recombination technology

1 is a BssHII-BglII base sequence containing an erythropoietin gene.

2 is a method for assembling plasmid pDW-2.

3 shows the assembly method of the plasmid vector pDW-M.

4 is a transgenic plasmid pDW-M 0931 containing an erythropoietin gene.

The present invention relates to the manipulation of genes and to the production of glycoprotein hormone by the recombinant method of human genes. More specifically, human erythropoietin gene sequences encoding human erythropoietin amino acid sequences having biological activity by recombinant genetic engineering techniques are inserted into plasmid vectors that self-replicate and transformed into appropriate eukaryotic or prokaryotic cells. The present invention relates to a method for continuously and stably producing human erythropoietin with high concentration. Erythropoietin is a glycoprotein hormone that is involved in the regulation of red blood cell counts in the blood and is promoted by carnot and deflandre (Compnot, Rend, Acad. Sci. 143; 384, 1906) to promote the formation of red blood cells after excess bleeding in the human body. Substances have been reported to be secreted into the blood, and experimental results with rats and observations of people living in highlands locally have supported this hypothesis experimentally (Stohlman. F., et al. Blood). 9, 721, 1954, Reissmann, KR, et al. Blood 5; 372, 1950.

Later, in 1969, the World Health Organization (WHO) set standards for biological quantification with erythropoietin derived from human urine (WHO, Expert Committee on Biological Standardization, Wld Hlth Orgtechn, Rep Ser no 413; 17, 17). 1969).

Erythrocyte production in humans occurs continuously throughout life, which is derived from erythrocyte progenitor cells, and erythropoietin promotes the differentiation and proliferation of these erythrocyte progenitor cells. Human erythropoietin is an acidic glycoprotein hormone that contains at least one disulfide bond and has α- and β-types with slightly different carbohydrate composition.

Carbohydrates make up 40% of the total molecular weight, and asialo-type erythropoietin (asialo-EPO) is inactive. In healthy people, 10 to 20 milliunits / milliliters are present in the blood, and the oxygen levels in the human body are reduced in high-land residents, large amounts of bleeding, erythrocyte destruction by some causes, and anemia. The production of ethine is stimulated and increased up to 1,000 times.

In the human body, erythropoietin is produced mostly in the epithelial cells of the kidneys. In other words, it is known by Jacobson et al. (Jacobson, LO, et al., Nature, 179; 633, 1657) that acute anemia is caused by a sharp decrease in erythropoietin when the market is damaged by the removal of the kidneys or by the binding of the ureters. .

Therefore, the area in which erythropoietin can be used is the treatment of anemia caused by sickle cell anemia, erythrolytic anemia, pre- and post-operative hyperbleeding and chronic renal failure due to a great damage to the kidneys, resulting in failure to produce erythropoietin.

These patients with anemia must receive blood transfusions regularly to receive insufficient red blood cells, which may lead to infections such as inflammatory virus or immunodeficiency syndrome virus, iron overload, and severe immunological side effects.

Thus, many attempts have been made to obtain erythropoietin in a relatively pure high yield, but it has not been successful, and the amount of erythropoietin to be purified is not enough to cure such a disease. Sherwood et al. (Sherwood, JB et al., Endocrinology, 99; 504, 1976) attempted to collect erythropoietin in tumor cells of the kidney but did not obtain satisfactory results, and Mlyake, T., et al. , J. Bioi. Chem. 252 (15); 5558, 1977) have isolated and purified relatively pure and high titers of erythropoietin from the urine of aplastic anemia patients.

This was followed by 7 steps including DEAE-ion exchange resin to finally obtain erythropoietin having a titer of 70,400-82,700 units / mg in 21% yield. Yanagawa et al. (Yanagawa, SI, et al., J. Biol. Chem. 259 (5), 2707, 1984) using monoclonal antibodies in a yield of 65% of erythropoietinol with a titer of 80,000 units / mg or more. Got it.

Erythropoietin isolated from plasma or urine is not enough to be used for therapeutic purposes. Purification of erythropoietin by single or multivalent antibodies may be useful for quantification and titer of. It was difficult to analyze the erythropoietin itself and obtain sufficient amount to be used clinically. Therefore, in order to produce erythropoietin in a large amount that can be used clinically with the biological characteristics of human erythropoietin, the EPO gene is isolated from human genomic DNA or cDNA, and the vector plasmid is well expressed in mammalian cells. Methods for producing large quantities of pure recombinant erythropoietin by transfection with mammalian cells after insertion are described in Jacobs (Jacobs, K, et al., Nature 313; 806, 1985); Lin, F.K., et al., Proc. Natl. Acad. Sci, USA 82; 7580, 1985; Powell (J. S. et al., Proc. Natl. Acad. Sci. USA, 83; 6465, 1986) and the like.

The present invention inserts a human erythropoietin gene sequence encoding a human erythropoietin amino acid sequence having biological activity from the human genome by recombinant genetic technology into a vector plasmid that is well expressed in eukaryotic or prokaryotic cells and then appropriately eukaryotic or The present invention relates to a method for producing human erythropoietin which is biologically active by transforming prokaryotic cells.

Recombinant human erythropoietin produced by the production method according to the present invention is essentially the same as human natural erythropoietin and can supply a sufficient amount to be used for clinical treatment.

In more detail, the separation of the DNA fragment containing the human erythropoietin gene was performed using probes of oligonucleotides labeled with radioactivity. Oligonucleotide probes are made based on the N- and C-terminal region sequences of erythropoietin proteins, and then, under specific conditions, DNAs similar to DNA sequences encoding human erythropoietin by simultaneously hybridizing N- and C-terminal probes simultaneously. Minimization of binding to sequences facilitates the isolation of DNA fragments containing erythropoietin genes. The fragment containing the isolated erythropoietin gene was treated with the restriction enzyme EcoRI, isolated on agalog gel, and used by the above-mentioned N-terminal probe by Southern method (J. Mol. Biol. 98; 503,1975). EcoRI fragments containing the erythropoietin gene were identified and inserted into the EcoRI site of the pBR 322 plasmid and transformed with E. coli HB101.

After plasmid separation only by density gradient centrifugation, BssHII-BglII restriction fragments treated with 5 'end and BglII end were collected and the sequence is shown in FIG. The erythropoietin DNA fragment of FIG. 1 digested with BssHII-BglII restriction enzyme is a secondary structure of mRNA for translational regulation that may be the result of transcription or DNA sequence at the 5 'end that controls the expression of the erythropoietin gene. Since the secondary structure is completely eliminated, the expression of the protein can be easily controlled and maximized according to the composition of the vector.

In addition, since the size of the erythropoietin gene containing the vector is small, the stability of the plasmid is expected to be enhanced in the host cell, and the synthesis of unnecessary mRNA sites is minimized, and thus the economical operation of the cell is also assumed. The BssHII-BglII restriction fragment of FIG. 1 is inserted into a mammalian expression plasmid vector and transformed into mammalian cells by calcium phosphate to transform mammalian cells into a large amount of recombinant human erythropoietin with biological activity. Created a transform.

The BssHII-BglII restriction fragments were directly linked to sequences in the vector plasmids engineered to maximize expression. The transcriptional promoter sequence of Simian virus 40 (SV40) in the vector plasmid promotes transcription of the gene (Sassone-Corsi, p, et al., Proc. Natl. Acad. Sci. USA 81; 308, 1984). The tripartite leader sequence of the virus enhances the translatability of the gene (Kaufmann, R, J., et al., Mol. Cell. Biol. 2; 1304,1982). The stability of gene expression was increased by adding SV40 early polyadenylation signal to existing regulatory sequences. The vector plasmid in which the erythropoietin gene is inserted as shown in Figure 2 of the present invention is recombined by linking sequences to enhance the transcription and translational ability of the gene at the 5 'and 3' ends of the erythropoietin gene having the shortest and most essential site. It can be said that the plasmid is constructed.

The usefulness of this recombinant plasmid can be seen by stably and continuously expressing high concentrations of erythropoietin after being transformed into mammalian animal cells, and erythropoietin by cotransfection and amplification of the DHFR gene. Gene stability and yield of erythropoietin can be greatly increased. Restriction enzymes, linkers and DNA used in the present invention were purchased from New England Biolabs unless otherwise noted. The invention is explained in more detail in the following examples, which do not limit the invention to the following examples.

Example 1

[Isolation of Genome Clones]

Lambda bacteriophage human genome library consisting of DNA fragments randomly isolated from human embryos under certain hybridization criteria such as Sugs (Proc. Natl. Acad. Sci USA 78; 6613, 1981). Genomic clones containing erythropoietin genes were isolated by hybridization using an oligonucleotide probe pool (Lawn, Cell 15; 1157, 1978). In other words, the oligonucleotide probe pool refers to the erythropoietin amino acid sequence of Jacobson (Nature, London 313; 806, 1985) and the sequence of -Glu-Ala-Glu-Asn-Ile at the N-terminal region and-at the C-terminal region. Reference was made to the sequence of Tyr-Thr-Gly-Glu-Ala. Codon selection for each amino acid is based on the rules of mammalian animal codons such as Nucleic acids Research 8 (43, 1981) and human amino acid sequence statistics of Radar (J. Mol. Biol. 183, 1, 1985). Was used.

The probe pool for the DNA sequence of Glu-Ala-Glu-Asn-Ile amino acid sequence (A / T / G) TA (A / G) TT (T / C) TC (A / T / C) CC (T / C) was synthesized with 15-mer oligonucleotides of TC and the probe pool for the DNA sequence of the Tyr-Thr-Gly-Glu-Ala amino acid sequence was (A / T / G / C) GC (T / C) TC (A / T-C) CC (A / T / G / C) GT (A / G) TA was synthesized with 15-mer oligonucleotides.

These oligonucleotides were labeled 5 ends with [γ- ³² P] ATP (ICN) using T4 polynucleotide kinase (T4 polynucleotide kinase, New England Biolabs Inc.).

Hybridization of the lambda bacteriophage with the human genome library and the probe pool was carried out by Belton (Science 196; 180, 1977). The bacterium for lambda bacteriophage infection was E. coli C620 (rm-) (Proc.Natl). Acad. Sci. USA 72; 3416, 1975). In order to prevent binding to very close sequences, including the substitution of a single base, the standard conditions of Sugs were introduced, and each of the three nitrocellulose filters on a lambda bacteriophage-induced plate ( erythrophilized by nitro-cellulose filter, hybridizing N-terminal probe to one replica, C-terminal probe to another, and N- and C-terminal probes to the other. Accuracy was given to the search for lambda bacteriophage with the poietin gene. Searching for approximately 7.0 × 10 ⁶ lambda bacteriophage identified three positive clones with simultaneous radioactivity in the N-terminal probe, the C-terminal probe, and the N- and C-terminal probes.

Example 2

[Isolation of Erythropoietin Gene Restriction Fragments]

Lambda bacteriophage DNA carrying the erythropoietin gene was isolated in large quantities by methods such as Maniatis (1982), and a portion thereof was completely cleaved with restriction enzyme EcoRI in 50 mmol sodium chloride, tris-hydrochloric acid buffer (pH 7.5), and denatured. Erythropoietin gene in EcoRI fragments by the method of Southern (J. Mol. Biol. 98; 503, 1975) using the probe of Example 1 which was isolated on 0.7% agarose gel and then radiolabeled. After identifying the fragments, the remaining lambda bacteriophage DNA was completely digested with EcoRI, separated and purified from 0.7% agarose, precipitated and concentrated with ethanol to collect the erythropoietin gene fragment, which was also digested completely with EcoRI, followed by bacterial alkaline phosphatase ( Bacterial alkaline phosphatase) was used to remove the phosphate groups from both ends of the pBR322 plasmid. Inserted. The hybrid plasmids were transformed into E. coli HB101 strain by the method of Maniatis, and then cultured in LB agar media containing 20 μg / ml and 50 μg / ml of ampicillin and tetracycline, respectively. Selected.

Selected E. coli HB101 transformed strains were confirmed by the presence of hybrid plasmid by mini-screen plasmid separation method, and the plasmid was isolated by mass screening by reselecting the E. coli HB101 transformed strain. That is, the E. coli HB101 transformed strain was cultured and collected in LB liquid medium, and the plasmid solution was purified by alkaline method (Maniatis, 1982) to collect etchium bromide-cesium chromide density gradient ultracentrifugation method ( Only plasmids were isolated by EtBr-CsCl density gradient ultracentrifugation. Ethium bromide and cesium chromide were removed by propanol treatment and dialysis on the plasmid separated by the super-centrifugation method, concentrated by precipitating with pure ethanol, and confirming the single band on 0.7% agarose gel. The base sequence was determined using dATP (α ³⁵ -S) by the method of (Proc. Natl. Acad. Sci. USA 74; 5463, 1977). The mass separated plasmid was manipulated in the following manner to obtain a fragment having the essential erythropoietin gene. Treated with BSSHII in Tris-HCl buffer pH 7.4, 50 ° C containing 25 mmol sodium chloride, concentrated with ethanol precipitation, and Bgl II in Tris-Yansam buffer solution containing 100 mmol sodium chloride (pH 7.4 37 ° C) Was processed and electrophoresed on 0.7% agarose gel to obtain a 2374 base pair (bp) fragment with full essential erythropoietin gene business (Figure 1).

Example 3

[Assembly of Plasmids Containing Erythropoietin Gene]

A 2374 base pair fragment (Fig. 1) of BssHII-BglII containing the complete human erythropoietin gene, prepared in Example 2, was inserted into a plasmid vector pDW-M that was genetically engineered to express well in mammalian animal cells. pDW-M was derived from pDW-2.

[Plasmid pDW-2 Assembly Method]

BamHI-EcoRI fragment (2.3 kb) present in plasmid pGA22 (J. Bacteriol. 140; 400, 1979) and having an E. coli replication origin (Ampicillin-resistant gene) After binding the BamHI-KpnI fragment (2.8 kb) in the Simian virus 40 (SV40) gene and having the site of SV40 replication and the Enhancer sequence (SVE), the type 2 adenovirus ( AluI-Hind III fragment (A1), which is present in the Adenovirus serotype 2 (Ad2) gene and has the major late promoter (MLP) of Ad2 and the first tripatite sequence of Ad2 (Ad2 tripartite leader seuqence 1, AL1) About 0.5kb) KpnI linker (linker) was linked by binding, both ends of the blunt end (blunt end) and then ligated by linking Hind III linker.

This plasmid, pDW-1, was transformed into E. coli HB101 and mass isolated according to the method described in Example 2, and then treated with BamHI to cleave the only BamHI site in pDW-1, and then linked to the EcoRI linker and religated. 5.6 kb of pDW-2 was assembled (Fig. 2).

[Assembling Method of Plaskid pDW-M]

Plasmid pDW-K (figure 1) was manipulated so that the tripartite leader sequences of the second and third Ad2 were located after the first tripartite leader sequence according to Buckner's method (Nucleic acid research 13; 841, 1985) from plasmid pDW-2 3) was made and plasmid pDW-L (Fig. 3) was assembled by replacing the SalI site present here with the Pstl site.

The HpaI-Pstl fragment (approximately 0.7 kb), which is then present in plasmid pCH104 (J. Mol. Appl. Genet 2; 101, 1983), has an SV40 early polyadenylation signal sequence (pA) of pDW The plasmid vector pDW-M (Fig. 3) was assembled by binding to the Pstl site of -L, making both ends the brut ends, and attaching and ligating a BamHI linker. pDW-M is the SV40 replication initiation site, SV40 electron promoter sequence (SVE), Ad2 important late regulatory genes (Ad2, MLP), Ad2 tripatite leader sequence (AL1, AL2, AL3), Ad2 5 ′ sparse code It has a sequence (5 splicing signal, 5 ss), SV40 initial polyadenylation coding sequence (pA), and the pGA22 fragment contains an E. coli replication initiation site and an ampicillin resistance gene.

[Assembling method of plasmid pDW-MO931]

The BssHII-BglII erythropoietin gene fragment prepared in Example 2 was a T4 DNA polymerase, and after filling the gaps at both ends, the BamHI linker was attached and the only BamHI site of pDW-M. Was inserted (Fig. 4).

The erythropoietin gene segment is located between Ad2 5 ′ ss and the SV40 initial polyadenylation sequence. The pDW-MO931 was about 9.05 kb in length, and was transformed to exist in E. coli HB101 and, if necessary, separated and used by the bulk separation method described in Example 2.

Example 4

Transformation of Erythropoietin Gene Insertion Vector The pDW-MO931 prepared in Example 3 was transformed into mammalian cells using the calcium phosphate deposition method (CaPO ₄ precipitation method) of Graham (Virology 52; 456, 1973).

That is, in order to establish a transformant that produces a high concentration of erythropoietin, plasmid pDHFR having dehydrofolate reductase (DHFR) cDNA derived from mouse mRNA together with pDW-MO931 (Nature 275; 617). , 1978) and carrier DNA (mouse liver DNA) were mixed at a ratio of 1: 1: 2, diluted in saline containing low concentrations of phosphoric acid, and calcium phosphate was added to allow calcium and phosphate to be deposited simultaneously. The suspension was added to Baby hamster kidney (BHK) cells and African green monkey kidney (COS) cells cultured in a single layer, followed by Eagle's BME medium lacking glycine, hypoxanthine and thymidine. Incubated in Modified Eagle's BME media). The viable colonies were separated into single cells with trypsin-EDTA solution and then treated with MTX (Methotrexate) to isolate erythropoietin with high yield. First, 50 nM, 10 nM, and 150 nM of MTX were sequentially treated to collect surviving cells, separated into single cells, and secondly, 5 μM, 10 μM, 25 μM, and 100 μM of MTX were selected to select a few cell clones that survived.

Cell clones surviving after the first MTX treatment were radioimmunoassy (RIA) using rabbit polyvalent anti-human erythropoietin antiserum to measure the concentration of erythropoietin in cell culture. It measured (Table 1). The erythropoietin concentration in the cell culture was increased by 30 to 50-fold during the MTX treatment, and secreted erythropoietin with persistent and stable biological activity.

Example 5

[Expression and Characteristics of Erythropoietin]

The transformed cells of Example 4 isolated recombinant erythropoietin, which has a very large amount of biological activity even before MTX treatment, into the culture medium, and its concentration was about 150-300 units / ml. Transformants that remained stable after MTX treatment were secreted up to 4,500-8,800 units per ml.

The determination of erythropoietin concentration in the culture of the transformed cells was carried out by the method of Yanagawa (Agric. Biol Chem. 47; 1311, 1983) in vitro, in which hepatocytes of mouse embryos were cultured in medium containing erythropoietin. The cells were suspended in a single cell state and stained with Benzidine to count cells stained with an inverse microscope.

In vivo measurement was performed using Kodes (Nature 191; 1965, 1961) hypoxic polycythemia mouse method. This is based on the principle that erythropoietin is controlled only by externally administered erythropoietin because the secretion of erythropoietin in the body is stopped when the mouse is grown under low pressure for 14 days to produce large amounts of red blood cells and then to normal pressure. . The concentration of erythropoietin administered was determined by measuring ⁵⁹ Fe uptake in the blood.

As standard erythropoietin, urine erythropoietin (Toyobo Co. Ltd. Japan, 67 unit / mg of protein) of aplastic anemia patients was used. The results of radioimmunoassay using rabbit polyvalent anti-human erythropoietin antiserum were consistent with Yanagawa's method. This indicates that recombinant erythropoietin is essentially the same as natural erythropoietin. Assuming that the natural erythropoietin and recombinant erythropoietin are the same, the transformant secretes 58-115 μg of recombinant erythropoietin per milliliter.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of natural erythropoietin and recombinant erythropoietin showed that the natural erythropoietin had a molecular weight of about 35 kd, and the recombinant erythropoietin of BHK cells was slightly wider (36-38 kd). It became permanent. Double diffusion experiments using anti-human erythropoietin antiserum in rabbits show that the recombinant and natural erythropoietin are immunologically identical because they show a single precipitation curve without overlapping lines. Recombinant erythropoietin treated with sialidase enhanced its activity in vitro, but the activity was near zero in invivo, indicating that sialic acid had a decisive effect on in vivo activity.

In the present invention, the transformant of BHK cells capable of expressing erythropoietin was deposited with the Korean spawn association on February 10, 1989 according to Article 2 of the Enforcement Decree of the Korean Patent Act. The accession number is KFCC-10670.

TABLE 1

Claims (5)

5 'end is treated with restriction enzyme BssHII and 3' end is treated with restriction enzyme BglII, human erythropoietin genomic DNA sequence of Figure 1, SV40 replication initiation sequence, SV40 transcriptional promoter sequence, SV40 early polyadenylation algo sequence, Recombinant pDW-MO931 vector comprising an Ad ₂ late regulatory gene, an Ad ₂ tripatite sequencer. A transformant having the ability to produce human erythropoietin, characterized in that the mammalian-derived kidney epithelial cells transformed with the recombinant DNA vector pDW-MO931 are transformants. The transformant is BHK (KFCC-10670) renal epithelial cell. Recombinant vector pDW-MO931 comprising a human erythropoietin genomic DNA sequence treated with the restriction enzyme BssHII-BglII of FIG. 1 was prepared, followed by culturing a transformant transformed into mammalian kidney epithelial cells stably to human erythropoie How to prepare ethyne. The method according to claim 4, wherein the transformant is BHK cells (KFCC-10670).

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