CN1224726A - High Level Expression of Human Erythropoietin Gene in Stably Transfected Mammalian Cells - Google Patents

Abstract

A method for expressing recombinant human erythropoietin, comprising transfecting host cells with DNA, RNA or nucleotide sequences mainly containing the 2.4kb ApaI restriction fragment of human erythropoietin gene, contacting transfected cells with culture medium, expressing And recover erythropoietin. and a method for expressing recombinant human erythropoietin by a cell line contacted with an incubating medium, comprising adding a cell line capable of producing erythropoietin in the incubating medium to said method, said cell line passing It is produced by transfecting host cell lines with DNA, RNA or nucleotide sequences mainly containing the 2.4kb ApaI restriction fragment of the human erythropoietin gene.

Description

High level expression of human erythropoietin gene in stably transfected mammalian cells

This application is a divisional application of the invention patent application with the application number 87104424.2, the filing date is June 26, 1987, and the title of the invention is "Method for preparing nucleotide sequence equivalent to human erythropoietin gene fragment".

The present invention generally relates to the field of genetic engineering, specifically to the expression of glycoprotein products of recombinant genes, and more specifically to the high-level expression of biologically active human erythropoietin from stably transfected cells.

Erythropoietin is a hormone that plays an important role in regulating erythropoiesis, the formation of red blood cells, and erythropoietin deficiency resulting from anemia. A detailed study of this hormone and attempts to use it as a replacement therapy have been difficult due to the lack of pure material.

Production of human red blood cells requires renal secretion of erythropoietin in the form of a mature glycoprotein. At steady state, the circulating blood concentration of this hormone is 10 to 18 milliunits per milliliter (128-230 picograms), and its levels can increase a thousand-fold under severe tissue hypoxia (hypoxia) stimulation . Increased hormone levels can trigger the proliferation and differentiation of stem cell populations in the bone marrow, activate the synthesis of hemoglobin in mature red blood cells, and accelerate the release of red blood cells from the bone marrow into circulation, thereby increasing the amount of red blood cells and improving the situation of insufficient oxygen supply. Patients lacking erythropoietin, such as chronic kidney patients, often suffer from severe anemia.

Erythropoietin is a 34-38 kilodalton glycoprotein, about 40% of its molecular weight is provided by carbohydrates. At least one disulfide bridge is required for activity, and little is known about the structure of this hormone, nor are the details of its synthesis fully understood. Recently isolated cDNA and genomic clones have provided the opportunity to analyze the regulatory control of erythropoietin production, however, biologically active human erythropoietin has not been expressed in sufficient quantities for replacement therapy.

According to the disclosure of the present invention, the biologically active human erythropoietin can be expressed at a high level (more than two million nominal titer units per liter of supernatant) by a stably transfected mammalian cell line, which is an important factor for clinical application. Pure human erythropoietin provides a great source. This surprisingly high level expression of erythropoietin was achieved by transfecting the host cell line with the ApaI restriction fragment of the human erythropoietin gene. The sense strand of the ApaI restriction fragment has the nucleotide sequence shown in Figure 1 .

Figure 1 shows the ApaI restriction fragment of 2426 base pairs containing the human erythropoietin gene sequence;

Figure 2 depicts a representative plasmid expression vector (pD11-Ep) containing an ApaI restriction fragment of 2426 base pairs;

Figure 3 depicts another expression vector (pBD-Ep) with an ApaI restriction fragment.

In a preferred embodiment, the ApaI restriction fragment constructed by genetic engineering as shown in Figure 1 is inserted into a mammalian expression vector as shown in Figure 2 and Figure 3, and then the expression vector is introduced into a mammalian cell line to develop Stably transfected cells produce large amounts of bioactive human erythropoietin. The selected ApaI restriction fragment of human erythropoietin gene should be able to transcribe erythropoietin messenger RNA with the highest efficiency, effectively translate RNA and carry out post-translational modification, so as to generate mature erythropoietin glycoprotein with biological activity. Specifically, it is important to remove interfering sequences at the 5' end of the erythropoietin gene while retaining the enhancer sequence. Introns in the ApaI restriction fragment were also preserved in order to preserve potentially valuable enhancer sequences. Certain 3' non-translated sequences were also retained at the 3' end of the gene to optimize putative regulatory sequences. The enhanced expression provided by the ApaI fragment is demonstrated in the following examples where use of this fragment with two promoters and two cell lines resulted in stable high levels of erythropoietin expression.

The following examples are provided to illustrate the advantages of the invention and to help ordinary skilled persons to grasp and apply the invention. These examples are not intended in any way to limit the scope of the present disclosure, or the protection afforded by this Certificate of Patent. Example 1. Extraction of gene clones

The human genome library (Cell, 15:1157-1174, 1978) constructed in λ phage was probed with low stringency hybridization conditions and the oligonucleotides described in Cell, 38: 287-297, 1984 (hereby cited). Screen the mixture.

The oligonucleotide mixture was prepared with an Applied Biosystems synthesizer and end-labeled with 32P-ATP and T4 polynucleotide kinase. The designed synthetic oligonucleotides correspond to the amino acid sequence of the amino-terminal part:

H2N-Ala-Pro-?-Arg-Leu-Ile-Leu-Asp-Ser-Arg-Val-Leu-Glu-Arg-

Tyr-Leu-Leu-Glu-Ala-Lys-Glu-Ala-Glu-?-Ile-Thr-Asp-Gly-Gly-Ala

24 This sequence was obtained from a human protein purified from the urine of aplastic anemia patients by Yanagawa et al. (J. Biol. Chem. 259:2707-2710, 1984). In order to reduce the degree of degeneracy of codons in this amino acid sequence, Grantham et al. (Nu-cleic Acids Research, 8:43-59, 1981) and Jaye et al. Codon usage rules. These rules take into account the relative rarity of the dinucleotide CPG in vertebrate DNA, and appropriately avoid potential A:G mismatches. Amino acid position 24 is most likely asparagine (J. Biol. Chem. 259:2707-2710, 1984). For the amino acid sequence glutamate-C-Lai-Glu-C-Glu-Asparagine, 2 groups of 72 codon sequences corresponding to expectations are synthesized, and the first group is a 20 nucleotide probe, which is TT(c/ t)TC(a/g/t)GC(c/t)TC(c/t)TT(a/g/t)-GCTTC, the second group replaces T with C at position 18. For the amino acid sequence glutamate-asparagine-isoleu-threo-aspart-glycine, a set of 23 nucleotide probes with the sequence AGC TCC TCC ATC AGT ATT ATT T(c/t) was constructed.

Plaques hybridized with oligonucleotide probes were rescreened at low density until purified. After the initial positive phage clones were purified by plaque, Jacobs et al. published further sequence data of the erythropoietin gene (Nature, 313:806-810, 1985). Based on this information, oligonucleotides were constructed and used to verify positive clones. Positive clones were digested with EcoRI restriction enzymes, and the insert DNA was purified by gel electrophoresis, and then ligated into the DNA by standard techniques. in the pUC13 plasmid. Determination of DNA sequence by means of dideoxynucleotide chain termination method (Proc.Natl.Acad.Sci.USA, 74:5463-5467,1977), this determination uses dATP(α- ^35S ) and universal 17 nucleotide primer , or in selected regions using specific oligonucleotide primers. ^32P -ATP was obtained from ICN and enzyme from New England Biolabs or Bethesda Research Laboratories.

About 4.8×10 ⁵ phage were screened by double nitrocellulose filter hybridization. Three different clones remained positive during plaque purification and their DNA inserts were subjected to restriction mapping and partial dideoxy sequencing. Two of the three clones apparently contained the full information for the erythropoietin gene. The restriction maps of these clones and their 2426-base-pair ApaI fragment sequences (see Figure 1) are basically identical to the human erythropoietin gene sequence recently published by Jacobs et al. (Nature, 313:806-810, 1985). The low frequency of erythropoietin gene-containing phages presented in this proliferating library—approximately 1 in 2 x ¹⁰⁶ phage—is consistent with the presence of the erythropoietin gene as a single copy in the human chromosomal genome. Using the ApaI fragment or other restriction fragments of the erythropoietin gene to do Southern blot hybridization with the total human chromosomal DNA, only a single hybridization band appeared, and there were no other highly homologous DNA regions. Example 2. Selection of ApaI restriction fragments

To construct the expression vector, the ApaI restriction fragment of the erythropoietin gene was prepared according to the technique described in Proc. Briefly, the extracted phage DNA was digested with restriction enzyme ApaI (Bethesda Research Laboratories), then separated on 1% agarose gel, eluted by electrophoresis, phenol extraction and ethanol precipitation. This fragment was verified by partial sequencing as in Example 1.

Referring to Figure 1, the inserted ApaI restriction fragment contains 58 base pairs of 5' untranslated sequence (0001-0058 nucleotides), followed by a deduced 27 amino acid signal peptide coding sequence, mature protein, four deduced Insert sequence and 222 base pairs of 3' non-coding DNA sequence. At the 5' end of the gene, the ApaI site is 58 base pairs upstream of the start codon ATG (0058) of the protein sequence. This 5' position (0001) was chosen to avoid a false start immediately upstream of the ApaI restriction site, while preserving regulatory sequences thought to be important for efficient ribosome binding and processing. The ApaI site at the 3' end (2426) was chosen to retain putative processing signals in most of the 3' untranslated region and to remove sequences further downstream. The use of the complete erythropoietin gene, including its intronic sequences, is intended to preserve potential regulatory and enhancer sequences within the introns that may play a role in the expression of the erythropoietin gene or in the modification and secretion of the protein. Example 3. Construction of Expression Plasmid with ApaⅠ Restriction Fragment

A 2426 base pair ApaI restriction fragment containing the complete human erythropoietin gene was inserted into two expression vectors pD11 and pBD, each based on a different mammalian promoter.

Plasmid expression vector pD11 is derived from the aforementioned plasmid (Nucleic Acids Research, 13:841-857, 1985, specifically cited here), containing the enhancer sequence and replication origin region of simian nephrovirus 40 (SV40), and adenovirus Major late promoter and triple leader sequence of virus 2. The ApaI fragment of the human erythropoietin genome sequence (Figure 1) was gel-purified, the single-stranded end was filled with T4 DNA polymerase, and the two blunt ends were connected with a BamHI linker, and the construct was inserted into the single BamHI restriction site of pD11 , to directly transcribe the erythropoietin gene from a strong promoter.

The structure of the constructed expression plasmid pD11-Ep with ApaI fragment (Ep) is shown in Fig. 2 . Plasmid pD11 is constructed by inserting the following components into the EcoRI (RI) site of pML, namely: the left end (0-1) of the adenovirus containing 350 base pairs, the SV-40 replication initiation region and the enhancer sequence (E ), major late promoter (MLP) of adenovirus, triple leader sequence (L1-3) of adenovirus 2, 5′ splice site (5′SS) of the third leader sequence, 3′ splice of immunoglobulin site (3'SS), and SV-40 late polyadenylation signal (pA). The recombinant plasmid was cloned in E.Coli HB101 and purified by cesium chloride isodensity centrifugation. The expression plasmid pD11-Ep was about 6500 base pairs, and the constructed product was verified by restriction map and partial dideoxy sequencing.

pBD contains a metallothionein I promoter sequence (MT-1, Glanville, Durham and Palmiter, Nature, 292:267-269, 1981 is hereby cited) and a pUC plasmid with a dihydrofolate reductase (DHFR) selectable marker sequence. Referring to Figure 3, the 2426 base pair ApaI restriction fragment (Ep) was inserted into the unique SmaI restriction site of pBD to form the expression plasmid pBD-Ep. The DHFR sequence of pBD-Ep is connected with the replication initiation sequence and enhancer sequence of SV-40 and the polyadenylation sequence of hepatitis virus B surface antigen. The construction of pBD-Ep was basically as described above for pD11-Ep.

Although plasmid vectors were used in these confirmatory experiments, it is also envisaged to use viral or retroviral expression vectors instead to introduce the ApaI erythropoietin gene fragment into host cell lines in a similar manner. DNA viruses suitable for this purpose include adenovirus or BPV (bovine papilloma virus), while suitable retroviruses are also known and readily available. It is clear that such retroviral (RNA) vectors will carry the antisense strand of the ApaI restriction fragment, ie the RNA transcript sequence corresponding to the sense strand of Figure 1, or its equivalent. Retroviral vectors will also include sequences for transchromosomal factors required for reverse transcription of the viral genome and integration of the viral DNA into the host genome. Example 4. Transfection of mammalian cells

Two mammalian cell lines, each transfected with pD11-Ep and pBD-Ep. Mammalian cell lines COS-7 (monkey kidney) and BHK (baby rat kidney) were maintained in modified Dulbecco's minimal medium with 10% fetal bovine serum. When the cells were subcultured to 50-70% confluence, they were transfected by the calcium phosphate method (Virology, 52:456-467, 1973). BHK is derived from renal epithelial cells. It is generally believed that they are most likely to produce natural erythropoietin in mammals when they are anemic or hypoxic. Therefore, these cells may recognize the key regulatory sequence on the ApaI fragment of erythropoietin. Efficiently processes and produces erythropoietin glycoprotein.

For temporary expression of cells, add 20 micrograms of total DNA, 10 micrograms of pD11-Ep plasmid with erythropoietin gene, and 10 micrograms of carrier salmon sperm DNA in a 100 mm culture dish, and collect the supernatant after 48 hours , centrifuged at 400g for 10 minutes to remove cells and particles, and frozen at -20°C. Cells were also collected separately. The temporary expression results of transfection with pD11-Ep alone are shown in Table 1. The data are obtained from three experiments for each type of cell.

Table 1

Erythropoietin mammalian cell protein per ml of culture (micrograms) Unit (in vitro bioassay)

BHK 3.4±0.2 270±16

COS-7 3.2±0.4 255±32

The observed secretion of erythropoietin into the supernatant by the mammalian cell lines COS-7 or BHK was approximately 80-fold higher than previously reported for either cDNA encoding erythropoietin or temporal expression using the entire erythropoietin gene.

To establish a stable cell line producing high levels of erythropoietin, COS-7 or BHK cells were co-transfected with pD11-Ep and pDHFR-1a plasmids (the latter is a similar mammalian expression vector containing the cDNA for dihydrofolate reductase) . The transfection step was changed to co-transfection with 5 micrograms of pD11-Ep plasmid, 5 micrograms of pDHFR-1a plasmid and 10 micrograms of carrier DNA, and continued incubation for 18-24 hours, and then different concentrations of methotrexate (10 nanomolar /L to 1 mmol/L) was added to the culture medium. Cells incorporating the DHFR gene survive on this selection medium. After several days of incubation, isolated methotrexate-resistant colonies, subcultured and screened for erythropoietin activity in the supernatant, about half of the detected methotrexate-resistant colonies secreted detectable erythropoietin vitality.

In order to establish a stable cell line with the expression vector pBD-Ep, BHK cells were transfected by the calcium phosphate method. After 18-24 hours, 1 μmol/L to 1 mmol/L of methotrexate was added to the medium, and cultured After several days, colonies surviving higher levels of methotrexate were isolated, passaged, and screened. In this series, all methotrexate-resistant colonies secreted measurable erythropoietin viability .

To optimize the expression of transcriptional units containing the erythropoietin gene and the DHFR gene, each BHK cell line secreting high levels of erythropoietin containing pBD-Ep or pD11-Ep was passaged in increasing concentrations of methotrexate times (Nature, 316:271-273, 1985). However, instead of exerting selective pressure on the cell lines by stepwise increases in methotrexate concentrations in small intervals, these cells were immediately challenged with very high concentrations of methotrexate (ie, 1 mmol/L). Only a very small number of cells survive, but these cells incorporate plasmid components into sites of DNA that are particularly favorable for expression (so-called hotspots) and/or possess many copies of the component transcription unit. Therefore, the cell line with the highest production can be selected in one step. If it is passed for more than 15 passages under the lack of methotrexate selection pressure, it still maintains a high production of erythropoietin. These cell lines (including F7.2 listed in Table 2) and S5.2) can be regarded as stable.

Erythropoietin activity cannot be quantified in cell pellets because there are many factors in cell extracts that inhibit detection. Therefore, the results listed in Table 2 are only the erythropoietin protein produced by the cell line and secreted into the supernatant, and the level of intracellular erythropoietin was not analyzed.

Table 2

    from a stably transfected BHK cell line

Expressed recombinant erythropoietin

PBD-EPF1.1 12.4 970F3.0 2500F6.6 6210F7.2 84.2 84.2 84.1 6728

Table 2 (continued)

The red blood cells in the clearing fluid in the clearing fluid PD11-EP protein (microphone) unit (in vitro biological test) S1.2 6.4 500S2.4 64.2 5000S5.2 82.1 6400 Observation of red blood cell formation secretion is equivalent Nominal yield of seven million units. This nominal yield was obtained by multiplying the observed yield per milliliter by a factor of one thousand to give the expected yield per liter for large-scale production.

The erythropoietin production observed with the ApaI fragment was greater than that reported earlier for CHO cell lines stably transformed with different human erythropoietin gene fragments (Lin et al., Proc. Natl. Acad. Sci. USA, 82: 7580-7584, Nov.1985) more than three hundred times higher.

Control experiments in these transfection assays included supernatants of non-transfected cells, and parallel cultures of cells transfected with DNA encoding other proteins, including bacterial chloramphenicol transacetylase and human factor IX- The supernatants of these control cultures, mock-transfected or cultured cells transfected with other genes had no detectable erythropoietin activity. It should also be pointed out that in the experiment of each erythropoietin gene mentioned above, the expression level obtained from the selected cell line, and whether the selectable marker accompanying the erythropoietin gene was co-infected with it, or the ApaI-containing fragment was inserted before infection The plasmids are not related.

Other representative infection methods suitable for use in the present invention include DEAE-dextran mediated transfection techniques, lysozyme fusion or erythrocyte fusion, scraping, direct ingestion, osmotic shock or glucose shock methods, direct microinjection or indirect microinjection, techniques such as erythrocyte-mediated and/or subjecting host cells to an electrical current. Transfection refers to the transfer of genetic information, specifically the coding information of the ApaI restriction fragment of the human erythropoietin gene, into cells using extracted DNA, RNA or synthetic nucleotide polymers. The technique of transfection cannot be considered perfect, as other means of introducing genetic information into cells will no doubt be developed. Prior to transfection, typically, the ApaI restriction fragment can be spliced (linked) to other nucleic acid sequences, such as promoters, enhancers, and polyadenylation sequences. While host cell lines of mammalian origin are described, with particular reference to renal epithelial cells, it is contemplated that other eukaryotic and prokaryotic (bacterial or yeast) host cell lines may be used with the present invention. Recent successes in introducing mammalian genes into plant cells offer the potential to exploit plant and algal cells. Example 5. Expression of erythropoietin from transfected cell lines

Erythropoietin secreted into supernatants of transfected cell lines is bioactive, secreting large amounts of the hormone up to seven thousand units per milliliter.

The in vitro determination of erythropoietin activity is based on the formation of red colonies (CFU-E, red colony-forming cells) (CFU-E, red colony-forming cells) of mouse bone marrow cells on the plasma clot medium (BloodCells, 4:89-103, 1978). The sensitivity of this assay is about 5 milliunits/ml. The erythropoietin used as assay standard was a preparation obtained from partially purified anemic sheep plasma (Connaught, EP of the third step, Lot No. 3026), which was grown on fresh medium without methotrexate for 24 hours. The supernatant of the passaged cell line containing 2× ¹⁰⁹ bone marrow cells per ml, 10% citrated bovine serum, 20% fetal bovine serum, 1% bovine serum albumin and 1.6% fetal bovine extract (Gibco) assay medium, add 1 to 10 microliters of the supernatant diluted 1:200 with the medium, and incubate for 36 to 48 hours. After 36 to 48 hours, the plasma clot is fixed on a glass slide and stained with benzidine for hemoglobin , Count the number of red blood cell colonies. When no erythropoietin was added, no colonies produced by CFU-E could be detected. Typically, 50 milliunits of erythropoietin (0.64 ng) per milliliter of culture was used to observe optimum colony growth (100-150 CFU-E per 2 x ¹⁰⁴ bone marrow cells). As shown in Tables 1 and 2, a large amount of the erythropoietin hormone was secreted into the supernatant of the transfected cell line, up to 7000 units per ml, assuming that the specific activity of the recombinant erythropoietin was equivalent to that of the native erythropoietin (per mg protein 78000 units), this bioassay is equivalent to about 80 micrograms of erythropoietin protein per ml.

In addition, a competitive radioimmunoassay was performed using multivalent anti-human erythropoietin rabbit antiserum (J. Cell. physiol. 118:87-96, 1984) to detect immune responses in supernatants of selected cell lines Active erythropoietin. The amount of protein detected by radioimmunoassay was equivalent to the protein level estimated by the bioassay. These data indicate that about 98% or more of the erythropoietin protein expressed and secreted by the transfected cell lines is viable.

The recombinant erythropoietin produced by the transfected cells was further examined. To confirm that the cells secreted by these cells are natural hormones, selected cell lines were used for in vivo detection of erythropoietin in mice with severe hypoxia-induced erythrocytosis (Nature, 191:1069-1087, 1961). The supernatants secreted by the cell lines were tested in mice with severe hypoxia-induced erythrocytosis, and had strong in vivo biological activity. In experiments with partially purified native erythropoietin, it has long been noted that treatment with sialidase completely abolishes erythropoietin activity when tested in whole animals (J. Biol. Chem. 247:5159 -5160, 1958). This loss of activity is presumed to be the result of enhanced hepatic clearance of the asialo hormone, since sialidase-treated erythropoietin retains its full activity in vitro. The strong bioactivity observed in vivo is indicative of post-translational modification in transfected mammalian cell lines. Appropriate sugars and terminal sialic acids are added to the erythropoietin protein.

In a separate experiment, in vitro bioassays of erythropoietin activity were also neutralized by anti-human erythropoietin antibodies added to the culture medium.

Erythropoietin secreted into the supernatants of representative transfected cell lines was also examined for their proliferative effects on other myeloid progenitor cells. To detect the effect of recombinant erythropoietin on various progenitor cells derived from human and mouse bone marrow, including: red colony-forming cells (CFU-E), red burst-forming cells (erythroid burst-forvming cells, BFU-E) , granulocyte-macrophage precursor (CFU-GM) and mixed cell colony forming cells (CFU-Mix) (J.Cell.Physiol.Suppl.1:79-85,1982; J.Cell.Phy-siol. 118:87-96, 1984). Red stem cells exhibited a proliferative effect on recombinant erythropoietin in a dose-parallel manner to the effect on native erythropoietin. Increased concentrations of recombinant erythropoietin up to 10 units per ml of cell culture tested for CFU-GM and CFU-Mix did not show any proliferative effect.

When analyzed by SDS-PAGE under reducing or non-reducing conditions, the electrophoresis of purified recombinant erythropoietin was the same as that of purified erythropoietin in the urine of patients with aplastic anemia. These proteins show the same microscopic heterogeneity, with a major component at a molecular weight of 34 kilodaltons.

While the invention has been described with reference to the presented examples, those of ordinary skill, having read the above examples, will be able to make various modifications, substitute equivalents, or make other changes to the above-mentioned components and methods. Accordingly, it is our intention to limit the scope of protection of this Patent Certificate only as defined by the appended claims and their equivalents.

Claims (8)

1. A method for expressing biologically active recombinant human erythropoietin, comprising transfecting host cells with DNA, RNA or nucleotide sequences mainly containing the 2.4kb ApaI restriction fragment of human erythropoietin gene, making transfected cells and culturing The substrate is contacted to allow the cells to express erythropoietin, and the expressed erythropoietin is recovered. 2. 2. The method of claim 1, wherein the ApaI restriction fragment is carried on a plasmid or virus. 3. The method of claim 1, wherein the host cell line is a mammalian cell. 4. A method for expressing biologically active recombinant human erythropoietin by a cell line in contact with an incubated medium, the improvement comprising adding to said method a cell line capable of producing erythropoietin in an incubated medium , said cell line is produced by transfecting a host cell line with a DNA, RNA or nucleotide sequence primarily containing the 2.4 kb ApaI restriction fragment of the human erythropoietin gene. 5. The method for expressing biologically active recombinant human erythropoietin in a cell line contacted with an incubation medium according to claim 4, wherein said cell line is capable of producing at least two million units of erythropoietin per liter of incubation medium Nominal production of erythropoietin. 6. The method of claim 4, wherein the ApaI restriction fragment is carried by a plasmid. 7. 4. The method of claim 4, wherein the ApaI restriction fragment is carried by a virus. 8. The method of claim 4, wherein the host cell line is a mammalian cell.

Images