CN1224726A - Human erythropoietin gene: high level expression in stably transfected mammalian cells - Google Patents

Abstract

A method of expressing recombination human erythropoietin includes transfecting host cells using DNA, RNA or or a nucleotide sequence which consisting essentially of a 2.4 kb Apa I restriction fragment of a human erythropoietin gene, bringing the transfected cells contact with the culture, expressing and recalling erythropoietin. And a method of expressing recombination human erythropoietin by a cell line contacted with the heat retaining cultures comprises adding the cell line which can generate erythropoietin in the heat retaining cultures in the said method, the said cell line generated by transfecting host cells using DNA, RNA or or a nucleotide sequence which consisting essentially of a 2.4 kb Apa I restriction fragment of a human erythropoietin gene.

Description

The high level expression of human erythropoietin gene in the mammalian cell of stable transfection

The application is that application number is 87104424.2, and the applying date is on June 26th, 87, and denomination of invention is divided an application for the application for a patent for invention of " preparation is equivalent to the method for the segmental nucleotide sequence of human erythropoietin gene ".

Relate generally to genetically engineered of the present invention field is expressed relevant, more particularly relevant with the human erythropoietin of expressing biologos from the cell of stable transfection high-levelly with the glycoprotein product of recombination specifically.

This hormone of erythropoietin regulate that red blood corpuscle generates, the formation of red blood cell and the erythropoietin that caused by anaemia play an important role in lacking.Owing to lack pure material, this hormone done studying in great detail and attempt with its treatment of substituting all very difficult always.

The generation of people's red blood cell needs the erythropoietin of the ripe glycoprotein form of renal secretion.When permanent attitude, this hormone concentration in blood circulation is every milliliter 10 to 18 milliunit (128-230 pico-gram), and serious organizing under oxygen supply deficiency (anoxic) stimulation, its level can increase by 1,000 times.Improve hormonal readiness and can trigger hyperplasia and the differentiation of experiencing population of stem cells in the marrow, oxyphorase is synthetic in the activation mature erythrocyte, quickens red corpuscle and discharges into circulation from marrow, thereby increase red cell volume, improves the insufficient situation of oxygen supply.Lack the patient such as the chronic kidney disease people of erythropoietin, often suffer from serious anemia.

Erythropoietin is the glycoprotein of a 34-38 kilodalton, and about 40% of molecular weight is that carbohydrate provides.At least one disulphide bridges is that vigor is required, and the structure of this hormone is known little, and its synthetic details are all clear Chu not also.CDNA that is separated to recently and genomic clone, the chance that the adjusting that provides the analysis erythropoietin to produce is controlled, still, the human erythropoietin that biologos is arranged of also not expressing sufficient amount is to be used for the substitute treatment.

According to content disclosed by the invention, have the human erythropoietin of biologos can be carried out high level expression (whenever go up clear liquid and surpass 2,000,000 nominal titre units) by the mammal cell line of stable transfection, this just provides huge source for the pure human erythropoietin of clinical application.The high level expression that this of erythropoietin is surprising is to realize by the Apa I restricted fragment transfection host cell system with human erythropoietin gene.The meaning chain of Apa I restricted fragment has nucleotide sequence shown in Figure 1.

Fig. 1 represents to contain the Apa I restricted fragment of 2426 base pairs of human erythropoietin gene sequence;

Fig. 2 has drawn the representational plasmid expression vector (pD11-Ep) of the Apa I restricted fragment that contains 2426 base pairs;

What Fig. 3 described is another expression vector that has Apa I restricted fragment (pBD-Ep).

In embodiment preferably, the Apa I restricted fragment that genetically engineered makes up of passing through as shown in Figure 1 is inserted in as shown in Figures 2 and 3 the mammalian expression vector, then expression vector is introduced mammal cell line with the development stability cells transfected, produced the human erythropoietin that biologos is arranged in a large number.The Apa I restricted fragment of selected human erythropoietin gene is wanted and can be transcribed the erythropoietin messenger RNA(mRNA) to top efficiency, the modification after translating RNA effectively and translating, the ripe erythropoietin glycoprotein that has biologos with generation.Specifically, importantly erythropoietin gene 5 ' end interference sequence is removed, and kept enhancer sequence.In order to preserve the enhancer sequence of potential value, the intron in the Apa I restricted fragment also is retained.Also be retained in the non-sequence of translating of some 3 ＇ of 3 of gene ' end, make the regulating and controlling sequence optimization of supposition.The enhancing that is provided by Apa I fragment is expressed as following example and is confirmed that in following example, this fragment and two promotors and two clones are share the erythropoietin that all obtains stable high level expression.

Proposing following example is the superiority of inventing in order to set forth, and helps common technical skill personnel to grasp and application the present invention.These examples in no case are intended scopes disclosed by the invention, or the protection that provided of this patent certificate.The extraction of example 1. gene clones

The people's gene group library (Cell, 15:1157-1174,1978) that makes up in lambda particles phage reaches at Cell with the low stringency hybridization conditions, 38:287-297, and the oligonucleotide probe mixture described in 1984 (the quoting as proof hereby) screens.

Oligonucleotide mixture system makes end mark with the preparation of Applied Biosystems synthesizer with 32P-ATP and T4 polynucleotide kinase.The synthetic oligonucleotide of design is corresponding to the aminoacid sequence of N-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 these sequences derive from the human protein of people such as Yanagawa (J.Biol.Chem.259:2707-2710,1984) purifying from aplastic anemia patient urine.In order to reduce the degeneracy degree of this section aminoacid sequence codon, (Nucleic Acids Research such as (Nu-cleic Acids Research, 8:43-59,1981) such as Grantham and Jaye have been adopted, 11:2325-2335,1983). codon laws of use.These rules have been considered among the vertebrates DNA that dinucleotides CPG is relatively rare and have suitably been avoided the potential mispairing of A:G.The 24th amino acids is amino-succinamic acid (J.Biol.Chem.259:2707-2710,1984) most likely.For aminoacid sequence paddy-third-Lai-paddy-third-paddy-amino-succinamic acid, 2 groups of each 72 kinds codon sequences corresponding to expection are synthesized, first group is 20 nucleotide probes, is TT (c/t) TC (a/g/t) GC (c/t) TC (c/t) TT (a/g/t)-GCTTC, and second group is changed to C at the 18th with T.For aminoacid sequence paddy-amino-succinamic acid-different bright-Su-Men Dong-Gan, make up 23 nucleotide probes that one group of sequence is AGC TCC TCC ATC AGT ATT ATT T (c/t).

Plaque with oligonucleotide probe hybridization arrives heavily screens up to purifying under low density.By behind the plaque purifying, Jaco bs etc. has delivered the further sequence data (Nature, 313:806-810,1985) of erythropoietin gene at initial positive phage clones.Made up oligonucleotide according to this data, and be used to verify positive colony, with EcoR I Restriction Enzyme enzymolysis positive colony, it inserts the sheet segment DNA with the gel electrophoresis purifying, links in the PUC13 plasmid of using EcoR I limited enzymatic hydrolysis in advance with standard technique again.Measure dna sequence dna (Proc.Natl.Acad.Sci.USA, 74:5463-5467,1977) by the dideoxy nucleotide chain cessation method, this mensuration use dATP (α- ³⁵S) and 17 general nucleotide primers, or in selected zone use special Oligonucleolide primers to carry out. ³²P-ATP obtains from ICN, and enzyme obtains from New EnglandBiolabs or Bethesda Research Laboratories.

About 4.8 * 10 ⁵Individual phage is screened by double nitrocellulose filter hybrid method.Three different being cloned in keep positive in the plaque purge process, its DNA inserts fragment and makes estriction map and part dideoxy sequencing.Two full details that obviously contain erythropoietin gene are arranged among three clones.The human erythropoietin gene sequence (Nature, 313:806-810,1985) that the Apa I fragments sequence (see figure 1) of these clones' estriction map and 2426 base pairs thereof and nearest Jacobs etc. deliver is basic identical.The phage frequency that contains erythropoietin gene that in the library of this propagation, proposes low by-Yue 2 * 10 ⁶Have in the individual phage 1-conform to the saying that single copy exists in human chromogene group with erythropoietin gene.Apa I fragment or other restricted fragment with erythropoietin gene are made the Southern blot hybridization with total human chromosome DNA, only manifest single hybridization region band, do not have other height homologous DNA zone.The selection of example 2.Apa I restricted fragment

Be construction of expression vector, the Apa I restricted fragment of erythropoietin gene is pressed Proc.Natl.Acad.Sci.USA 74:5463-6467, and 1977 described technology (quoting as proof hereby) prepare.In brief, the phage DNA of extraction separates electrophoresis elution, phenol extracting and ethanol sedimentation then with restriction enzyme Apa I (Bethesda Research Laboratories) enzymolysis on 1% sepharose.This fragment is by verifying as the part sequencing in the example 1.

With reference to Fig. 1, the Apa I restricted fragment that inserts contains the 5 ' non-translated sequence (0001-0058 Nucleotide) of 58 base pairs, and the back is 27 amino acid whose signal coding sequences, maturation protein, the insertion sequence of four suppositions and the 3 ' noncoding DNA sequence of inferring of 222 base pairs.At 5 of gene ' end, Apa I site is 58 the base pair places, initiator codon ATG (0058) upstream at protein sequence.Selecting this 5 ' end site (0001) is for fear of a false starting point near Apa I restriction site upstream, has preserved again simultaneously to be considered to rrna combination and the very important regulating and controlling sequence of processing effectively.Select the Apa I site (2426) of 3 ' end,, and remove the more sequence in downstream with the processing signal of inferring in the reservation of great majority 3 ' non-translational region.Using complete erythropoietin gene, comprise its intron sequences, is to regulate and enhancer sequence in order to be retained in the intron potential, and they may play a role aspect the expression of erythropoietin gene or proteinic modification and the secretion.Example 3. has the structure of the expression plasmid of Apa I restricted fragment

The Apa I restricted fragment that comprises 2426 base pairs of complete human erythropoietin gene is inserted into two expression vector pD11 and pBD, and the two is respectively based on different mammalian promoters.

Plasmid expression vector pD11 is from aforesaid plasmid derivative (Nucleic AcidsResearch, 13:841-857,1985, the spy gives and quoting as proof herein), the enhancer sequence and the ori that contain simian virus 40 (SV40), and the major late promoter of adenovirus 2 and triple homing sequence.The Apa I fragment (Fig. 1) of human erythropoietin gene group sequence is a gel-purified, strand is terminal with T4 archaeal dna polymerase polishing, connect two flat ends with BamH I connexon, with setting up the single BamH I restriction site that thing inserts pD11, directly to transcribe the gene of erythropoietin from a strong promoter.

The structure of the expression plasmid pD11-Ep of setting up that has Apa I fragment (Ep) is painted as Fig. 2.Plasmid pD11 inserts following one-tenth branch by the EcoR I of pML (R I) site to constitute, that is: contain the adenovirus left distal end (0-1) of 350 base pairs, SV-40 ori and enhancer sequence (E), the major late promoter of adenovirus (MLP), triple homing sequences (L1-3) of adenovirus 2,5 ' splicing site of the 3rd homing sequence (5 ' SS), 3 ' splicing site of immunoglobulin (Ig) (3 ' SS), and SV-40 polyadenylic acid in late period signal (pA).The plasmid of reorganization is cloned in E.Coli HB101, cesium chloride isopycnic centrifugation purifying, and expression plasmid pD11-Ep is approximately 6500 base pairs, and making up product is estriction map and the checking of part dideoxy sequencing method.

PBD contains metallothionein(MT) I promoter sequence (Durham and Palmiter, Nature, 292:267-269,1981 quote as proof hereby for MT-1, Glanvi11e) and has the pUC plasmid of Tetrahydrofolate dehydrogenase (DHFR) selective marker sequence.With reference to Fig. 3, the Apa I restricted fragment (Ep) of this 2426 base pair inserts unique Sma I restriction site of pBD, forms expression plasmid pBD-Ep.The DHFR sequence of pBD-Ep links to each other with enhancer sequence and hepatitis virus B surface antigen polyadenylic acid sequence with the replication initiation sequence of SV-40.Basic as the above-mentioned pD11-Ep of the structure of pBD-Ep.

Though in these confirmatory tests, use plasmid vector, also imagined and used virus or inverse virus expression vector instead, in a similar manner Apa I erythropoietin gene fragment is introduced host cell system.The dna virus that is fit to this purpose comprises adenovirus or BPV (bovine papilloma virus), and suitable retroviral also is known and ready-made.Very clear such retroviral (RNA) carrier will carry the antisense strand of Apa I restricted fragment, promptly corresponding to the rna transcription product sequence of the meaning chain of Fig. 1, or its Equivalent.Retroviral vector also will comprise with the viral genome reverse transcription and with viral DNA and be integrated into the required sequence of striding the karyomit(e) acting factor of host's genome.The transfection of example 4. mammalian cells

Two kinds of mammal cell lines, each uses pD11-Ep and pBD-Ep transfection.Mammal cell line COS-7 (monkey kidney) and BHK (young kidney of rats) are kept in the improvement Dul becco minimum medium that contains 10% foetal calf serum.When passage merges to 50-70%, with calcium phosphate method transfection (Virology, 52:456-467,1973).BHK system comes from renal epithelial cell, it is generally acknowledged that they are that most probable produces the native state erythropoietin when Mammals anaemia or anoxic, therefore, these cells might be discerned key regulating and controlling sequence on the erythropoietin Apa I fragment, process and produce erythropoietin glycoprotein effectively.

For doing the interim expression of cell, add the total DNA of 20 micrograms in 100 millimeters culture dish, wherein 10 micrograms have the pD11-Ep plasmid of erythropoietin gene, and 10 microgram carrier salmon sperm dnas, after 48 hours, collect supernatant liquor, centrifugal 10 minutes of 400g removes cell and particle, freezing in-20 ℃, cell is also collected respectively, the results are shown in Table 1 with interim expression of pD11-Ep transfection separately, and data are each three times experiments of every type cell.

Table 1

Erythropoietin mammalian cell albumen (microgram) unit (external biological detection) in every milliliter of culture

BHK 3.4±0.2 270±16

COS-7 3.2±0.4 255±32

Observed mammal cell line COS-7 or BHK are secreted into the erythropoietin in the supernatant liquor, than the coding erythropoietin cDNA that reported in the past or all high about 80 times with the interim expression of whole erythropoietin gene work.

Produce for setting up the stable cell lines of high-level erythropoietin, COS-7 or bhk cell are with pD11-Ep and the while transfection (latter is the similar mammalian expression vector that comprises Tetrahydrofolate dehydrogenase cDNA) of pDHFR-1a plasmid.The transfection step changes into 5 microgram pD11-Ep plasmids, and 5 microgram pDHFR-1a plasmids and 10 microgram carrier DNAs are done common transfection, continues insulation 18-24 hour, then the methotrexate of different concns (10 nanomoles/rise to, 1 mmole/liter) is added substratum.Mixing the cell of DHFR gene selects to survive on the substratum at this.After insulation a couple of days, isolate the colony of anti-methotrexate, go down to posterity and screen erythropoietin vigor in the supernatant liquor, in the colony of detected anti-methotrexate, only about half of secretion has detectable erythropoietin vigor.

For setting up stable clone with expression vector pBD-Ep, with calcium phosphate method transfection bhk cell, after 18-24 hour, add in the substratum 1 micromoles per liter to 1 mmole/liter methotrexate, after cultivating a couple of days, isolate the colony of in higher methotrexate, surviving, go down to posterity and screen, in these were a series of, the colony of the anti-methotrexate that all are detected was all secreted the vigor that detectable erythropoietin is arranged.

Contain erythropoietin gene and DHFR gene transcription unit expression optimization in order to make, each bhk cell that will contain the secreting high levels erythropoietin of pBD-Ep or pD11-Ep ties up to the (Nature for several times that goes down to posterity in the methotrexate that concentration increases progressively, 316:271-273,1985).Yet, be not progressively to increase pair cell system by oxygen methopterin-A concentration closely-spaced to apply selective pressure, but make these cells be subjected to the challenge of the very high methotrexate of concentration (i.e. 1 mmole/liter) immediately.Only the only a few cell survival gets off, but plasmid establishment thing is incorporated into position (so-called focus) that is particularly conducive to expression among the DNA and/or the duplicate that has the transcription unit of many establishments in these cells.Therefore, once going on foot the clone that can choose high yield, if pass more than 15 generations lacking under the methotrexate selective pressure, still keep the high yield of erythropoietin, it is stable that these clones (being included in listed F7.2 of table 2 and S5.2) can be regarded as.

Can not be in cell granulations the quantitative vigor of erythropoietin because the factor that has many inhibition to detect in the cell extract.Produce and be secreted into erythropoietin albumen in the supernatant liquor so the listed result of table 2 is a clone, do not analyze the level of the erythropoietin in the born of the same parents.

Table 2

From the bhk cell system of stable transfection

The reorganization erythropoietin of expressing

The pBD-EpF1.1 of erythropoietin clone albumen (microgram) unit (external biological detection) 12.4 970F3.4 32.0 2500F6.1 79.6 6210F7.2 84.1 6728 in every milliliter of supernatant liquor

Table 2 (continuing)

The S1.2 of erythropoietin clone pD11-Ep albumen (microgram) unit (external biological detection) 6.4 500S2.4 64.2 5000S5.2 82.1 6400 observed erythropoietins secretions in every milliliter of supernatant liquor are equivalent to the nominal productive rate of 7,000,000 units up to every liter.This nominal productive rate takes advantage of 1,000 times to obtain observed every milliliter of output, every liter output when providing the scale operation of expection.

Use the viewed erythropoietin output of Apa I fragment, output (the Lin etc. that the Chinese hamster ovary celI system of the human erythropoietin gene fragment stable conversion more different than the use of previous report obtains, Proc.Nat1.Acad.Sci.USA, 82:7580-7584, Nov.1985) Senior Three is more than hundred times.

Control experiment during these transfection things are measured, the supernatant liquor that comprises non-transfected cell, with the paraxin transacetylase that comprises bacterium with other protein one of coding and humanclottingfactor-the supernatant liquor of the parallel culture of DNA cells transfected, these control cultures, mimic transfection or with the culturing cell of other gene transfection does not all have detectable erythropoietin vigor.Also must point out, in the experiment of above-mentioned each erythropoietin gene, the expression level that obtains from the clone selected, with the selected marker of following erythropoietin gene be co-infected with it, still insertion earlier contains the segmental plasmid of Apa I before infection, and irrelevant.

Be suitable for being useful for other representational infection method of this invention, comprise DEAE-dextran mesomeric rotaring dyeing technology, N,O-Diacetylmuramidase fusion method or red corpuscle fusion method, facing, direct absorption method, osmotic shock or glucose shock method, direct microinjection or indirect microinjection place the medium method of electric current such as red corpuscle mesomeric technology and/or host cell.Transfection is meant genetic information, specifically with the coded message of human erythropoietin gene Apa I restricted fragment, utilize DNA, the RNA or the synthetic Nucleotide polymer that extract to be transferred in the cell, above listed rotaring dyeing technology can not calculate perfect because will develop out undoubtedly other means of genetic information transfered cell.Before carrying out transfection, say that typically Apa I restricted fragment can be connected to (being connected to) other sequence of nucleic acid usually, such as promotor, enhanser and polyadenylic acid sequence.Though narrated each host cell system in Mammals source, and mentioned the kidney epithelial cell especially, considered that also the present invention can use other eucaryon and protokaryon (bacterium or yeast) host cell system.With the success of mammalian genes introduced plant cell, provide the potential possibility of utilizing plant and alga cells recently.Example 5. is expressed erythropoietin from cells transfected system

Being secreted into the erythropoietin that cells transfected fastens in the clear liquid has biologos, secrete a large amount of hormones up to every milliliter seven kilounits.

The external test of erythropoietin vigor is the formation (CFU-E according to bone marrow cells in mice red colony on the plasma clot substratum, red colony forming cell) (BloodCells, 4:89-103,1978) sensitivity of this mensuration is about 5 milliunit/milliliters.Erythropoietin as bioassay standard is the preparation (Connaught that is obtained by anaemia sheep plasma partial purification, the EP in the 3rd step, lot number 3026), what be used to measure is the supernatant liquor of 24 hours continuous cell line of growth on the fresh culture that does not contain methotrexate, and every milliliter contains 2 * 10 ⁹In the mensuration substratum of bovine serum, 20% foetal calf serum, 1% bovine serum albumin and 1.6% tire ox extract (Gibco) that individual medullary cell, 10% adding citric acid salt are handled, add 1 to 10 microlitre and press the supernatant liquor of dilution in 1: 200 with substratum, cultivate after 36 to 48 hours, plasma clot is fixing on slide glass, do oxyphorase dyeing with p-diaminodiphenyl, the number of number red corpuscle colony.When not adding erythropoietin, do not detect the colony that produces by CFU-E.Usually, every milliliter of culture can be observed only colony growth (per 2 * 10 with the erythropoietin (0.64 nanogram(ng)) of 50 milliunits ⁴Individual medullary cell can record 100-150 CFU-E).As shown in Table 1 and Table 2, a large amount of erythropoietin hormone secretions are in the transfectional cell series supernatant liquor, 7000 units up to every milliliter, suppose the ratio vigor and the natural erythropoietin equivalence (every milligram of protein 78000 units) of the erythropoietin of reorganization, this bioassay results promptly is equivalent to the erythropoietin protein of every milliliter of about 80 micrograms.

In addition, use the anti-human erythropoietin rabbit anti-serum of polyvalent (J.Cell.physiol.118:87-96,1984) to do competitive radioimmunoassay, the erythropoietin of immune response vigor is arranged in the clone supernatant liquor of selecting with detection.With the detected protein content of radioimmunoassay, the protein level of estimating with biological detection equates.These data declaration transfectional cell series expression and excretory erythropoietin albumen are about to be great-hearted more than 98%.

The reorganization erythropoietin that cells transfected produces is done further inspection.With the pillbox that confirms these emiocytosises is natural hormone, and the clone of selecting is used in severe hypoxia and causes interior detect (Nature, 191:1069-1087,1961) of body of doing erythropoietin in the mouse of red blood cell proliferation.Clone excretory supernatant liquor causes that with severe hypoxia the mouse of red blood cell proliferation detects, and has biologos in the intensive body.In the experiment that the natural erythropoietin of applying portion purifying is done, notice that for a long time when detecting, the processing of sialidase can be eliminated the vigor (J.Biol.Chem.247:5159-5160,1958) of erythropoietin fully in whole animal.The forfeiture of this vigor infers it is owing to the removing enhanced results of liver to the asialoglycoprotein hormone, because the erythropoietin that sialidase is handled can be possessed its whole vigor in vitro detection.Viewed intravital intensive biologos, illustrated mammalian cells transfected tie up to translate the back modification stage.On adding on the erythropoietin albumen suitable sugar and terminal sialic acid.

In the experiment that separates, the vigor of the detected erythropoietin of external biological is also neutralized by the anti-human erythropoietin antibody that is added in the substratum.

Those representational transfectional cell series are secreted into the erythropoietin in the supernatant liquor, have also detected their proliferative effects to other marrow founder subculture cell.Detect the effect of the erythropoietin of reorganization to the various progenitors in people and mouse bone marrow cells source, comprise: red group founder cell (CFU-E), red burst form cell (erythroid burst-forvming cells, BFU-E), granulocyte-macrophage precursor (CFU-GM) and mixed-cell colony founder cell (CFU-Mix) (J.Cell.Physiol.Suppl.1:79-85,1982; J.Cell.Phy-siol.118:87-96,1984).The proliferative effect that red stem cell shows the reorganization erythropoietin has parallel dose relationship with the effect that natural erythropoietin is shown.By for CFU-GM and CFU-Mix, the concentration of reorganization erythropoietin is increased to every milliliter and detects 10 units of cell culture, does not show any proliferative effect.

When doing the SDS-PAGE analysis under reductive or non-reduced condition, the reorganization erythropoietin of purifying is identical with the erythropoietin electrophoresis situation of purifying in the aplastic anemia patient urine.These albumen demonstrate same microheterogeneity, and its main component is all at molecular weight 34 kilodalton places.

Although the present invention narrates with the embodiment of proposition, common technical skill personnel can make various modifications after having read above-mentioned example, use Equivalent to replace, or component above-mentioned and method are done other change.Therefore, we plan the protection domain of this patent certificate is only limited by appended claim and the defined content of part of equal value thereof.

Claims (8)

1. an expression has the method for the recombinant human erythropoietin of biologos, comprise with DNA, the RNA or the nucleotide sequence transfection host cell that mainly contain the 2.4kb Apa I restricted fragment of human erythropoietin gene, transfectional cell is contacted with substratum, make the cell expressing erythropoietin, and reclaim the erythropoietin of expressing.

2. the process of claim 1 wherein that Apa I restricted fragment is carried on plasmid or the virus.

3. the process of claim 1 wherein that host cell is a mammalian cell.

4. one kind by having the method for the recombinant human erythropoietin of biologos with the expression of cell lines that contacts of substratum of insulation, the improvement of making is included in and adds the clone that can produce erythropoietin in the heat insulating culture base in the said method, and said clone produces by DNA, RNA or the nucleotide sequence transfection host cell system with the 2.4kb Apa I restricted fragment that mainly contains human erythropoietin gene.

According to claim 4 with clone that the heat insulating culture base contacts in express the method for the recombinant human erythropoietin that biologos is arranged, wherein said clone can make the nominal yield of the erythropoietin that at least two hundred ten thousand units are arranged in the substratum of every liter of insulation.

6. the method for claim 4, wherein Apa I restricted fragment is that plasmid is entrained.

7. the method for claim 4, wherein Apa I restricted fragment is that virus is entrained.

8. the method for claim 4, wherein host cell is a mammalian cell.

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