CN101337988B - New erythrocyte-stimulating factor analogues - Google Patents

Abstract

The invention relates to an erythropoietin analog with at least one extra glycosylation sites or a variant thereof. The invention also relates to a DNA sequence for coding the erythropoietin analog or the variant thereof, and a recombinant plasmid and a host cell for expressing the analog or the variant thereof.

Description

A kind of new erythrocyte-stimulating factor analogues

Technical field

The invention belongs to gene engineering technology field, relate in particular to a kind of erythrocyte-stimulating factor analogues or its variant with at least one extra glycosylation site.The invention still further relates to the dna sequence dna of the described erythrocyte-stimulating factor analogues of coding or its variant, and recombinant plasmid and the host cell of expressing this analogue or its variant.

Background of invention

Erythropoietin (erythropoietin, EPO) is that first is found and is applied to clinical hemopoieticgrowth factor.As far back as 1906, after losing blood, the discovery rabbits such as Carnot can in peripheral blood, produce a kind of hemopoietic system that acts on to accelerate erythropoietic material, propose thus to exist a kind of humoral factor to regulate hemopoietic with feedback system.This viewpoint is confirmed that just this factor is named as erythropoietin after more than 30 years.Erythropoietin claims again hematopoietin, red corpuscle stimulating factor, and the genus acid glycoprotein is that red blood cell concentration is in the interior main hormone of normal physiological scope in adjusting precursor red corpuscle propagation, differentiation and the maintenance peripheral blood.It can with the EPO receptors bind on red corpuscle precursor cell surface, promote the synthetic of its oxyphorase to make it the proliferation and differentiation protoerythrocyte, thus the physiological equilibrium of red corpuscle and oxyphorase in the control agent.EPO is a strong hemopoieticgrowth factor, and analyzed in vitro has dose-dependent effect when being presented at the concentration of 0.05～1U/ml.EPO can stimulate CFU-E (BFU-E) and the ripe red corpuscle colony of early inmature red corpuscle (CFU-E) formation.EPO all without effect, is the hemopoieticgrowth factor of thinking that so far effect is the most single to other hematopoietic cells except acting on marrow macronucleus precursor cell.Certainly, to the complete adjusting of red corpuscle hematopoiesis, except EPO, the synergy that also needs other factors comprises Multi-CSF, GM-CSF and IL-1, and these factors impel stem cell to become BFU-E, and combined stimulation breeds red corpuscle in early days, is only subsequently the proliferation function of EPO.

1977, Miyake etc. extracted the sterling that has obtained EPO from Severe Aplastic Anemia anaemia patient's urine, because the scarcity in initial source is difficult to obtain a large amount of sterlings with its biology of abundant research and molecules character.1984, scientists is first take the EPO mRNA of human renal carcinoma cell as template, through the synthetic cDNA of reverse transcription and in intestinal bacteria, cloned and express, obtain on this basis the complete genome of EPO and in eukaryotic cell, efficiently expressed, be applied to clinical laying a good foundation for the biological effect of further investigation EPO and with it.Can use gene engineering method Restruction erythropoietin (rhEPO) both at home and abroad now, and be used for the multiple oligocythemia of clinical treatment, have good curative effect.

Many cell surface proteins and secretory protein that eukaryotic cell produces are all by one or more oligosaccharides base group modifications.This physico-chemical property that is called glycosylated modification can strong effect protein also can play an important role to stability, secretion and the Subcellular Localization of protein.Correct glycosylation is that biological activity is necessary sometimes.Some Eukaryotic gene makes in the bacterium (such as intestinal bacteria) of the cell processes of protein glycosylation in shortage and expresses, and the albumen that is recovered to is not because the shortage glycosylation has or almost do not have activity.

Glycosylation appears at the special position of polypeptide backbone, usually has two classes: O to connect glycosylation and is connected glycosylation with N.The oligosaccharides that O connects is connected on Serine or the threonine residues, and the oligosaccharides that N connects then is connected on the asparagine residue as a sequence A sn-X-Ser/Thr part, and wherein X can be any amino acid except proline(Pro).The saccharide residue that N connects existence in the oligosaccharide structure be connected with O and every type all is different.A class sugar that jointly is present in two types is N-acetylneuraminic acid (hereinafter to be referred as sialic acid).Sialic acid normally N connects the terminal residue that is connected oligosaccharides with O, because it is electronegative, may make glycoprotein have acidity.

Ripe EPO is comprised of 166 amino acid, and aminoacid sequence is referring to SEQ ID NO:1, and peptide chain structure is seen Fig. 1.Two disulfide linkage are arranged in the EPO molecule, 3 N-key glycosylation sites and 1 O-key glycosylation site, wherein two disulfide linkage are absolutely necessary in sex change EPO renaturation and when being folded into the biological activity configuration.The natural human erythropoietin is connected human erythropoietin and is all contained three N and connect oligonucleotide chains and is connected an O and connect an oligonucleotide chain with mammalian cell expression, they account for 40% of albumen total molecular weight altogether.N connects glycosylation and appears at 24,38,83 asparagine residue, and O connects glycosylation and then appears at 126 serine residue (Lai etc., J.Biol.Chem.261:3116,1986; Broundy etc., Arch.Biochem.Biophys.265:329,1988)).Proved that oligonucleotide chain modified by terminal sialic acid residues.Glycosylation erythropoietin is carried out that enzyme is processed and after removing sialic acid residues, causes the activity in vivo forfeiture, but do not affect external activity (Lowy etc., Nature 185:102,1960; Goldwasser etc., J.Biol.Chem.249:4202,1974).This phenomenon once was interpreted as the asialo erythropoietin owing to remove rapidly (Morrell etc., J.Bio.Chem.243:155,1968 from circulation with liver asialoglycoprotein binding protein interactions; The Am.J.Physiol.227:1385 such as Briggs, 1974; Ashwell, Methods Enzymol.50:287,1978).Thereby therefore erythropoietin only avoided by the sialic acid acidifying by liver in conjunction with the time just have an in vivo bioactivity.

In the oligosaccharides of erythropoietin, its effect is also very not definite.Prove that the erythropoietin of part de-glycosylation is compared with the glycosylation form, activity in vivo reduces greatly, but has kept external activity (Dordal etc., Endocrinology 116:2293,1985).But another research then shows, undergo mutation if make as l-asparagine or the serine residue of glycosylation site, thereby remove separately or jointly the oligonucleotide chain that N connects or O connects, then can make the external activity of the mutain that in mammalian cell, the produces (Dube etc. that sharply descend, J.Biol.Chem.263:17516,1988).

Glycoprotein such as erythropoietin can be used such as technology such as isoelectrofocusing (IEF) and be separated into different charged forms.The existing in many ways report of IEF research (Lukosky etc., J.Biochem.50:909,1972 of rough and partially purified erythropoietin prepared product; Shelton etc., Biochem.Med.12:45,1975; Fuhr etc., Biochem.Biophys.Res.Comm.98:930,1981).Urinating from the people the Purification of Human erythropoietin that the people such as Miyake discuss, two erythropoietin components that obtain by the hydroxylapatite chromatography method.These two components are named is II, IIIA.The carbohydrate analysis that subsequently II and IIIA is carried out shows that the sialic acid content of component I I is higher than component III A.

Further research also shows, the degree of glycosylation in a site depends on its on every side amino acid whose composition (Kasturi etc., Biochem.J.323:415,1997 to a great extent; Elliott etc., JBC.279:16854,2004), therefore, changing near the amino acid composition of N-glycosylation site can affect glycosylated degree to a great extent.

Increase the number of erythropoietin carbohydrate, therefore and increase the sialic acid number of each erythropoietin molecule, can produce some superior character, for example solubleness improves, more tolerates proteolyzing, immunogenicity decline, serum half-life prolongation, biological activity raising.

The present invention increases the performance that N-glycosylation site number has improved the EPO analogue by several amino-acid residues in the sudden change natural EPO sequence.

Summary of the invention

The present invention relates to a kind of natural human erythrocyte-stimulating factor analogues or its variant with erythropoietin activity, this analogue comprises at least one extra N-glycosylation site, its site scope is positioned at the 1-9 of human erythropoietin aminoacid sequence, 28-32,40-55,86-92,112-133, the 162-166 position, and this variant has one or several amino acid whose disappearance, replacement or interpolation in the aminoacid sequence of this analogue, and have the activity that this analogue is equal to.In a preferred embodiment, extra N-glycosylation site is positioned at the 1-6 of human erythropoietin aminoacid sequence, 28-32,87-90 position.In another preferred embodiment, the number of extra N-glycosylation site is 1-3.In another preferred embodiment, l-asparagine has been replaced in any position of analogue of the present invention in 2,3,4,28,30,88 of natural human erythropoietin aminoacid sequence.In another preferred embodiment, Threonine has been replaced in any position of analogue of the present invention in 4,5,6,30 of neutral red erythropoietin aminoacid sequence.In another preferred embodiment, Serine has been replaced in analogue of the present invention any one position in 4,5,6,30,32,90 of neutral red erythropoietin aminoacid sequence.In another preferred embodiment, analogue of the present invention has been changed L-Ala, leucine, Isoleucine, α-amino-isovaleric acid, Threonine or Serine in 1,2,3,27,87 positions of neutral red erythropoietin aminoacid sequence.In another preferred embodiment, Isoleucine, phenylalanine, glycine, arginine, Serine, Threonine, l-asparagine, aspartic acid or Histidine have been replaced in analogue of the present invention any one position in 3,4,5,31,89 of natural human erythropoietin aminoacid sequence.In another preferred embodiment, analogue of the present invention is selected from Z ¹Asn ²Y ³X ⁴EPO, Z ²Asn ³Y ⁴X ⁵EPO, Z ³Asn ⁴Y ⁵X ⁶EPO, Z ²⁷Asn ²⁸X ³⁰EPO, Asn ³⁰Y ³¹X ³²EPO, Z ⁸⁷Asn ⁸⁸Y ⁸⁹Ser ⁹⁰EPO or their any two kinds or any three kinds combination, wherein X is selected from Serine and Threonine; And Y and Z are selected from L-Ala, leucine, Isoleucine, α-amino-isovaleric acid, proline(Pro), Threonine and Serine.

The invention still further relates to a kind of dna sequence dna, the human erythropoietin analogue that this dna sequence encoding is of the present invention or its variant.

The invention still further relates to a kind of eukaryotic host cell, its conversion has the carrier that comprises dna sequence dna of the present invention.In a preferred embodiment, this eukaryotic host cell is selected from CHO, COS-7 or BHK.

The invention further relates to a kind of have the natural human erythrocyte-stimulating factor analogues of erythropoietin activity or the production method of its variant, comprise step: (i) cultivate eukaryotic host cell of the present invention, and (ii) from culture, collect the glycoprotein with erythropoietin activity.

The invention further relates to the erythropoietic pharmaceutical composition of a kind of increase, said composition comprises human erythropoietin analogue of the present invention or its variant and pharmaceutically useful thinner, auxiliary agent or the carrier for the treatment of significant quantity.This pharmaceutical composition is preferred for promoting erythropoiesis, prevention or treatment anaemia.

Particularly, the carrier that comprises dna sequence dna of the present invention changes in the eukaryotic cell (such as Chinese hamster ovary celI, COS-7 cell, BHK) expresses, and preferentially selects Chinese hamster ovary celI.Add serum free medium in the Chinese hamster ovary celI, comprise the carrier of dna sequence dna of the present invention, the transfection mixed solution that lipofectamine forms, in substratum, add methotrexate (MTX) screening resistance clone after cultivating for some time.Improve gradually subsequently the resistance clone that MTX concentration is screened high expression level.Utilize enzyme-linked immunosorbent assay to confirm that resulting cell can express the erythropoietin analogue.

Can express the cell amplification of erythropoietin analogue, cultivate, collect nutrient solution.Then, contain the cell culture fluid of recombiant erythropoietin analogue and be splined on the post of the similar ion-exchange chromatography media filling such as QSepharose Fast flow or DEAE Sepharose Fast flow (GE) after concentrated, after the damping fluid flushing with low pH value damping fluid drip washing pillar.At last, the damping fluid by containing salt with the erythropoietin analogue determined by eluting on the post.The above-mentioned erythropoietin analogue of collecting further passes through the reversed phase chromatography purifying, and the reversed phase chromatography posts such as loading C4, C8, Source utilize ethanol, Virahol or the acetonitrile of 0-100% gradient that erythropoietin analogue is eluted.

Those skilled in the art can expect, lack in the aminoacid sequence of erythrocyte-stimulating factor analogues of the present invention, replace or add one or several amino acid, and the variant of generation still has the activity that this analogue is equal to.For example, at the non-key position of analogue of the present invention one or several amino acid of disappearance; Mutual replacement between the amino acid of the same type (non-conservation amino acid), for example, the replacement between the acidic amino acid, for example Glu and Asp, the replacement between the basic aminoacids, for example Arg and Lys, the replacement between the die aromatischen Aminosaeuren, for example Phe and Tyr; Replacement between the hydroxyl polare Aminosaeren, for example Thr and Ser; Add one or several amino acid in the front and back of analogue full length amino acid sequence, for example, add His label or secretory signal sequence at the N end, or add polyA tail etc. at the C end.

Erythrocyte-stimulating factor analogues of the present invention comprises the aminoacid sequence of at least one extra glycosylation site.Extra glycosylation site can cause the sialic acid content than the more carbohydrate chain of natural human erythropoietin number and Geng Gao.The analogue that sialic acid content is higher than the natural human erythropoietin produces by adding glycosylation site, and these glycosylation sites do not disturb the required secondary of biological activity or three grades of conformations.The human erythropoietin analogue preferably has 1,2 or 3 extra N-glycosylation site, connects carbohydrate chain thereby add 1,2 or 3 extra N.For example, with the proline(Pro) of 2 of l-asparagine displacements, the arginine with 4 of Serine displacements obtains sequence A sn-Pro-Ser, and this sequence just becomes the 4th N-glycosylation site.A kind of like this variation can provide 4 extra sialic acids by per molecule usually.It will be recognized by those skilled in the art, the present invention includes many other human erythropoietin analogues with extra glycosylation site.

The present invention has analyzed the situation that carbohydrate increases in the erythrocyte-stimulating factor analogues that increases respectively 1-3 extra glycosylation site.Concrete grammar is, gets the expression supernatant of erythropoietin analogue, uses the 15%SDS-Polyacrylamide Gel Electrophoresis, transfers on the nitrocellulose (referring to document: Burnette etc., Anal.Biochem.112:195-203,1981; Elliott etc., Gen 79:167-180,1989), generate plain monoclonal antibody with the mouse anti-erythrocyte and carry out Western Blot analysis.

The iso-electric point of having analyzed the erythrocyte-stimulating factor analogues that increases respectively 1-3 extra glycosylation site changes.Use respectively 0.1M phosphoric acid and 0.1M sodium hydroxide as anolyte and catholyte, employing contains 10% polyacrylamide gel of pH 2.5-5 and pH 3-10 amphotericeledrolyte and 7M urea, will be according to after containing of obtaining of the various erythrocyte-stimulating factor analogues dialysis of above-mentioned cultivation and purification process, 800V, 50mA, 30W electrophoresis 2 hours.After electrophoresis finishes, be transferred on the nitrocellulose filter, generate with the mouse anti-erythrocyte that plain monoclonal antibody be combined again and with sheep anti mouse enzyme labelled antibody mark, add substrate solution and develop the color.

The present invention also provides the method for measuring above-mentioned erythrocyte-stimulating factor analogues purified sialic acid content.The method summary utilizes Bradford analysis of protein method (Bradford, Anal.biochem.72,248. (1976)) to determine the protein concentration of each erythrocyte-stimulating factor analogues purified and it is adjusted to 0.2-0.4mg/ml.Add Resorcinol, copper sulfate, hydrochloric acid soln respectively in sample and standard substance, boiled 30 minutes, add butylacetate-butanol solution (4: 1), fully mixing is got organic phase and is detected behind the standing demix.Measure absorbancy at the 580nm wavelength.Concentration with the sialic acid reference substance solution is done straight-line regression to its corresponding absorbancy, obtains the sialic absorbancy of 5 μ g by linear regression equation.Calculate erythrocyte-stimulating factor analogues purified sialic acid content (mol/mol albumen)=(A according to formula ₂* 5 * 3.24 * W * D)/(A ₁* P * 100), A ₁Be the sialic absorbancys of 5 μ g, A ₂Absorbancy for the erythrocyte-stimulating factor analogues purified, D is the extension rate of erythrocyte-stimulating factor analogues purified, P is the protein content (μ g/ μ l) of erythrocyte-stimulating factor analogues purified, W is the amount (amount that does not comprise carbohydrate) of 1nmol erythrocyte-stimulating factor analogues, is equivalent to 18.2 μ g.

The present invention also provides the method for the erythrocyte-stimulating factor analogues isomer mixture (embodiment 10) that a kind of separation has different iso-electric point scopes, the simple description, to contain the cell culture fluid of erythrocyte-stimulating factor analogues is splined on the post of Q Sepharose Fast flow (GE) filling after concentrated, behind the application of sample, wash pillar with damping fluid.Afterwards with low pH value damping fluid drip washing pillar.At last by with the damping fluid that contains 0-400mM NaCl respectively with the erythropoietin analogue isomer of different components by eluting on the post.

The present invention has also analyzed above-mentioned erythrocyte-stimulating factor analogues isomer mixture stimulates erythropoietic effect to change in vivo.Can stimulate erythropoietic effect according to erythropoietin, according to basic, normal, high three dosage groups, give mouse (Balb/c) subcutaneous injection EPO.Because erythropoietin (EPO) can promote the release of the reticulocyte in the marrow, so that the rising of the ratio of the reticulocyte counts in the Mouse Blood and RBC number, and lift-off value and injected dose are linear.Detect the EPO Biological acdtivity in vivo by the dose-response parallel method.(this standard substance derives from Nat'l Pharmaceutical ﹠ Biological Products Control Institute with the EPO biological reference standard, be used for measuring in vivo bioactivity) be diluted to concentration 80IU/ml, 40IU/ml and 20IU/ml, the erythrocyte-stimulating factor analogues isomer mixture also is diluted to respectively near above-mentioned three kinds of concentration.Mouse is divided into standard group and to be checked group at random.Each group is established basic, normal, high three dosage.According to basic, normal, high three dosage groups difference veutro subcutaneous injection mouse.Used EDTA-K in the mouse orbit blood sampling in the 4th day after the injection ₂(physiological saline that contains 1%EDTA) anticoagulant tube anti-freezing.Get anticoagulation in R-500 analyser (Japanese Sysmex company) Auto-counting red corpuscle sum, reticulocyte is total and reticulocyte counts to the ratio (Ret) of red corpuscle sum.In Ret value input computer, program is calculated the biologic activity of test sample automatically.Specific activity is the observed value of specific activity in the relative body in the body described here, is not the absolute interior specific activity observed value of body.Specific activity only is used for the relatively relative reactivity of isomer, and the mensuration of isomer adopts identical measuring method, adopts the same terms, and the animal of identical type has for the identical data analysis of calculating specific activity, measures the same analysis method of protein content.

The present invention has also studied above-mentioned erythrocyte-stimulating factor analogues and isomer is injected rear reticulocyte content over time to the mouse single dose.

The change detection method of reticulocyte content behind injection erythrocyte-stimulating factor analogues or its isomer: erythrocyte-stimulating factor analogues or its isomer with the dosage subcutaneous injection of 5 μ g/kg to the Balb/c mouse, and take the recombinant human erythropoietin of same dose as reference substance, establish simultaneously the solvent control group.Blood sampling in per 24 hours after the injection detects reticulocyte content with reticulocyte numeration instrument, detects reticulocyte content temporal evolution situation.

The present invention has also studied above-mentioned erythrocyte-stimulating factor analogues isomer increases situation at Pharmacokinetics in Rat and Mouse Blood specific volume of cell.

The pharmacokinetics measuring method

Select male SD (Sprague Dawley) rat, intravenous injection ¹²⁵The erythrocyte-stimulating factor analogues isomer mixture 0.05 μ g/kg that obtains among the erythropoietin of I mark and the embodiment 10, according to the timed interval (5 minutes to 10 hours) the eye socket venous blood collection of setting, collect serum, calculate pharmacokinetic parameter.

The erythrocyte-stimulating factor analogues isomer is on the impact of Mouse Blood specific volume of cell

The increase of degree of glycosylation causes erythrocyte-stimulating factor analogues elimination Increased Plasma Half-life in vivo, and this becomes possibility for improving dosing interval.Adopt the isomer mixture that obtains among the erythropoietin of various dose and the embodiment 10, respectively with weekly and on every Wendesdays time subcutaneous injection to BALB/c, continuous 4 weeks, the changing conditions of monitoring hematocrit.

Description of drawings

Fig. 1 has shown and has had 3 Natively glycosylated sites in the human erythropoietin aminoacid sequence, lay respectively at 24,38 and 83, and marked the position of impelling erythropoietin to produce extra glycosylation site, laid respectively at 2,3,4,28,30 and 88.

Fig. 2 has shown the plasmid construction method.After natural EPO cDNA fragment is taken turns PCR by rite-directed mutagenesis is several, native sequences is introduced in the primer sudden change.The total length EPO that recovery obtains suddenling change, use the BamHI enzymic digestion, use simultaneously BamHI enzymic digestion carrier for expression of eukaryon pEC4, reclaim fragment, PCR product and carrier after cutting with T4DNA ligase enzyme connection BamHI enzyme, room temperature 5 minutes, transform competent escherichia coli cell DH5 α, select positive colony, identify the clone with the enzymic digestion method, and with correct cloning and sequencing, obtain containing the carrier pEC4 EPO of mutant clon.

Fig. 3 has shown the molecular weight situation of EPO and analogue expression product thereof.Swimming lane 1 is the recombinant human epo, and swimming lane 2-4 is the EPO analogue that contains an extra glycosylation site, and swimming lane 5 is for containing the EPO analogue of two extra glycosylation sites, and swimming lane 6 is for containing [the Asn of three extra glycosylation sites ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰] EPO, swimming lane 7 is mutant [Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰Ala ¹⁶⁴] EPO.The result shows, increase glycosylation site after, molecular weight of product all increases to some extent, and molecular weight increasing and increase with glycosylation site.From having an extra glycosylation site [Asn ²Gly ³Thr ⁴] EPO, [Asn ³Phe ⁴Ser ⁵] EPO and [Val ²Asn ³Phe ⁴Ser ⁵] EPO, because the difference of the last amino acids of glycosylation site, molecular weight increases different, [Val ²Asn ³Phe ⁴Ser ⁵] EPO expression product [Asn ²Gly ³Thr ⁴] EPO and [Asn ³Phe ⁴Ser ⁵] EPO expression product molecular weight is large.Mutant [Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰Ala ¹⁶⁴] EPO expression product molecular weight and the [Asn with three extra glycosylation sites ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰] EPO analog molecules amount is consistent, illustrates that the mutation expression product in non-glycosylated site does not have extra sugar chain synthetic, on the not impact of its molecular weight.

Fig. 4 has shown erythrocyte-stimulating factor analogues culture expression product IEF immunoblotting assay result.Swimming lane 8 is the recombinant human epo, and swimming lane 1-3 is the EPO analogue that contains an extra glycosylation site, and swimming lane 4-5 is the EPO analogue that contains two extra glycosylation sites, and swimming lane 6 is for containing [the Asn of three extra glycosylation sites ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰] EPO, swimming lane 7 is mutant [Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰Ala ¹⁶⁴] EPO.The result shows that the expression product acidic molecular all increases to some extent, and acidic molecular increasing and increase with glycosylation site.From having an extra glycosylation site [Asn ²Gly ³Thr ⁴] EPO, [Asn ³Phe ⁴Ser ⁵] EPO and [Val ²Asn ³Phe ⁴Ser ⁵] EPO since the position of glycosylation site with and the amino acid whose difference of last position, site, it is different that acidic molecular increases proportion, [Val ²Asn ³Phe ⁴Ser ⁵] EPO expression product [Asn ²Gly ³Thr ⁴] EPO, [Asn ³Phe ⁴Ser ⁵] EPO expression product acidic molecular is many.Mutant [Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰Ala ¹⁶⁴] EPO expression product and [Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰] acidic molecular of EPO expression product is consistent, illustrates that non-glycosylated site mutation expression product does not have extra sugar chain synthetic, on the not impact of its iso-electric point.

Fig. 5 has shown by the SDS electrophoretic analysiss to the high isomer mixture of the erythrocyte-stimulating factor analogues that contains different extra N-glycosylation sites.After the increase glycosylation site had been described, the molecular weight of product increased.The extra analog molecules amount that increases by three glycosylation sites is greater than two glycosylation sites of extra increase, and the analog molecules amount that additionally increases by two glycosylation sites is higher than glycosylation site of extra increase, and an extra glycosylation site [Val ²Asn ³Phe ⁴Ser ⁵] the EPO molecular weight of product is than having equally an extra glycosylation site [Asn ²Gly ³Thr ⁴] EPO and [Asn ³Phe ⁴Ser ⁵] EPO is large.The last amino acids that glycosylation site is described has certain impact to it.[Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰] EPO and [Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰Ala ¹⁶⁴] the EPO molecular weight is consistent.[Asn is described ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰Ala ¹⁶⁴] sudden change of EPO in non-glycosylated site do not have extra sugar chain synthetic, on the not impact of its molecular weight.

Fig. 6 has shown by the IEF electrophoretic analysiss to the high isomer mixture of the erythrocyte-stimulating factor analogues that contains different extra N-glycosylation sites.After the increase glycosylation site had been described, the acidity band of product increased.The extra acid carbohydrate of analogue of three glycosylation sites that increases is more than two glycosylation sites of extra increase, the extra acid carbohydrate of analogue that increases by two glycosylation sites increases a glycosylation site more than extra, and an extra glycosylation site [Val ²Asn ³Phe ⁴Ser ⁵] the acid band of EPO product is than having equally an extra glycosylation site [Asn ²Gly ³Thr ⁴] EPO and [Asn ³Phe ⁴Ser ⁵] EPO increases.The last amino acids that glycosylation site is described has certain impact to it.[Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰] EPO and [Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Glv ⁸⁹Ser ⁹⁰Ala ¹⁶⁴] the acid band of EPO unanimously.[Asn is described ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰Ala ¹⁶⁴] the non-glycosylated site mutation expression product of EPO do not have extra sugar chain synthetic, on the not impact of its iso-electric point.

After Fig. 7 showed the erythrocyte-stimulating factor analogues single dose injection mouse of different catastrophe points, reticulocyte over time in the body.In this research, with the purified of erythropoietin analogue, to the female BALB/c mouse of body weight as 20 ± 2, and take the recombinant human erythropoietin of same dose as reference substance, establish simultaneously the solvent control group take 5 μ g/kg subcutaneous injections.Blood sampling in per 24 hours after the injection detects reticulocyte content with reticulocyte numeration instrument, investigates reticulocyte content temporal evolution situation.The result shows, increase with extra glycosylation site, erythrocyte-stimulating factor analogues prolongs the action time in Mice Body, and the prolongation of time and glycosylation site increase positive correlation, action intensity (in the area under curve) enhancing of analogue in Mice Body.And action time and intensity are subject to the last bit amino effect of acid of glycosylation site.

Fig. 8 has shown respectively the 10 described method preparation [Asn with embodiment ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰] the IEF collection of illustrative plates of EPO analogue isomer and liquid collecting.Swimming lane 1 is natural EPO, and swimming lane 2-5 is respectively isomer 1-5,2-6,3-6,4-8.Result's demonstration is along with acid band among isomer and the liquid collecting IEF increases, and activity in vivo increases.Illustrate that extra glycosylation has increased the acid carbohydrate in the molecule, the variation of this molecular structure causes activity in vivo to raise.

Fig. 9 has shown erythropoietin and has prepared [Asn with embodiment 10 described methods ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰] mixture of EPO isomer 3-6 to injected in mice after reticulocyte curve over time.With erythropoietin isomer 3-6 mixture, to the female Balb/c mouse of body weight as 20 ± 2, and take the recombinant human erythropoietin of same dose as reference substance, establish simultaneously the solvent control group take 5 μ g/kg subcutaneous injections.Blood sampling in per 24 hours after the injection detects reticulocyte content with reticulocyte numeration instrument, investigates reticulocyte content temporal evolution situation.The result shows that erythropoietin group second day RET% after medication begins to raise, and reaches the peak, and continues about 4 days in the 4th day; Erythropoietin analogue group RET% also begins to raise in second day, reaches the peak, and continues about 7 days in the 6th day.This shows, compare isomer 3-6 mixture with the recombinant human erythropoietin and obviously prolong the action time in Mice Body that action intensity (in area under curve) strengthens.

Figure 10 has shown erythropoietin and [Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰] the mixture intravenous injection serum level temporal evolution curve of EPO isomer 3-6, select 10～11 weeks of male SD rat (SpragueDawley), body weight 300 ± 20g, 6/group.Intravenous injection ¹²⁵The erythropoietin of I mark and erythropoietin analogue isomer 3-6 mixture, every mouse 1 μ ci ¹²⁵The erythropoietin of I mark or analogue isomer according to the timed interval (5 minutes to 10 hours) the eye socket venous blood collection of setting, are collected serum.Get 0.1ml serum and add the cold dehydrated alcohol of 0.9ml (20 ℃), put 4 ℃ of overnight incubation, centrifugal 15 minutes of 12000rpm detects with the coffee detector, calculates pharmacokinetic parameter.Compare with erythropoietin, removing speed obviously reduces in the isomer 3-6 mixing object, cause eliminating in the body Increased Plasma Half-life, the transformation period is respectively 8.5h and 2.6h behind the intravenous injection rat of the two, and the elimination transformation period of erythrocyte-stimulating factor analogues is 3.3 times of erythropoietin.

Figure 11 and Figure 12 are injected in mice erythropoietin and [Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰] hematocrit temporal evolution situation behind the mixture of EPO isomer 3-6.Laboratory animal is female BALB/c mouse, the cleaning level, age in male and female half and half, 8 week, body weight 18～22g, principle grouping according to random packet, every group 10, adopt erythropoietin and the erythropoietin analogue isomer 3-6 mixture of various dose, respectively with weekly and on every Wendesdays time subcutaneous injection to BALB/c, the solvent of control group injection equal volume, continuous 4 weeks.The changing conditions of monitoring hematocrit, 2 times/week, administration same day is the 1st day (1d), gets blood respectively at the kapillary that the 4th day (4d) and 7 days (7d) in 1 week processes with heparinization, centrifuging detection hematocrit is adopted in sealing.The result shows, after weekly and on every Wendesdays time administration, isomer 3-6 mixture obviously strengthens the rising effect of Mouse Blood specific volume of cell, and prompting clinical application erythrocyte-stimulating factor analogues can anemia, the weekly injection can reach the result for the treatment of of expection.

Embodiment

Illustrate in greater detail the present invention by the following example, but these embodiment should not think it is to limit the scope of the invention by mistake.Used erythropoietin standard substance is recombinant human erythropoietin's standard substance among the embodiment, and this standard substance derives from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, is used for measuring in vivo bioactivity.

Embodiment 1: the structure of human erythropoietin analogue dna fragmentation

Have 3 natural N-glycosylation sites in the human erythropoietin aminoacid sequence, lay respectively at 24,38 and 83, the position of impelling erythropoietin to produce extra glycosylation site can be 2,3,4,28,30,88 (seeing Fig. 1).

To suddenly change the combination between the group of above site, as making up [Asn ²Gly ³Thr ⁴], [Asn ²⁸Thr ³⁰], [Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰] combination, add the N-glycosylation site of 2,28,88 3 positions, and strengthen the glycosylation of this position by the aminoacid sequence of or the last position of glycosylation site in the middle of changing.

The extra primer of N-glycosylation site sudden change part that adds is referring to SEQ ID NOS:2-85.Utilize these primers, carry out the erythrocyte-stimulating factor analogues dna fragmentation after polymerase chain reaction (PCR) can obtain to suddenly change take erythropoietin DNA as template.Template DNA derives from the natural EPO cDNA library.The structure in library is summarized as follows, utilize Trizol from the tire hepatic tissue, to extract total RNA, use Oligo (dT) Cellulose (Cat.No.15940-026, Lifetechnologies, USA), by the method that producer provides, extract PolyA+RNA, use afterwards cDNA synthetic agent box (ZAP-cDNA

Synthesis Kit, Cat.No.200400-200402, Stratagene, USA) synthesize the double-stranded cDNA of people's tire liver, angle from above-mentioned cDNA library according to US Pat.4703008 design primer and get natural erythropoietin dna fragmentation.

Take above-mentioned EPO dna fragmentation as template, the primer of design SEQ ID NOS:86-93:

3 ' end: the 2-4 position changes NST into by PPR

Primer 1:SEQ ID NO:86

5 ' end 2-4 position primer

Primer 2: SEQ ID NO:87

3 ' end 30-32 position changes NRS into by AEH

Primer 3:SEQ ID NO:88

5 ' end 30-32 position primer

Primer 4:SEQ ID NO:89

3 ' end 87-90 position changes VNIS into by PWEP

Primer 5:SEQ ID NO:90

5 ' end 87-90 position primer

Primer 6:SEQ ID NO:91

Design EPO two ends primer is used for PCR

5 ' end adds BamHI site and GCCACC

Primer 7:SEQ ID NO:92

3 ' end primer adds the BamHI site

Primer 8:SEQ ID NO:93

(the PCR instrument is ABI 2700, and reagent is the Supermix of Invitrogen company to carry out first round PCR with SEQ ID NO:86+92,87+88,89+90,91+93 ^TM), condition is 95 ℃ of 5min, (95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s) * 30 circulation, 72 ℃ of 7min.The dna fragmentation of gained carries out agarose gel electrophoresis, the eZNA that utilizes manufacturing firm (Omiga company) to provide ^TMGel reclaims test kit and method reclaims fragment.Respectively take SEQ ID NO:86+92 and 87+88,89+90,91+93 as template, SEQ ID NO:88+92,89+93 are primer, carry out second and take turns PCR again, and condition is the same, reclaim fragment.Then take SEQ ID NO:92+88 and SEQ ID NO:89+93 as template, SEQ ID NO:92+93 is primer, the total length EPO that obtains suddenling change with identical PCR condition.Reclaim fragment, with BamHI (Invitrogen) enzymic digestion, use simultaneously BamHI enzymic digestion carrier for expression of eukaryon pEC4, reclaim fragment, PCR product and carrier after cutting with T4DNA ligase enzyme (Invitrogen) connection BamHI enzyme, room temperature 5 minutes, transform competent escherichia coli cell DH5 α, select positive colony, identify the clone with the enzymic digestion method, and with correct cloning and sequencing, obtain containing the carrier pEC4 EPO of mutant clon.Expression vector pEC4 imports pcDNA3.1 (Invitrogen) with little mouse dihydrofolate reductase (DHFR) gene fragment and forms, and mouse DHFR gene fragment is to synthesize according to the sequence that GenebankNucleotide AH001899 announces.Aforesaid method is summarized in Fig. 2.

Make up erythrocyte-stimulating factor analogues with aforesaid method.The dna sequence dna that has provided the part analogue in the table 1 changes, otherwise is exactly for the sequence of the Oligonucleolide primers that suddenlys change and the sequence complementation of human erythropoietin.

Table 1: part has the erythrocyte-stimulating factor analogues of N-glycosylation site

The numbering amino-acid substitution sequentially changes

N1 Asn ²Gly ³Thr ⁴ CCA CCA CGC→AAT GGC ACT

N2 Asn ³Phe ⁴Ser ⁵ CCA CGC CTC→AAT TTC TCC

N3 Val ²Asn ³Phe ⁴Ser ⁵ CCA CCA CGC CTC→GTC AAT TTC TCC

N4 Asn ⁴Lys ⁵Thr ⁶ CGC CTC ATC→AAT AAG ACT

N5 Asn ²⁸Thr ³⁰ GGC→AAT，GCT→ACT

N6 Asn ²⁸Ser ³⁰ GGC→AAT，GCT→TCC

N7 Asn ³⁰Thr ³¹Ser ³² GCT GAA CAC→AAT ACT TCC

N8 Asn ³⁰Gly ³¹Ser ³² GCT GAA CAC→AAT GGC TCC

N9 Asn ³⁰Lys ³¹Ser ³² GCT GAA CAC→AAT AAG TCC

N10 Asn ³⁰Ile ³¹Ser ³² GCT GAA CAC→AAT ATC TCC

N11 Asn ³⁰Asp ³¹Ser ³² GCT GAA CAC→AAT GAC TCC

N12 Val ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰ CCG TGG GAG CCC→GTC AAT GGC TCC

N13 Val ⁸⁷Asn ⁸⁸Ser ⁸⁹Ser ⁹⁰ CCG TGG GAG CCC→GTC AAT TCC TCC

N14 Val ⁸⁷Asn ⁸⁸Ile ⁸⁹Ser ⁹⁰ CCG TGG GAG CCC→GTC AAT ATC TCC

N15 Val ⁸⁷Asn ⁸⁸Asp ⁸⁹Ser ⁹⁰ CCG TGG GAG CCC→GTC AAT GAC TCC

N16 Val ⁸⁷Asn ⁸⁸Ile ⁸⁹Ser ⁹⁰ CCG TGG GAG CCC→GTC AAT ATC TCC

N17 Val ⁸⁷Asn ⁸⁸Lys ⁸⁹Ser ⁹⁰ CCG TGG GAG CCC→GTC AAT AAG TCC

N18 Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰ CCG TGG GAG CCC→TCC AAT GGC TCC

N19 Ser ⁸⁷Asn ⁸⁸Lys ⁸⁹Ser ⁹⁰ CCG TGG GAG CCC→TCC AAT AAG TCC

N20 Ser ⁸⁷Asn ⁸⁸Asn ⁸⁹Ser ⁹⁰ CCG TGG GAG CCC→TCC AAT AAT TCC

N21 Ser ⁸⁷Asn ⁸⁸Phe ⁸⁹Ser ⁹⁰ CCG TGG GAG CCC→TCC AAT TTC TCC

N22 Ser ⁸⁷Asn ⁸⁸Asp ⁸⁹Ser ⁹⁰ CCG TGG GAG CCC→TCC AAT GAC TCC

N23 Ser ⁸⁷Asn ⁸⁸His ⁸⁹Ser ⁹⁰ CCG TGG GAG CCC→TCC AAT CAC TCC

N24 Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Lys ⁸⁹Ser ⁹⁰ N5+N19

N25 Asn ²⁸Ser ³⁰Ser ⁸⁷Asn ⁸⁸Asn ⁸⁹Ser ⁹⁰ N6+N20

N26 Asn ²⁸Thr ³⁰Val ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰ N5+N12

N27 Asn ²⁸Ser ³⁰Ser ⁸⁷Asn ⁸⁸Asp ⁸⁹Ser ⁹⁰ N6+N22

N28 Asn ²⁸Thr ³⁰Val ⁸⁷Asn ⁸⁸Asp ⁸⁹Ser ⁹⁰ N5+N15

N29 Asn ³⁰Thr ³¹Ser ³²Val ⁸⁷Asn ⁸⁸Ser ⁸⁹Ser ⁹⁰ N7+N13

N30 Asn ³⁰Lys ³¹Ser ³²Val ⁸⁷Asn ⁸⁸Ile ⁸⁹Ser ⁹⁰ N9+N16

N31 Asn ³⁰Ile ³¹Ser ³²Ser ⁸⁷Asn ⁸⁸Asn ⁸⁹Ser ⁹⁰ N10+N20

N32 Asn ³⁰Asp ³¹Ser ³²Ser ⁸⁷Asn ⁸⁸Asp ⁸⁹Ser ⁹⁰ N11+N22

N33 Asn ³⁰Thr ³¹Ser ³²Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰ N7+N18

N34 Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰ N5+N18

N35 Asn ³⁰Gly ³¹Ser ³²Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰ N8+N18

N36 Asn ³⁰Thr ³¹Ser ³²Val ⁸⁷Asn ⁸⁸Ser ⁸⁹Ser ⁹⁰ N7+N13

N37 Asn ²⁸Ser ³⁰Val ⁸⁷Asn ⁸⁸Ser ⁸⁹Ser ⁹⁰ N6+N13

N38 Asn ³⁰Gly ³¹Ser ³²Val ⁸⁷Asn ⁸⁸Ser ⁸⁹Ser ⁹⁰ N8+N13

N39 Asn ²Gly ³Thr ⁴Asn ²⁸Ser ³⁰ N1+N6

N40 Asn ⁴Gly ⁵Ser ⁶

N41 Asn ³Phe ⁴Ser ⁵Asn ³⁰Ile ³¹Ser ³² N2+N10

N42 Asn ⁴Lys ⁵Thr ⁶Asn ³⁰Gly ³¹Ser ³² N4+N8

N43 Asn ²Gly ³Thr ⁴Asn ³⁰Thr ³¹Ser ³² N1+N7

N44 Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰ N1+N34

N45 Asn ³Phe ⁴Ser ⁵Asn ²⁸Thr ³⁰Val ⁸⁷Asn ⁸⁸Ser ⁸⁹Ser ⁹⁰ N2+N5+N13

N46 Asn ⁴Lys ⁵Thr ⁶Asn ²⁸Thr ³⁰Val ⁸⁷Asn ⁸⁸Asp ⁸⁹Ser ⁹⁰ N4+N28

N47 Asn ²Gly ³Thr ⁴Asn ³⁰Lys ³¹Ser ³²Val ⁸⁷Asn ⁸⁸Ile ⁸⁹Ser ⁹⁰ N1+N30

N48 Asn ³Phe ⁴Ser ⁵Asn ³⁰Ile ³¹Ser ³²Ser ⁸⁷Asn ⁸⁸Asn ⁸⁹Ser ⁹⁰ N2+N31

N49 Asn ⁴Lys ⁵Thr ⁶Asn ³⁰Asp ³¹Ser ³²Ser ⁸⁷Asn ⁸⁸Asp ⁸⁹Ser ⁹⁰ N4+N32

N50 Asn ⁴Gly ⁵Ser ⁶Asn ³⁰Gly ³¹Ser ³² N8+N40

N51 Asn ³Gly ⁴Thr ⁵Asn ³⁰Gly ³¹Ser ³²Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰

N52 Asn ⁴Gly ⁵Thr ⁶Asn ³⁰Lys ³¹Ser ³²Val ⁸⁷Asn ⁸⁸Ile ⁸⁹Ser ⁹⁰

N53 Asn ²Gly ³Thr ⁴Asn ²⁸Ser ³⁰Ser ⁸⁷Asn ⁸⁸Asp ⁸⁹Ser ⁹⁰ N1+N27

N54 Asn ³Gly ⁴Thr ⁵Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Lys ⁸⁹Ser ⁹⁰

N55 Asn ⁴Gly ⁵Thr ⁶Asn ²⁸Ser ³⁰Ser ⁸⁷Asn ⁸⁸Asn ⁸⁹Ser ⁹⁰

N56 Asn ⁴Gly ⁵Thr ⁶Asn ³⁰Thr ³¹Ser ³²Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰

N57 Asn ²Gly ³Thr ⁴Asn ³⁰Thr ³¹Ser ³²Val ⁸⁷Asn ⁸⁸Ser ⁸⁹Ser ⁹⁰ N1+N29

N58 Asn ³Gly ⁴Thr ⁵Asn ²⁸Ser ³⁰Val ⁸⁷Asn ⁸⁸Ser ⁸⁹Ser ⁹⁰

N59 Asn ⁴Gly ⁵Thr ⁶Asn ³⁰Gly ³¹Ser ³²Val ⁸⁷Asn ⁸⁸Ser ⁸⁹Ser ⁹⁰

N60 Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰Ala ¹⁶⁴

With above-mentioned cloning vector numbering called after pEC4-X (X is the analogue numbering).

Embodiment 2:Asn ²Gly ³Thr ⁴The preparation of EPO

A. the structure of engineering cell, screening and cultivation

According to the plasmid pEC4-N1 transfection CHO cell that the method for embodiment 1 makes up, host cell is Tetrahydrofolate dehydrogenase deficient Chinese Hamster (CHO dhfr-) available from American type culture collection (ATCC).With this cell cultures in the 100mm culture dish, treat that cell grows to 60～95% when full, with serum free medium drip washing cell, the transfection mixed solution that adds 5ml serum free medium, 10 μ g pEC4-N1,60 μ g lipofectamine composition, cultivated 4 hours for 37 ℃, the sucking-off substratum adds the MEM substratum that contains 5% foetal calf serum, 37 ℃ of overnight incubation.In substratum, add subsequently MTX, continue to cultivate and to occur to resistance clone in 10-14 days.Cell so that the trysinization resistance clone is turned out increases MTX concentration, the screening resistance clone.Utilize enzyme-linked immunosorbent assay to confirm that resulting cell can express erythropoietin analogue N1.With expressing cell amplification, the cultivation of erythropoietin analogue N1, collect nutrient solution, carry out purifying.

B.Asn ²Gly ³Thr ⁴The purifying of EPO

Use the three-step approach purifying EPO analogue N1 that is formed by ion-exchange chromatography, reverse chromatograms and gel filtration chromatography.

Detailed process is as follows:

1. collect to express the cho cell conditioned medium of EPO analogue N1, use with the ultra-filtration membrane of 5k molecular retention amount after its concentrated 15 times, damping fluid is replaced into 10mM Tris-HClpH7.0.

2. the flow velocity that the solution after will concentrating divides with 0.5cm/ is added on the Q Sepharose Fast flow post, and this post is used 10mM Tris-HCl pH7.0 balance mistake in advance.

3. after application of sample finishes, wash post with the 10mM Tris-HCl pH7.0 of 5 column volumes, and use the acetic acid by 40mM, the 1mM glycine, the solution that 6M urea forms washes the post bed, subsequently with 10mM Tris-HCl, 200mM NaCl, pH7.0 is with Asn ²Gly ³Thr ⁴EPO is by eluting on the post.

4. with the Asn that collects ²Gly ³Thr ⁴The EPO component is added to VydacC with the flow velocity that 3cm/ divides ₄On the reversed-phase resin post, this post has been used 20% ethanol/10mM Tris-HCl, pH7.0 balance mistake.With 20%-95% ethanol/10mM Tris-HCl, pH7.0 progressively launches wash-out.Through 50-75% ethanol/10mM Tris-HCl, the component of pH7.0 wash-out is used 10mM Tris-HClpH7.0 according to 1: 2.5, after the dilution, use concentrated 20 times of the little thickener of pall nanosep 10K omega, and the displacement damping fluid is 10mM Tris-HCl pH7.0.

5. concentrated solution is carried out purifying with the S-200 post of crossing with 20mM citrate buffer solution pH6.8 balance, collecting corresponding monomer analogue peak.

Embodiment 3:Asn ³Phe ⁴Ser ⁵The preparation of EPO

A. the structure of engineering cell, screening and cultivation

According to the plasmid pEC4-N2 transfection CHO cell that the method for embodiment 1 makes up, host cell is Tetrahydrofolate dehydrogenase deficient Chinese Hamster (CHO dhfr-) available from American type culture collection (ATCC).With this cell cultures in the 100mm culture dish, treat that cell grows to 60～95% when full, with serum free medium drip washing cell, the transfection mixed solution that adds 5ml serum free medium, 10 μ g pEC4-N2,60 μ g lipoFectamin composition, cultivated 4 hours for 37 ℃, the sucking-off substratum adds the MEM substratum that contains 5% foetal calf serum, 37 ℃ of overnight incubation.In substratum, add subsequently MTX, continue to cultivate and to occur to resistance clone in 10-14 days.Cell so that the trysinization resistance clone is turned out increases MTX concentration, the screening resistance clone.Utilize enzyme-linked immunosorbent assay to confirm that resulting cell can express erythropoietin analogue N2.With expressing cell amplification, the cultivation of erythropoietin analogue N2, collect nutrient solution, carry out purifying.

B.Asn ³Phe ⁴Ser ⁵The purifying of EPO

Use the three-step approach purifying EPO analogue N2 that is formed by ion-exchange chromatography, reverse chromatograms and gel filtration chromatography.

Detailed process is as follows:

1. collect to express the cho cell conditioned medium of EPO analogue N2, use with the ultra-filtration membrane of 5k molecular retention amount after its concentrated 15 times, damping fluid is replaced into 10mM Tris-HClpH7.0.

2. the flow velocity that the solution after will concentrating divides with 0.5cm/ is added on the Q Sepharose Fast flow post, and this post is used 10mM Tris-HCl pH7.0 balance mistake in advance.

3. after application of sample finishes, wash post with the 10mM Tris-HCl pH7.0 of 5 column volumes, and use the acetic acid by 40mM, the 1mM glycine, the solution flushing post bed that 6M urea forms is subsequently with 10mM Tris-HCl, 200mM NaCl, pH7.0 with EPO analogue N2 by eluting on the post.

4. the flow velocity that the EPO analogue N2 component of collecting is divided with 3cm/ is added to Vydac C ₄On the reversed-phase resin post, this post has been used 20% ethanol/10mM Tris-HCl, pH7.0 balance mistake.With 20%-95% ethanol/10mM Tris-HCl, pH7.0 progressively launches wash-out.Through 50-75% ethanol/10mM Tris-HCl, the component of pH7.0 wash-out is used 10mM Tris-HClpH7.0 according to 1: 2.5, after the dilution, use concentrated 20 times of the little thickener of pall nanosep 10K omega, and the displacement damping fluid is 10mM Tris-HCl pH7.0.

5. concentrated solution is carried out purifying with the S-200 post of crossing with 20mM citrate buffer solution pH6.8 balance, collecting corresponding monomer analogue peak.

Embodiment 4:Val ²Asn ³Phe ⁴Ser ⁵The preparation of EPO

A. the structure of engineering cell, screening and cultivation

According to the plasmid pEC4-N3 transfection CHO cell that the method for embodiment 1 makes up, host cell is Tetrahydrofolate dehydrogenase deficient Chinese Hamster (CHO dhfr-) available from American type culture collection (ATCC).With this cell cultures in the 100mm culture dish, treat that cell grows to 60～95% when full, with serum free medium drip washing cell, the transfection mixed solution that adds 5ml serum free medium, 10 μ g pEC4-N3,60 μ g lipoFectamin composition, cultivated 4 hours for 37 ℃, the sucking-off substratum adds the MEM substratum that contains 5% foetal calf serum, 37 ℃ of overnight incubation.In substratum, add subsequently MTX, continue to cultivate and to occur to resistance clone in 10-14 days.Cell so that the trysinization resistance clone is turned out increases MTX concentration, the screening resistance clone.Utilize enzyme-linked immunosorbent assay to confirm that resulting cell can express erythropoietin analogue N3.With expressing cell amplification, the cultivation of erythropoietin analogue N3, collect nutrient solution, carry out purifying.

B.Val ²Asn ³Phe ⁴Ser ⁵The purifying of EPO

Use the three-step approach purifying EPO analogue N3 that is formed by ion-exchange chromatography, reverse chromatograms and gel filtration chromatography.

Detailed process is as follows:

1. collect to express the cho cell conditioned medium of EPO analogue N3, use with the ultra-filtration membrane of 5k molecular retention amount after its concentrated 15 times, damping fluid is replaced into 10mM Tris-HClpH7.0.

2. the flow velocity that the solution after will concentrating divides with 0.5cm/ is added on the Q Sepharose Fast flow post, and this post is used 10mM Tris-HCl pH7.0 balance mistake in advance.

3. after application of sample finishes, wash post with the 10mM Tris-HCl pH7.0 of 5 column volumes, and use the acetic acid by 40mM, the 1mM glycine, the solution flushing post bed that 6M urea forms is subsequently with 10mM Tris-HCl, 200mM NaCl, pH7.0 with EPO analogue N3 by eluting on the post.

4. the flow velocity that the analogue N3 component of collecting is divided with 3cm/ is added to Vydac C ₄On the reversed-phase resin post, this post has been used 20% ethanol/10mM Tris-HCl, pH7.0 balance mistake.With 20%-95% ethanol/10mM Tris-HCl, pH7.0 progressively launches wash-out.Through 50-75% ethanol/10mM Tris-HCl, the component of pH7.0 wash-out is used 10mM Tris-HClpH7.0 according to 1: 2.5, after the dilution, use concentrated 20 times of the little thickener of pall nanosep 10K omega, and the displacement damping fluid is 10mM Tris-HCl pH7.0.

5. concentrated solution is carried out purifying with the S-200 post of crossing with 20mM citrate buffer solution pH6.8 balance, collecting corresponding monomer analogue peak.

Embodiment 5:Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰The preparation of EPO

A. the structure of engineering cell, screening and cultivation

According to the plasmid pEC4-N34 transfection CHO cell that the method for embodiment 1 makes up, host cell is Tetrahydrofolate dehydrogenase deficient Chinese Hamster (CHO dhfr-) available from American type culture collection (ATCC).With this cell cultures in the 100mm culture dish, treat that cell grows to 60～95% when full, with serum free medium drip washing cell, the transfection mixed solution that adds 5ml serum free medium, 10 μ g pEC4-N34,60 μ g lipoFectamin composition, cultivated 4 hours for 37 ℃, the sucking-off substratum adds the MEM substratum that contains 5% foetal calf serum, 37 ℃ of overnight incubation.In substratum, add subsequently MTX, continue to cultivate and to occur to resistance clone in 10-14 days.Cell so that the trysinization resistance clone is turned out increases MTX concentration, the screening resistance clone.Utilize enzyme-linked immunosorbent assay to confirm that resulting cell can express erythropoietin analogue N34.With expressing cell amplification, the cultivation of erythropoietin analogue N34, collect nutrient solution, carry out purifying.

B.Asn ²⁸Thr ³⁰Serw ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰The purifying of EPO

Use the three-step approach purifying EPO analogue N34 that is formed by ion-exchange chromatography, reverse chromatograms and gel filtration chromatography.

Detailed process is as follows:

1. collect to express the cho cell conditioned medium of EPO analogue N34, use with the ultra-filtration membrane of 5k molecular retention amount after its concentrated 15 times, damping fluid is replaced into 10mM Tris-HClpH7.0.

2. the flow velocity that the solution after will concentrating divides with 0.5cm/ is added on the Q Sepharose Fast flow post, and this post is used 10mM Tris-HCl pH7.0 balance mistake in advance.

3. after application of sample finishes, wash post with the 10mM Tris-HCl pH7.0 of 5 column volumes, and use the acetic acid by 40mM, the 1mM glycine, the solution that 6M urea forms washes the post bed, subsequently with 10mM Tris-HCl, 200mM NaCl, pH7.0 is with Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰EPO is by eluting on the post.

4. with the Asn that collects ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰The flow velocity that the EPO component is divided with 3cm/ is added to Vydac C ₄On the reversed-phase resin post, this post has been used 20% ethanol/10mMTris-HCl, pH7.0 balance mistake.With 20%-95% ethanol/10mM Tris-HCl, pH7.0 progressively launches wash-out.Through 50-75% ethanol/10mM Tris-HCl, the component of pH7.0 wash-out is according to 1: 2.5 usefulness 10mM Tris-HCl pH7.0, after the dilution, use concentrated 20 times of the little thickener of pall nanosep 10K omega, and the displacement damping fluid is 10mM Tris-HCl pH7.0.

5. concentrated solution is carried out purifying with the S-200 post of crossing with 20mM citrate buffer solution pH6.8 balance, collecting corresponding monomer analogue peak.

Embodiment 6:Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰The preparation of EPO

A. the structure of engineering cell, screening and cultivation

According to the plasmid pEC4-N44 transfection CHO cell that the method for embodiment 1 makes up, host cell is Tetrahydrofolate dehydrogenase deficient Chinese Hamster (CHO dhfr-) available from American type culture collection (ATCC).With this cell cultures in the 100mm culture dish, treat that cell grows to 60～95% when full, with serum free medium drip washing cell, the transfection mixed solution that adds 5ml serum free medium, 10 μ g pEC4-N44,60 μ g lipoFectamin composition, cultivated 4 hours for 37 ℃, the sucking-off substratum adds the MEM substratum that contains 5% foetal calf serum, 37 ℃ of overnight incubation.In substratum, add subsequently MTX, continue to cultivate and to occur to resistance clone in 10-14 days.Cell so that the trysinization resistance clone is turned out increases MTX concentration, the screening resistance clone.Utilize enzyme-linked immunosorbent assay to confirm that resulting cell can express erythropoietin analogue N44.With expressing cell amplification, the cultivation of erythropoietin analogue N44, collect nutrient solution, carry out purifying.

B.Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰The purifying of EPO

Use the three-step approach purifying EPO analogue N44 that is formed by ion-exchange chromatography, reverse chromatograms and gel filtration chromatography.

Detailed process is as follows:

1. collect to express the cho cell conditioned medium of EPO analogue N44, use with the ultra-filtration membrane of 5k molecular retention amount after its concentrated 15 times, damping fluid is replaced into 10mM Tris-HClpH7.0.

2. the flow velocity that the solution after will concentrating divides with 0.5cm/ is added on the Q Sepharose Fast flow post, and this post is used 10mM Tris-HCl pH7.0 balance mistake in advance.

3. after application of sample finishes, wash post with the 10mM Tris-HCl pH7.0 of 5 column volumes, and use the acetic acid by 40mM, the 1mM glycine, the solution that 6M urea forms washes the post bed, subsequently with 10mM Tris-HCl, 200mM NaCl, pH7.0 is with Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰EPO is by eluting on the post.

4. with the Asn that collects ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰The flow velocity that the EPO component is divided with 3cm/ is added to Vydac C ₄On the reversed-phase resin post, this post has been used 20% ethanol/10mM Tris-HCl, pH7.0 balance mistake.With 20%-95% ethanol/10mM Tris-HCl, pH7.0 progressively launches wash-out.Through 50-75% ethanol/10mM Tris-HCl, the component of pH7.0 wash-out is according to 1: 2.5 usefulness 10mM Tris-HCl pH7.0, after the dilution, use concentrated 20 times of the little thickener of pall nanosep 10Komega, and the displacement damping fluid is 10mM Tris-HCl pH7.0.

5. concentrated solution is carried out purifying with the S-200 post of crossing with 20mM citrate buffer solution pH6.8 balance, collecting corresponding monomer analogue peak.

Embodiment 7:Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰Ala ¹⁶⁴The preparation of EPO

A. the structure of engineering cell, screening and cultivation

According to the plasmid pEC4-N60 transfection CHO cell that the method for embodiment 1 makes up, host cell is Tetrahydrofolate dehydrogenase deficient Chinese Hamster (CHO dhfr-) available from American type culture collection (ATCC).With this cell cultures in the 100mm culture dish, treat that cell grows to 60～95% when full, with serum free medium drip washing cell, the transfection mixed solution that adds 5ml serum free medium, 10 μ g pEC4-N60,60 μ g lipoFectamin composition, cultivated 4 hours for 37 ℃, the sucking-off substratum adds the MEM substratum that contains 5% foetal calf serum, 37 ℃ of overnight incubation.In substratum, add subsequently MTX, continue to cultivate and to occur to resistance clone in 10-14 days.Cell so that the trysinization resistance clone is turned out increases MTX concentration, the screening resistance clone.Utilize enzyme-linked immunosorbent assay to confirm that resulting cell can express erythropoietin analogue N60.With expressing cell amplification, the cultivation of erythropoietin analogue N60, collect nutrient solution, carry out purifying.

B.Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰Ala ¹⁶⁴The purifying of EPO

Use the three-step approach purifying ASN30THR32ASN114THR116EPO that is formed by ion-exchange chromatography, reverse chromatograms and gel filtration chromatography.

Detailed process is as follows:

1. collect to express the cho cell conditioned medium of ASN30THR32ASN114THR116EPO, use with the ultra-filtration membrane of 5k molecular retention amount after its concentrated 15 times, damping fluid is replaced into 10mM Tris-HClpH7.0.

2. the flow velocity that the solution after will concentrating divides with 0.5cm/ is added on the Q Sepharose Fast flow post, and this post is used 10mM Tris-HCl pH7.0 balance mistake in advance.

3. after application of sample finishes, wash post with the 10mM Tris-HCl pH7.0 of 5 column volumes, and use the acetic acid by 40mM, the 1mM glycine, the solution flushing post bed that 6M urea forms is subsequently with 10mM Tris-HCl, 200mM NaCl, pH7.0 with ASN30THR32ASN114THR116EPO by eluting on the post.

4. the flow velocity that the ASN30THR32ASN114THR116EPO component of collecting is divided with 3cm/ is added on the VydacC4 reversed-phase resin post, and this post has been used 20% ethanol/10mM Tris-HCl, pH7.0 balance mistake.With 20%-95% ethanol/10mM Tris-HCl, pH7.0 progressively launches wash-out.Through 50-75% ethanol/10mM Tris-HCl, the component of pH7.0 wash-out is used 10mM Tris-HClpH7.0 according to 1: 2.5, after the dilution, use concentrated 20 times of the little thickener of pall nanosep 10K omega, and the displacement damping fluid is 10mM Tris-HCl pH7.0.

5. concentrated solution is carried out purifying with the S-200 post of crossing with 20mM citrate buffer solution pH6.8 balance, collecting corresponding monomer analogue peak.

Embodiment 8: the mensuration of erythrocyte-stimulating factor analogues

A. erythrocyte-stimulating factor analogues culture expression product S DS-PAGE immunoblotting assay

According to the nutrient solution 5ml that contains each expression product that the described method of embodiment 2-7A obtains, use the little thickener of pall nanosep 10K omega to be concentrated into respectively 20 μ l.With 15% polyacrylamide gel at 0.375M Tris-HCl, 0.1%SDS, vertical slab electrophoresis is 2.5 hours in the pH8.8 electrolytic solution.After electrophoresis finishes, cut one and shift filter paper with gel nitrocellulose filter of a size and 12 Whatman 3MM, with it with after shifting liquid and infiltrating, on MultiphorElectrophoresis System with 0.8mA/cm ²Individual hour of half-dried transferase 12, shift and finish rear PBST solution closing membrane with containing 10% bovine serum albumin 1 hour, adding the mouse anti-erythrocyte generates plain monoclonal antibody and hatched 2 hours again, hatched 1 hour with the sheep anti mouse enzyme labelled antibody more at last, after PBST cleans 3 times, final substrate solution colour developing with containing DAB.Swimming lane 1-7 is respectively normal EPO and contains the EPO analogue of one, two, three extra glycosylation site as shown in Figure 3, and the result shows, increase glycosylation site after, molecular weight of product increases, and molecular weight increasing and increase with glycosylation site.

B. erythrocyte-stimulating factor analogues culture expression product IEF immunoblotting assay

The nutrient solution 6ml that contains each expression product according to the described method of embodiment 2-7A obtains after 18 hours, uses the little thickener of pall nanosep 10Komega to being concentrated into respectively 20 μ l through molecular retention amount 5k dialysis membrane pure water dialyzed overnight.Use respectively 0.1M phosphoric acid and 0.1M sodium hydroxide as anolyte and catholyte, the 10% polyacrylamide gel 15ml that will contain pH3-10 and pH2.5-5 amphotericeledrolyte and 7M urea, pour in the vertical slab electrophoresis groove after gelling is poly-, 600V, 50mA, 30W prerunning 30 minutes, sample and sample buffer mix and add in the gel sample groove at 1: 1 after will dialysing, 800V, 50mA, 30W electrophoresis 2 hours.Electrophoresis carries out immunoblotting assay according to the described method of embodiment 8A after finishing.As shown in Figure 4, swimming lane 8 is normal EPO, and swimming lane 1-7 is respectively the EPO analogue that contains one, two, three extra glycosylation site, and the result shows that behind the increase glycosylation site, the acidity band of product increases.

C. erythrocyte-stimulating factor analogues purified product SDS-PAGE analyzes

The purified erythrocyte-stimulating factor analogues 40 μ l that obtain according to the described method of embodiment 2-7B.With 15% polyacrylamide gel at 0.375M Tris-HCl, 0.1%SDS, vertical slab electrophoresis is 2.5 hours in the pH8.8 electrolytic solution.After finishing, electrophoresis carries out Coomassie brilliant blue dyeing.Swimming lane 1-7 is respectively normal EPO and the EPO analogue that contains one, two, three extra glycosylation site as shown in Figure 5, the result shows, after increasing glycosylation site, molecular weight of product increases, there is just extra glycosylation to produce, and molecular weight increases and increases with glycosylation site, illustrates that the glycosylation amount increases progressively.

D. erythrocyte-stimulating factor analogues purified product IEF analyzes

According to the purified erythrocyte-stimulating factor analogues 100 μ l that the described method of embodiment 2-7B obtains, process molecular retention amount 5k dialysis membrane pure water dialyzed overnight 18 hours.Use respectively 0.1M phosphoric acid and 0.1M sodium hydroxide as anolyte and catholyte, the 10% polyacrylamide gel 15ml that will contain pH3-10 and pH2.5-5 amphotericeledrolyte and 7M urea, pour in the vertical slab electrophoresis groove after gelling is poly-, 600V, 50mA, 30W prerunning 30 minutes, sample and sample buffer mix and add in the gel sample groove at 1: 1 after will dialysing, 800V, 50mA, 30W electrophoresis 2 hours.After finishing, electrophoresis carries out Coomassie brilliant blue dyeing.Swimming lane 1-7 is respectively normal EPO and the EPO analogue purified that contains one, two, three extra glycosylation site as shown in Figure 6, the result shows, along with increasing of glycosylation site, the acidity band of product increases, illustrating has increased extra acid carbohydrate in the molecule, and the quantity that increases increasing and increase with glycosylation site.

E. sialic acid content analysis in the erythrocyte-stimulating factor analogues purified product

Sialic acid mole number with every mole of erythrocyte-stimulating factor analogues represents.At first will be diluted to 0.2-0.4mg/ml according to described each the erythrocyte-stimulating factor analogues purified product protein concentration that obtains of embodiment 2-7B with purified water.In order to determine existing erythrocyte-stimulating factor analogues purified protein content, analytical reagent and the micromethod step of using Bio-Rad to provide, with recombiant erythropoietin with erythropoietin aminoacid sequence as standard, carry out Bradford analysis of protein (Bradford, Anal, biochem.72,248. (1976)).Then get sialic acid working standard liquid, purified water and erythrocyte-stimulating factor analogues purified solution according to table 2.Add respectively in the 10ml glass test tube, mixing, add in every pipe and contain 0.2% Resorcinol, 250 μ M copper sulfate, 20% hydrochloric acid soln 1ml, add a cover, boiling water boiled 30 minutes, took out ice bath after 3 minutes, and every pipe adds butylacetate-butanol solution (4: 1) 2ml, abundant mixing, room temperature was placed 10 minutes.Measure absorbancy at the 580nm wavelength.Concentration with the sialic acid reference substance solution is done straight-line regression to its corresponding absorbancy, obtains the sialic absorbancy of 5 μ g by linear regression equation.Calculate erythrocyte-stimulating factor analogues purified sialic acid content (mol/mol albumen)=(A according to formula ₂* 5 * 3.24 * W * D)/(A ₁* P * 100), A ₁Be the sialic absorbancys of 5 μ g, A ₂Absorbancy for the erythrocyte-stimulating factor analogues purified, D is the extension rate of erythrocyte-stimulating factor analogues purified, P is erythrocyte-stimulating factor analogues purified protein content (μ g/ μ l), W is the amount (amount that does not comprise carbohydrate) of 1nmol erythrocyte-stimulating factor analogues purified, is equivalent to 18.2 μ g.The results are shown in table 3.

Table 2

Table 3

Erythrocyte-stimulating factor analogues	The mole number of sialic mole number/erythropoietin
		[Asn 2Gly 3Thr 4]EPO	10.8±0.5
[Asn 3Phe 4Ser 5]EPO	10.8±0.3
		[Val 2Asn 3Phe 4Ser 5]EPO	11.5±0.4
[Asn 28Ser 30Asn 88Ser 90]EPO	12.8±0.4
		[Asn 2Gly 3Thr 4Asn 28Thr 30Ser 87Asn 88Gly 89Ser 90]EPO	13.6±0.4
[Asn 2Gly 3Thr 4Asn 28Thr 30Ser 87Asn 88Gly 89Ser 90Ala 164]EPO	13.7±0.6

Embodiment 9: behind the erythrocyte-stimulating factor analogues single dose injection mouse, reticulocyte over time in the body

The purified erythrocyte-stimulating factor analogues that obtains according to the described method of embodiment 2-7B, take 5 μ g/kg subcutaneous injections to the female Balb/c mouse of body weight as 20 ± 2, and take the recombinant human erythropoietin of same dose as reference substance, establish simultaneously the solvent control group.Blood sampling in per 24 hours detects reticulocyte content with reticulocyte numeration instrument after the injection, detects reticulocyte content temporal evolution situation, Fig. 7 be the promoting erythrocyte analogue to injected in mice after reticulocyte curve over time.The result shows, increase with extra glycosylation site, erythrocyte-stimulating factor analogues prolongs the action time in Mice Body, and the prolongation of time and glycosylation site increase positive correlation, action intensity (in the area under curve) enhancing of analogue in Mice Body.And action time and intensity are subject to the last bit amino effect of acid of glycosylation site.

Embodiment 10:Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰The acquisition of EPO isomer

The method that provides according to embodiment 6A obtains contains Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰The nutrient solution of EPO is used the three-step approach purifying that is comprised of ion-exchange chromatography, reverse-phase chromatography and gel filtration chromatography and is isolated Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰The different isomerization body component of EPO.

Detailed process is as follows:

1. collect and express Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰The cho cell conditioned medium of EPO analogue is used with the ultra-filtration membrane of 5k molecular retention amount after its concentrated 15 times, and damping fluid is replaced into 10mM Tris-HCl pH7.0.

2. the flow velocity that the solution after will concentrating divides with 0.5cm/ is added on the Q Sepharose Fast flow post, and this post is used 10mM Tris-HCl pH7.0 balance mistake in advance.

3. after application of sample finishes, use respectively the 10mM Tris-HCl of 5 volumes, pH7.0 and by 40mM acetic acid, the 1mM glycine, the solution flushing post bed that 6M urea forms, and use 0-0.4MNaCl/10mM Tris-HCl, 15 column volumes of pH7.0 gradient elution are collected take 1.5 column volumes as a component.

4. sampling is carried out the IEF analysis according to the described method of embodiment 8D from each component, collects 4 main ingredients, contains respectively the also liquid collecting of isomer 1-5,2-6,3-6,4-8.

5. the flow velocity that isomer 3-6 and liquid collecting is divided with 3cm/ is added to Vydac C ₄On the reversed-phase resin post, this post has been used 20% ethanol/10mM Tris-HCl, pH7.0 balance mistake.With 20% ethanol/10mM Tris-HCl, pH7.0 and 95% ethanol/10mM Tris-HCl, pH7.0 progressively launches wash-out.Through 50-75% ethanol/10mM Tris-HCl, the component of pH7.0 wash-out after usefulness pH7.010mM Tris-HCl dilution in 1: 2.5, is used concentrated 20 times of the little thickener of pall nanosep 10K omega.

With concentrated solution to use the 20mM citrate buffer solution, carry out purifying on the S-200 post that pH 7.0 balances are crossed, collect corresponding analogue isomer 3-6.

Embodiment 11 Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰The biological property of EPO isomer

A. erythrocyte-stimulating factor analogues isomer IEF analyzes

Obtain each 100 μ l of erythrocyte-stimulating factor analogues isomer component according to embodiment 10 described methods, process molecular retention amount 5k dialysis membrane pure water dialyzed overnight 18 hours.Use respectively 0.1M phosphoric acid and 0.1M sodium hydroxide as anolyte and catholyte, the 10% polyacrylamide gel 15ml that will contain pH3-10 amphotericeledrolyte and 7M urea, pour in the vertical slab electrophoresis groove after gelling is poly-, 600V, 50mA, 30W prerunning 30 minutes, sample and sample buffer mix and add in the gel sample groove at 1: 1 after will dialysing, 800V, 50mA, 30W electrophoresis 2 hours.After finishing, electrophoresis carries out Coomassie brilliant blue dyeing.Swimming lane 1-5 is respectively normal EPO and the also liquid collecting that contains isomer 1-5,2-6,3-6,4-8 as shown in Figure 8, and the result has shown along with having increased access to of salt concn the isomer component that a series of acid carbohydrate contents increase progressively.

B. the mensuration of erythrocyte-stimulating factor analogues isomer activity in vivo

This law can stimulate erythropoietic effect according to erythropoietin, according to basic, normal, high three dosage groups, gives mouse (BALB/C) subcutaneous injection EPO.Because erythropoietin (EPO) can promote the release of the reticulocyte in the marrow, so that the rising of the ratio of the reticulocyte counts in the Mouse Blood and RBC number, and lift-off value and injected dose are linear.Detect the EPO Biological acdtivity in vivo by the dose-response parallel method.With containing 0.1% bovine serum albumin physiological sodium chloride diluent, (this standard substance derives from Nat'l Pharmaceutical ﹠ Biological Products Control Institute with the EPO biological reference standard, be used for measuring in vivo bioactivity) for measuring in vivo bioactivity) being diluted to concentration 80IU/ml, 40IU/ml and 20IU/ml, the erythrocyte-stimulating factor analogues isomer mixture also is diluted to respectively near above-mentioned three kinds of concentration.With the mouse random packet, 18 every group, be divided into standard group and to be checked group.Each group is established basic, normal, high three dosage, and each dosage group is 6 mouse, carries out mark with picric acid.According to basic, normal, high three dosage groups difference veutro subcutaneous injection mouse.The volume injected of every mouse is 0.5ml.Used EDTA-K in the mouse orbit blood sampling in the 4th day after injection ₂(physiological saline that contains 1%EDTA) anticoagulant tube anti-freezing, blood sampling volume: 4-5 drips/mouse.Get anticoagulation, put upside down 5 times under every pipe reading is front upper, make its abundant mixing, careful uncap, insert test tube in R-500 analyser (the Japanese Sysmex company) suction needle and press beginning, instrument Auto-counting red corpuscle sum, reticulocyte is total and reticulocyte counts to the ratio (Ret) of red corpuscle sum.In Ret value input computer, program is calculated the biologic activity of test sample automatically.The activity in vivo that table 4 shows the erythropoietin isomer increases and raises with acid carbohydrate.Specific activity is the observed value of specific activity in the relative body in the body described here, is not the absolute interior specific activity observed value of body.Specific activity only is used for the relatively relative reactivity of erythrocyte-stimulating factor analogues, the mensuration of analogue adopts identical measuring method, adopts the same terms, the animal of identical type, have for the identical data analysis of calculating specific activity, measure the same analysis method of protein content.Present embodiment does not want to represent with specific activity value in any body of any analogue of being reported the intrinsic or absolute value of this analogue.

Table 4

The erythrocyte-stimulating factor analogues isomer	Activity in vivo (U/mg polypeptide)
		Isomer 1-5	157,600±1400
Isomer 2-6	201,100±1800
		Isomer 3-6	254,700±1700
Isomer 4-8	288,500±3200

C. behind erythrocyte-stimulating factor analogues isomer 3-6 and the liquid collecting single dose injection mouse, reticulocyte over time in the body

The erythrocyte-stimulating factor analogues isomer 3-6 that obtains according to embodiment 10 described methods, take 5 μ g/kg subcutaneous injections to the female Balb/c mouse of body weight as 20 ± 2, and take the recombinant human erythropoietin of same dose as reference substance, establish simultaneously the solvent control group.Blood sampling in per 24 hours after the injection, detect reticulocyte content with reticulocyte numeration instrument, detect reticulocyte content temporal evolution situation, Fig. 9 be erythrocyte-stimulating factor analogues isomer 3-6 to injected in mice after reticulocyte curve over time.The result shows that erythropoietin group second day RET% after medication begins to raise, and reaches the peak, and continues about 4 days in the 4th day; Erythropoietin analogue group RET% also begins to raise in second day, reaches the peak, and continues about 7 days in the 6th day.This shows that shift with sialic acid content increase, iso-electric point oxytropism, prolong action time in vivo.

D. erythrocyte-stimulating factor analogues isomer 3-6 union vena axillaris is injected pharmacokinetic

Select male SD (Sprague Dawley) 10～11 all rats, body weight 300 ± 20g, 6/group.Intravenous injection ¹²⁵The erythropoietin of I mark and erythropoietin analogue isomer 3-6 mixture, 0.05 μ g/kg according to the timed interval (5 minutes to 10 hours) the eye socket venous blood collection of setting, collects serum.Get 0.1ml serum and add the cold dehydrated alcohol of 0.9ml (20 ℃), put 4 ℃ of overnight incubation, centrifugal 15 minutes of 12000rpm detects with the coffee detector, calculates pharmacokinetic parameter.Figure 10 is the mixture Plasma Concentration temporal evolution curve of erythropoietin and isomer 3-6, compare with erythropoietin, removing speed obviously reduces in the isomer 3-6 mixing object, cause eliminating in the body Increased Plasma Half-life, the transformation period is respectively 8.5h and 2.6h behind the intravenous injection rat of the two, and the elimination transformation period of erythrocyte-stimulating factor analogues is 3.3 times of erythropoietin.

E. erythrocyte-stimulating factor analogues isomer 3-6 and liquid collecting are on the impact of Mouse Blood specific volume of cell

The increase of degree of glycosylation causes erythrocyte-stimulating factor analogues elimination Increased Plasma Half-life in vivo, and this becomes possibility for improving dosing interval.Laboratory animal is female BALB/c mouse, the cleaning level, 8 ages in week, body weight 18～22g is according to the principle grouping of random packet, 10 every group, the isomer 3-6 that obtains among the natural erythropoietin of employing various dose and the embodiment 10, so that inferior subcutaneous injection is to BALB/c once in a week and on every Wendesdays, control group is injected the solvent of equal volume, continuous 4 weeks respectively.The changing conditions of monitoring hematocrit, 2 times/week, administration same day is the 1st day (1d), gets blood respectively at the kapillary that the 4th day (4d) and 7 days (7d) in 1 week processes with heparinization, centrifuging detection hematocrit is adopted in sealing.Figure 11-the 12nd, hematocrit temporal evolution situation is seen after injected in mice erythropoietin and erythropoietin analogue isomer 3-6 and the liquid collecting.The result shows that after weekly and on every Wendesdays time administration, isomer 3-6 mixture obviously strengthens the rising effect of Mouse Blood specific volume of cell, and prompting clinical application erythrocyte-stimulating factor analogues can anemia.In the identical situation of weekly dose, to the rising effect of Mouse Blood specific volume of cell and the on every Wendesdays time injection of natural human erythropoietin no significant difference as a result, inject once in a week after the weekly injection of isomer mixture, can reach the result for the treatment of of expection.

Sequence table

Claims (11)

1. natural human erythrocyte-stimulating factor analogues with erythropoietin activity, this analogue comprises 1 extra N-glycosylation site, wherein changed l-asparagine in the 2nd position of natural human erythropoietin aminoacid sequence, glycine has been changed in the 3rd position, and Threonine has been changed in the 4th position; I.e. [Asn ²Gly ³Thr ⁴] EPO; Described extra N-glycosylation site is the l-asparagine of the 2nd of aminoacid sequence.

2. natural human erythrocyte-stimulating factor analogues with erythropoietin activity, this analogue comprises 3 extra N-glycosylation sites, wherein changed l-asparagine in the 2nd position of natural human erythropoietin aminoacid sequence, glycine has been changed in the 3rd position, Threonine has been changed in the 4th position, and has further replaced l-asparagine, Threonine, Serine, l-asparagine, glycine, Serine the 28th, 30,87,88,89,90 difference; I.e. [Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰] EPO; Described 3 extra N-glycosylation sites are respectively aminoacid sequence the 2nd, 28,88 l-asparagine.

3. natural human erythrocyte-stimulating factor analogues with erythropoietin activity, this analogue comprises 3 extra N-glycosylation sites, wherein changed l-asparagine in the 2nd position of natural human erythropoietin aminoacid sequence, glycine has been changed in the 3rd position, Threonine has been changed in the 4th position, and has further replaced l-asparagine, Threonine, Serine, l-asparagine, glycine, Serine, L-Ala the 28th, 30,87,88,89,90,164 difference; I.e. [Asn ²Gly ³Thr ⁴Asn ²⁸Thr ³⁰Ser ⁸⁷Asn ⁸⁸Gly ⁸⁹Ser ⁹⁰Ala ¹⁶⁴] EPO; Described 3 extra N-glycosylation sites are respectively aminoacid sequence the 2nd, 28,88 l-asparagine.

4. dna sequence dna, each human erythropoietin analogue of this dna sequence encoding claim 1-3.

5. expression vector, this expression vector contains the dna sequence dna of claim 4.

6. eukaryotic host cell, this host cell is with the expression vector transfection of claim 5.

7. the eukaryotic host cell of claim 6, it is selected from CHO, COS-7 or BHK.

8. production method with natural human erythrocyte-stimulating factor analogues of erythropoietin activity comprises step:

(i) cultivate the eukaryotic host cell of claim 6 or 7, and

(ii) from culture, collect the glycoprotein with erythropoietin activity.

9. one kind increases erythropoietic pharmaceutical composition, and said composition comprises each human erythropoietin analogue and the pharmaceutically useful auxiliary agent of claim 1-3 for the treatment of significant quantity.

10. the pharmaceutical composition of claim 9, described auxiliary agent is diluent or carrier.

11. the pharmaceutical composition of claim 9, it is used for promoting erythropoiesis, prevention or treatment anaemia.

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