CN103113464B - Natural human erythropoietin analogue - Google Patents

Natural human erythropoietin analogue Download PDF

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CN103113464B
CN103113464B CN201310016616.3A CN201310016616A CN103113464B CN 103113464 B CN103113464 B CN 103113464B CN 201310016616 A CN201310016616 A CN 201310016616A CN 103113464 B CN103113464 B CN 103113464B
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asn
epo
erythropoietin
analogue
ser
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CN103113464A (en
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娄竞
耿建玲
张�杰
侯绪凤
赵会林
陆军
苏冬梅
胡金东
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SHENYANG SUNSHINE PHARMACEUTICAL CO Ltd
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SHENYANG SUNSHINE PHARMACEUTICAL CO Ltd
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Abstract

The invention relates to an erythropoietin analogue containing at least one extra glycosylation site or a variant of the erythropoietin analogue. The invention also relates to a DNA (Deoxyribonucleic Acid) sequence for encoding the erythropoietin analogue or the variant thereof as well as a recombinant plasmid and a host cell which are supplied for the analogue or the variant thereof to express.

Description

Natural human erythrocyte-stimulating factor analogues
The application is to be on July 2nd, 2007 applying date, and application number is 200710127362.7, and name is called the divisional application of the application for a patent for invention of " 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 express recombinant plasmid and the host cell of this analogue or its variant.
Background technology
Erythropoietin (erythropoietin, EPO) is that first is found and is applied to clinical hemopoieticgrowth factor.As far back as 1906, after the discovery rabbits such as Carnot lose blood, can in peripheral blood, produce one and can act on hemopoietic system to accelerate erythropoietic material, propose thus to exist a kind of humoral factor to regulate hemopoietic with feedback system.After more than 30 years, this viewpoint is just confirmed, this factor is named as erythropoietin.Erythropoietin claims again hematopoietin, red corpuscle stimulating factor, belongs to acid glycoprotein, is regulate precursor red corpuscle propagation, differentiation and keep the main hormone of red blood cell concentration within the scope of normal physiological in peripheral blood.It can with the EPO receptors bind on red corpuscle precursor cell surface, promote the synthetic of its oxyphorase to make it proliferation and differentiation protoerythrocyte, thus the physiological equilibrium of red corpuscle and oxyphorase in control agent.EPO is a strong hemopoieticgrowth factor, and analyzed in vitro has dose-dependent effect while being presented at the concentration of 0.05～1U/ml.EPO can stimulate CFU-E (BFU-E) and inmature red corpuscle morning (CFU-E) to form ripe red corpuscle colony.EPO, except acting on marrow macronucleus precursor cell, all without effect, is the hemopoieticgrowth factor of thinking that so far effect is the most single to other hematopoietic cells.Certainly, to the complete adjusting of red corpuscle hematopoiesis, except EPO, also need the synergy of other factors, comprise Multi-CSF, GM-CSF and IL-1, these factors impel stem cell to become BFU-E, and combined stimulation breeds red corpuscle in early days, be 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, due to the scarcity in initial source, were difficult to obtain a large amount of sterlings fully to study its biology and molecules character.1984, scientists is first taking the EPO mRNA of human renal carcinoma cell as template, synthesize cDNA and in intestinal bacteria, cloned and express through reverse transcription, obtain on this basis the complete genome of EPO and in eukaryotic cell, carried out high efficient expression, for furtheing investigate the biological effect of EPO and it being applied to clinical laying a good foundation.Can use now gene engineering method Restruction erythropoietin (rhEPO) both at home and abroad, and for the multiple oligocythemia of clinical treatment, there is good curative effect.
Many cell surface proteins and secretory protein that eukaryotic cell produces, all by one or more oligosaccharides base group modifications.This physico-chemical property that is called glycosylated modification energy strong effect protein, stability, secretion and Subcellular Localization to protein also can play an important role.Correct glycosylation is that biological activity is necessary sometimes.Some Eukaryotic gene makes to express in the bacterium (as intestinal bacteria) of the cell processes of protein glycosylation in shortage, and the albumen being recovered to does not have or almost do not have activity owing to lacking glycosylation.
Glycosylation appears at the special position of polypeptide backbone, conventionally has two classes: O to connect glycosylation and is connected glycosylation with N.The oligosaccharides that O connects is connected in Serine or threonine residues, and the oligosaccharides that N connects 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).It is all different that N connects the saccharide residue existing in the oligosaccharide structure that is connected with O and every type.The class sugar being jointly 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 made up of 166 amino acid, and aminoacid sequence is referring to SEQ ID NO:1, and peptide chain structure is shown in Fig. 1.In EPO molecule, there are two disulfide linkage, 3 N-key glycosylation sites and 1 O-key glycosylation site, wherein two disulfide linkage are absolutely necessary in sex change EPO renaturation and while being folded into biological activity configuration.The human erythropoietin of natural human erythropoietin and mammalian cell expression all contains three N and is connected oligonucleotide chain and is connected oligonucleotide chain with an O, and they account for 40% of albumen total molecular weight altogether.N connects glycosylation and appears at the asparagine residue of 24,38,83, and O connects glycosylation and appears at serine residue (Lai etc., J.Biol.Chem.261:3116,1986 of 126; Broundy etc., Arch.Biochem.Biophys.265:329,1988)).Prove that oligonucleotide chain modified by end sialic acid residues.Glycosylation erythropoietin is carried out enzyme processing and removed after sialic acid residues, cause activity in vivo to be lost, but do not affect external activity (Lowy etc., Nature185:102,1960; Goldwasser etc. .J.Biol.Chem.249:4202,1974).This phenomenon was once interpreted as asialo erythropoietin owing to removing 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 sialic acid acidifying by liver in conjunction with time just there is in vivo bioactivity.
In the oligosaccharides of erythropoietin, its effect is also very not definite.Prove, the erythropoietin of part de-glycosylation is compared with glycosylation form, and activity in vivo reduces greatly, but has retained external activity (Dordal etc., Endocrinology116:2293,1985).But another research shows, undergo mutation as l-asparagine or the serine residue of glycosylation site if made, thereby remove separately or jointly the oligonucleotide chain that N connects or O connects, can make the external activity of the mutain producing (Dube etc. that sharply decline in mammalian cell, J.Biol.Chem.263:17516,1988).
Glycoprotein, as erythropoietin, can be used and be separated into different charged forms technology such as isoelectrofocusing (IEF).The existing report in many ways 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).That discusses people such as Miyake urinates Purification of Human erythropoietin from people, two erythropoietin components that obtain by hydroxylapatite chromatography method.These two components are named as II, IIIA.The carbohydrate analysis subsequently II and IIIA being carried out shows, 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 around 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, and therefore 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 has improved the performance of EPO analogue by several amino-acid residues increases N-glycosylation site number in 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, 28-32,40-55,86-92,112-133,162-166 position, and this variant has one or several amino acid whose disappearance, replacement or interpolation in the aminoacid sequence of this analogue, and there is 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 90the combination of EPO or their any two kinds or any three kinds, 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, this DNA sequence encoding human erythropoietin analogue 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 a kind of erythropoietic pharmaceutical composition that increases, 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 proceeds in eukaryotic cell (as Chinese hamster ovary celI, COS-7 cell, BHK) and expresses, and preferentially selects Chinese hamster ovary celI.To the transfection mixed solution of the carrier that adds serum free medium in Chinese hamster ovary celI, comprises DNA sequence dna of the present invention, lipofectamine composition, in substratum, add methotrexate (MTX) screening resistance clone after cultivating for some time.Improve gradually MTX concentration subsequently and screen the resistance clone of high expression level.Utilize enzyme-linked immunosorbent assay to confirm that the cell obtaining can express erythropoietin analogue.
Can express the cell amplification of erythropoietin analogue, cultivate, collect nutrient solution.Then, after concentrated containing the cell culture fluid of recombiant erythropoietin analogue, be splined on the post of the similar ion-exchange chromatography media filling such as Q Sepharose Fast flow or DEAE Sepharose Fast flow (GE), after damping fluid rinses with low pH value damping fluid drip washing pillar.Finally, the damping fluid by containing salt by definite erythropoietin analogue by eluting on post.The above-mentioned erythropoietin analogue of collecting further passes through 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, in the aminoacid sequence of erythrocyte-stimulating factor analogues of the present invention, lack, and 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 amino acid of the same type (non-conservation amino acid), for example, the replacement between acidic amino acid, for example Glu and Asp, the replacement between basic aminoacids, for example Arg and Lys, replacement between die aromatischen Aminosaeuren, for example Phe and Tyr; Replacement between 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 N end, or add polyA tail etc. at C end.
The aminoacid sequence that erythrocyte-stimulating factor analogues of the present invention comprises at least one extra glycosylation site.Extra glycosylation site can cause than the sialic acid content of the more carbohydrate chain of natural human erythropoietin number and Geng Gao.Sialic acid content produces by adding glycosylation site higher than the analogue of natural human erythropoietin, and these glycosylation sites do not disturb the required secondary of biological activity or three grades of conformations.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, with the arginine of 4 of Serine displacements, obtain sequence A sn-Pro-Ser, this sequence just becomes the 4th N-glycosylation site.A kind of variation can provide 4 extra sialic acids by per molecule conventionally like this.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 in the erythrocyte-stimulating factor analogues that increases respectively 1-3 extra glycosylation site, carbohydrate increases.Concrete grammar is, gets the expression supernatant of erythropoietin analogue, uses 15%SDS-Polyacrylamide Gel Electrophoresis, transfers on nitrocellulose (referring to document: Burnette etc., Anal.Biochem.112:195-203,1981; Elliott etc., Gen79:167-180,1989), generate plain monoclonal antibody with 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, 10% polyacrylamide gel that employing contains pH2.5-5 and pH3-10 amphotericeledrolyte and 7M urea, by containing after various erythrocyte-stimulating factor analogues dialysis of obtaining according to above-mentioned cultivation and purification process, 800V, 50mA, 30W electrophoresis 2 hours.After electrophoresis finishes, be transferred on nitrocellulose filter, then generate plain monoclonal antibody with mouse anti-erythrocyte and be combined and use sheep anti mouse enzyme labelled antibody mark, add substrate solution colour developing.
The present invention also provides the method for measuring above-mentioned erythrocyte-stimulating factor analogues purified sialic acid content.Method summary, utilizes Bradford analysis of protein method (Bradford, Anal.biochem.72,248. (1976)) determine the protein concentration of each erythrocyte-stimulating factor analogues purified and adjusted to 0.2-0.4mg/ml.In sample and standard substance, add Resorcinol, copper sulfate, hydrochloric acid soln respectively, boil 30 minutes, add butylacetate-butanol solution (4:1), fully mix, after stratification, get organic phase and detect.Measure absorbancy at 580nm wavelength.By the concentration of sialic acid reference substance solution, its corresponding absorbancy is done to straight-line regression, obtain 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 absorbancy of 5 μ g, A 2for the absorbancy of erythrocyte-stimulating factor analogues purified, D is the extension rate of erythrocyte-stimulating factor analogues purified, P is that (μ g/ μ l) for the protein content of erythrocyte-stimulating factor analogues purified, W is the amount (not comprising the amount of 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, simple description, after cell culture fluid containing erythrocyte-stimulating factor analogues is concentrated, be splined on the post of QSepharose Fast flow (GE) filling, after application of sample, rinse pillar with damping fluid.Afterwards with low pH value damping fluid drip washing pillar.The damping fluid that finally contains 0-400mM NaCl by use respectively by the erythropoietin analogue isomer of different components by eluting on 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 marrow, reticulocyte counts in Mouse Blood and the ratio of RBC number are raise, and lift-off value and injected dose linear.Detect EPO Biological acdtivity in vivo by dose-response parallel method.By EPO biological reference standard, (this standard substance derives from Nat'l Pharmaceutical & Biological Products Control Institute, be used for measuring in vivo bioactivity) be diluted to concentration 80IU/ml, 40IU/ml and 20IU/ml, erythrocyte-stimulating factor analogues isomer mixture is also diluted to respectively and approaches 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 veutro subcutaneous injection mouse respectively.Within the 4th day after injection, take a blood sample in mouse orbit, use EDTA-K 2(containing the physiological saline of 1%EDTA) anticoagulant tube anti-freezing.Get anticoagulation Auto-counting red corpuscle sum, reticulocyte sum and reticulocyte counts ratio (Ret) to red corpuscle sum in R-500 analyser (Japanese Sysmex company).By in Ret value input computer, program is calculated the biologic activity that detects sample automatically.In body described here, specific activity is the observed value of specific activity in relative body, is not the absolute interior specific activity observed value of body.Specific activity is only for comparing the relative reactivity of isomer, and the mensuration of isomer adopts identical measuring method, adopts the same terms, and the animal of identical type, has the identical data analysis for 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 mouse single dose.
The change detection method of reticulocyte content after 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 Balb/c mouse, and taking the recombinant human erythropoietin of same dose as reference substance, establish solvent control group simultaneously.Blood sampling in every 24 hours after injection, with reticulocyte numeration instrument detection reticulocyte content, 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.
Pharmacokinetics measuring method
Select male SD (Sprague Dawley) rat, intravenous injection 125the erythrocyte-stimulating factor analogues isomer mixture 0.05 μ g/kg obtaining in the erythropoietin of I mark and 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 impact of erythrocyte-stimulating factor analogues isomer on 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 obtaining in the erythropoietin of various dose and embodiment 10, respectively with weekly and time subcutaneous injection on every Wendesdays to BALB/c, continuous 4 weeks, the changing conditions of monitoring hematocrit.
Brief description of the drawings
Fig. 1 has shown and in human erythropoietin aminoacid sequence, has had 3 Natively glycosylated sites, lay respectively at 24,38 and 83, and mark the position of impelling erythropoietin to produce extra glycosylation site, laid respectively at 2,3,4,28,30 and 88.
Fig. 2 has shown plasmid construction method.Natural EPO cDNA fragment is taken turns after PCR by rite-directed mutagenesis is several, and primer sudden change is introduced to native sequences.Recovery obtains the total length EPO of sudden change, use BamHI enzymic digestion, use BamHI enzymic digestion carrier for expression of eukaryon pEC4 simultaneously, 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, with enzymic digestion method qualification clone, and by correct cloning and sequencing, obtain the carrier pEC4EPO containing mutant clon.
Fig. 3 has shown the molecular weight situation of EPO and analogue expression product thereof.Swimming lane 1 is recombinant human epo, and swimming lane 2-4 is the EPO analogue that contains an extra glycosylation site, the EPO analogue of swimming lane 5 for containing two extra glycosylation sites, and swimming lane 6 is the [Asn containing 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 tM] EPO.Result shows, increases after glycosylation site, and molecular weight of product all increases to some extent, and molecular weight increasing and increase with glycosylation site.From thering is an extra glycosylation site [Asn 2gly 3thr 4] EPO, [Asn 3phe 4ser 5] EPO and [Val 2asn 3phe 4ser 5] EPO, due to the difference of the last amino acids of glycosylation site, molecular weight increases different, [Val 2asn 3phe 4ser 5] [Asn of EPO expression product 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, its molecular weight is not affected.
Fig. 4 has shown erythrocyte-stimulating factor analogues culture expression product IEF immunoblotting assay result.Swimming lane 8 is 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 the [Asn containing 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.Result shows, expression product acidic molecular all increases to some extent, and acidic molecular increasing and increase with glycosylation site.From thering is an extra glycosylation site [Asn 2gly 3thr 4] EPO, [Asn 3phe 4ser 5] EPO and [Val 2asn 3phe 4ser 5] EPO, due to 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] [Asn of EPO expression product 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, does not affect its iso-electric point.
Fig. 5 has shown by the SDS electrophoretic analysis of the high isomer mixture to the erythrocyte-stimulating factor analogues that contains different extra N-glycosylation sites.Illustrated after increase glycosylation site, the molecular weight of product increases.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 increases a glycosylation site higher than extra, and an extra glycosylation site [Val 2asn 3phe 4ser 5] 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 a certain impact to its tool.[Asn 2gly 3thr 4asn 28thr 30ser 87asn 88gly 89ser 90] EPO and [Asn 2gly 3thr 4asn 28thr 30ser 87asn 88gly 89ser 90ala 164] EPO molecular weight is consistent.[Asn is described 2gly 3thr 4asn 28thr 30ser 87asn 88gly 89ser 90ala 164] EPO do not have extra sugar chain synthetic in the sudden change in non-glycosylated site, its molecular weight is not affected.
Fig. 6 has shown by the IEF electrophoretic analysis of the high isomer mixture to the erythrocyte-stimulating factor analogues that contains different extra N-glycosylation sites.Illustrated after increase glycosylation site, the acidity band of product increases.The extra acid carbohydrate of analogue that increases by three glycosylation sites 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 is once many.The last amino acids that glycosylation site is described has a certain impact to its tool.[Asn 2gly 3thr 4asn 28thr 30ser 87asn 88gly 89ser 90] EPO and [Asn 2gly 3thr 4asn 28thr 30ser 87asn 88gly 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, its iso-electric point is not affected.
Fig. 7 shows that after the erythrocyte-stimulating factor analogues single dose injection mouse of different catastrophe points, in body, reticulocyte over time.In this research, by the purified of erythropoietin analogue, taking 5 μ g/kg subcutaneous injections to body weight the female BALB/c mouse as 20 ± 2, and taking the recombinant human erythropoietin of same dose as reference substance, establish solvent control group simultaneously.Blood sampling in every 24 hours after injection, with reticulocyte numeration instrument detection reticulocyte content, investigates reticulocyte content temporal evolution situation.Result shows, increase with extra glycosylation site, erythrocyte-stimulating factor analogues extends 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 impact of the last amino acids of glycosylation site.
Fig. 8 has shown respectively with method preparation [Asn described in embodiment 10 2gly 3thr 4asn 28thr 30ser 87asn 88gly 89ser 90] the IEF collection of illustrative plates of EPO analogue isomer 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 demonstration is along with acid band in isomer liquid collecting IEF increases, and activity in vivo increases.Illustrate that extra glycosylation has increased the acid carbohydrate in molecule, the variation of this molecular structure causes activity in vivo to raise.
Fig. 9 has shown erythropoietin and with method preparation [Asn described in embodiment 10 2gly 3thr 4asn 28thr 30ser 87asn 88gly 89ser 90] mixture of EPO isomer 3-6 to injected in mice after reticulocyte curve over time.By erythropoietin isomer 3-6 mixture, taking 5 μ g/kg subcutaneous injections to body weight the female Balb/c mouse as 20 ± 2, and taking the recombinant human erythropoietin of same dose as reference substance, establish solvent control group simultaneously.Blood sampling in every 24 hours after injection, with reticulocyte numeration instrument detection reticulocyte content, investigates reticulocyte content temporal evolution situation.Result demonstration, erythropoietin group second day RET% after medication starts to raise, and within the 4th day, reaches peak, continues approximately 4 days; Erythropoietin analogue group RET% also started in second day to raise, and within the 6th day, reached peak, continued approximately 7 days.As can be seen here, isomer 3-6 mixture obviously extends the action time in Mice Body compared with recombinant human erythropoietin, and 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 male SD rat (Sprague Dawley) 10～11 weeks, 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, collect serum.Get 0.1ml serum and add the cold dehydrated alcohol of 0.9ml (20 DEG C), put 4 DEG C of overnight incubation, centrifugal 15 minutes of 12000rpm, with the detection of coffee detector, calculates pharmacokinetic parameter.Compared with erythropoietin, in isomer 3-6 mixing object, removing speed obviously reduces, cause elimination Increased Plasma Half-life in body, after the intravenous injection rat of the two, the transformation period is respectively 8.5h and 2.6h, 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 after the mixture of EPO isomer 3-6.Laboratory animal is female BALB/c mouse, clean level, half and half, 8 week age of male and female, body weight 18～22g, according to the principle grouping of random packet, every group 10, adopt erythropoietin and the erythropoietin analogue isomer 3-6 mixture of various dose, respectively with weekly and time subcutaneous injection on every Wendesdays to BALB/c, the solvent of control group injection same 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 4th day (4d) and 7 days (7d) in 1 week with the kapillary of heparinization processing, sealing, adopts centrifuging to detect hematocrit.Result shows, after weekly and time administration on every Wendesdays, 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, 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.In embodiment, erythropoietin standard substance used is recombinant human erythropoietin's standard substance, and this standard substance derives from Nat'l Pharmaceutical & Biological Products Control Institute, for measuring in vivo bioactivity.
Embodiment 1: the structure of human erythropoietin analogue DNA fragmentation
In human erythropoietin aminoacid sequence, have 3 natural N-glycosylation sites, 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).
The combination between sudden change group is carried out in above site, as built [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 that adds N-glycosylation site sudden change part is referring to SEQ ID NOS:2-85.Utilize these primers, carry out polymerase chain reaction (PCR) taking erythropoietin DNA as template and can obtain the erythrocyte-stimulating factor analogues DNA fragmentation after sudden change.Template DNA derives from natural EPO cDNA library.The structure in library is summarized as follows, utilize Trizol from tire hepatic tissue, to extract total RNA, application Oligo (dT) Cel lulose (Cat.No.15940-026, Lifetechnologies, USA), the method providing by producer, extract PolyA+RNA, apply afterwards cDNA synthetic agent box ( kit, Cat.No.200400-200402, Stratagene, USA) the double-stranded cDNA of synthetic people's tire liver angles and gets natural erythropoietin DNA fragmentation according to US Pat.4703008 design primer from above-mentioned cDNA library.
Taking above-mentioned EPO DNA fragmentation as template, the primer of design SEQ ID NOS:86-93:
3 ' end: 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 for PCR
5 ' end adds BamHI site, and GCCACC
Primer 7:SEQ ID NO:92
3 ' end primer, adds BamHI site
Primer 8:SEQ ID NO:93
(PCR instrument is ABI2700, 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 DEG C of 5min, (95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s) × 30 circulation, 72 DEG C 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 taking 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, taking SEQ ID NO:92+88 and SEQ ID NO:89+93 as template, SEQ ID NO:92+93 is primer, obtains the total length EPO suddenling change by identical PCR condition.Reclaim fragment, with BamHI (Invitrogen) enzymic digestion, use BamHI enzymic digestion carrier for expression of eukaryon pEC4 simultaneously, 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, with enzymic digestion method qualification clone, and by correct cloning and sequencing, obtain the carrier pEC4EPO containing mutant clon.Expression vector pEC4 imports pcDNA3.1 (Invitrogen) by little mouse dihydrofolate reductase (DHFR) gene fragment and forms, and mouse DHFR gene fragment is that the sequence of announcing according to Genebank Nucleotide AH001899 is synthetic.Aforesaid method is summarized in Fig. 2.
Build erythrocyte-stimulating factor analogues with aforesaid method.The DNA sequence dna that has provided part analogue in table 1 changes, otherwise is exactly the sequence of Oligonucleolide primers and the sequence complementation of human erythropoietin for suddenling change.
Table 1: part has the erythrocyte-stimulating factor analogues of N-glycosylation site
By above-mentioned cloning vector numbering called after pEC4-X (X is analogue numbering).
Embodiment 2:Asn 2gly 3thr 4the preparation of EPO
A. the structure of engineering cell, screening and cultivation
The plasmid pEC4-N1 transfection CHO cell building according to the method for embodiment 1, host cell, purchased from American type culture collection (ATCC), is Tetrahydrofolate dehydrogenase deficient Chinese Hamster (CHO dhfr-).By this cell cultures in 100mm culture dish, grow to 60～95% when full until cell, with serum free medium drip washing cell, add the transfection mixed solution of 5ml serum free medium, 10 μ g pEC4-N1,60 μ g lipofectamine compositions, cultivate 4 hours for 37 DEG C, sucking-off substratum, adds the MEM substratum containing 5% foetal calf serum, 37 DEG C of overnight incubation.In substratum, add subsequently MTX, continue to cultivate and occur to resistance clone for 10-14 days.The cell of turning out with trysinization resistance clone, increases MTX concentration, screening resistance clone.Utilize enzyme-linked immunosorbent assay to confirm that the cell obtaining can express erythropoietin analogue N1.By expressing cell amplification, the cultivation of erythropoietin analogue N1, collect nutrient solution, carry out purifying.
B.Asn 2gly 3thr 4the purifying of EPO
Purify EPO analogue N1 by the three-step approach being formed by ion-exchange chromatography, reverse chromatograms and gel filtration chromatography.
Detailed process is as follows:
1. collect the cho cell conditioned medium of expressing EPO analogue N1, use with the ultra-filtration membrane of 5k molecular retention amount and concentrated after 15 times, damping fluid is replaced into 10mM Tris-HCl pH7.0.
2. the flow velocity solution after concentrated being divided with 0.5cm/ is added on 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, 1mM glycine, the solution of 6M urea composition rinses post bed, subsequently with 10mM Tris-HCl, 200mM NaCl, pH7.0 is by Asn 2gly 3thr 4ePO is by eluting on post.
4. by the Asn collecting 2gly 3thr 4the flow velocity that EPO component is divided with 3cm/ is added to Vydac C 4on 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, according to 10mM Tris-HCl pH7.0 for 1:2.5, after dilution, use concentrated 20 times of pall nanosep10K omega micro-thickener, and to replace damping fluid be 10mM Tris-HCl pH7.0.
5. concentrated solution is carried out to purifying on the S-200 post to cross by 20mM citrate buffer solution pH6.8 balance, collect corresponding monomer analogue peak.
Embodiment 3:Asn 3phe 4ser 5the preparation of EPO
A. the structure of engineering cell, screening and cultivation
The plasmid pEC4-N2 transfection CHO cell building according to the method for embodiment 1, host cell, purchased from American type culture collection (ATCC), is Tetrahydrofolate dehydrogenase deficient Chinese Hamster (CHO dhfr-).By this cell cultures in 100mm culture dish, grow to 60～95% when full until cell, with serum free medium drip washing cell, add the transfection mixed solution of 5ml serum free medium, 10 μ g pEC4-N2,60 μ g lipoFectamin compositions, cultivate 4 hours for 37 DEG C, sucking-off substratum, adds the MEM substratum containing 5% foetal calf serum, 37 DEG C of overnight incubation.In substratum, add subsequently MTX, continue to cultivate and occur to resistance clone for 10-14 days.The cell of turning out with trysinization resistance clone, increases MTX concentration, screening resistance clone.Utilize enzyme-linked immunosorbent assay to confirm that the cell obtaining can express erythropoietin analogue N2.By expressing cell amplification, the cultivation of erythropoietin analogue N2, collect nutrient solution, carry out purifying.
B.Asn 3phe 4ser 5the purifying of EPO
Purify EPO analogue N2 by the three-step approach being formed by ion-exchange chromatography, reverse chromatograms and gel filtration chromatography.
Detailed process is as follows:
1. collect the cho cell conditioned medium of expressing EPO analogue N2, use with the ultra-filtration membrane of 5k molecular retention amount and concentrated after 15 times, damping fluid is replaced into 10mM Tris-HCl pH7.0.
2. the flow velocity solution after concentrated being divided with 0.5em/ is added on 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, 1mM glycine, the solution of 6M urea composition rinses post bed, subsequently with 10mM Tris-HCl, 200mM NaCl, pH7.0 by EPO analogue N2 by eluting on post.
4. the flow velocity EPO analogue N2 component of collecting being divided with 3cm/ is added to Vydac C 4on 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, according to 1:2.5 10mMTris-HCl pH7.0, after dilution, use concentrated 20 times of pall nanosep10K omega micro-thickener, and to replace damping fluid be 10mM Tris-HCl pH7.0.
5. concentrated solution is carried out to purifying on the S-200 post to cross by 20mM citrate buffer solution pH6.8 balance, collect corresponding monomer analogue peak.
Embodiment 4:Val 2asn 3phe 4ser 5the preparation of EPO
A. the structure of engineering cell, screening and cultivation
The plasmid pEC4-N3 transfection CHO cell building according to the method for embodiment 1, host cell, purchased from American type culture collection (ATCC), is Tetrahydrofolate dehydrogenase deficient Chinese Hamster (CHO dhfr-).By this cell cultures in 100mm culture dish, grow to 60～95% when full until cell, with serum free medium drip washing cell, add the transfection mixed solution of 5ml serum free medium, 10 μ g pEC4-N3,60 μ g lipoFectamin compositions, cultivate 4 hours for 37 DEG C, sucking-off substratum, adds the MEM substratum containing 5% foetal calf serum, 37 DEG C of overnight incubation.In substratum, add subsequently MTX, continue to cultivate and occur to resistance clone for 10-14 days.The cell of turning out with trysinization resistance clone, increases MTX concentration, screening resistance clone.Utilize enzyme-linked immunosorbent assay to confirm that the cell obtaining can express erythropoietin analogue N3.By expressing cell amplification, the cultivation of erythropoietin analogue N3, collect nutrient solution, carry out purifying.
B.Val 2asn 3phe 4ser 5the purifying of EPO
Purify EPO analogue N3 by the three-step approach being formed by ion-exchange chromatography, reverse chromatograms and gel filtration chromatography.
Detailed process is as follows:
1. collect the cho cell conditioned medium of expressing EPO analogue N3, use with the ultra-filtration membrane of 5k molecular retention amount and concentrated after 15 times, damping fluid is replaced into 10mM Tris-HCl pH7.0.
2. the flow velocity solution after concentrated being divided with 0.5cm/ is added on 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, 1mM glycine, the solution of 6M urea composition rinses post bed, subsequently with 10mM Tris-HCl, 200mM NaCl, pH7.0 by EPO analogue N3 by eluting on post.
4. the flow velocity analogue N3 component of collecting being divided with 3cm/ is added to Vydac C 4on 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, according to 10mM Tris-HCl pH7.0 for 1:2.5, after dilution, use concentrated 20 times of pall nanosep10K omega micro-thickener, and to replace damping fluid be 10mMTris-HCl pH7.0.
5. concentrated solution is carried out to purifying on the S-200 post to cross by 20mM citrate buffer solution pH6.8 balance, collect 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
The plasmid pEC4-N34 transfection CHO cell building according to the method for embodiment 1, host cell, purchased from American type culture collection (ATCC), is Tetrahydrofolate dehydrogenase deficient Chinese Hamster (CHO dhfr-).By this cell cultures in 100mm culture dish, grow to 60～95% when full until cell, with serum free medium drip washing cell, add the transfection mixed solution of 5ml serum free medium, 10 μ g pEC4-N34,60 μ g lipoFectamin compositions, cultivate 4 hours for 37 DEG C, sucking-off substratum, adds the MEM substratum containing 5% foetal calf serum, 37 DEG C of overnight incubation.In substratum, add subsequently MTX, continue to cultivate and occur to resistance clone for 10-14 days.The cell of turning out with trysinization resistance clone, increases MTX concentration, screening resistance clone.Utilize enzyme-linked immunosorbent assay to confirm that the cell obtaining can express erythropoietin analogue N34.By expressing cell amplification, the cultivation of erythropoietin analogue N34, collect nutrient solution, carry out purifying.
B.Asn 28thr 30ser 87asn 88gly 89ser 90the purifying of EPO
Purify EPO analogue N34 by the three-step approach being formed by ion-exchange chromatography, reverse chromatograms and gel filtration chromatography.
Detailed process is as follows:
1. collect the cho cell conditioned medium of expressing EPO analogue N34, use with the ultra-filtration membrane of 5k molecular retention amount and concentrated after 15 times, damping fluid is replaced into 10mM Tris-HCl pH7.0.
2. the flow velocity solution after concentrated being divided with 0.5cm/ is added on 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, 1mM glycine, the solution of 6M urea composition rinses post bed, subsequently with 10mM Tris-HCl, 200mM NaCl, pH7.0 is by Asn 28thr 30ser 87asn 88gly 89ser 90ePO is by eluting on post.
4. by the Asn collecting 28thr 30ser 87asn 88gly 89ser 90the flow velocity that EPO component is divided with 3cm/ is added to Vydac C 4on reversed-phase resin post, this post has been used 20% ethanol/10mM Tris-HCl, pH7.0 balance mistake.With 20%-95% ethanol/10mMTris-HCl, pH7.0 progressively launches wash-out.Through 50-75% ethanol/10mM Tris-HCl, the component of pH7.0 wash-out, according to 10mM Tris-HCl pH7.0 for 1:2.5, after dilution, use concentrated 20 times of pall nanosep10K omega micro-thickener, and to replace damping fluid be 10mM Tris-HCl pH7.0.
5. concentrated solution is carried out to purifying on the S-200 post to cross by 20mM citrate buffer solution pH6.8 balance, collect 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
The plasmid pEC4-N44 transfection CHO cell building according to the method for embodiment 1, host cell, purchased from American type culture collection (ATCC), is Tetrahydrofolate dehydrogenase deficient Chinese Hamster (CHO dhfr-).By this cell cultures in 100mm culture dish, grow to 60～95% when full until cell, with serum free medium drip washing cell, add the transfection mixed solution of 5ml serum free medium, 10 μ g pEC4-N44,60 μ g lipoFectamin compositions, cultivate 4 hours for 37 DEG C, sucking-off substratum, adds the MEM substratum containing 5% foetal calf serum, 37 DEG C of overnight incubation.In substratum, add subsequently MTX, continue to cultivate and occur to resistance clone for 10-14 days.The cell of turning out with trysinization resistance clone, increases MTX concentration, screening resistance clone.Utilize enzyme-linked immunosorbent assay to confirm that the cell obtaining can express erythropoietin analogue N44.By 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
Purify EPO analogue N44 by the three-step approach being formed by ion-exchange chromatography, reverse chromatograms and gel filtration chromatography.
Detailed process is as follows:
1. collect the cho cell conditioned medium of expressing EPO analogue N44, use with the ultra-filtration membrane of 5k molecular retention amount and concentrated after 15 times, damping fluid is replaced into 10mM Tris-HCl pH7.0.
2. the flow velocity solution after concentrated being divided with 0.5cm/ is added on 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, 1mM glycine, the solution of 6M urea composition rinses post bed, subsequently with 10mM Tris-HCl, 200mM NaCl, pH7.0 is by Asn 2gly 3thr 4asn 28thr 30ser 87asn 88gly 89ser 90ePO is by eluting on post.
4. by the Asn collecting 2gly 3thr 4asn 28thr 30ser 87asn 88gly 89ser 90the flow velocity that EPO component is divided with 3cm/ is added to Vydac C 4on 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, according to 10mM Tris-HCl pH7.0 for 1:2.5, after dilution, use concentrated 20 times of pall nanosep10K omega micro-thickener, and to replace damping fluid be 10mM Tris-HCl pH7.0.
5. concentrated solution is carried out to purifying on the S-200 post to cross by 20mM citrate buffer solution pH6.8 balance, collect 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
The plasmid pEC4-N60 transfection CHO cell building according to the method for embodiment 1, host cell, purchased from American type culture collection (ATCC), is Tetrahydrofolate dehydrogenase deficient Chinese Hamster (CHO dhfr-).By this cell cultures in 100mm culture dish, grow to 60～95% when full until cell, with serum free medium drip washing cell, add the transfection mixed solution of 5ml serum free medium, 10 μ g pEC4-N60,60 μ g lipoFectamin compositions, cultivate 4 hours for 37 DEG C, sucking-off substratum, adds the MEM substratum containing 5% foetal calf serum, 37 DEG C of overnight incubation.In substratum, add subsequently MTX, continue to cultivate and occur to resistance clone for 10-14 days.The cell of turning out with trysinization resistance clone, increases MTX concentration, screening resistance clone.Utilize enzyme-linked immunosorbent assay to confirm that the cell obtaining can express erythropoietin analogue N60.By 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
Purify ASN30THR32ASN114THR116EPO by the three-step approach being formed by ion-exchange chromatography, reverse chromatograms and gel filtration chromatography.
Detailed process is as follows:
1. collect the cho cell conditioned medium of expressing ASN30THR32ASN114THR116EPO, use with the ultra-filtration membrane of 5k molecular retention amount and concentrated after 15 times, damping fluid is replaced into 10mM Tris-HCl pH7.0.
2. the flow velocity solution after concentrated being divided with 0.5cm/ is added on 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, 1mM glycine, the solution of 6M urea composition rinses post bed, subsequently with 10mM Tris-HCl, 200mM NaCl, pH7.0 by ASN30THR32ASN114THR116EPO by eluting on post.
4. the flow velocity ASN30THR32ASN114THR116EPO component of collecting being divided with 3cm/ is added to Vydac C 4on 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, according to 10mM Tris-HCl pH7.0 for 1:2.5, after dilution, use concentrated 20 times of pall nanosep10K omega micro-thickener, and to replace damping fluid be 10mM Tris-HCl pH7.0.
5. concentrated solution is carried out to purifying on the S-200 post to cross by 20mM citrate buffer solution pH6.8 balance, collect 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
The nutrient solution 5ml containing each expression product obtaining according to method described in embodiment 2-7A, uses the micro-thickener of pall nanosep10K omega to be concentrated into respectively 20 μ l.With 15% polyacrylamide gel at 0.375M Tris-HCl, 0.0%SDS, vertical slab electrophoresis 2.5 hours in pH8.8 electrolytic solution.After electrophoresis finishes, cut one and shift filter paper with gel nitrocellulose filter of a size and 12 Whatman3MM, after use and shifting immersion and moisten, on Multiphor Electrophoresis System with 0.8mA/cm 2individual hour of half-dried transferase 12, transfer finishes rear use containing the PBST solution closing membrane of 10% bovine serum albumin 1 hour, adding mouse anti-erythrocyte to generate plain monoclonal antibody hatches 2 hours again, finally hatch 1 hour with sheep anti mouse enzyme labelled antibody again, PBST cleans after 3 times, final with the substrate solution colour developing containing DAB.Swimming lane 1-7 is respectively normal EPO and the EPO analogue that contains one, two, three extra glycosylation site as shown in Figure 3, and result shows, increases after glycosylation site, and 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 containing each expression product obtaining according to method described in embodiment 2-7A, after 18 hours, uses the micro-thickener of pal l nanosep10K omega 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, 10% polyacrylamide gel 15ml of pH3-10 and pH2.5-5 amphotericeledrolyte and 7M urea will be contained, pour in vertical slab electrophoresis groove after gelling is poly-, 600V, 50mA, 30W prerunning 30 minutes, sample after dialysis and sample buffer 1:1 are mixed and added in gel sample groove, 800V, 50mA, 30W electrophoresis 2 hours.After electrophoresis finishes, carry out immunoblotting assay according to method described in embodiment 8A.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 result shows, increases after glycosylation site, and 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 method described in embodiment 2-7B.With 15% polyacrylamide gel at 0.375M Tris-HCl, 0.i%SDS, vertical slab electrophoresis 2.5 hours in pH8.8 electrolytic solution.After finishing, electrophoresis carries out Coomassie brilliant blue dyeing.The EPO analogue that swimming lane 1-7 is respectively normal EPO and contains one, two, three extra glycosylation site as shown in Figure 5, result shows, increase after 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 glycosylation amount increases progressively.
D. erythrocyte-stimulating factor analogues purified product IEF analyzes
The purified erythrocyte-stimulating factor analogues 100 μ l that obtain according to method described in embodiment 2-7B, 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, 10% polyacrylamide gel 15ml of pH3-10 and pH2.5-5 amphotericeledrolyte and 7M urea will be contained, pour in vertical slab electrophoresis groove after gelling is poly-, 600V, 50mA, 30W prerunning 30 minutes, sample after dialysis and sample buffer 1:1 are mixed and added in gel sample groove, 800V, 50mA, 30W electrophoresis 2 hours.After finishing, electrophoresis carries out Coomassie brilliant blue dyeing.The EPO analogue purified that swimming lane 1-7 is respectively normal EPO and contains one, two, three extra glycosylation site as shown in Figure 6, result shows, along with increasing of glycosylation site, the acidity band of product increases, illustrate and in molecule, increased extra acid carbohydrate, and the quantity increasing increasing and increase with glycosylation site.
E. sialic acid content analysis in erythrocyte-stimulating factor analogues purified product
Sialic acid mole number with every mole of erythrocyte-stimulating factor analogues represents.First will be diluted to 0.2-0.4mg/ml according to the each erythrocyte-stimulating factor analogues purified product protein concentration obtaining described in embodiment 2-7B by purified water.In order to determine existing erythrocyte-stimulating factor analogues purified protein content, the analytical reagent and the micromethod step that use Bio-Rad to provide, using the 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 10ml glass test tube, mix, in every pipe, add containing 0.2% Resorcinol 250 μ M copper sulfate, 20% hydrochloric acid soln 1ml, add a cover, boiling water boils 30 minutes, takes out ice bath after 3 minutes, and every pipe adds butylacetate-butanol solution (4:1) 2ml, fully mix, room temperature is placed 10 minutes.Measure absorbancy at 580nm wavelength.By the concentration of sialic acid reference substance solution, its corresponding absorbancy is done to straight-line regression, obtain 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 absorbancy of 5 μ g, A 2for the absorbancy of erythrocyte-stimulating factor analogues purified, D is the extension rate of erythrocyte-stimulating factor analogues purified, P is that (μ g/ μ l) for erythrocyte-stimulating factor analogues purified protein content, W is the amount (not comprising the amount of 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
Embodiment 9: after erythrocyte-stimulating factor analogues single dose injection mouse, in body, reticulocyte over time
The purified erythrocyte-stimulating factor analogues obtaining according to method described in embodiment 2-7B, taking 5 μ g/kg subcutaneous injections to body weight the female Balb/c mouse as 20 ± 2, and taking the recombinant human erythropoietin of same dose as reference substance, establish solvent control group simultaneously.Blood sampling in every 24 hours after injection, detects reticulocyte content with reticulocyte numeration instrument, detects reticulocyte content temporal evolution situation, Fig. 7 be promoting erythrocyte analogue to injected in mice after reticulocyte curve over time.Result shows, increase with extra glycosylation site, erythrocyte-stimulating factor analogues extends 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 impact of the last amino acids of glycosylation site.
Embodiment 10:Asn 2gly 3thr 4asn 28thr 30ser 87asn 88gly 89ser 90the acquisition of EPO isomer
What the method providing according to embodiment 6A obtained contains Asn 2gly 3thr 4asn 28thr 30ser 87asn 88gly 89ser 90the nutrient solution of EPO is purified by the three-step approach being made up 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, uses with the ultra-filtration membrane of 5k molecular retention amount and is concentrated after 15 times, and damping fluid is replaced into 10mM Tris-HCl pH7.0.
2. the flow velocity solution after concentrated being divided with 0.5cm/ is added on 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, 1mM glycine, the solution of 6M urea composition rinses post bed, and with 0-0.4M NaCl/10mM Tris-HCl, 15 column volumes of pH7.0 gradient elution, collect taking 1.5 column volumes as a component.
4. from each component, sampling is carried out IEF analysis according to method described in embodiment 8D, collects 4 main ingredients, contains respectively the also liquid collecting of isomer 1-5,2-6,3-6,4-8.
5. flow velocity isomer 3-6 liquid collecting being divided with 3cm/ is added to Vydac C 4on 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, with after pH7.010mM Tris-HCl dilution, is used concentrated 20 times of the micro-thickener of pall nanosep10K omega according to 1:2.5.
By concentrated solution to use 20mM citrate buffer solution, on the S-200 post that pH7.0 balance is crossed, carry out purifying, collect corresponding analogue isomer 3-6.
Embodiment 11Asn 2gly 3thr 4asn 28thr 30ser 87asn 88gly 89ser 90the biological property of EPO isomer
A. erythrocyte-stimulating factor analogues isomer IEF analyzes
Obtain the each 100 μ l of erythrocyte-stimulating factor analogues isomer component according to method described in embodiment 10, 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, 10% polyacrylamide gel 15ml of pH3-10 amphotericeledrolyte and 7M urea will be contained, pour in vertical slab electrophoresis groove after gelling is poly-, 600V, 50mA, 30W prerunning 30 minutes, sample after dialysis and sample buffer 1:1 are mixed and added in gel sample groove, 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 containing isomer 1-5,2-6,3-6,4-8 as shown in Figure 8, and 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 marrow, reticulocyte counts in Mouse Blood and the ratio of RBC number are raise, and lift-off value and injected dose linear.Detect EPO Biological acdtivity in vivo by dose-response parallel method.With containing 0.1% bovine serum albumin physiological sodium chloride diluent, by EPO biological reference standard, (this standard substance derives from Nat'l Pharmaceutical & Biological Products Control Institute, be used for measuring in vivo bioactivity) for measuring in vivo bioactivity) being diluted to concentration 80IU/ml, 40IU/ml and 20IU/ml, erythrocyte-stimulating factor analogues isomer mixture is also diluted to respectively and approaches above-mentioned three kinds of concentration.By 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 veutro subcutaneous injection mouse respectively.The volume injected of every mouse is 0.5ml.Within the 4th day after injection, take a blood sample in mouse orbit, use EDTA-K 2(containing the physiological saline of i%EDTA) anticoagulant tube anti-freezing, drip/mouse of blood sampling volume: 4-5.Get anticoagulation, before every pipe reading, turn upside down 5 times, it is fully mixed, careful uncap, test tube is inserted in R-500 analyser (Japanese Sysmex company) suction needle and press beginning, instrument Auto-counting red corpuscle sum, reticulocyte sum and the reticulocyte counts ratio (Ret) to red corpuscle sum.By in Ret value input computer, program is calculated the biologic activity that detects sample automatically.The activity in vivo that table 4 shows erythropoietin isomer increases and raises with acid carbohydrate.In body described here, specific activity is the observed value of specific activity in relative body, is not the absolute interior specific activity observed value of body.Specific activity is only for comparing the relative reactivity of erythrocyte-stimulating factor analogues, the mensuration of analogue adopts identical measuring method, adopts the same terms, the animal of identical type, there is the identical data analysis for calculating specific activity, measure the same analysis method of protein content.The present embodiment does not want that specific activity value in any body of any analogue to be reported represents the intrinsic or absolute value of this analogue.
Table 4
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. after erythrocyte-stimulating factor analogues isomer 3-6 liquid collecting single dose injection mouse, in body, reticulocyte over time
The erythrocyte-stimulating factor analogues isomer 3-6 obtaining according to method described in embodiment 10, taking 5 μ g/kg subcutaneous injections to body weight the female Balb/c mouse as 20 ± 2, and taking the recombinant human erythropoietin of same dose as reference substance, establish solvent control group simultaneously.Blood sampling in every 24 hours after injection, with reticulocyte numeration instrument detection reticulocyte content, 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.Result demonstration, erythropoietin group second day RET% after medication starts to raise, and within the 4th day, reaches peak, continues approximately 4 days; Erythropoietin analogue group RET% also started in second day to raise, and within the 6th day, reached peak, continued approximately 7 days.As can be seen here, shift with sialic acid content increase, iso-electric point oxytropism, extend action time in vivo.
D. erythrocyte-stimulating factor analogues isomer 3-6 union vena axillaris injection pharmacokinetic
Select 10～11 weeks rats of male SD (Sprague Dawley), 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 DEG C), put 4 DEG C of overnight incubation, centrifugal 15 minutes of 12000rpm, with the detection of coffee detector, calculates pharmacokinetic parameter.Figure 10 is the mixture Plasma Concentration temporal evolution curve of erythropoietin and isomer 3-6, compared with erythropoietin, in isomer 3-6 mixing object, removing speed obviously reduces, cause elimination Increased Plasma Half-life in body, after the intravenous injection rat of the two, the transformation period is respectively 8.5h and 2.6h, and the elimination transformation period of erythrocyte-stimulating factor analogues is 3.3 times of erythropoietin.
E. erythrocyte-stimulating factor analogues isomer 3-6 the liquid collecting impact on 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, clean level, 8 week age, body weight 18～22g, according to the principle grouping of random packet, 10 every group, the isomer 3-6 obtaining in the natural erythropoietin of employing various dose and 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 same 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 4th day (4d) and 7 days (7d) in 1 week with the kapillary of heparinization processing, sealing, adopts centrifuging to detect hematocrit.Figure 11-12nd, after injected in mice erythropoietin and erythropoietin analogue isomer 3-6 liquid collecting, hematocrit temporal evolution situation is shown in.Result demonstration, after weekly and time administration on every Wendesdays, 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 situation that weekly dose is identical, the rising effect to Mouse Blood specific volume of cell after the weekly injection of isomer mixture and natural human erythropoietin be time injection result no significant difference on every Wendesdays, injects once in a week, can reach the result for the treatment of of expection.

Claims (9)

1. one kind has the natural human erythrocyte-stimulating factor analogues of erythropoietin activity, this analogue comprises 1 extra N-glycosylation site, wherein change l-asparagine in the 3rd position of natural human erythropoietin aminoacid sequence, phenylalanine has been changed in the 4th position, and Serine has been changed in the 5th position; I.e. [Asn 3phe 4ser 5] EPO; Described extra N-glycosylation site is the l-asparagine of the 3rd of aminoacid sequence.
2. one kind has the natural human erythrocyte-stimulating factor analogues of erythropoietin activity, this analogue comprises 1 extra N-glycosylation site, wherein change α-amino-isovaleric acid in the 2nd position of natural human erythropoietin aminoacid sequence, l-asparagine has been changed in the 3rd position, phenylalanine has been changed in the 4th position, and Serine has been changed in the 5th position; I.e. [Val 2asn 3phe 4ser 5] EPO; Described extra N-glycosylation site is the l-asparagine of the 3rd of aminoacid sequence.
3. a DNA sequence dna, the human erythropoietin analogue of this DNA sequence encoding claim 1-2 any one.
4. an expression vector, the DNA sequence dna that this expression vector contains claim 3.
5. an eukaryotic host cell, the expression vector transfection of claim 4 for this host cell.
6. the eukaryotic host cell of claim 5, it is selected from CHO, COS-7 or BHK.
7. a production method with the natural human erythrocyte-stimulating factor analogues of erythropoietin activity, comprises step:
(i) eukaryotic host cell of cultivation claim 5 or 6, and
(ii) from culture, collect the glycoprotein with erythropoietin activity.
8. increase an erythropoietic pharmaceutical composition, the human erythropoietin analogue that said composition comprises the claim 1-2 any one for the treatment of significant quantity and pharmaceutically useful thinner, auxiliary agent or carrier.
9. the pharmaceutical composition of claim 8, it prevents or treatment anaemia for promoting erythropoiesis.
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