CN100362019C - Recombinant erythropoietin with intracorporeal physical activity and modified by macrogol - Google Patents
Recombinant erythropoietin with intracorporeal physical activity and modified by macrogol Download PDFInfo
- Publication number
- CN100362019C CN100362019C CNB2005100520223A CN200510052022A CN100362019C CN 100362019 C CN100362019 C CN 100362019C CN B2005100520223 A CNB2005100520223 A CN B2005100520223A CN 200510052022 A CN200510052022 A CN 200510052022A CN 100362019 C CN100362019 C CN 100362019C
- Authority
- CN
- China
- Prior art keywords
- epop
- epo
- peg
- leu
- erythropoietin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 title claims abstract description 66
- 102000003951 Erythropoietin Human genes 0.000 title claims abstract description 17
- 108090000394 Erythropoietin Proteins 0.000 title claims abstract description 17
- 229940105423 erythropoietin Drugs 0.000 title claims abstract description 17
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 title abstract description 6
- 229960003511 macrogol Drugs 0.000 title abstract 2
- 230000037081 physical activity Effects 0.000 title abstract 2
- 102100036508 Elongin BC and Polycomb repressive complex 2-associated protein Human genes 0.000 claims abstract description 30
- 101000852151 Homo sapiens Elongin BC and Polycomb repressive complex 2-associated protein Proteins 0.000 claims abstract description 30
- 208000007502 anemia Diseases 0.000 claims abstract description 20
- 239000003814 drug Substances 0.000 claims abstract description 14
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 11
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 9
- 230000009465 prokaryotic expression Effects 0.000 claims abstract description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 6
- 238000001727 in vivo Methods 0.000 claims description 22
- 230000000694 effects Effects 0.000 claims description 21
- 239000000126 substance Substances 0.000 claims description 21
- 238000011282 treatment Methods 0.000 claims description 19
- 230000014509 gene expression Effects 0.000 claims description 16
- 101000987586 Homo sapiens Eosinophil peroxidase Proteins 0.000 claims description 9
- 101000920686 Homo sapiens Erythropoietin Proteins 0.000 claims description 9
- 230000004048 modification Effects 0.000 claims description 9
- 238000012986 modification Methods 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- 102000044890 human EPO Human genes 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 230000000630 rising effect Effects 0.000 claims description 2
- 241000588722 Escherichia Species 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 25
- 238000000034 method Methods 0.000 abstract description 24
- 210000003743 erythrocyte Anatomy 0.000 abstract description 13
- 102000004169 proteins and genes Human genes 0.000 abstract description 12
- 230000001965 increasing effect Effects 0.000 abstract description 3
- 102100031939 Erythropoietin Human genes 0.000 description 52
- 108010092408 Eosinophil Peroxidase Proteins 0.000 description 51
- 101150002621 EPO gene Proteins 0.000 description 30
- 108090000765 processed proteins & peptides Proteins 0.000 description 24
- 239000000523 sample Substances 0.000 description 21
- 150000001413 amino acids Chemical class 0.000 description 17
- 235000001014 amino acid Nutrition 0.000 description 14
- 210000004369 blood Anatomy 0.000 description 14
- 239000008280 blood Substances 0.000 description 14
- 241000894006 Bacteria Species 0.000 description 13
- 239000000047 product Substances 0.000 description 12
- 230000004071 biological effect Effects 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- 230000001737 promoting effect Effects 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 10
- 241001465754 Metazoa Species 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 9
- 229930182817 methionine Natural products 0.000 description 8
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 8
- 108020004705 Codon Proteins 0.000 description 7
- 238000006206 glycosylation reaction Methods 0.000 description 7
- 230000000968 intestinal effect Effects 0.000 description 7
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 6
- 102220369445 c.668T>C Human genes 0.000 description 6
- 230000013595 glycosylation Effects 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 210000003527 eukaryotic cell Anatomy 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000001976 improved effect Effects 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 239000004475 Arginine Substances 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 229920001427 mPEG Polymers 0.000 description 4
- 210000002381 plasma Anatomy 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000003442 weekly effect Effects 0.000 description 4
- 208000030507 AIDS Diseases 0.000 description 3
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 3
- 238000010241 blood sampling Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000013068 control sample Substances 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000001995 reticulocyte Anatomy 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 108010074604 Epoetin Alfa Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 102220369447 c.1352G>A Human genes 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 208000020832 chronic kidney disease Diseases 0.000 description 2
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000006011 modification reaction Methods 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- -1 polyoxyethylene Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000008521 reorganization Effects 0.000 description 2
- 102220023256 rs387907547 Human genes 0.000 description 2
- 102220023258 rs387907548 Human genes 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 201000000736 Amenorrhea Diseases 0.000 description 1
- 206010002068 Anaemia neonatal Diseases 0.000 description 1
- 208000030760 Anaemia of chronic disease Diseases 0.000 description 1
- 208000032467 Aplastic anaemia Diseases 0.000 description 1
- 108010002913 Asialoglycoproteins Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 241000258916 Leptinotarsa decemlineata Species 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000219492 Quercus Species 0.000 description 1
- 108020001027 Ribosomal DNA Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 102000000763 Survivin Human genes 0.000 description 1
- 108010002687 Survivin Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 208000022400 anemia due to chronic disease Diseases 0.000 description 1
- 201000000975 anemia of prematurity Diseases 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 210000003995 blood forming stem cell Anatomy 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000011082 depyrogenation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 229960003388 epoetin alfa Drugs 0.000 description 1
- 229940089118 epogen Drugs 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000006052 feed supplement Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 229940124541 immunological agent Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 150000002742 methionines Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 210000003360 nephrocyte Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 210000004279 orbit Anatomy 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Substances [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 230000005070 ripening Effects 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Abstract
The invention relates to a polyethylene glycol conjugate (PEG-EPOP) of the hematopoietin protein, which generates a recombinant erythropoietin (rhEPOP) with intracorporeal physical activity and modified by macrogol. The invention discloses the method of obtaining EPOP with low cost by the prokaryotic expression system so as to obtain polyethylene glycol conjugate (PEG-EPOP) with low cost for producing the medicine for treating anemic diseases and increasing erythrocyte.
Description
Technical field
The present invention relates to the recombinant erythropoietin albumen (EPOP) of no metabolically active be modified with polyoxyethylene glycol, and the binding substances (PEG-EPOP) of the metabolically active with promoting erythrocyte nucleus formation that obtains.
Background of invention
(human Erythropoietin is a kind of glycoprotein hormones that is mainly synthesized justacrine by kidney hEPO) to erythropoietin, and EPO results from liver in embryonic stage, and being born to change in back 4 months is produced by kidney.Its physiological action is differentiation and the propagation that stimulates marrow red corpuscle precursor cell, plays an important role in the red blood cell development ripening process, is the endogenous conditioning agent of red corpuscle growth.
In one's early years from aplastic amenia people's urine, can obtain more hEPO in the nutrient solution of tire nephrocyte, but can not satisfy clinical demand.1985, the applying gene recombinant technology obtained recombinant human epo (rhEPO), had begun the history of suitability for industrialized production EPO, made EPO widespread use clinically become possibility.The rhEPO that in June, 1989, U.S. Amgen company produced formally obtains the U.S. FDA registration permission, and commodity are called " EPOGEN
EPOETINALFA ", be used for the treatment of chronic renal failure (CRF) and merge anemia, obtained great success.The existing in the world at present Development and Production rhEPO of many companies, the result of the clinical application of nearly more than ten years shows the evident in efficacy of rhEPO, side effect is little, is the specific medicament of all kinds of anaemias of treatment, also is current the most successful genetically engineered medicine.Along with people are deep day by day to the research of EPO, its clinical indication will further enlarge, be applicable at present: (1) chronic kidney hypofunction merges the treatment of anaemia: a major cause of this type of anaemia is that EPO generates shortage, and EPO is the replacement therapy method, and its curative effect reaches more than 95%, content of hemoglobin (Hb) and pcv (Hct) are increased, anaemia is improved, and the bleeding time shortens, and quality of life improves, the blood transfusion number of times reduces the patient who also helps implementing renal transplantation; (2) acquired immune deficiency syndrome (AIDS) (AIDS) patient accompanies anaemia, the patient Chang Yi that especially the uses AZT treatment anaemia that occurs together, and the EPO treatment is effectively; (3) anaemia due to the tumour patient chemotherapy is especially used the obvious anaemia of the normal appearance of cis-platinum, and the EPO treatment effectively; (4) anemia of chronic disease as rheumatoid arthritis, tumour patient, needs blood transfusion to keep the curer when occurring together anaemia, and EPO can make the part patient reduce the number of times of blood transfusion; (5) anaemia of hemopoietic stem cell disease, as the patient of myelodysplastic syndrome (MDS), aplastic anemia etc., blood transfusion also is difficult to the Sustainer if need repeatedly, can try out EPO, and about 25%～30% patient can make anaemia improve, and reduces the blood transfusion number of times; (6) from body blood supply infusion: the American association of blood banks regulation, the patient who chooses date for operation should take from blood as the hand orthosis patient and store, in order to when operation infusion, preceding 3 weeks of art begin to use EPO, and Hb rises and gets blood 400ml storage when obvious, gather 3 times standby, can satisfy patient's needs.Use EPO can improve storage blood quality, reduce storage blood quantity; (7) control of anemia of prematurity: 50～100IU/kg 3 times weekly, begins 4 weeks of logotype after birth.Can estimate: EPO will be a line medicine of treatment for anemia in a quite long period.
In adopting the pharmaceutical grade protein therapeutic process, plasma half-life that Chang Yinqi is short and its bioavailability is reduced, this shows particularly evidently on the such hormones protein of EPO.Body is regulated the secretory volume of EPO by the oxygen partial pressure susceptor on the kidney cell, be discharged into EPO in the blood plasma by the liver cell quick inactivating, set up thus and keep the sensitive regulation mechanism of the relative constant of pcv: both can when anaemia, promote erythrocytic release in a large number, and can prevent that again excessive release red corpuscle from causing blood viscosity to cross high negative consequence.Use rhEPO to the anaemia treatment of diseases in, this mechanism has lowered the bioavailability of rhEPO greatly, causes the increase of dosage, and because the price of rhEPO costliness also causes the increase of medical expense; Simultaneously because the quantitative limitation of EPO acceptor number, this mechanism has also limited the ability that dosage obtains higher result of treatment that strengthens, in the treatment of reality, in most cases need adopt 3 doses treatment plan weekly, only the visual situation of keeping in the treatment after Anemia is corrected reduces dosage and the medication frequency to 2 doses or 1 dose weekly weekly.
The EPO molecule is a kind of sialic acid glycoprotein that contains, and is comparatively stable to heat (unchangeability under 80 ℃ of conditions) and soda acid (stable range is between PH3.5～10.0).Cys in the protein peptide chain
161With Cys
7, Cys
29And Cys
33Between form two couples of disulfide linkage, wherein Cys respectively
7And Cys
161Between disulfide linkage most important for the biological activity of EPO, if disulfide linkage is reduced, EPO will lose its biological activity.Four glycosylation sites of EPO molecule are respectively at Asn
38, Asn
24, Asn
83And Ser
126On, former three is the N-sugar chain, the latter is the O-sugar chain.The branch degree difference of sugar chain has two tips, three tips or four tips, and wherein four tips are the most common.Result of study shows that the branch degree of sugar chain is influential to EPO biological activity in vivo, and the height branch of N-end sugar chain keeps to it that biological activity is necessary in organism.Asialoglycoprotein or de-glycosylation do not influence in external biological activity, but have shortened the transformation period in vivo greatly (mainly through liver cell absorption metabolism), and the result has completely lost biologic activity in vivo.After having only the EPO molecule by abundant glycosylation, could show biological activity in vivo.Therefore the existence of sugar chain structure is most important to EPO biological activity in vivo.That is to say the EPO biologically active just in vivo that has only eukaryotic cell expression.Since EPO glycosyl structure division account for whole molecular weight 30～40% and have the height unhomogeneity, the difference of its sugar chain branch degree makes molecular weight (Wasley LC between 30～40KD of EPO, Linderberg G, Fishman L, et al.The importance of N-and O-linked oligosaccharides for thebiosynthesis and in vitro and vivo biological activities of erythropoietin.Blood, 1991,77 (3): 419-434.).Numerous researchs about EPO have all confirmed above conclusion, and therefore general viewpoint thinks that recombinant epo must be with eukaryotic cell expression, as document Ma Peiqi.Erythropoietin and clinical application thereof and market outlook." Chinese Medicine information " 1999 the 5th the 1st phases of volume, P11).
Based on above understanding to natural hEPO, the rhEPO that is used for drug manufacture at present all adopts the eukaryotic cell expression system, adopts on the rhEPO that the eukaryotic cell expression system obtained and EPO characteristic and the improved research of drug effect also concentrated on.For increasing the drug effect of rhEPO, there are two kinds at present than successful method, the one, existing rhEPO is carried out chemically modified, as in 00898956 patent application, the conjugate of a kind of EPO and PEG is disclosed, it is by modifying covalently bound 1～3 the lower alkoxy polyoxyethylene glycol of EPO glycoprotein (PEG) group, and the circulating half-life and the blood plasma residence time of having improved EPO, and reduced the clearance rate of EPO and improved its activity in vivo.Another is to increase glycosylation site and then to increase degree of glycosylation by the part amino acid in the engineered method change EPO peptide chain, referring to U.S. Pat 5856298 and Amgen Inc.One Amgen Center Drive Thousand Oaks, California product A ranesp
TMSpecification sheets.
Although above improvement has obtained the obvious increase rhEPO effect of plasma half-life, it still is to adopt the used eukaryotic cell expression system of existing rhEPO pharmaceutical production technology, and producing should be suitable with existing product.
(application number: 200410021859.7) in another patent application early of applicant, redesign by base sequence known hEPO gene, eliminate the intestinal bacteria rare codon, and replace the intestinal bacteria preference codon, and adjust GC content in right amount, successfully made up the recombination and the carrier system that can efficiently express prokaryotic system (for example in the intestinal bacteria).Though the recombinant human erythropoietin albumen (EPOP) of expressing does not have physiologically active in vivo owing to lack glycosylation modified, but the prokaryotic system production cost is significantly less than eukaryotic system, and the EPOP of Zhi Bei no activity in vivo can be used for for example preparing EPO peptide chain protein reagent, also can be used for the EPO antigen of immune animal like this; Or be prepared into EPO positive control standard substance, be used for the test kit of EPO immunodetection, as methods such as anti-phase blood clotting, radioimmunity, enzyme linked immunologicals.
Summary of the invention
The invention provides a kind of pEG-EpOp binding substances, adopt sugar basedization thereby do not have promoting erythrocyte survivin (EPO) albumen (EPOP) of activity in vivo, as: provided by the invention by bioactive epo protein in the no body of prokaryotic expression system acquisition, utilize PEG that EPOP is carried out chemically modified again, obtain to have the product (PEG-EPOP) of promoting erythrocyte nucleus formation physiologically active in the body.
The present invention also provides the purposes of PEG-EPOP binding substances in preparation treatment anaemia disease medicament and rising red corpuscle medicine.
The PEG-EPOP binding substances of formula (I) structure is provided according to an aspect of the present invention:
mPEG-X-EPOP
(I)
In the formula (I), the molecular structural formula of mPEG is CH
3 CH
2CH
2O
k
Wherein k is 100～1000 integer, and molecular weight is 5000～40000 dalton;
X is NH or O, shows the covalently bound mode of PEG to the EPOP chemically modified;
EPOP is the reorganization epo protein, be bioactive epo protein in the no body, expressing the recombinant epo that obtains with natural EPO or eukaryotic expression system compares and lacks sugar chain partly, in experimentation on animals, do not observe the metabolically active that promoting erythrocyte generates, learn active but have the distinctive cell in vitro of EPO.
Indication does not have metabolically active or does not have biological activity in the body among the present invention, be meant by the general treatment using dosage, with present known detection method, can not detect the physiological action of tangible promoting erythrocyte growth in experimental animals, promptly EPOP does not possess the value as the anemiaing correcting drug use; Certainly, do not get rid of the possibility that the more heavy dose of or more highly sensitive detection method of employing can detect fainter activity in vivo.
In the present invention, the EPOP of no metabolically active can have multiple source, the homology peptide chain that all peptide chain structures and natural EPO are close, no matter be synthetic, still express by prokaryotic system or eukaryotic system, even the process transformation, for example in the embodiment of the invention 1,2, have 166 amino acid or 167 amino acid whose peptide chains by what prokaryotic system was expressed, do not possess metabolically active in default of sugar chain, the raw material that can modify as PEG of the present invention, thus obtain to have the product of metabolically active.
In order to obtain the epo protein of a large amount of no activity in vivo at low cost, in one embodiment of the invention, at first make up the recombinant epo gene and the engineering bacteria that can utilize the intestinal bacteria system high efficiency to express, the recombinant epo P of its expression does not have activity in vivo, based on this EPOP, use PEG then
2NHS-20K carries out chemically modified to it, and the PEG-EPOP binding substances after modified shows the metabolically active that tangible promoting erythrocyte generates in animal body.
The present invention adopts prokaryotic expression system to obtain bioactive EPOP in the no body, adopts PEG that EPOP is carried out chemically modified again, acquisition PEG-EPOP binding substances, and in experimentation on animals, observe and have activity in vivo.As make the medicine of treatment anaemia disease with this PEG-EPOP binding substances, because of prokaryotic expression system significantly reduces on production cost and scale cost, the huge advantage than eukaryotic expression system has can reduce patient's medicine for treatment expense significantly.
Description of drawings
Fig. 1 is reorganization EPOP prokaryotic expression system plasmid construction figure.
Fig. 2 PEG-EPO metabolically active test experience, use R-500 analysis-e/or determining reticulocyte percentage ratio:
Among the figure: ordinate zou is a reticulocyte percentage ratio, and X-coordinate is blood sampling time (1,2,3,4 represent blood sampling on the fourth, fifth, six, seven after injected sample respectively)
0#: negative control
The 1#:rhEPO positive control
2#: recombinant epo P, unmodified contrast
The 3#:PEG-EPOP binding substances is modified after product
Fig. 3 is that the epo protein of two seed amino acid sequences (167AA and 166AA) is expressed contrast figure;
Among the figure: 1: after password is revised, the 167AA of expression (containing 167 Arg) sample
2: after password is revised, the 166AA of expression (not containing 167 Arg) sample
Control sample before the abduction delivering of 3:1
Control sample before the abduction delivering of 4:2
Embodiment
Below in conjunction with accompanying drawing,, illustrate but do not limit the present invention by detailed description to better embodiment of the present invention.
[embodiment 1] is by the preparation of the recombinant epo P of prokaryotic expression system expression
1. bacterial strain and plasmid
Plasmid PBV
220Total length 3.66Kb, series connection phage P
LP
RPromotor, downstream are the ribosomal gene termination signal, amicillin resistance, and the preparation method sees and contains P
LP
RThe establishment and the application thereof of the protokaryon efficient expression vector of promotor, viral journal, 1990,62): 11, this plasmid is given by the author.Coli strain DH
5 αAvailable from the DH of GIBCO company
5 αType strain.
2. toolenzyme and reagent
Restriction enzyme BamH I, EcoRI, T4 ligase enzyme and Taq archaeal dna polymerase are purchased the company in Roche, plasmid purification test kit (Wizard Plus Minipreps DNA Purification System) is purchased the company in Promega, the molecular weight of albumen mark is purchased in magnificent company, and other reagent is homemade analytical reagent.
3. human erythropoietin gene is synthetic
The EPO molecule of expressing in the human body is the glycosylated protein that is made of 166 amino acid, the terminal Arg of its C-in the post-treatment modification
166The position is cut, becomes the ripe hEPO that is made up of 165 amino acid.The cDNA sequence of hEPO shown in SEQID NO.1, the aminoacid sequence of its prlmary structure of protein such as SEQ ID NO.2.Wherein the ratio of intestinal bacteria rare codon accounts for 12%.In order to allow the gene optimizing express, we are by to the redesign of gene and synthetic, eliminate rare codon and utilize the optimizing codon.Most rare codons among the hEPO are replaced to the intestinal bacteria preference codon, and adjust GC content in right amount, increase by 3 restriction enzyme sites at the two ends of target gene fragment simultaneously: EcoR I:GAATTC; Nde I:CATATG; BamH I:GGATCC.It is as shown in table 1 that password is replaced cross-reference, and encoding sequence is shown in SEQ ID NO.3 in the synthetic sequence of replacement back.Because it is initial with methionine(Met) that bacillus coli gene is expressed, the actual peptide chain that obtains is at the many methionine(Met)s of SEQ ID NO.2 sequence N end, totally 167 amino acid whose peptide chains, and the albumen primary structure does not have other change.
Table 1 hEPO gene-code is replaced the cross-reference table
4. sequence synthetic method:
Method is with reference to Jelenkovic G, Billings S, Chen Q, et al.Transformation of eggplant withsynthetic ccryIIIA gene produces a high level of resistance to the Colorado potatobeetle.J Amer Soc Hort Sci, 1998.123 (1): 19～25, adopt full gene synthetic method, at first use the design software design length to be about the oligo of 80nt, form the overlap of about 18nt between these oligo mutually, carry out then obtaining full-length gene behind many wheel pcr amplifications, electrophoresis is cloned after reclaiming, and order-checking obtains the correct clone of sequence again.
For the synthetic gene order of expressing 165 amino acid (lacking the 166th arginine) of ripe hEPO by the aminoacid sequence shown in the SEQ ID NO.2, design following two primers, from the plasmid that contains the EPO gene after password is replaced, amplify the EPO gene (165 amino acid whose gene orders of the expression shown in SEQ ID NO.4) that lacks the 166th arginine password (CGT) with the PCR method again, obtain equally as stated above and efficiently express, see Fig. 3.Equally, the peptide chain at expression in escherichia coli is that the N end increases a methionine(Met) totally 166 amino acid whose peptide chains.
Primer: 5 ' GAG GAA TTC ATA TGG CTC CGC CGC GTC TG 3 '
5’CAG?CTG?GGA?TCC?TCA?ATC?ACC?GGT?ACG?GCA?3’
5. construction of recombinant plasmid and Screening and Identification
To obtain encoding sequence (SEQ ID NO.4) with restriction enzyme BamH I and EcoR I double digestion through the correct goal gene that checks order, reclaim back and the PBV that uses restriction enzyme BamH I and the same double digestion of EcoR I behind the electrophoresis
220Plasmid connects.Connect product transformed into escherichia coli DH
5 α, coating contains the dull and stereotyped transformant that gets of LB of penbritin.The picking transformant extracts plasmid, analyzes with restriction enzyme BamH I and EcoR I double digestion, and screening contains recombinant plasmid transformed of hEPO gene fragment.
The construction of recombinant plasmid route is seen Fig. 1.
6.EPO the expression of gene in intestinal bacteria
Recon is inoculated in the 5ml LB substratum, and at 30 ℃, the 180rpm shaking table is cultivated about 14h.Change in the LB substratum with 1: 30 ratio then, cultivate (A about 4h for 30 ℃
600During ≈ 0.8).Again with 42 ℃ of inducing culture 4h.Get culture 1ml, carry out SDS-PAGE and Western blot after centrifugal and identify expressed protein.
7. substratum and the culture condition cultivated of high density fermentation:
7.1.LB seed culture medium: every liter contains peptone 10g, yeast powder 5g, and NaCl 5g, PH7.0, autoclaving adds penbritin to 100ug/ml during use.
7.2. substratum is used in fermentation: every 10L contains K
2HPO
412g, KH
2PO
43H
2O 20g, NaCl 20g, MgSO
47H
2O 10g, glucose 500g, yeast powder 480g, peptone 430g uses behind the autoclaving.
7.3. fermentation culture: the bacterial classification of preserving is drawn plate, and the single bacterium colony of picking is inoculated in the LB seed culture medium, cultivate 14～16h after, again with 1: 30 ratio enlarged culturing 12h.Be inoculated in then in the NBSMPP-40 fermentor tank and cultivate, the feed supplement in 1 hour of every interval once and is adjusted PH and oxygen solubility (DO) according to the thalli growth situation in culturing process.
8. purifying:
Tunning is according to method known to those skilled in the art separation, purifying protein, as document Asn to Lysmutations at three sites which are N-glycosylated in the mammalian protein decreasethe aggregation of Escherichia coli-derived erythropoietin, Protein Engineering, Vol.14 no.2 pp.135-140,2001, Linda O.Narhi etc. obtains the recombinant epo (EPOP) from prokaryotic expression system.
9.EPOP cell in vitro is learned determination of activity
9.1. method: measure the active comparison of rhEPO extracorporeal biology [cell and molecular immunology magazine (J Cell Mol Immunol) 2000 with reference to MTT colorimetry and ELISA method; 16 (5)] Han Lei, Han Weiyue, results thalline are centrifugal after with the broken bacterium of ultrasonic wave.Supernatant liquor directly carries out the UT7 cytoactive and detects, and carries out the UT7 cytoactive after the 7M urea dissolving of precipitation with 6 times of volumes and detects.Standard substance are sharp blood treasured (kylin roc company) 3000IU/ bottle (2ml)
9.2. result: all do not record the UT7 cytoactive in the carrier bacterium supernatant of contrast and the resolution of precipitate liquid.The broken bacterium supernatant liquor of the engineering bacteria that makes up records 8IU/ml, records 700IU/ml after 400 times of dilutions of resolution of precipitate liquid.
Therefore the recombinant epo P that obtains has the activity of keeping the growth of UT7 cell, proves that the recombinant epo P peptide chain that is obtained has the distinctive cell in vitro of EPO and learns active.
The preparation of [embodiment 2] PEG-EPOP binding substances
The present invention relates to modify EPOP with PEG2NHS-20K, the chemical modification reaction formula of being carried out is as follows:
Wherein X is NH or O, represents the covalently bound mode of PEG to the EPOP chemically modified, is generally the side chain amino (NH) of Methionin in the EPO peptide chain or the N of EPO and does not hold amino, also can be as the pendant hydroxyl group (OH) of Serine in the EPO peptide chain.
Wherein i, j are integer, represent the length of PEG carbochain;
According to the degree of modification of PEG to EPOP, n is 1～5 integer, is preferably 1;
The molecular structural formula of mPEG is CH
3 CH
2CH
2O
k
Wherein k is 100～1000 integer, is i and j sum, and making the mPEG part molecular weight of binding substances is 5000～40000 dalton, preferred 10000～20000 dalton.
Can adopt following method that the recombinant epo that obtains is carried out chemically modified:
1. exchange buffering system (ultrafiltration process):
Concentrate the recombinant human erythropoietin work in-process with the ultra-fine filter of handling through the 0.5mol/LNaOH depyrogenation in advance, and to PH8.5, the saturating filter of 50.0mmol/L phosphate buffered saline buffer 4 times, and to adjust protein concentration with same buffer be 1mg/ml.
2. the chemical modification reaction of recombinant epo
Get recombinant epo sample (1mg/ml) 20ml, add 100 milligrams of (PEG of PEG2NHS-20K
2NHS is a NEKTAR company product, and the product operation instruction is seen Nektar Molecule Engingeering CATALOG2003), 25 ℃ are slowly stirred reaction 30min down, add Gly400 milligram termination reaction.
In chemically modified, the protein concentration of recombinant epo sample can adopt 0.01～5mg/ml, preferred 0.1～1mg/ml, 1mg/ml most preferably in the present invention.The activated PEG ester be 1: 1～1: 10 or higher by the mol ratio of modified protein, as adopting PEG
2The NHS-20K weight ratio is about 2: 1～and 1: 5, preferred weight ratio is 1: 5 in the present invention.
3. separate and purifying
3.1.Superdex 200 molecular sieve chromatographies: the post size (26cm * 100cm)
3.2. clean and balance: wash chromatography column with pyrogen-free, flow velocity 8～10ml/min cleans with 5 times of column volumes, uses level pad (the 0.2mol/L NaCl 20mmol/L citrate buffer solution PH7.0) balance columns of 5 times of column volumes then.
3.3. last sample and wash-out: with erythropoietin modification reaction blend sample sample introduction, with damping fluid (0.2mol.dm-3 NaCl 20mmol.dm-3 citrate buffer solution PH7.0) wash-out, OD280nm detects behind the sample introduction.Collect first peak (PEG-EPOP binding substances) and second peak (unreacted EPOP) respectively.
Promoting erythrocyte generates one of active mensuration in [embodiment 3] PEG-EPOP binding substances animal body
1. experimental technique: with reference to " detect biologic activity in the EPO body the reticulocyte method sets up Wang Qingzhou, Cheng Ya qin Chinese biological goods are learned 1997 the 10th the 1st phases of volume of magazine ", with slightly different in the literary composition be to recommend by the author, the method for potion blood sampling on the 4th is only injected in existing employing.
1.1. recombinant epo P sample: according to the epo protein purification sample that the EXAMPLE l method is expressed preparation, contain the methionine(Met) of N end and the arginine of C end, long altogether 167 amino acid whose peptide chains, it is 44000IU/ml that UT-7 detects cytoactive;
1.2.PEG-EPOP binding substances sample: with reference to the PEG-EPOP binding substances sample after the PEG modification of embodiment 2 methods preparation, concrete grammar in this experiment is: add 50 milligrams of PEG2NHS-20K in the aforementioned recombinant epo P of the 5ML sample, modification reaction back is fully put 4 degree preservations and is used for experimentation on animals without separating and purifying.
1.3. laboratory animal: Balb/C mouse inbred lines, about 20 grams of body weight.
1.4. sample is with 50 times of dilutions of 20mM phosphate buffered saline buffer, each sample sets is with back subcutaneous injection 0.2ml, 3 every group, and eye socket venous blood collection on the 4th after the administration, smear staining, survey grid are knitted red percentage ratio.
2. result:
2.1. recombinant epo P:2.6%, 3.4%, 3.7%, average is 3.2%
2.2.PEG-EPOP:9.7%, 7.9%, 7.5%, average is 8.4%
2.3. negative control: 2.3%, 3.9%, 3.4%, average is 3.2% (history value)
In experimentation on animals, prokaryotic expression recombinant EPOP sample fails to detect the promoting erythrocyte nucleus formation, and the PEG of same amount modification back PEG-EPOP binding substances sample has tangible promoting erythrocyte nucleus formation.Illustrate that recombinant epo P sample can obtain the activity in vivo of the former erythropoietin that does not have by the PEG modification.Therefore, the explanation of this result of experiment does not have activity in vivo and has the active epo protein of cell in vitro to modify the activity in vivo that obtains erythropoietin by PEG.
Promoting erythrocyte generates two of active mensuration in [embodiment 4] PEG-EPOP binding substances animal body
1. experimental technique: reference example 3, every abdominal part hypodermic 0.2ml replaces the smear staining survey grid to knit red corpuscle percentage ratio with the R-500 analyser, and measures fourth, fifth, six, seven day the result in injection back.
1.1.0#: totally 4 of negative controls, injecting normal saline;
1.2.1#:rhEPO positive control adopts the Shanghai multiple star science and technology biological cloning rhEPO of company product to make a gift of treasured, lot number 2004010l, and the 2000IU/ bottle, the 2ml physiological saline solution is got 200ul, adds physiological saline 4.8ml dilution, and concentration is 40IU/ml, divides 4 groups every group 4.
1.3.2#: recombinant epo P, unmodified control sample.According to the epo protein purification sample that embodiment 1 method is expressed preparation, the methionine(Met) that contains the N end does not contain the arginine of C end, long altogether 166 amino acid whose peptide chains, and it is 30000IU/ml that UT-7 detects cytoactive, 100 times of dilutions, concentration 300IU/ml.Divide 4 groups every group 4.
1.4.3#:PEG-EPOP binding substances, the modification after product of aforementioned 2# sample, modifying method divide into groups with the 2# diluted sample with embodiment 3.
2. experimental result:
See Fig. 2, recombinant epo P only detects faint activity in vivo in this experiment, and measures tangible activity in vivo in the PEG-EPOP binding substances.
The foregoing description shows, the EPOP of no activity in vivo, and the product through after the inventive method modification has the activity that promoting erythrocyte generates in the good body, because with low cost, application prospect is good.
The peptide chain that the present invention relates to comprises the peptide chain with natural EPO amino-acid sequence and is the improved homology peptide chain of various purposes, as: cut Arg in the simulation post-treatment by natural expressed sequence that proposes among the present invention and expression with 166 amino acid (should be 167 amino acid if start at from first methionine(Met) MET of N end, embodiments of the invention show that the EPOP with this methionine(Met) still has the metabolically active after due in theory cell in vitro is learned activity and PEG and modified)
166Natural peptide chain, or for its plants the peptide chain that purpose changes partial amino-acid, as: reduce glycosylation site to increase peptide chain stability or the like, as long as this EPO homology peptide chain can not show or only can show faint EPO physiologically active in vivo, after modifying, PEG obtains stronger metabolically active, all within the scope of the invention.
The modification reagent that the present invention relates to is the activated PEG ester, also can adopt the peptide chain covalent coupling of other method with PEG chain and EPOP, the activating group that comprises other kind, or the corresponding site of EPOP peptide chain also activated, and the mPEG of the multiple structure of strand, two strands or multichain, equally within the scope of the invention.
Obviously, above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes or distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the defined scope of claims of the present invention.
Recombinant human erythropoietin .ST25
SEQUENCE?LISTING
＜110〉Chengdu Inst. of Biological Products
＜120〉recombinant erythropoietin that has metabolically active after polyethyleneglycol modified
<130>2905
<160>4
<170>PatentIn?version?3.2
<210>1
<211>501
<212>DNA
＜213〉Artificial (artificial sequence)
<220>
<221>CDS
<222>(1)..(501)
＜223〉coding people recombination human erythropoietin gene
<400>1
gcc?cca?cca?cgc?ctc?atc?tgt?gac?agc?cga?gtc?ctg?gag?agg?tac?ctc 48
Ala?Pro?Pro?Arg?Leu?Ile?Cys?Asp?Ser?Arg?Val?Leu?Glu?Arg?Tyr?Leu
1 5 10 15
ttg?gag?gcc?aag?gag?gcc?gag?aat?atc?acg?acg?ggc?tgt?gct?gaa?cac 96
Leu?Glu?Ala?Lys?Glu?Ala?Glu?Asn?Ile?Thr?Thr?Gly?Cys?Ala?Glu?His
20 25 30
tgc?agc?ttg?aat?gag?aat?atc?act?gtc?cca?gac?acc?aaa?gtt?aat?ttc 144
Cys?Ser?Leu?Asn?Glu?Asn?Ile?Thr?Val?Pro?Asp?Thr?Lys?Val?Asn?Phe
35 40 45
tat?gcc?tgg?aag?agg?atg?gag?gtc?ggg?cag?cag?gcc?gta?gaa?gtc?tgg 192
Tyr?Ala?Trp?Lys?Arg?Met?Glu?Val?Gly?Gln?Gln?Ala?Val?Glu?Val?Trp
50 55 60
cag?ggc?ctg?gcc?ctg?ctg?tcg?gaa?gct?gtc?ctg?cgg?ggc?cag?gcc?ctg 240
Gln?Gly?Leu?Ala?Leu?Leu?Ser?Glu?Ala?Val?Leu?Arg?Gly?Gln?Ala?Leu
65 70 75 80
ttg?gtc?aac?tct?tcc?cag?ccg?tgg?gag?ccc?ctg?cag?ctg?cat?gtg?gat 288
Leu?Val?Asn?Ser?Ser?Gln?Pro?Trp?Glu?Pro?Leu?Gln?Leu?His?Val?Asp
85 90 95
aaa?gcc?gtc?agt?ggc?ctt?cgc?agc?ctc?acc?act?ctg?ctt?cgg?gct?ctg 336
Lys?Ala?Val?Ser?Gly?Leu?Arg?Ser?Leu?Thr?Thr?Leu?Leu?Arg?Ala?Leu
100 105 110
gga?gcc?cag?aag?gaa?gcc?atc?tcc?cct?cca?gat?gcg?gcc?tca?gct?gct 384
Gly?Ala?Gln?Lys?Glu?Ala?Ile?Ser?Pro?Pro?Asp?Ala?Ala?Ser?Ala?Ala
115 120 125
cca?ctc?cga?aca?atc?act?gct?gac?act?ttc?cgc?aaa?ctc?ttc?cga?gtc 432
Pro?Leu?Arg?Thr?Ile?Thr?Ala?Asp?Thr?Phe?Arg?Lys?Leu?Phe?Arg?Val
130 135 140
tac?tcc?aat?ttc?ctc?cgg?gga?aag?ctg?aag?ctg?tac?aca?ggg?gag?gcc 480
Tyr?Ser?Asn?Phe?Leu?Arg?Gly?Lys?Leu?Lys?Leu?Tyr?Thr?Gly?Glu?Ala
145 150 155 160
tgc?agg?aca?ggg?gac?aga?tga 501
Cys?Arg?Thr?Gly?Asp?Arg
165
<210>2
<211>166
<212>PRT
＜213〉Artificial (artificial sequence)
<400>2
Recombinant human erythropoietin .ST25
Ala?Pro?Pro?Arg?Leu?Ile?Cys?Asp?Ser?Arg?Val?Leu?Glu?Arg?Tyr?Leu
1 5 10 15
Leu?Glu?Ala?Lys?Glu?Ala?Glu?Asn?Ile?Thr?Thr?Gly?Cys?Ala?Glu?His
20 25 30
Cys?Ser?Leu?Asn?Glu?Asn?Ile?Thr?Val?Pro?Asp?Thr?Lys?Val?Asn?Phe
35 40 45
Tyr?Ala?Trp?Lys?Arg?Met?Glu?Val?Gly?Gln?Gln?Ala?Val?Glu?Val?Trp
50 55 60
Gln?Gly?Leu?Ala?Leu?Leu?Ser?Glu?Ala?Val?Leu?Arg?Gly?Gln?Ala?Leu
65 70 75 80
Leu?Val?Asn?Ser?Ser?Gln?Pro?Trp?Glu?Pro?Leu?Gln?Leu?His?Val?Asp
85 90 95
Lys?Ala?Val?Ser?Gly?Leu?Arg?Ser?Leu?Thr?Thr?Leu?Leu?Arg?Ala?Leu
100 105 110
Gly?Ala?Gln?Lys?Glu?Ala?Ile?Ser?Pro?Pro?Asp?Ala?Ala?Ser?Ala?Ala
115 120 125
Pro?Leu?Arg?Thr?Ile?Thr?Ala?Asp?Thr?Phe?Arg?Lys?Leu?Phe?Arg?Val
130 135 140
Tyr?Ser?Asn?Phe?Leu?Arg?Gly?Lys?Leu?Lys?Leu?Tyr?Thr?Gly?Glu?Ala
145 150 155 160
Cys?Arg?Thr?Gly?Asp?Arg
165
<210>3
<211>501
<212>DNA
＜213〉Artificial (artificial sequence)
<220>
<221>misc_feature
<222>(1)..(501)
<400>3
gctccgccgc?gtctgatctg?tgatagccgt?gttctggaac?gttacctgct?ggaagctaaa 60
gaagctgaaa?acatcacgac?gggctgtgct?gaacactgca?gcctgaacga?aaacatcact 120
gttccggata?ccaaagttaa?cttctatgct?tggaaacgta?tggaagttgg?tcagcaggct 180
gtagaagttt?ggcagggcct?ggctctgctg?tcggaagctg?ttctgcgtgg?ccaggctctg 240
ctggttaact?ctagccagcc?gtgggaaccc?ctgcagctgc?acgtggataa?agctgttagt 300
ggcctgcgta?gcctgaccac?tctgctgcgt?gctctgggag?ctcagaaaga?agctatcagc 360
cctccggatg?cggcttcagc?tgctccgctg?cgtaccatca?ctgctgatac?tttccgtaaa 420
ctgttccgtg?tttacagcaa?cttcctgcgt?ggaaaactga?aactgtacac?cggtgaagct 480
tgccgtaccg?gtgatcgttg?a 501
Recombinant human erythropoietin .ST25
<210>4
<211>498
<212>DNA
＜213〉Artificial (artificial sequence)
<220>
<221>misc_feature
<222>(1)..(498)
<400>4
gctccgccgc?gtctgatctg?tgatagccgt?gttctggaac?gttacctgct?ggaagctaaa 60
gaagctgaaa?acatcacgac?gggctgtgct?gaacactgca?gcctgaacga?aaacatcact 120
gttccggata?ccaaagttaa?cttctatgct?tggaaacgta?tggaagttgg?tcagcaggct 180
gtagaagttt?ggcagggcct?ggctctgctg?tcggaagctg?ttctgcgtgg?ccaggctctg 240
ctggttaact?ctagccagcc?gtgggaaccc?ctgcagctgc?acgtggataa?agctgttagt 300
ggcctgcgta?gcctgaccac?tctgctgcgt?gctctgggag?ctcagaaaga?agctatcagc 360
cctccggatg?cggcttcagc?tgctccgctg?cgtaccatca?ctgctgatac?tttccgtaaa 420
ctgttccgtg?tttacagcaa?cttcctgcgt?ggaaaactga?aactgtacac?cggtgaagct 480
tgccgtaccg?gtgattga 498
Claims (5)
1. proteic polyethylene glycol conjugate of erythropoietin is characterized in that it has formula (I) structure:
In the formula (I), n is 1～5 integer, represents the degree of modification of PEG to EPOP;
MPEG part total molecular weight in the binding substances is 5000～40000 dalton;
X is NH or O, represents the covalently bound mode of PEG to the EPOP chemically modified;
The recombinant human erythropoietin albumen of the no activity in vivo of EPOP representative.
2. the proteic polyethylene glycol conjugate of the described erythropoietin of claim 1 is characterized in that described EPOP is expressed by prokaryotic expression system.
3. the proteic polyethylene glycol conjugate of the described erythropoietin of claim 2 is characterized in that described EPOP is expressed by escherichia expression system.
4. the application of the proteic polyethylene glycol conjugate of the described erythropoietin of claim 1 in preparation treatment anaemia disease medicament.
5. the application of the proteic polyethylene glycol conjugate of the described erythropoietin of claim 1 in preparation rising red corpuscle medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100520223A CN100362019C (en) | 2004-03-02 | 2005-03-01 | Recombinant erythropoietin with intracorporeal physical activity and modified by macrogol |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200410021953 | 2004-03-02 | ||
| CN200410021953.2 | 2004-03-02 | ||
| CNB2005100520223A CN100362019C (en) | 2004-03-02 | 2005-03-01 | Recombinant erythropoietin with intracorporeal physical activity and modified by macrogol |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1680449A CN1680449A (en) | 2005-10-12 |
| CN100362019C true CN100362019C (en) | 2008-01-16 |
Family
ID=35067236
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100520223A Expired - Fee Related CN100362019C (en) | 2004-03-02 | 2005-03-01 | Recombinant erythropoietin with intracorporeal physical activity and modified by macrogol |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100362019C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101455844B (en) * | 2007-12-10 | 2011-09-14 | 江苏豪森药业股份有限公司 | PEG-erythrocyte-stimulating factor and preparation method and use thereof |
| CN101456911A (en) * | 2007-12-12 | 2009-06-17 | 江苏豪森药业股份有限公司 | Erythrocyte-stimulating factor mimic peptide derivative, medical salts thereof, preparation method and use thereof |
| CN101376676B (en) * | 2008-10-09 | 2012-04-25 | 天津派格生物技术有限公司 | PEGylated erythropoietin protein long-acting preparation |
-
2005
- 2005-03-01 CN CNB2005100520223A patent/CN100362019C/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| Chemistry for peptide and protein PEGylation. M.J. Roberts et al.Advanced Drug Delivery Reviews,Vol.54 . 2002 * |
| 聚乙二醇化重组人红细胞生成素在动物体内的作用. 沈富兵等.中国生物制品学杂志，第16卷第6期. 2003 * |
| 重组人促红细胞生成素聚乙二醇修饰及纯化研究. 刘兰军等.中国生物制品学杂志，第17卷第1期. 2004 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1680449A (en) | 2005-10-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2005084711A1 (en) | A pegylated recombinant erythropoietin that has in vivo activity | |
| CN100528235C (en) | Conjugates of erythropoietin | |
| CN1346369B (en) | Method and compositions for the prevention and treatment of anamia | |
| CN100365117C (en) | Pegylated and diglycosylated erythropoietin | |
| TWI235667B (en) | Erythropoietin derivatives | |
| EP2292651A1 (en) | Methods and compositions for the prevention and treatment of anemia | |
| KR100694994B1 (en) | Human Granulocyte-Colony Stimulating Factor Isoforms | |
| US20080102492A1 (en) | Methods and compositions for the prevention and treatment of anemia | |
| SE509359C2 (en) | Use of stabilized protein or peptide conjugates for the preparation of a drug | |
| CN102153653B (en) | Fusion protein of tumor blood vessel targeted polypeptide and tissue factor and preparation method thereof | |
| US6187564B1 (en) | DNA encoding erythropoietin multimers having modified 5′ and 3′ sequences and its use to prepare EPO therapeutics | |
| CN100362019C (en) | Recombinant erythropoietin with intracorporeal physical activity and modified by macrogol | |
| KR20090046923A (en) | Modified erythropoietin | |
| CN104558198A (en) | Preparation method and application of fusion protein of GLP-1 analogue and amylin analogue | |
| KR20040020816A (en) | Glycosylated human granulocyte colony stimulating factor(g-csf) isoform | |
| CN101376676B (en) | PEGylated erythropoietin protein long-acting preparation | |
| CN112225820B (en) | Recombinant human serum albumin-collagen binding domain fusion protein of tumor specific targeting matrix and application thereof | |
| CN108218978A (en) | A kind of recombinant interleukin 18 and preparation method and application | |
| CN1718739A (en) | Preparation method of recombination human granular cell colony stimulating factor | |
| CN102260352B (en) | Targeted interleukin fusion protein as well as preparation method thereof and application thereof | |
| CN101456913B (en) | Anti-tumor fusion protein and use thereof | |
| WO2006061853A2 (en) | Novel erythropoietic compounds and a process for producing erythropoietic compounds | |
| CN101812135B (en) | Fusion protein with high-efficient platelet-increasing role and preparation method thereof | |
| CN101352573A (en) | Recombinant human granulocyte colony stimulating factor lysine defect body modified by polyethyleneglycol | |
| CN102585013B (en) | Fusion protein containing omega interferon and method for preparing same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20080116 |
|
| RR01 | Reinstatement of patent right |
Former decision: cessation of patent right due to non-payment of the annual fee Former decision publication date: 20100728 |
|
| C56 | Change in the name or address of the patentee |
Owner name: CHENGDU INSTITUTE OF BIOLOGICAL PRODUCTS CO., LTD. Free format text: FORMER NAME: CHENGDU INST. OF BIOLOGICAL PRODUCTS |
|
| CP01 | Change in the name or title of a patent holder |
Address after: 610023 Jiangqiao biological products research institute, Chengdu, Chengdu, Sichuan Patentee after: Chengdu Institute of Biological Products Co., Ltd. Address before: 610023 Jiangqiao biological products research institute, Chengdu, Chengdu, Sichuan Patentee before: Chengdu Institute of Biological Products |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20080116 Termination date: 20210301 |