CN101121753A - Human serum albumin recombination fusion protein with continuous repairing function to multifarious skin cell - Google Patents
Human serum albumin recombination fusion protein with continuous repairing function to multifarious skin cell Download PDFInfo
- Publication number
- CN101121753A CN101121753A CNA2007100575719A CN200710057571A CN101121753A CN 101121753 A CN101121753 A CN 101121753A CN A2007100575719 A CNA2007100575719 A CN A2007100575719A CN 200710057571 A CN200710057571 A CN 200710057571A CN 101121753 A CN101121753 A CN 101121753A
- Authority
- CN
- China
- Prior art keywords
- hsa
- fusion rotein
- growth factor
- yeast
- serum albumin
- 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.)
- Granted
Links
- 102000008100 Human Serum Albumin Human genes 0.000 title claims abstract description 243
- 108091006905 Human Serum Albumin Proteins 0.000 title claims abstract description 243
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 24
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 21
- 238000005215 recombination Methods 0.000 title abstract description 8
- 230000006798 recombination Effects 0.000 title abstract description 8
- 210000004927 skin cell Anatomy 0.000 title description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 92
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 92
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 32
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 25
- 201000010099 disease Diseases 0.000 claims abstract description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 8
- 239000003102 growth factor Substances 0.000 claims description 187
- 230000004927 fusion Effects 0.000 claims description 178
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 80
- 239000002773 nucleotide Substances 0.000 claims description 67
- 125000003729 nucleotide group Chemical group 0.000 claims description 67
- 210000004027 cell Anatomy 0.000 claims description 57
- 102000004169 proteins and genes Human genes 0.000 claims description 53
- 230000010261 cell growth Effects 0.000 claims description 41
- 241000235648 Pichia Species 0.000 claims description 26
- 150000001413 amino acids Chemical class 0.000 claims description 25
- 241000894006 Bacteria Species 0.000 claims description 23
- 102000003972 Fibroblast growth factor 7 Human genes 0.000 claims description 23
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 claims description 23
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 18
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 16
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 16
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 claims description 16
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 claims description 16
- 229940126864 fibroblast growth factor Drugs 0.000 claims description 16
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 claims description 14
- 102000013275 Somatomedins Human genes 0.000 claims description 14
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims description 8
- 230000003796 beauty Effects 0.000 claims description 6
- 210000003491 skin Anatomy 0.000 claims description 6
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 5
- 241000196324 Embryophyta Species 0.000 claims description 4
- 241000238631 Hexapoda Species 0.000 claims description 4
- 238000010361 transduction Methods 0.000 claims description 4
- 230000026683 transduction Effects 0.000 claims description 4
- 241000251468 Actinopterygii Species 0.000 claims description 3
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 3
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 3
- 238000001890 transfection Methods 0.000 claims description 3
- 241000699802 Cricetulus griseus Species 0.000 claims description 2
- 101000851176 Homo sapiens Pro-epidermal growth factor Proteins 0.000 claims description 2
- 241001123227 Saccharomyces pastorianus Species 0.000 claims description 2
- 241000700605 Viruses Species 0.000 claims description 2
- 241000235347 Schizosaccharomyces pombe Species 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000006870 function Effects 0.000 abstract description 15
- 239000007788 liquid Substances 0.000 abstract description 12
- 238000010353 genetic engineering Methods 0.000 abstract description 11
- 229920001184 polypeptide Polymers 0.000 abstract description 10
- 150000001875 compounds Chemical class 0.000 abstract description 8
- 238000001727 in vivo Methods 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 230000008439 repair process Effects 0.000 abstract description 6
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract description 5
- 238000000855 fermentation Methods 0.000 abstract description 5
- 230000004151 fermentation Effects 0.000 abstract description 5
- 230000012010 growth Effects 0.000 abstract description 5
- 238000012545 processing Methods 0.000 abstract description 4
- 208000014674 injury Diseases 0.000 abstract description 2
- 230000008733 trauma Effects 0.000 abstract description 2
- 101100286588 Mus musculus Igfl gene Proteins 0.000 abstract 1
- 230000002844 continuous effect Effects 0.000 abstract 1
- 230000005180 public health Effects 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 47
- 238000000034 method Methods 0.000 description 34
- 230000000694 effects Effects 0.000 description 30
- 102000009027 Albumins Human genes 0.000 description 26
- 108010088751 Albumins Proteins 0.000 description 26
- 230000014509 gene expression Effects 0.000 description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- 239000013612 plasmid Substances 0.000 description 24
- 239000000047 product Substances 0.000 description 22
- 206010052428 Wound Diseases 0.000 description 19
- 208000027418 Wounds and injury Diseases 0.000 description 19
- 230000004071 biological effect Effects 0.000 description 19
- 239000000243 solution Substances 0.000 description 18
- 230000009466 transformation Effects 0.000 description 18
- 101001060261 Homo sapiens Fibroblast growth factor 7 Proteins 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 16
- 238000003752 polymerase chain reaction Methods 0.000 description 16
- 102000057239 human FGF7 Human genes 0.000 description 15
- 108091028043 Nucleic acid sequence Proteins 0.000 description 14
- 210000004369 blood Anatomy 0.000 description 14
- 239000008280 blood Substances 0.000 description 14
- 239000000178 monomer Substances 0.000 description 14
- 238000012360 testing method Methods 0.000 description 13
- 239000013598 vector Substances 0.000 description 12
- 241000283973 Oryctolagus cuniculus Species 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 238000002347 injection Methods 0.000 description 11
- 241001465754 Metazoa Species 0.000 description 10
- 108091034117 Oligonucleotide Proteins 0.000 description 10
- 206010020718 hyperplasia Diseases 0.000 description 10
- 235000015097 nutrients Nutrition 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 108010025188 Alcohol oxidase Proteins 0.000 description 8
- 238000013461 design Methods 0.000 description 8
- 239000013604 expression vector Substances 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 101150061183 AOX1 gene Proteins 0.000 description 7
- 108090000695 Cytokines Proteins 0.000 description 7
- 102000004127 Cytokines Human genes 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 230000009471 action Effects 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000003119 immunoblot Methods 0.000 description 6
- 230000008521 reorganization Effects 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 238000003757 reverse transcription PCR Methods 0.000 description 6
- 238000003860 storage Methods 0.000 description 6
- 230000002195 synergetic effect Effects 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 5
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 5
- 102000007562 Serum Albumin Human genes 0.000 description 5
- 108010071390 Serum Albumin Proteins 0.000 description 5
- 208000025865 Ulcer Diseases 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 231100000397 ulcer Toxicity 0.000 description 5
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 4
- 238000012300 Sequence Analysis Methods 0.000 description 4
- 108010084455 Zeocin Proteins 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000002537 cosmetic Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000003248 secreting effect Effects 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 108010081589 Becaplermin Proteins 0.000 description 3
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 3
- 241000235649 Kluyveromyces Species 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- -1 glycosyl seminose Chemical compound 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 230000002608 insulinlike Effects 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 101150006240 AOX2 gene Proteins 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 241000235036 Debaryomyces hansenii Species 0.000 description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 101000846416 Homo sapiens Fibroblast growth factor 1 Proteins 0.000 description 2
- 101001052035 Homo sapiens Fibroblast growth factor 2 Proteins 0.000 description 2
- 102000048143 Insulin-Like Growth Factor II Human genes 0.000 description 2
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 description 2
- 101001055320 Myxine glutinosa Insulin-like growth factor Proteins 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 102000015336 Nerve Growth Factor Human genes 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- ULQISTXYYBZJSJ-UHFFFAOYSA-N R-12-HOA Natural products CCCCCCC(O)CCCCCCCCCCC(O)=O ULQISTXYYBZJSJ-UHFFFAOYSA-N 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 241000235346 Schizosaccharomyces Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 210000000795 conjunctiva Anatomy 0.000 description 2
- 210000004087 cornea Anatomy 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000010612 desalination reaction Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 102000035122 glycosylated proteins Human genes 0.000 description 2
- 108091005608 glycosylated proteins Proteins 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 101150026546 hsa gene Proteins 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000007794 irritation Effects 0.000 description 2
- 210000002510 keratinocyte Anatomy 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000000394 mitotic effect Effects 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 229940053128 nerve growth factor Drugs 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 230000037303 wrinkles Effects 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- BLSQLHNBWJLIBQ-OZXSUGGESA-N (2R,4S)-terconazole Chemical compound C1CN(C(C)C)CCN1C(C=C1)=CC=C1OC[C@@H]1O[C@@](CN2N=CN=C2)(C=2C(=CC(Cl)=CC=2)Cl)OC1 BLSQLHNBWJLIBQ-OZXSUGGESA-N 0.000 description 1
- FQVLRGLGWNWPSS-BXBUPLCLSA-N (4r,7s,10s,13s,16r)-16-acetamido-13-(1h-imidazol-5-ylmethyl)-10-methyl-6,9,12,15-tetraoxo-7-propan-2-yl-1,2-dithia-5,8,11,14-tetrazacycloheptadecane-4-carboxamide Chemical compound N1C(=O)[C@@H](NC(C)=O)CSSC[C@@H](C(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@@H]1CC1=CN=CN1 FQVLRGLGWNWPSS-BXBUPLCLSA-N 0.000 description 1
- PAZZVPKITDJCPV-UHFFFAOYSA-N 10HSA Natural products CCCCCCCCC(O)CCCCCCCCC(O)=O PAZZVPKITDJCPV-UHFFFAOYSA-N 0.000 description 1
- RJMZIUFNDNYWDU-UHFFFAOYSA-N 3-chloro-2-hydroxy-5-phenylbenzoic acid Chemical compound ClC1=C(O)C(C(=O)O)=CC(C=2C=CC=CC=2)=C1 RJMZIUFNDNYWDU-UHFFFAOYSA-N 0.000 description 1
- 102100030310 5,6-dihydroxyindole-2-carboxylic acid oxidase Human genes 0.000 description 1
- 101150078509 ADH2 gene Proteins 0.000 description 1
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- 102100022900 Actin, cytoplasmic 1 Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 101150021974 Adh1 gene Proteins 0.000 description 1
- 101710134784 Agnoprotein Proteins 0.000 description 1
- 102100034035 Alcohol dehydrogenase 1A Human genes 0.000 description 1
- 241000893451 Arthroderma Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
- 241001508787 Citeromyces Species 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000003712 Complement factor B Human genes 0.000 description 1
- 108090000056 Complement factor B Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 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
- 241000235035 Debaryomyces Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102100028412 Fibroblast growth factor 10 Human genes 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 206010017553 Furuncle Diseases 0.000 description 1
- 108010001498 Galectin 1 Proteins 0.000 description 1
- 102100021736 Galectin-1 Human genes 0.000 description 1
- 206010017711 Gangrene Diseases 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 description 1
- 101000917237 Homo sapiens Fibroblast growth factor 10 Proteins 0.000 description 1
- 101000579123 Homo sapiens Phosphoglycerate kinase 1 Proteins 0.000 description 1
- 102000002265 Human Growth Hormone Human genes 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- KJWZYMMLVHIVSU-IYCNHOCDSA-N PGK1 Chemical compound CCCCC[C@H](O)\C=C\[C@@H]1[C@@H](CCCCCCC(O)=O)C(=O)CC1=O KJWZYMMLVHIVSU-IYCNHOCDSA-N 0.000 description 1
- 102100028251 Phosphoglycerate kinase 1 Human genes 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 206010063669 Photoelectric conjunctivitis Diseases 0.000 description 1
- 208000004210 Pressure Ulcer Diseases 0.000 description 1
- 101710124584 Probable DNA-binding protein Proteins 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 101150085390 RPM1 gene Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 208000019155 Radiation injury Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108010046685 Rho Factor Proteins 0.000 description 1
- 241000223252 Rhodotorula Species 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 241000235003 Saccharomycopsis Species 0.000 description 1
- 101100379247 Salmo trutta apoa1 gene Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 206010040943 Skin Ulcer Diseases 0.000 description 1
- 208000028990 Skin injury Diseases 0.000 description 1
- 206010040880 Skin irritation Diseases 0.000 description 1
- 206010072170 Skin wound Diseases 0.000 description 1
- 101800004225 Somatomedin-B Proteins 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 101150006914 TRP1 gene Proteins 0.000 description 1
- 206010053615 Thermal burn Diseases 0.000 description 1
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 1
- 206010046996 Varicose vein Diseases 0.000 description 1
- 102100035140 Vitronectin Human genes 0.000 description 1
- IXKSXJFAGXLQOQ-XISFHERQSA-N WHWLQLKPGQPMY Chemical compound C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 IXKSXJFAGXLQOQ-XISFHERQSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 230000007059 acute toxicity Effects 0.000 description 1
- 231100000403 acute toxicity Toxicity 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000035578 autophosphorylation Effects 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000000837 carbohydrate group Chemical group 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 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 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000003328 fibroblastic effect Effects 0.000 description 1
- 210000000630 fibrocyte Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 229940089256 fungistat Drugs 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000002641 glycemic effect Effects 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 238000009499 grossing Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 229940116978 human epidermal growth factor Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000000976 ink Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Substances N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 230000008558 metabolic pathway by substance Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 231100000286 mucous membrane, eye irritation or corrosion testing Toxicity 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000107 myocyte Anatomy 0.000 description 1
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 1
- 230000000508 neurotrophic effect Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002417 nutraceutical Substances 0.000 description 1
- 235000021436 nutraceutical agent Nutrition 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 101710135378 pH 6 antigen Proteins 0.000 description 1
- 239000003182 parenteral nutrition solution Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000019624 protein content Nutrition 0.000 description 1
- 230000005664 protein glycosylation in endoplasmic reticulum Effects 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000003488 releasing hormone Substances 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 208000017443 reproductive system disease Diseases 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000036556 skin irritation Effects 0.000 description 1
- 231100000475 skin irritation Toxicity 0.000 description 1
- 230000036560 skin regeneration Effects 0.000 description 1
- 231100000019 skin ulcer Toxicity 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
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 208000027185 varicose disease Diseases 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Images
Abstract
The invention provides a recombinant fusion protein which can stimulate the skin to repair the relevant cells and has the continuous function; the recombinant fusion protein can be used to improve the public health, hairdressing or the treatment of trauma and disease. In particular, the invention provides a human serum albumin (HSA) and a cell gene polypeptide (EGF and FGF, KGF, HGH, HGF, PDGF or IGFL); the genetic engineering method is used to recombine to form the fusion protein. 1) the fusion protein can be singly or combined for use to stimulate the repair of the cells, particularly for the development and update of each cell in the human epidermal system; 2) the fusion protein can greatly extend the service life of the cell gene in vivo and vitro to achieve the maximum stability of the product containing the cell gene and the continuous effects; 3) the cell gene formed by the recombination can be combined for use; the compound can get the functions brought by the cell gene to the greatest extent to stimulate the growth of the cells, to continuously repair and to increase the efficiency; 4) the fermentation liquid produced with the use of the yeast fermentation can be directly used in the product manufacture with the beautifying as the purpose after the simple processing. The invention also provides a manufacturing process and method of the high or low cost by using the yeast to produce the recombinant fusion protein.
Description
Technical field
The present invention is the continuation application of Chinese patent ZL021428816 and ZL2004100428148.The present invention relates to human serum albumin fusion proteins of gene recombination and expression and preparation method thereof, and relate to the method that single fusion rotein or different fusion rotein are used in combination.Be particularly related to and utilize yeast expressed production by human serum albumin and the formed fusion rotein of the human cell growth factor (HSA/GF).Cell growth factor comprises: epithelical cell growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF) and insulin-like growth factor (IGF) etc.
Background technology
Human serum albumin (HSA) is a kind of soluble and monomeric protein, constitutes half of Tot Prot in the blood.Albumin is carried and is transmitted lipid acid, steroid and hormone molecule etc. as a kind of underlying carrier, and its stable inert nature is to keep an important factor of blood pressure.Serum albumin is that a kind of spherical nonglycosylated, molecular weight is 65 kilodaltons, has 585 amino acid whose serum protein.This albumen (albumin precursor) by the conversion processing of golgi body, is removed the guiding polypeptide, and is secreted into the extracellular again.Serum albumin has 35 halfcystines, and in blood, albumin is a monomer (referring to Brown JR, " albuminous structure, function and application " Pergamon, New York, 1977) that 17 disulfide linkage are arranged.When not having secrete polypeptide, the albumin product in yeast cell is the mispairing state, with the antigenicity (comparing with state of nature albumin in the blood plasma) that loses 90%, and forms insoluble albumin polymer, does not have albuminous biological activity.Being used for clinical albumin now all extracts from human plasma.Utilize the production of the recombinant expressed albumin of microorganism (rHSA) open in patent EP330451, EP361991 and Chinese patent application CN2004010057313.7.
Albumin is the main component in the blood, is that every liter of blood contains 40 grams at people's in-vivo content, and its life-span transformation period is 14-20 days.In sum, after therapeutic protein and albumin formed fusion rotein, fusion rotein will have great advantage made it to resist enzymolysis in the organism, and the external in vivo life-span of therapeutic protein is improved greatly, can also higher dosage use.
The use in cosmetics of people source or recombination human serum albumin has also obtained very significant practical application, and concrete description is arranged in patent CN01810411.8.
In pharmaceutical preparation was formed, albumin also was often used as the stablizer of medicine, especially biological medicine.
Utilize genetic engineering technique, human serum albumin and therapeutic protein gene fusion are expressed as fusion rotein in yeast, can increase stability and the long transformation period of therapeutic protein in serum and when storing, reach long-actingization of protein drug.Modification common techniques platform is open in Yu Zailin, rich rock China granted patent ZL02142881.6 and ZL200410042814.8 in this protein drug body, draws at this to be reference.
Summary of the invention
But the present inventor experimental results show that the fusion rotein irritation cell hyperplasia that human serum albumin and cell growth factor form by the vivo and vitro of animal and cell, and having the time longer with external stop in vivo (long half-lift and persistence) than monomeric cell growth factor, cell finds also that with experimentation on animals fusion rotein has the mole biological activity identical with the human cytokines monomer.During the combination of the fusion rotein (HSA/GF) that forms by HSA and different somatomedins when compound use, can have the reparation and the tool synergistic function that stimulate the various skin cell simultaneously, and be applicable to beauty treatment, wound and treatment of diseases, finish the present invention thus.
Cell growth factor involved in the present invention (GF) including, but not limited to, epithelical cell growth factor (EGF), fibroblast growth factor (FGF), Thr6 PDGF BB (PDGF), keratinocyte growth factor (KGF-2) and para-insulin cell growth factor (IGF).Be summarized as follows respectively with regard to its characteristic below:
1. (Epidiuen cell Growth Factor, EGF): be that EGF is the protein of the biologically active of being made up of 53 amino acid of human secretory, it extensively is distributed in the body fluid such as blood, saliva, urine epithelical cell growth factor.It has biologic activity widely, can promote the division of epidermic cell, quickens the metabolism of cell, makes newborn epidermic cell substitute aged cell (Carpenter, experimental cell research, 164:1-10,1986) rapidly.
2. keratinocyte growth factor (Keratinocyte Growth Factor, KGF, KGF-2): have multiple keratinocyte growth factor to exist in the animal body.With KGF-2 (also being FGF10) is a kind of basic protein of being made up of 208 amino acid, derive from embryo's epithelial cell, new life or grow up between the fibroblastic KGF of matter.KGF-2 has the mitotic effect of the epithelial cell of promotion.Can divide a word with a hyphen at the end of a line to the wound by attracting inoblast and reticular tissue, promote the reparation of damage epithelium, show as gentle short cell regeneration effect (Rubin JS etc., institute of NAS reports 86:802-806,1989; CN00809802.6).
3. (Platelet Derived Growth Factor, PDGF): Thr6 PDGF BB is a kind of somatomedin with neurotrophic activity to Thr6 PDGF BB.People PDGF is the dimer that is connected by disulfide linkage by A chain and B chain.Can form dimer (PDGFAA) between the A chain of PDGF.Find that in the recent period PDGFAA is to the growth decisive role of male mice reproductive system, prompting PDGFAA may be significant for the diagnosis and the treatment of some disease of reproductive system.Also can form dimer (PDGFBB) between the B chain of PDGF, reparation for increment, regeneration and the damaged tissue of epidermic cell has extremely strong effect, effectively promote various kinds of cell division and new skin regeneration, repair damaged tissue, promote the generation (Josephs etc. of collagen protein, science, 225 (4662): 636-639,1984; Li Peiwang etc., life science, 9:346-350,2005).
4. fibroblast growth factor (Fibroblast Growth Factor, FGF): mainly contain acidity (aFGF) and alkalescence (bFGF) two kinds of molecules.FGF derives from mesoderm and neuroectodermal multifunctional cytokine (Zhao etc., transplanting, 56,1177-1182,1993; Florkiewicz etc., New York science annual report, 638:109-126,1991).The mitotic division effect that tool is stronger.Vascular endothelial cell, inoblast etc. all there is short splitting action.Studies show that,, observe FGF isoreactivity polypeptide and have remarkable inducing cell reparation, quicken wound healing, reduce synulotic effect by different animal models such as mouse, rat, rabbit.BFGF can also promote the division of body melanocyte specifically.Animal experiment proof aFGF can promote the new life of ischemic tissue, but owing to the transformation period of aFGF in blood is too short, and in clinical trial, show obvious effects.
5. Insulin-Like cell growth factor (Insulin-like Growth Factors, IGFs): be with the Regular Insulin portion homologous and have the polypeptide of ILA on the class formation, existing short cytodifferentiation and proliferation activity, have ILA again, all play an important role keeping the adjusting of normal insulin sensitivity and glycaemic homeostasis.Domestic and international a lot of research shows that all the generation of IGFs and chronic complicating diseases of diabetes is closely related.The promotion of IGF-1 (somatomedin C) is grown, and the effect report of aspects such as substance metabolism is more.IGF-2 (somatomedin A; GenBank:MIM 147470), IGF-3 (somatomedin B; GenBank:MIM 193190) new discovering also arranged.
Except that the above cell growth factor of sketching, also have other somatomedin to form fusion rotein with human serum albumin, they can independent or compound use, can with albumin monomer or the independent or compound use of albumin fusion protein, and in order to cytothesis to reach the purpose of beauty treatment or wound, disease treatment.
Cell growth factor is unstable in preservation.With EGF is example, and in actual use, EGF also is extremely unstable in the skin wound environment, the protease hydrolysis inactivation that it is existed in the wound, and only 1 hour transformation period, but in the agglutination after the skin injury, the synthetic of DNA but needs 8-12 hour.Therefore, single uses EGF that healing is not had obvious promoter action on skin or corneal wound.Have only repeatedly to use repeatedly and could promote curative effect having healed.Therefore, in the practical application of medical science and cosmetic field be very limited (Manning etc., study of pharmacy, 6:903-917,1989; CN03011136).
Therefore, the present invention relates to following several aspect:
1) HSA/GF fusion rotein
The invention provides a kind of human serum albumin (HSA) and cell growth factor (GF) fusion rotein of producing with gene engineering method, this fusion rotein is the fusion rotein that is formed by the HSA of nucleotide coding and a kind of GF, i.e. HSA/GF fusion rotein.The method according to this invention, any albumin or its varient all can form a kind of fusion rotein with a kind of GF.
GF can be any albumen, but the differentiation of its irritation cell, hyperplasia or reparation.The most typical example of GF is cell growth factor (GF) application clinically, is approved for an example of the clinical treatment of wound and ulcer respectively as hEGF and hKGF.
GF of the present invention can be any a member in the following protein classification, including, but not limited to, epithelical cell growth factor (EGF), pHGF (HGF), the nerve growth factor (NGF), fibroblast growth factor (FGF), endothelial cell growth factor (ECGF) (VEGF), Insulin-Like cell growth factor (IGF), stem cell factor (SGF), keratinocyte growth factor (KGF), platelet-derived cell growth factor (PDGF), promotes growth releasing hormone (GHRF-6) etc.
GF can directly link to each other with the formation fusion rotein with the C-of HSA is terminal with N-terminal, or can increase a connection peptides (Linker) between HSA and GF, to constitute fusion rotein: HSA/L/GF or GF/L/HSA (L=connection peptides).Connection peptides length can be 2-100, and commonly used is 10-50 amino acid, preferably 14-30 amino acid.Connect small peptide and can make and bigger variation space is arranged between HSA and the GF and better snappiness or rigidity are arranged, or make GF easier because of bigger variation space with combining of acceptor.The structure of connection peptides can be (G4S) 3-4.The adding of connection peptides may make fusion rotein produce extra immunogenicity as the use of medicine the time, does not most preferably have connection peptides.
Fusion rotein can be a secretor type, and it can combine with AHS's albumin antibody of specific recognition.Fusion rotein also can combine with the antibody of the GF of specific recognition.If but polypeptide that the sero-abluminous secretion peptide of secretion peptide end user of fusion rotein or its occurring in nature exist or synthetic have the polypeptide that fusion rotein is secreted into the outer function of host cell.
Particularly, the present invention utilizes genetic engineering method to make up a nucleotide chain, this nucleotide chain encode a kind of HSA and the formed fusion rotein of people KGF-2 (HSA/hKGF-2), the nucleotide chain and the Seq ID No.1 of this fusion rotein of encoding has at least 90% sequence homology, and preferably this nucleotide chain and Seq ID No.1 have 95% sequence homology.By this nucleotide chain encoded protein matter amino acid sequence of polypeptide is Seq ID No.2.
Particularly, the present invention utilizes genetic engineering method to make up a nucleotide chain, the fusion rotein (HSA/hEGF) that a kind of HSA of this nucleotide chain codified and people EGF form, the nucleotide chain and the Seq ID No.3 of this fusion rotein of encoding has at least 90% sequence homology, and preferably this nucleotide chain and Seq ID No.3 have at least 95% sequence homology.Preferably, the aminoacid sequence of this nucleotide sequence coded protein and peptide is Seq ID No.4.
Particularly, the present invention utilizes genetic engineering method to make up a nucleotide chain, the fusion rotein (HSA/hIGF1) that a kind of HSA of this nucleotide chain codified and people IGF1 form, the nucleotide chain of this fusion rotein of encoding and Seq ID No.5 have at least 90% sequence homology.Preferred this nucleotide chain and Seq ID No.5 have at least 95% sequence homology.Preferably, the protein amino acid sequence of this nucleotide coding is Seq ID No.6.
Particularly, the present invention utilizes genetic engineering method to make up a nucleotide chain, the fusion rotein (HSA/hbFGF) that a kind of HSA of this nucleotide chain codified and people bFGF form.The nucleotide chain and the Seq ID No.7 of this fusion rotein of encoding has at least 90% sequence homology, and preferably, this nucleotide sequence and Seq ID No.7 have at least 95% sequence homology.Preferably, this nucleotide sequence coded protein amino acid sequence is Seq ID No.8.
Particularly, the present invention utilizes genetic engineering method to make up a nucleotide chain, a kind of HSA of this nucleotide chain codified and the formed fusion rotein of people aFGF (HSA/haFGF).The nucleotide chain and the Seq ID No.9 of this fusion rotein of encoding has at least 90% sequence homology, and preferably, this nucleotide sequence and Seq ID 9 have at least 95% sequence homology.Preferably, this nucleotide sequence coded protein amino acid sequence is Seq ID No.10.
Again particularly, the present invention utilizes genetic engineering method to make up a nucleotide chain, a kind of HSA of this nucleotide chain codified and the formed fusion rotein of PDGF-B (HSA/hPDGFB).The nucleotide chain and the Seq ID No.11 of this fusion rotein of encoding has at least 90% sequence homology, and preferably, this nucleotide sequence and Seq ID 11 have at least 95% sequence homology.Preferably, this nucleotide sequence coded protein amino acid sequence is Seq ID No.12.
Have the Nucleotide of sequence similarity to a certain degree to be meant with above-mentioned Nucleotide: for example, a kind of HSA/GF fusion rotein of encoding, and between similar nucleotide sequence, have the Nucleotide of at least 95% sequence homology.It comprises that per hundred Nucleotide have 5 point mutation on the nucleotide chain, and also is the nucleotide sequence of coding HSA/GF fusion rotein.In other words, the nucleotide chain of any nucleotide chain and above-mentioned coding HSA/GF fusion rotein has 95% sequence homology, that is, even if having nearly 5% Nucleotide individuality can be replaced, delete or insertion etc., all the nucleotide sequence with indication of the present invention is identical.No matter be at 5 ' or 3 ' end, or the sudden change that takes place of any site between two ends, still the sudden change that takes place with monomer or many bodies all within the scope of the invention.
In the practice, any nucleic acid molecule that refers in particular to is as long as have 90%, 95%, 96%, 97%, 98% or 99% similarity, just in protection scope of the present invention.And also can obtain as Bestfit software (Wisconsin) sequence analysis software bag by the known computer software of routine by nucleotide sequence coded HSA/GF fusion rotein.(2:482-489 (1981) seeks homologous sequence best between two sequences to Bestfit application region homology comparative approach for document: Smith and Waterman, applied mathematics progress.When using that this software or other are any to have similar dna sequence dna comparative analysis, a certain specific sequence all is considered as and sequence identical sources of the present invention as with sequence proposed by the invention sequence homology more than 95% being arranged.
The GF gene order can the method with pcr amplification obtain from total RNA of different tissue sources, if also can obtain with the method for synthetic full DNA sequence.The gene of synthetic can with Yi Anjisuanshi at its nucleotide coding, adopt the Nucleotide of expression system hobby, HSA/hEGF expressing fusion protein recombination microzyme, be deposited in China Committee for Culture Collection of Microorganisms of Microbe Inst., Chinese Academy of Sciences and obtained preserving number: CGMCC-2072, as the masterpiece of the HSA/GF fusion rotein among the present invention.
Under room temperature or higher temperature, HSA compares with the GF monomer with the fusion rotein that GF forms, and under similarity condition, has more than 2 times, preferably reaches 5 times or 10 times or more preferably reaches storage period and transformation period more than 20 times.
The invention still further relates to albumin as carrier, carry curative pharmaceutical grade protein, improve looks and treat various wounds, disease or unusual, or those are the people of purpose to strengthen hyperplasia, to repair, as wound, ulcer patient as GF.In the present invention, the HSA/GF fusion rotein can be used for vertebrates, preferably human, and pass through different approaches, including, but not limited to smear, in the spraying, oral, parenteral, abdominal cavity film, in intravenously, intra-arterial, subcutaneous, hypogloeeis, intramuscular, the intestines, in the sialisterium, nose, PEGization, lipid method, by suction, injection, vagina, the administration of intraocular method.HSA/GF also can discharge (as conduit or Stent) by locality, comprise subcutaneous, fat down, the joint is down and in the film, and can make sustained release dosage.Particularly, fusion rotein can be by smearing, spray administration.
The present invention relates to an albumin molecule or its mutation or fragment and a GF molecule or its mutation or fragment are merged by genetically engineered, formed fusion rotein has prolonged the transformation period without the GF that merges.For simplicity, the present invention only mentions human serum albumin (HSA) and cell growth factor (GF), i.e. the fusion rotein of formation such as people KGF, people EGF, people IGF, people PDGF, people VEGF and people SGF.Certainly the fusion rotein of other vertebrate albumin and GF formation all can be included.Preferably, at GF or its mutation or segmental N-terminal portions, behind HSA or its mutation or segmental C-terminal portions formation fusion rotein, all negative effects reach and minimize when fusion rotein and receptors bind.Have the fusion rotein that forms by GF of signal conduction, receptors bind effect equally, perhaps contain a GF or one variant or segmental fusion rotein and include in the present invention.
HSA/GF fusion rotein among the present invention or its prescription all can pass through traditional method, comprise non-mode administration through gi tract (as subcutaneous or muscle) injection or venoclysis.Treatment is included in and uses single dose or compound dosage in for some time.Fusion rotein HSA/GF among the present invention can use separately, and preferably, can become the form of pharmaceutical preparation to use with one or more acceptable carriers or another kind of HSA/GF or HSA or multiple HSA/GF.Particularly another kind of HSA/GF of described carrier or multiple HSA/GF must be compatible and receptor autophosphorylation do not produced the carrier of harmful effect." acceptable " typical carrier is water and salt solution, and it should be that aseptic, pyrogen-free does not have additionally immunogenic yet.Between the different HSA/GF,, also can consider common use when having consistency, synergetic or only rising and assist preparation to do the time spent.
The HSA/GF fusion rotein can dosage form or the easy way administration of dosage unit, also can be known and the method preparation of approval via any pharmaceutical field, and this method comprises the step that HSA/GF and the carrier with one or more auxiliary compositions are combined.Briefly say, with activeconstituents and liquid phase carrier or combination solid phase carrier, auxiliary agent or other reagent equably in conjunction with and the preparation preparation, then as required, with formed product.
Be suitable for the aseptic parenteral solution that the non-HSA/GF preparation that uses through the gi tract approach comprises water or do not contain water, can contain antioxidant, damping fluid, fungistat and solubility promoter or increase drug permeability and poiser.Moisture or water-free sterile suspensions comprises suspension agent and thickening material.Said preparation can be stored in the container of dosage unit or polynary dosage, as in the ampoule of sealing and in the tubule, stores under dried (lyophilization) condition of ice.Add aseptic liquid carrier before using,, make provisional injection liquid and suspension as water for injection.
After sending into the HSA/GF fusion rotein in the animal body, compare with the GF monomer, the transformation period of HSA/GF in blood has and is about 2 times, preferably is about 4 times, more preferably for being about 6 times, more preferably is about 10 times transformation period again.HSA/GF fusion rotein of the present invention can use jointly with the human serum albumin of or recombinant type isolating by nature.Preferably form dosage and ratio with curative effect with the human serum albumin of reorganization.
After can believing fusion, therapeutic protein has the higher transformation period and in use stops the long time at site of action, storage requirement and requirements on transport reduce when making product, can reduce cost, simultaneously than the long half-lift also can reduce using dosage and the access times of GF.
Identical strategy also can be used for researching and developing the stable product that contains fusion rotein with beautification function effect.The for example beauty treatment of skin, preserve moisture, smoothing wrinkle, wrinkle resistant, whiten.Can contain a kind of fusion rotein or two or more fusion rotein in the product combination, or other component (as HSA) also can there be ropiness increasing agent, wetting Agent for Printing Inks, emulsifying agent, sanitas etc. as host.
2) be used for the host system of HSA/GF expressing fusion protein
HSA/GF Nucleotide among the present invention can utilize the recombinant clone technology to introduce host cell, so that fusion rotein is expressed.In general, host cell changes in the host system with the mode of infection, virus infection forms such as " phages " through the various HSA/GF vector plasmids that may make up that genetic engineering (transduction or conversion or transfection) method will carry among the present invention to be mentioned.The engineering host cell can be cultivated in containing conventional nutraceutical substratum, and is beneficial to promotor through suitably revising.Select the culture condition of the nucleotide chain of transformant or amplification coding HSA/GF with the control of suitable operating method, as temperature, pH and select express cell.
By of the present invention, recombinant vectors carries the Nucleotide that comprises coding HSA/GF fusion rotein.Recombinant vectors can be an expression vector, can come expressed fusion protein by the nucleotide coding that carries in host cell.Form can be, but is not limited to, HSA/CPSA, GF/HSA, HSA/L/GF or GF/L/HSA (L=connection peptides).Host organisms and somatocyte include, but are not limited to, vertebrates (as people, monkey, mouse, rabbit etc.) fish, chicken, insect, plant, yeast, fungi and bacterium etc.
The Nucleotide of coding HSA/GF is to express the HSA/GF fusion rotein under suitable promotor effect.Available suitable promotor includes, but are not limited to, and adenovirus promoter is as the late promoter of adenovirus major; Or allogeneic promoter, as CMV promotor and RSV promotor; Inducible promoter can have MMT promotor, thermal stimulus promotor, albumin promoter, ApoAI promotor and human immunoglobulin promotor; Virus Thymine deoxyriboside enzyme promotor then has simplexvirus thymus gland kinase promoter; Retrovirus LTR promotor comprises the LTR promotor after modified; The beta-actin promotor; The human growth hormone promotor.Also available natural promoter is controlled nucleotide coding and is expressed the HSA/GF fusion rotein.
According to the present invention, reconstitution cell has the ability of expressing coding HSA/GF fusion rotein nucleotide sequence.The recombined engineering cell can be constantly or is being had or do not having under the existence of inductor and express HSA/GF, HSA/L/GF or GF/L/HSA.The recombined engineering cells form includes, but are not limited to cells such as vertebrates (being people, ox, pig, monkey, mouse, rabbit, fish, chicken etc.) insect, plant, yeast, fungi and bacterium.
The yeast belong host who preferably is used to express HSA/GF comprises, but be not limited to, yeast saccharomyces cerevisiae belongs to (Saccharomyces), pichia yeast belongs to (Phichia), genus kluyveromyces (Kluyveromyces), Candida (Candida), saccharomyces hansenii (Hancenula), spore garden yeast belong (Tarulaspora) and Schizosaccharomyces (Schizosaromyces) etc.More preferably, host system can be pichia yeast and belongs to the pasteur bacterial classification.Transfer vector plasmid can be pPICZ-A, B or C specifically.
According to selecting for use different hosts to express the HSA/GF fusion rotein.The expressed proteins polypeptide can be glycosylation or nonglycosylated among the present invention.Preferably, when obtaining expressing in host system, HSA/GF obtains expressed proteins in vertebrate cells such as Chinese hamster (CHO) be glycosylated protein.In being expressed in the pichia yeast bacterium, then be the non-glycosylated or glycosylated albumen of part.
As mentioned above, the albumin fusion protein of mentioning in the present invention preferably uses genetic engineering technique to make up and expresses.The preferred method that obtains fusion rotein is to utilize vector plasmid, comes expressed fusion protein by the mode of conversion and transfection or infection.But particularly utilize the expression vector of transformed yeast, transform pichia yeast, and fusion rotein is secreted in the nutrient solution.
Utilize the advantage of yeast expressed HSA/GF to be, the yeast system can produce the sophisticated fusion rotein of high quality, and can be secreted in the nutrient solution and be convenient to purifying.
The progress of yeast genetic engineering makes foreign gene can obtain expressing the justacrine protein product to the extracellular in yeast.Utilize the advantage of yeast expressed secreted protein to be, but be not limited to, high expression level output, protein are folding solubility, correct and are easy to scale operation and purifying.
Via albumin natural signals peptide, the HSA/GF fusion rotein can be secreted in the yeast nutrient solution.The polypeptide of HSA/GF fusion rotein can be dominated and be obtained processing by Secretory Pathway by signal peptide.Albuminous leader peptide sequences can be used in the yeast, and fusion rotein is secreted into outside the yeast.Other secretes peptide, and the α-factor secreting signal peptide as natural yeast saccharomyces cerevisiae also can be used for secreting the fusion rotein among the present invention.
Preferred embodiment is united for application saccharomyces pastorianus fungus strain and is expressed the HSA/GF fusion rotein of mentioning among the present invention.It is better than utilizing other expression system.The pichia yeast bacterium has the advantage that many higher eucaryotic cells expression systems are had, for example proteinic processing, folding, the modification after transcribing and as the large scale culturing that is easy to of culturing bacterium or yeast saccharomyces cerevisiae, with other system such as baculovirus, vertebrate cells cultivate compare have more simple and direct, express fast, output is higher.It is to have the doubly high expression level of 10-100 that pichia yeast also has the another one advantage.These characteristics make pichia yeast become very strong protein expression system.
Compare with S. cervisiae, the pichia yeast bacterium is carrying out that to excretory protein bigger advantage is arranged on the glycosylated degree.That is: pichia yeast does not have the phenomenon of excessive glycosylation.Yeast saccharomyces cerevisiae all has N-to be connected the modification of glycosyl seminose with pichia yeast.Yet few saccharides chain is added on the expressed proteins, has only 8-14 glycosyl in the pichia yeast bacterium, far is shorter than the 50-150 sweet dew sugar chain in yeast saccharomyces cerevisiae.The less glycosylated that connects in the pichia yeast bacterium takes place.The core glycosylation of yeast saccharomyces cerevisiae has the terminal thing that α-1,3 connects, and does not then have in the pichia yeast bacterium.Studies show that the glycosylated protein that is produced by yeast saccharomyces cerevisiae has stronger antigen-reactive, and make these albumen be unsuitable for being used in the use on the therapeutic purpose particularly.Certainly this also shows to have reduced with the pichia yeast bacterium and comes the worry of expressing protein in glycosylation.
Utilize pichia yeast to have many test kits to use as expression system.Easily select EasySelect as Invitrogen
TMPichia yeast is expressed the examination box.There is an AOX1 promotor can make foreign gene in pichia yeast, utilize methyl alcohol to come abduction delivering on the expression vector.Also has microbiotic Zeocin resistant gene, the mark of recon alternatively simultaneously.In the present invention, the expressed fusion protein promotor is an important factors.
In the pichia yeast system, the AOX1 gene promoter is very powerful promotor.Particularly in the pichia yeast bacterium, the two kinds of alcohol oxidases of in pichia yeast, encoding altogether, AOX1 and AOX2.AOX1 has the activity that produces synthetic a large amount of alcohol oxidases (AOX1) in cell.The AOX1 expression of gene is subjected to closely control and is reached high level by methanol induction.Typically when using methyl alcohol as sole carbon source, the product of AOX1 gene just reaches 30% in all soluble proteinss.The AOX1 gene has separated and has obtained.The AOX1 gene promoter also is used for expression (Ellis etc., 1985 of vector plasmid of the present invention in order to the foreign protein of driving coding goal gene; Koutz etc., 1989, Tschopp etc., 1987a).And AOX2 and AOX1 gene have 97% homology, and its speed of growth in methyl alcohol is slower than AOX1 gene.This growth conditions slowly can be separated to Mut
+(AOX1) strain.Except the AOX1 gene promoter, other promotor in the yeast also can be used for driving the HSA/GF fusion rotein.These promotors include, but are not limited to PGK1, GAPDH, Gal1, Cal10, Cyc1, PH05, TRP1, ADH1 or ADH2 gene promoter.
Expression plasmid also can be used for host bacterium simultaneously, in colibacillus DH5 α (Gibcol/BRL) and yeast host.Microbiotic Zeocin, histidine defect type substratum are applied in the various embodiments of the present invention as selective marker.
Expression vector contains the Nucleotide of coding HSA or HSA/GF fusion rotein, presses the described method transformed yeast of Invitrogen test kit bacterium.Select the yeast bacterium colony of conversion through resistance.These cells can be expressed the HSA/GF fusion rotein, in the time of in being inoculated in suitable nutrient solution, analyze the ability of these yeast expressed secretion fusion roteins in substratum.Proteinic results can be carried out in the cell cultured continuously.Or cultivate in batches finish after results in the lump, fusion rotein of the present invention through cultivate yeast expressed after, come in addition separation and purification with the albumen method of purification that can keep protein-active and drug activity.
What this also need mention be, other expression system also can be used for HSA/GF Expression of Fusion Protein of the present invention, comprise, but be not limited to, bacterium, Bacillus subtilus (B.Subtitis), yeast saccharomyces cerevisiae, kluyveromyces spp, saccharomyces hansenii, Candida, the spore torulopsis, Schizosaccharomyces, Citeromycesbaodingensis belongs to (Citeromyces), Pachysoler, Debaryomyces (Debaromyces), the strange yeast belong of plum (Metschumikowia), red teliosporeae (Rhodosporidium), colourless basidiospore belongs to (Leucosporiduum), Portugal's shape arthroderma (Botryoascus), lock is thrown yeast belong (Sporidiobolcus), Endomycopsis (Endomyucopsis), animal, plant and insect cell etc.
3) the HSA/GF fusion rotein is used separately and applied in any combination
Outside any albumen among the present invention can be used separately, also provide a kind of applied in any combination of different HSA/GF fusion roteins.
Hyperplasia, differentiation and maturation or have a certain certain kinds cell is had synergistic function when the combination of the fusion rotein of this uniqueness can provide the various kinds of cell of user soma.Especially, the different fusion roteins of HSA among the present invention and different cell growth factors formation, the result that common combination is used is hyperplasia and the maturation that stimulates various kinds of cell simultaneously.The directive action of using HSA/GF is in the signal transduction pathway of dissimilar cells or the generation approach of various kinds of cell, can only need once be used in the user on one's body after, make such as epidermic cell, keratinocyte and myocyte, fibrocyte etc. and all significantly repaired and hyperplasia.
In the present invention, the treatment function of albuminous blood plasma propagation function and GF obtains integrating because of albumen takes place to merge.When circulation time in blood, owing to merging the super stable of giving GF opposing proteasome degradation with albumin, the result has prolonged the transformation period of GF in blood greatly.Because a large amount of albuminous existence, different GF and albumin form fusion rotein, through making up the mutual interference phenomenon that can reduce and form on the less biological function.This interference phenomenon often appears at the combination between " GF monomer ".Furthermore, a GF who merges with albumin can stop the quite a long time and slowly discharged in blood system, thereby has lowered under ordinary method, when using heavy dose of single GF separately, and the acute reaction that brings, toxicity and side effect.The slow release action form of this fusion rotein, with the using dosage among the present invention, frequency injection reduces, and more selects a step to reduce the side effect of GF injection.
According to the present invention, after serum albumin and this type of GF form fusion rotein, can in entering body, reduce above-mentioned limitation because of slowly discharging in the back.In addition, such fusion rotein can make up with relative a large amount of albumin, can further alleviate central nervous system direct injection GF is entered in the body, caused strong foreseeable reaction.
Known, the cytokine of storing " exposing " is a rather unstable, and in storage the life-span extremely short.Be fully aware ofly, the stability of the fragility like this that cytokine has external be quite inconvenience, be very expensive and inconvenient to the user.In fact albumin also Chang Zuowei additive and cytokine together, it is stable to guarantee to contain the product that the product of cytokine has in long shelf storage time and the transportation preservation, reaches the useful effect intensity of cytokine with this.
The invention enables HSA/GF fusion rotein transformation period in blood to prolong, stability increases.Compound HSA/GFs is HSA and hEGF, hKGF, hIGF and the hPDGF reparation and the differentiation of various kinds of cell in the skin irritation jointly.
Particularly, the HSA/hEGF fusion rotein can form compound acting on trauma patient with the HSA/hKGF fusion rotein, and it is differentiation hyperplasia such as patient's epidermic cell and keratinocyte as a result, and the result repairs wound fast.
Alternative, according to user's needs, a kind of HSA/GF can with another kind of or multiple HSA/GF with simultaneously or mode administration successively.This dosage that is used in combination is to reach treatment or obviously have cosmetic purpose dosage, and effect is the effective dose with synergistic function.
The invention provides the description and the practice of complete use HSA/GF combined therapy, complete combination comprises first HSA/GF and second HSA/GF identical or different with first GF.For example, GF can be KGF among first HSA/GF, and second GF is EGF; Or first GF is KGF-2, and second GF is PDGF; Or first GF is IGF-1, and second GF can be FGF etc.The HSA monomer also can be used as first or second protein ingredient constitutes preparation, is used for combination preparation.
HSF/GF fusion rotein among the present invention separately or their combination can be used for treating various diseases, including, but not limited to, burn, scald, (the shallow II degree of burning, dark II degree, granulation wound), the fresh surface of a wound (comprises various knife wounds, wound, wound, the beauty and shaping surface of a wound), intractable ulcer due to diabetes or the varix etc. and gangrene, ulcer wound surface (comprising oral cavity and various skin ulcers etc.), sore carbuncle class disease is (as bedsore, acne, furuncle etc.), the cornea wound, ulcer, electric ophthalmia, the skin donor site surface of a wound, the radiation injury surface of a wound and histoorgan transplanting etc.Preferably, fusion rotein should not contain the protein sequence that can cause the human immunity reaction.
The present invention also provides needing a kind of using method of GF patient, and this method is with HSA and GF gene fusion and form fusion rotein and combine effective compound formulation by certain proportioning.This pharmaceutical formulations can use the vehicle accepted on the pharmaceutics and formulation to reach the stability that increases HSA/GF.Certainly medically receptible formulation also comprises HSA and or another or the multiple HSA/GF fusion rotein of natural and reorganization.
4) in addition, the present invention also provides and has utilized yeast more effectively to reach the HSA/GF albumen that the fusion of recombinating is manufactured on cost saving ground.The present invention also provides the method for fermentation liquor treatment, can be used for the separation, purifying of fusion rotein or the fermented liquid that simple program is handled is directly used in the making and the application of beauty and skin care, makeup.
Description of drawings
The synoptic diagram of accompanying drawing 1. synthetic hEGF and hIGF-1 gene.
Accompanying drawing 2. contains the yeast expressed plasmid DNA of HSA gene, and to form the antigen-4 fusion protein gene expression vector plasmid that has connection peptides (Linker) between this plasmid (pZYHSA-L) structure HSA and the cell growth factor.
Accompanying drawing 3.A) the pichia yeast bacterium (b) that inserts of protein standard molecular weight (a), plasmid-free, yeast expressed HSA/hEGF fusion rotein (c) and the hEGF albumen (d) of escherichia coli expression; B) yeast expressed HSA/hKGF-2 fusion rotein (e), HSA/haFGF fusion rotein (f) and HSA (g); C) the protein immunoblotting experiment of being done with mouse-anti human serum albumin monoclonal antibody (Sigma company).Yeast expressed HSA/hEGF (73kd) is fusion rotein (h); Yeast expressed human serum albumin (65kd) (i).
Accompanying drawing 4. contains the mean value of the fermented liquid of the different HSA/PDGF-B that measure to 3 results of the biological activity determination of BalBC3T3 cell, shows that HSA/PDGF-B has the function of stimulate cell growth.
Accompanying drawing 5. biological activity determination results are presented at 37 ℃ (A) or 50 ℃ (B) these fusion roteins survival time under differing temps down.Series 1 is the hEGF monomer; Series 2 is the HSA/hEGF fusion rotein; Series 3 is the HSA/hKGF-2 fusion rotein; Series 4 is the combination (each 5000IU) of HSA/hIGF and HSA/hEGF.All samples all are 10000IU.HSA/hEGF, HSA/hIGF-1 fusion rotein stimulate the biological activity determination of BalB/C3T3 hyperplasia.Measure hyperplasia quantity and synergistic function when containing GF monomer or fusion rotein or applied in any combination in the nutrient solution.
Injection experimental observation HSA/GF fusion rotein transformation period in blood in the accompanying drawing 6. animal body endosomes.
Embodiment
Embodiment 1: the molecule clone technology summary
Conventional molecule clone technology comprises the extraction of DNA, RNA, sepharose and polyacrylamide gel electrophoresis, the connection of dna fragmentation, digestion with restriction enzyme reacts equal reference literature (Maniatis, et al., " molecular cloning laboratory manual " cold spring harbor laboratory publishes, the cold spring port, New York, 1982).The purifying of plasmid DNA, the recovery of dna fragmentation etc. all adopt the preparation of commodity purification column.Enzyme that archaeal dna polymerase chain reaction (PCR) (reference literature Saikiet al., science, 230:1350,1985) is used and the PCR instrument that reacts required are Perkin Elmer product.And with reference to producer's schedule of operation.The oligonucleotide primer of dna sequencing and the required usefulness of DNA cloning is finished by functional body.Changeing buied by GIBCO/BRL company by the attitude intestinal bacteria.Yeast bacterial classification used in the present invention is all available from Invitrogen company.The transformed yeast bacterium is to be undertaken by the electricimpulse mode, and with reference to the schedule of operation of producer (Bio-Rad).
Embodiment 2: the structure of human serum albumin (HSA) genetic expression and vector plasmid
Use HSA cloning process and the test-results of contriver described in its patent of invention ZL02142881.6, ZL200410042814.8 and CN200410057313.7.The HSA sequence of GenBank retrieval numbering AY728024 is used for the present invention's HSA gene order and cell growth factor gene order producer recombination fusion protein.Use AY728024 sequence encoding HSA, can make fusion rotein obtain beyond thought the efficiently expressing in yeast.Expression plasmid (seeing Chinese patent ZL021428816 accompanying drawing 2) also is used for embodiments of the invention.
Embodiment 3: the gene Fusion of people KGF-2, EGF, IGF1, PDGF-B and bFGF and the structure of expression plasmid
The RT-PCR method that human keratinocyte Growth Factor (hKGF2), platelet-derived cell growth factor-B (hPDGF), Prostatropin (hbFGF) applications similar are explained in embodiment 1 increases from total RNA prepared product in difference source.The molecular cloning of epithelical cell growth factor (hEGF) and Insulin-Like cell growth factor-1 (hIGF-1) adopts dna sequence dna (PCR method) complete synthesizing process.The PCR product is all cloned in the pCRII vector plasmid, and through the dna sequencing analysis.
Utilize the RT-PCR technology, from the total RNA of the human embryonic fibroblast of vitro culture, amplify the gene order of human keratinocyte Growth Factor (hKGF-2), and it is cloned in the PCR product vector plasmid.The synthetic oligonucleotide primer of PCR is respectively:
Seq.ID NO.13:5 '-GCCTTAGGCTTA
GCCCTTGGTCAGGACATGG-3 ' and
Seq.ID?NO.14：5’-CTCGAG
TCATGAGTGGACCACCATTGG-3’
Primer design is to design according to BenBank retrieval numbering NM_004465.The PCR product includes only the maturation protein peptide coding zone of hKGF-2.And 5 ' end introduce HSA 3 ' end sequence comprises the Bsu36I restriction endonuclease sites.3 ' end at fusion rotein is introduced the XhoI site.Be convenient to the hKGF-2 gene is inserted the pZY-HSA carrier, form the structure of HSA/hKGF-2 fusion gene at Yeast expression carrier.The dna sequence dna of HSA and hKGF-2 gene fusion (Seq ID NO.1) is listed in sequence table A, and fusion rotein aminoacid sequence (Seq ID NO.2) is listed in sequence table B.
The hEGF full length sequence is to design according to GenBank retrieval numbering X04571, and adopts yeast hobby amino acid coding base, by the synthetic pcr amplification.The dna sequence dna of hEGF oligonucleotide primer is respectively:
Seq.ID?NO.15：5’-G
CCTTAGGCTTAaacagcgactctgaatgtcc-3’，
Seq.ID?NO.16：
5’-GGCAGTAACCGTCGTGGGACAGCGGACATTCAGAGTCGCTGTT-3’
Seq.ID?NO.17：
5’-CCCACGACGGTTACTGCCTGCACGACGGTGTTTGCATGTACATCGAAGCTC-3’
Seq.ID?NO.18：
5’-GCCAACAACACAGTTGCATGCATACTTGTCCAGAGCTTCGATGTACATGC-3’
Seq.ID?NO.19：
5 '-GCATGCAACTGTGTTGTTGGCTACATCGGTGAACGTTGTCAGTACCGTGACC-3 ' and
Seq.ID?NO.20：
5’-A
GCGGCCGCTCAACGCAGTTCCCACCATTTCAGGTCACGGTACTGACAACG-3’
DNA synthetic route and method are seen shown in Figure 2.The product of PCR is gone in the pCRII vector plasmid through the amplification rear clone, through dna sequence analysis, proves that it is identical with bibliographical information.Restriction endonuclease Bsu36I and NotI recognition site are added in the two ends (underscore) of hEGF gene respectively.The synthetic gene order is inserted in the pZY-HSA carrier.The dna sequence dna of HSA/hEGF fusion gene (Seq ID NO.3) is listed in sequence table C, and fusion rotein aminoacid sequence (Seq ID NO.4) is listed in sequence table D.This dna nucleotide sequence specially is deposited in the culture presevation council of Chinese Institute of Micro-biology with the reorganization pichia yeast bacterium that can express this fusion rotein, obtains preserving number: 2072, and as a representative of the HSA/GF fusion rotein among the present invention.
HbFGF full length gene sequence is from the total RNA of human fibroblasts, gets with the method amplification of RT-PCR, clonal expansion.The required primed DNA sequence of PCR is according to the GenBank retrieve encoded: S81809) design.The oligonucleotide primer sequence is:
Seq.ID NO.21:5 '
GCCTTAGGCTTACtgggggacc gcgggcgcgg-3 ' and
Seq.ID?NO.22：5’-AGCGGCCGCTCA
GTGAGGGTCGCTCTTCTCCC-3’
Bsu36I and NotI recognition site people are the two ends that are added in clone's hbFGF gene, and the hbFGF gene DNA sequence through increasing is inserted in the pZY-HSA expression vector.Fusion rotein/hbFGF nucleotide sequence (Seq ID NO.5) is listed in sequence table E, and fusion rotein aminoacid sequence (Seq ID NO.6) is listed in sequence table F.
The hIGF-1 full length gene is in Fig. 1 mode, and is manually complete synthesis.The PCR oligonucleotide primer sequence of design is based on the GenBank retrieve encoded: NM_000618 designs:
Seq.ID?NO.23：
5’-ACCTTAGGCTTAggaccggagacgctctgcggggctgagctggtggatgctcttc-3’；
Seq.ID?NO.24：
5’-cttgttgaaataaaagcccctgtctccacacacgaactgaagagcatccaccagc-3’；
Seq.ID?NO.25：
5’-ggcttttatttcaacaagcccacagggtatggctccagcagtcggagggcgcctcag-3’；
Seq.ID NO.26：
5’-acagctccggaagcagcactcatccacgatgcctgtctgaggcgccctccgactgc-3’；
Seq.ID?NO.27：
5’-gctgcttccggagctgtgatctaaggaggctggagatgtattgcgcacccctca-3’；
Seq.ID?NO.28：
5’-
AGCGGCCGCtcaagctgacttggcaggcttgaggggtgcgcaataca-3’
The RT-PCR product is cloned the carrier in pCRII after agarose gel electrophoresis is identified, and inserts the Bsu36I and the NotI site of the yeast expressed carrier of pZY-HSA after dna sequence analysis is identified.Obtain the pZY-HSA/hIGF-1 plasmid.The dna nucleotide sequence of fusion gene (Seq ID NO.7) is listed in sequence table G, and fusion rotein aminoacid sequence (Seq ID NO.8) is listed in sequence table H.
HaFGF full length gene sequence is from the total RNA of human brain, gets with the method amplification of RT-PCR, clonal expansion.The required primed DNA sequence of PCR is according to the GenBank retrieve encoded: S67291 designs.The oligonucleotide primer sequence is:
Seq ID No.29:5 '-GCCTTAGGCTTAgctgaaggggaaatcacc-3 ' and
Seq?ID?No.30：5’-AGCGGCCGCTCAGAAGAGACTGGCAGG-3’
The RT-PCR product is cloned the carrier in pCRII after agarose gel electrophoresis is identified, and inserts the Bsu36I and the NotI site of the yeast expressed carrier of pZY-HSA after dna sequence analysis is identified.Obtain the pZY-HSA/haFGF plasmid.The dna nucleotide sequence of fusion gene (Seq ID NO.9) is listed in sequence table I, and fusion rotein aminoacid sequence (Seq ID NO.10) is listed in sequence table J.
HPDGF-B full length gene sequence is from the DNA plasmid that contains this full length gene that the contriver has, and gets with the method amplification of PCR, clonal expansion.The required primed DNA sequence of PCR is according to the GenBank retrieve encoded: AH002986 designs.The oligonucleotide primer sequence is:
Seq ID No.31:5 '-GCCTTAGGCTTAaatcgctgctgggcgctc-3 ' and
Seq?ID?No.32：5’-AGCGGCCGCTAGGCTCCAAGGGTCTCC-3’
The PCR product is cloned the carrier in pCRII after agarose gel electrophoresis is identified, and inserts the Bsu36I and the NotI site of the yeast expressed carrier of pZY-HSA after dna sequence analysis is identified.Obtain the pZY-HSA/hPDGF-B plasmid.The dna nucleotide sequence of fusion gene (Seq ID NO.11) is listed in sequence table K, and fusion rotein aminoacid sequence (Seq ID NO.12) is listed in sequence table L.
Contain a restriction enzyme Bsu36I recognition site at the C-of HSA end, cell growth factor: hKGF-2, hEGF, hbFGF, hIGF-1, aFGF and hPDGF-B etc. all can link to each other connection peptides sequence: 3 (GGGGS) by the genetically engineered mode with HSA C-terminal protein matter sequence, and form pZYHSA-L expression vector plasmid.Way is that the BamHI site that at first will exist on the pZY-HSA vector plasmid utilizes the BamHI enzyme to cut, and enzyme is treated to blunt end then, connects the back transduction bacterium again, reaches the disappearance in BamHI site.Special oligonucleotide sequence with synthetic:
Seq ID No.33:5 '-ttaggcttaggaggaggaggatcaggaggaggaggatcaggaggaggaggatccgc-3 ' and
Seq?ID?No.34：5’-ggccgcggatcctcctcctcctgatcctcctcctcctcctgatcctcctcctcctcctaagcc-3’。
Moles such as Seq ID No.33 and 34 two oligonucleotide chains is mixed, and heating slowly cools to room temperature then.Recombinate under the oligonucleotide that contains connection peptides and the effect of pZYHSA carrier linear plasmid at ligase enzyme of cutting through Bsu36I and NotI enzyme.So just made up pZYHSA-L expression vector plasmid (accompanying drawing 1).To various hyperplasia stimulating factor genes, modify 5 ' of various cell growth factor genes respectively with PCR method and terminally be the BamHI restriction enzyme site.Directly be cloned into through the Bsu36I/BamHI double digestion again and get in the pYZ-HSA vector plasmid.The sequence of new each rho factor that inserts is connected with the sequence order of the HSA C-terminal protein matter of encoding, and all genes all can enter same reading frame and form intergenic fusion and form new HSA/L/GF dna molecular sequence like this.It can express the HSA fusion rotein of band connection peptides, and has the biological function of cell growth factor.
With pichia yeast bacteria strain GS115 or X33 colony inoculation in the 50ml substratum that contains 5ml YPD nutrient solution, with 250 rev/mins speed in 30 ℃ of following overnight incubation.Get the 0.2ml overnight culture next day and transfer again in the nutrient solution of 500ml YPD, place 2 liters triangle culturing bottle.At 30 ℃ of following rotating and culturing 2-3 hours, make cell density reach OD600=1.3-1.5.Yeast is collected through centrifugal method, is resuspended in washed twice in the sterilized water of 500ml ice precooling again.Then yeast be suspended from the precooling of 20ml ice 1M Sortbitol solution washing once.
Plasmid pZY-HSA/hKGF-2, pZY-HSA/hEGF, pZY-HSA/hIGF, pZY-HSA/hbGF, pZY-HSA/aFGF or the pZY-HSA/PDGF-B that makes up in example 3 and embodiment 4 all after Pme I restriction enzyme is handled, forms the linear plasmid molecule.Yeast after getting 5ug linear plasmid DNA and 80ul handling mixes mutually and places in the pole cup of 0.2 cm thick, places on the pulse instrument.The electricimpulse condition is voltage 7500V/CM, and the electrode space time is 5-10 (ms).After electric shock was handled, the 1M Sorbitol solution that adds the precooling of 1ml ice immediately changed in the 15ml test tube in yeast then.The yeast that transforms places 30 ℃ of incubators to place 2 hours, and inoculation is coated on and contains on the antibiotic YPD plate culture medium of Zeocin then.The clone who grows through the resistance selection identifies the insertion of its gene again with molecular biology method.Protein expression is then done the protein immunoblotting detection with SDS-PAGE or with specific antibody with secretion.Different pichia yeast bacteria strains, as X-33, KM71 and protease-deficient yeast strain all can be used to express and secrete the HSA/GF fusion rotein of reorganization as SMD1168.
The characteristic of embodiment 6 yeast expressed and excretory HSA and HSA/GF fusion rotein
Several are contained remain the yeast colony of expressing gene and containing the Zeocin microbiotic respectively, have in the basic culture solution of surge capability and glycerine and cultivate.Be cultured to cell density with 300 rev/mins speed and reach OD
600=2-6.Culture is through 1500 rev/mins, centrifugal collection thalline under 15 minutes conditions, and thalline is resuspended in basic culture solution of the same race again but does not contain glycerine, changes to contain 0.5% methyl alcohol, continues to cultivate, and cell density reaches OD
600=1.0.Yeast is under the inducing of methyl alcohol, and foreign protein begins to express under the effect of promotor.Thereafter, per 24 hours add 100% methyl alcohol to ultimate density is 0.5%.Collect culture supernatant respectively at different time points.Use SDS-PAGE denaturing polyacrylamide gel electrophoresis or protein immunoblotting method to measure the HSA/GF Expression of Fusion Protein.
The result shows, the HSA/GF fusion rotein is by yeast expressed, and is secreted in the nutrient solution.The monoclonal antibody of mouse-anti human serum albumin (Sigma company) determines by expressed protein to be HSA or HSA/GF fusion rotein with the protein immunoblotting experiment.
Typical protein immunoblotting experiment is with the isolating protein of sex change (SDS) gel electrophoresis, protein transduction is moved on on nylon or the cellulose acetate film through electrophoresis apparatus, discerns corresponding protein with specific antibody (first antibody) again.First antibody is discerned and be incorporated into to the second antibody that has the fluorescent functional gene then, and through the whole specificity bonded of fluorescence developing mixture, promptly specific protein stays the marking on the X-ray sheet.The standard protein molecular weight is used to determine the molecular weight of agnoprotein.To through yeast expressed excretory HSA and HSA/hKGF-1, HSA/hEGF, HSA/hIGF-1, HSA/bFGF and HSA/hPDGF-B, can be respectively with the antigenicity of commercially available recombinant protein standard substance (middle inspection institute) relatively.The protein immunoblotting experiment of being done with the monoclonal antibody of anti-people EGF shows fusion rotein HSA/EGF and pure product hEGF (R﹠amp; D System company) has identical antigenicity, and show between the two on immune response intensity and molecular size to have identical ratio.In like manner, detect with anti-people GF antibody, identical result also is confirmed.Accompanying drawing 3 is expressed fusion protein fermented liquid electrophoresis result and end user's serum albumin specific antibody (Sigma) and human serum albumin/human epidermal growth factor (HSA/hEGF) and human serum albumin (HSA) monomer, the Western blot result who is done.On the molecular weight of the two, show the mobility of two molecules of 73Kd of the 65Kd of HSA and HSA/hEGF fairly obviously.
Purifying and the characteristic of embodiment 7 secretor type HSA/GF
Contain oozy sero-abluminous fusion rotein HSA/GF in the yeast cell (ZY-HSA/GF) of reorganization or the culture supernatant of CHO mammalian cell expression.Behind the centrifugation thalline, supernatant liquor is through the 2-10% activated carbon treatment, after collecting, concentrate and reducing salt concn, adjusts about PH to 7.5 the Affi-Gel Blue-Gel chromatography column that concentrated solution is made by Bio-Rad company.HSA or HSA/GF then combine and hang on the cylinder with function group on the chromatography column.Through washing, HSA or HSA/GF then can obtain having the pure protein formulation of 75-85% via 1-5M NaCl gradient elution.Then further by molecular sieve chromatography, can get purity and be increased to 95-99% or purer protein formulation if needed.Be used for zooperal sample then with possible thermal source such as intracellular toxin, remove, to meet the in vivo test needs by Affi-Preppolymyxin Support chromatography column (Bio-Rad).Utilize ordinary method to decide proteinic concentration, as the determination of protein concentration test kit of Bio-Rad production.The protein of final purification by the 0.2uM filter membrane to reach aseptic requirement.
The cell bio-activity determination test of embodiment 8 people HSA/GF
Testing used cell strain is the BalB/C3T3 cell strain.The EGF standard substance: purchase in Chinese biological goods calibrating institute, activity is 6800IU.RPMI-1640 substratum (pulvis): Gibco-BRL perfect medium: the R-1640 substratum that contains 10% foetal calf serum (standard HyCloneSH), 4 ℃ of refrigerators are preserved, keep substratum: contain the R-1640 substratum of 0.5% foetal calf serum (standard HyCloneSH), 4 ℃ of refrigerators are preserved.MTT solution (Sigma): be made into the solution of 5.0mg/ml with phosphate buffered saline buffer, the degerming of 0.22um membrane filtration, 4 ℃ of refrigerators are preserved; Go down to posterity the back be used in 24-48 hour titration 96 porocyte culture plates (flat, Costar), DMSO (Sigma).The BalB/C3T3 cell of taking the logarithm vegetative period makes 5.0 * 10 with perfect medium
4The single cell suspension of individual cell/ml adds in 96 orifice plates, and every hole 100ul cultivated 24 hours.Discard perfect medium, change into and keep substratum, continue to cultivate 24 hours.Standard substance and dilution of sample: respectively standard substance and sample are carried out 4 times of doubling dilutions with keeping substratum, standard substance are done 8 extent of dilution since 100 times; Sample is done 6 extent of dilution, and extension rate adjusted accordingly according to survey the situation of living in the past.Discard and keep substratum, standard solution and sample solution that dilution is good add in the 96 porocyte culture plates, and every hole 100ul, each extent of dilution do 2 repetitions, and blank only adds keeps substratum, continues to cultivate 48 hours.Add MTT solution, every hole 20ul continues to cultivate 4 hours.Discard substratum, every hole adds 100ul DMSO lysate.Measure absorption value on the inherent microplate reader in back 5 minutes in colour developing, measure wavelength 490nm.Calculate with return law of the straight line, that calculates each sample respectively partly imitates extension rate (can cause the diluted sample multiple of standard substance 50% maximum effect), and by following formula calculation result: sample tires=standard substance tire * sample partly imitate extension rate/standard substance partly imitate extension rate.
Fermented liquid can directly be carried out the biological activity determination of HSA/GF fusion rotein after activated carbon treatment, desalination, degerming.Accompanying drawing 4 is to contain the mean value of the fermented liquid of the different HSA/PDGF-B that measure to 3 results of the biological activity determination of BalBC3T3 cell, shows that HSA/PDGF-B has the function of stimulate cell growth.Accompanying drawing 5 has shown various samples (fusion rotein of fermented liquid or purifying) (as heating) front and back under the different treatment condition, the stimulating organism activity of pair cell differentiation.Test shows that the biological activity of fusion rotein and pure product hEGF numerically has the same molar ratio activity.Than living and the corresponding to ratio of its molecular weight size, this result shows that the HSA/hEGF fusion rotein has identical biological activity with pure product hEGF.Similar measuring method is used for HSA/hKGF-2, HSA/bFGF, HSA/aFGF, HSA/PDGF-B measure.Clone can be with BalB/C3T3 or BalB/MK.
Embodiment 9 measures fusion rotein (HSA/GF) in the active stability of external biological
With HSA/FGF is example, and the HSA/GF fusion rotein under 37 ℃ and 50 ℃ of conditions, in the different residence time, is measured the stability of HSA/GF.Get 10000 units by the two combination (series 4) sample of the people EGF of bacterial expression (series 1) and 10000 HSA/hEGF of unit (series 2) or 10000 HSA/hKGF-2 of unit (series 3) or two fusion rotein HSA/hEGF and HSA/hIGF-1 5000 units respectively, place and contain the 200 microlitre thin-walled test tubes that 200 microlitres do not contain the RPM1 nutrient solution of serum and other component, every temperature has 10 repetitions.Wherein one group places under 37 ℃ of conditions and is incubated, and another group places 50 ℃ of water-soluble insulations.From each group, took out one, and be placed on immediately in-80 ℃ of refrigerators and preserve in per 7 days.After treating whole sample collections, the BalBC3T3 cell is carried out biological activity determination with these samples.Test the contrast that sets up standard simultaneously.After the result shows that GF monomer and HSA form fusion molecule, biological activity difference under different storage conditions.7 week back HSA/GF still preserve its original big biological activity (accompanying drawing 5A) under 37 ℃.And the EGF monomer promptly completely loses biological activity in 1 week.Biological activity is near transformation period (accompanying drawing 5B) during 50 ℃ times 4 weeks.After showed cell somatomedin (GF) merges with serum albumin as a result, make it to preserve, the storage time prolongs.Environment there are stronger resistance and biologically stable.HSA/hKGF, HSA/hEGF or HSA/PDGF obtain similar test-results.
Being used in combination of embodiment 10HSA/GF has synergistic function
When HSA/hEGF and HSA/hIGF are used in combination, carry out measuring as the cytoactive of embodiment 8, the result show the biological activity that is used in combination HSA/hEGF greater than with the monomeric HSA/hEGF of amount (waiting mole number) to the stimulate cell growth activity.(accompanying drawing 5) has synergism (two 5000IU merge when being used in combination, and cell bio-activity is greater than 10000IU) to the cell growth-stimulating biological activity of EGF when as seen being used in combination in the presence of the IGF-1 cell growth factor.There are HSA, hEGF, hIGF, HSA/hEGF and HSA/hIGF equivalent cell to divide other cell growth-stimulating test in the contrast.
The animal toxicity test of embodiment 11HSA/GF
The rabbit acute eye irritation test is undertaken by China's " cosmetics health standard ".Rabbit 2-3kg, healthy male and female all have.0.1ml, wherein contain fusion rotein 150 micrograms of 80% purity for test agent, directly splash into rabbit conjunctiva of left eye capsule, closed gently.Right eye splashes into stroke-physiological saline solution 0.1ml.At 1,24,48,72,96 hour with 1 week the time checked right and left eyes respectively, carrying out an acute toxicity and an eye stimulus intensity ranking respectively, with 4 for the most serious, is non-stimulated with 0.Then the result shows eye conjunctiva, the iris of experimental animal and cornea under for test agent or physiological saline effect, to the stimulus intensity comparative figure of eye all between 0-0.5.The result proves nonirritant and the nontoxicity of HSA/hGF fusion rotein to animal.Tangible stimulation toxicity is not seen in the preliminary experiment that the eye that carries out with the fermented liquid that contains HSA/GF of gac, desalination and degerming stimulates yet.
The residence time (transformation period mensuration) of embodiment 12HSA/GF in animal blood
Select 2.3-2.6 kilogram rabbit for use, respectively at first day, injection 0.5ml fusion rotein injection liquid.Rabbit A injects 1,000,000 hEGF of unit (about 10 microgram protein contents); Rabbit B injects the reorganization HSA/hEGF of 1,000,000 units fusion rotein; Rabbit C injects the HSA/haFGF of 1,000,000 units; Rabbit D injects 100 microgram people HSA.
After the injection, collect blood sample every day once, survey work with the BalBC3T3 cell by the method for embodiment 8 then.Rough determination the results are shown in accompanying drawing 6.The EGF monomer is when the 1st week, and biological activity just drops to basic point.Then the transformation period was extended for about 4 weeks the fusion rotein of HSA and GF greatly.
These results show that the HSA/GF fusion rotein has the longer transformation period than exposed GF in vivo.Show that in view of the above the present invention has tangible effect on the GF of protection " exposing ".Especially, DeGrain during aFGF tested in the existing clinical II phase, it is too short that possible reason is considered to the aFGF transformation period, needs the multiple injection just can (Stegmann etc., Cardiac Vascular Regeneration, 1: 5-10,2000).GF has the long transformation period, and this should have great using value when making makeup clinically.
Embodiment 11 application of fermentation technology are expressed preparation HSA/GF fusion rotein on a large scale
Show in the experiment that the application pichia yeast is expressed and the large-scale production recombination fusion protein all will be easy to many compared with other any system.After recombinant strain was separated to, the expression of recombinant proteins strain can have Mut
+And Mut
sTwo kinds of forms.From cultivating on a small scale, methanol induction is in the proteic expression of different incubation time point sampling and testings.All analyze with the SDS-PAGE method for the content that secreted protein reaches in the nutrient solution in cell, to the biological activity of expression product, expression level and purity were all monitored in each step.The result shows that various HSA/GFs Expression of Fusion Protein levels in the different fermentations liquid are respectively at 50-300mg/L.
Human serum albumin (HSA) and cell growth factor (GF) form the DNA Nucleotide and the protein sequence table of fusion rotein
A.Seq ID No.1HSA/hKGF-2 fusion rotein nucleotide sequence
atgaagtgggtaacctttatttcccttctttttctctttagctcggcttattccaggggtgtgtttcgtcgagatgcac
acaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctca
gtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgct
gatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtg
aaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatga
caacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacattt
ttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggt
ataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcggga
tgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagca
tgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttacca
aagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtga
aaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaa
gtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaact
atgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgct
gctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgcc
aaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagc
ttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgt
agaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaa
gactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgct
gcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaa
tgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcactt
gttgagcttgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtag
agaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgc
cttaggctta
caagcccttggtcaggacatggtgtcaccagaggccaccaactcttcttcctcctccttctcctctcct
tccagcgcgggaaggcatgtgcggagctacaatcaccttcaaggagatgtccgctggagaaagctattctctttcacca
agtactttctcaagattgagaagaacgggaaggtcagcgggaccaagaaggagaactgcccgtacagcatcctggagat
aacatcagtagaaatcggagttgttgccgtcaaagccattaacagcaactattacttagccatgaacaagaaggggaaa
ctctatggctcaaaagaatttaacaatgactgtaagctgaaggagaggatagaggaaaatggatacaatacctatgcat
catttaactggcagcataatgggaggcaaatgtatgtggcattgaatggaaaaggagctccaaggagaggacagaaaac
acgaaggaaaaacacctctgctcactttcttccaatggtggtacactcatag
B.Seq ID No.2 HSA/hKGF-2 fusion rotein aminoacid sequence (756AA, MW:85.8Kd)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG
DKLCTVATLRETYGEMADCCAKQEPGRNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEI
ARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKA
WAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLL
EKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYET
TLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEV
SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETY
VPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCF
AEEGKKLVAASQAALGL
QALGQDMVSPEATNSSSSSFSSPSSAGRHVRSYNHLQGDVRWRKLFSFTKYFLK
IEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYN
TYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMVVHS.
C.Seq ID No.3HSA-hEGF fusion rotein nucleotide sequence
atgaagtgggtaacctttatttcccttctttttctctttagctcggcttattccaggggtgtgtttcgtcgagatgcac
acaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctca
gtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgct
gatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtg
aaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatga
caacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacattt
ttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggt
ataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcggga
tgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagca
tgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttacca
aagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtga
aaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaa
gtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaact
atgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgct
gctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgcc
aaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagc
ttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgt
agaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaa
gactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgct
gcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaa
tgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcactt
gttgagcttgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtag
agaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgc
cttaggctta
aacagcgactctgaatgtccgctgtcccacgacggttactgcctgcacgacggtgtttgcatgtacatc
gaagctctggacaagtatgcatgcaactgtgttgttggctacatcggtgaacgttgtcagtaccgtgacctgaaatggt
gggaactgcgttga
D.Seq ID No.4 HSA-hEGF fusion rotein aminoacid sequence (638AA; MW:72.7KD)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVAT
LRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFA
KRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTD
LTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVC
KNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELF
EQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVT
KCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA
FVEKCCKADDKETCFAEEGKKLVAASQAALGL
NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDL
KWWELR.
E.Seq ID No.5, HSA/hIGF-1 fusion rotein nucleotide sequence
atgaagtgggtaacctttatttcccttctttttctctttagctcggcttattccaggggtgtgtttcgtcgagatgcac
acaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctca
gtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgct
gatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtg
aaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatga
caacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacattt
ttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggt
ataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcggga
tgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagca
tgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttacca
aagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtga
aaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaa
gtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaact
atgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgct
gctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgcc
aaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagc
ttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgt
agaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaa
gactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgct
gcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaa
tgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcactt
gttgagcttgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtag
agaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgc
cttaggctta
ggaccggagacgctctgcggggctgagctggtggatgctcttcagttcgtgtgtggagacaggggcttt
tatttcaacaagcccacagggtatggctccagcagtcggagggcgcctcagacaggcatcgtggatgagtgctgcttcc
ggagctgtgatctaaggaggctggagatgtattgcgcacccctcaagcctgccaagtcagcttga
F.Seq ID No.6HSA/hIGF-1 fusion rotein aminoacid sequence (655AA:MW:74KD)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVAT
LRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFA
KRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTD
LTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVC
KNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELF
EQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVT
KCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA
FVEKCCKADDKETCFAEEGKKLVAASQAAL
GLGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDEC
CFRSCDLRRLEMYCAPLKPAKSA
G.Seq ID No.7HSA/hbFGF fusion rotein nucleotide sequence
atgaagtgggtaacctttatttcccttctttttctctttagctcggcttattccaggggtgtgtttcgtcgagatgcac
acaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctca
gtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgct
gatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtg
aaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatga
caacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacattt
ttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggt
ataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcggga
tgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagca
tgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttacca
aagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtga
aaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaa
gtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaact
atgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgct
gctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgcc
aaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagc
ttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgt
agaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaa
gactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgct
gcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaa
tgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcactt
gttgagcttgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtag
agaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgc
cttaggctta
ctgggggaccgcgggcgcggccgcgcgctgccgggcgggaggctggggggccggggccggggccgtgcc
ccggagcgggtcggaggccggggccggggccgggggacggcggctccccgcgcggctccagcggctcggggatcccggc
cgggccccgcagggaccatggcagccgggagcatcaccacgctgcccgccttgcccgaggatggcggcagcggcgcctt
cccgcccggccacttcaaggaccccaagcggctgtactgcaaaaacgggggcttcttcctgcgcatccaccccgacggc
cgagttgacggggtccgggagaagagcgaccctcactga
H.Seq ID No.8 HSA/hbFGF fusion rotein aminoacid sequence (699AA:MW:78Kd)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVAT
LRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFA
KRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTD
LTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVC
KNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELF
EQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVT
KCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA
FVEKCCKADDKETCFAEEGKKLVAASQAALGL
LGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARG
SRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPH.
I.Seq ID NO.9 HSA/hPDGF-B fusion rotein nucleotide sequence
atgaagtgggtaacctttatttcccttctttttctctttagctcggcttattccaggggtgtgtttcgtcgagatgcac
acaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctca
gtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgct
gatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtg
aaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatga
caacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacattt
ttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggt
ataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcggga
tgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagca
tgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttacca
aagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtga
aaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaa
gtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaact
atgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgct
gctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgcc
aaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagc
ttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgt
agaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaa
gactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgct
gcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaa
tgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcactt
gttgagcttgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtag
agaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgc
cttaggctta
aatcgctgctgggcgctcttcctgtctctctgctgctacctgcgtctggtcagcgccgagggggacccc
attcccgaggagctttatgagatgctgagtgaccactcgatccgctcctttgatgatctccaacgcctgctgcacggag
accccggagaggaagatggggccgagttggacctgaacatgacccgctcccactctggaggcgagctggagagcttggc
tcgtggaagaaggagcctgggttccctgaccattgctgagccggccatgatcgccgagtgcaagacgcgcaccgaggtg
ttcgagatctcccggcgcctcatagaccgcaccaacgccaacttcctggtgtggccgccctgtgtggaggtgcagcgct
gctccggctgctgcaacaaccgcaacgtgcagtgccgccccacccaggtgcagctgcgacctgtccaggtgagaaagat
cgagattgtgcggaagaagccaatctttaagaaggccacggtgacgctggaagaccacctggcatgcaagtgtgagaca
gtggcagctgcacggcctgtgacccgaagcccggggggttcccaggagcagcgagccaaaacgccccaaactcgggtga
ccattcggacggtgcgagtccgccggccccccaagggcaagcaccggaaattcaagcacacgcatgacaagacggcact
gaaggagacccttggagcctag
J.Seq ID No.10 HSA/hPDGF-B fusion rotein aminoacid sequence (825AA; MW:93.6)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVAT
LRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFA
KRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTD
LTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVC
KNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELF
EQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVT
KCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA
FVEKCCKADDKETCFAEEGKKLVAASQAALGL
NRCWALFLSLCCYLRLVSAEGDPIPEELYEMLSDHSIRSFDDLQRLL
HGDPGEEDGAELDLNMTRSHSGGELESLARGRRSLGSLTIAEPAMIAECKTRTEVFEISRRLIDRTNANFLVWPPCVEV
QRCSGCCNNRNVQCRRTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVTRSPGGSQEQRAKTPQT
RVTIRTVRVRRPPKGKHRKFKHTHDKTALKETLGA.
K.Seq ID No.11 HSA/haFGF fusion rotein nucleotide sequence
atgaagtgggtaacctttatttcccttctttttctctttagctcggcttattccaggggtgtgtttcgtcgagatgcac
acaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttggtgttgattgcctttgctca
gtatcttcagcagtgtccatttgaagatcatgtaaaattagtgaatgaagtaactgaatttgcaaaaacatgtgttgct
gatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtg
aaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatga
caacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacattt
ttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggt
ataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcggga
tgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagca
tgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttacca
aagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtga
aaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaa
gtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaact
atgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgct
gctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgcc
aaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagc
ttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgt
agaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaa
gactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgct
gcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaa
tgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcactt
gttgagcttgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtag
agaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgc
cttaggctta
gctgaaggggaaatcaccaccttcacagccctgaccgagaagtttaatctgcctccagggaattacaag
aagcccaaactcctctactgtagcaacgggggccacttcctgaggatccttccggatggcacagtggatgggacaaggg
acaggagcgaccagcacattcagctgcagctcagtgcggaaagcgtgggggaggtgtatataaagagtaccgagactgg
ccagtacttggccatggacaccgacgggcttttatacggctcacagacaccaaatgaggaatgtttgttcctggaaagg
ctggaggagaaccattacaacacctatatatccaagaagcatgcagagaagaattggtttgttggcctcaagaagaatg
ggagctgcaaacgcggtcctcggactcactatggccagaaagcaatcttgtttctccccctgccagtctcttctga
L.Seq ID No.12 HSA/aFGF fusion rotein aminoacid sequence (738AA; MW:84Kd)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVAT
LRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFA
KRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTD
LTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVC
KNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELF
EQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVT
KCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAA
FVEKCCKADDKETCFAEEGKKLVAASQAALGL
AEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLRILPDGTVDG
TRDRSDQHIQLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEKNWFVGLK
KNGSCKRGPRTHYGQKAILFLPLPVSS
Claims (10)
1. the fusion rotein of a human serum albumin (HSA) and cell growth factor (GF) is characterized in that, its fusion rotein for being linked to each other and constitute with cell growth factor by human serum albumin.
2. human serum albumin as claimed in claim 1 and growth factor fusion protein is characterized in that, the protein amino acid sequence of this fusion rotein and Seq ID No.2,4,6,8,10 or 12 have at least 90% sequence homology.
3. the fusion rotein of human serum albumin as claimed in claim 1 and cell growth factor, it is characterized in that, described cell growth factor is selected from epithelical cell growth factor (EGF), endothelial cell growth factor (ECGF) (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF) and insulin-like growth factor (IGF).
4. human serum albumin as claimed in claim 1 and growth factor fusion protein is characterized in that, can link to each other with human serum albumin to constitute fusion rotein by a connection peptides.
5. human serum albumin as claimed in claim 4 and growth factor fusion protein is characterized in that, the length of described connection peptides is 2-50 amino acid, and preferred connection peptides is (G
4S) 3-4.
6. the fusion rotein of human serum albumin as claimed in claim 1 and cell growth factor is characterized in that described fusion rotein is a secretor type.
7. the fusion rotein of human serum albumin as claimed in claim 1 and cell growth factor is characterized in that, described fusion rotein can combine with the albuminous antibody of the AHS of specific recognition.
8. the nucleotide sequence of the fusion rotein of described human serum albumin of the claim 1 of encoding and cell growth factor has at least 90% sequence homology with Seq ID No.1,3,5,7,9 or 11.
9. carry the carrier of the nucleotide sequence of fusion rotein according to claim 1 of encoding, conversion, transfection or transduction host system, it comprises cell, yeast, virus or the bacterium of vertebrates, fish, insect, plant; Described vertebrate cells is a Chinese hamster cell; Described yeast is that yeast saccharomyces cerevisiae, pichia yeast, candida yeasts, Crewe tie up that inferior yeast, spore circle are female to belong to yeast or fission yeast; Described pichia yeast is the Bi Shi saccharomyces pastorianus, and its CGMCC preserving number is 2072.
10. preparation, contain at least a fusion rotein as claimed in claim 1, wherein said at least a HSA/GF, or two kinds of combinations that different fusion roteins is HSA/hEGF fusion rotein and HSA/KGF fusion rotein, the combination of HSA/IGF fusion rotein and HSA/PDGF fusion rotein, or the combination of HSA, HSA/IGF, HSA/FGF, HSA/EGF, HSA/PDGF, HSA/KGF, be used for beauty and skin care, wound, treatment of diseases and application.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100575719A CN101121753B (en) | 2007-06-06 | 2007-06-06 | Human serum albumin recombination fusion protein with continuous repairing function to multifarious skin cell |
| CN201310335662.XA CN105777908B (en) | 2007-06-06 | 2007-06-06 | Recombinant human serum albumin/keratinocyte growth factor fusion protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100575719A CN101121753B (en) | 2007-06-06 | 2007-06-06 | Human serum albumin recombination fusion protein with continuous repairing function to multifarious skin cell |
Related Child Applications (4)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310335662.XA Division CN105777908B (en) | 2007-06-06 | 2007-06-06 | Recombinant human serum albumin/keratinocyte growth factor fusion protein |
| CN201110211843A Division CN102311503A (en) | 2007-06-06 | 2007-06-06 | Recombinant human serum albumin / FGF fusion protein with continuous effect on restoration of a plurality of skin cells |
| CN201310335639.0A Division CN104004096A (en) | 2007-06-06 | 2007-06-06 | Recombinant human serum albumin/platelet-derived growth factor fusion protein |
| CN201310335689.9A Division CN104004097A (en) | 2007-06-06 | 2007-06-06 | Recombinant human serum albumin/insulin-like growth factor fusion protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101121753A true CN101121753A (en) | 2008-02-13 |
| CN101121753B CN101121753B (en) | 2011-11-16 |
Family
ID=39084271
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007100575719A Active CN101121753B (en) | 2007-06-06 | 2007-06-06 | Human serum albumin recombination fusion protein with continuous repairing function to multifarious skin cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101121753B (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009043277A1 (en) * | 2007-09-25 | 2009-04-09 | Tianjin Sinobiotech Ltd. | Skin care composition containing hsa fusion protein, method for preparation and use thereof |
| CN101693016B (en) * | 2009-11-02 | 2012-07-25 | 北京美福源生物医药科技有限公司 | Universal pharmaceutical formulation for recombined human serum albumin fusion proteins for injection |
| CN103491888A (en) * | 2010-12-13 | 2014-01-01 | 生物模拟治疗有限责任公司 | Compositions and methods for spine fusion procedures |
| CN105283541A (en) * | 2013-05-30 | 2016-01-27 | 味之素株式会社 | Medium for culturing stem cells |
| CN105457102A (en) * | 2014-09-05 | 2016-04-06 | 陕西艾美雅生物科技有限公司 | Purifying and concentrating method for composite bioactive factors and concentrated solution |
| CN106220724A (en) * | 2016-09-13 | 2016-12-14 | 河南师范大学 | Human fibroblastic growth factor 21 recombiant protein and its preparation method and application |
| CN106397607A (en) * | 2016-09-13 | 2017-02-15 | 河南师范大学 | Recombinant human fibroblast growth factor 21 fusion protein and application thereof in preparation of medicine for treating metabolic diseases |
| CN106432509A (en) * | 2016-09-13 | 2017-02-22 | 河南师范大学 | RhFGF-21(recombinant human fibroblast growth factor-21) fusion protein capable of treating metabolic diseases as well as preparation method and application of rhFGF-21 fusion protein |
| CN111484557A (en) * | 2019-01-25 | 2020-08-04 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying recombinant human serum albumin-epidermal growth factor fusion protein from genetically engineered rice seeds |
| CN113087808A (en) * | 2021-04-01 | 2021-07-09 | 芜湖英特菲尔生物制品产业研究院有限公司 | Brewing yeast expression human rKGF-1-HSA fusion protein and preparation method of standard substance thereof |
| CN113105557A (en) * | 2021-04-01 | 2021-07-13 | 芜湖英特菲尔生物制品产业研究院有限公司 | Saccharomyces cerevisiae expression human rVEGF-HSA fusion protein and preparation method of standard substance thereof |
| CN113105558A (en) * | 2021-04-01 | 2021-07-13 | 芜湖英特菲尔生物制品产业研究院有限公司 | Saccharomyces cerevisiae expression human rPDGF-HSA fusion protein and preparation method of standard substance thereof |
-
2007
- 2007-06-06 CN CN2007100575719A patent/CN101121753B/en active Active
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8603973B2 (en) | 2002-07-01 | 2013-12-10 | Tianjin Sinobiotech Ltd. | Skincare composition comprising HSA fusion protein, preparation method and uses thereof |
| WO2009043277A1 (en) * | 2007-09-25 | 2009-04-09 | Tianjin Sinobiotech Ltd. | Skin care composition containing hsa fusion protein, method for preparation and use thereof |
| CN101693016B (en) * | 2009-11-02 | 2012-07-25 | 北京美福源生物医药科技有限公司 | Universal pharmaceutical formulation for recombined human serum albumin fusion proteins for injection |
| CN103491888A (en) * | 2010-12-13 | 2014-01-01 | 生物模拟治疗有限责任公司 | Compositions and methods for spine fusion procedures |
| CN105283541B (en) * | 2013-05-30 | 2019-02-19 | 味之素株式会社 | Culture medium is used in stem cell culture |
| CN105283541A (en) * | 2013-05-30 | 2016-01-27 | 味之素株式会社 | Medium for culturing stem cells |
| US11859205B2 (en) | 2013-05-30 | 2024-01-02 | Ajinomoto Co., Inc. | Medium for culturing stem cells |
| CN105457102A (en) * | 2014-09-05 | 2016-04-06 | 陕西艾美雅生物科技有限公司 | Purifying and concentrating method for composite bioactive factors and concentrated solution |
| CN105457102B (en) * | 2014-09-05 | 2019-02-26 | 陕西艾美雅生物科技有限公司 | The purification method for concentration and concentrate of the composite bio-active factor |
| CN106432509A (en) * | 2016-09-13 | 2017-02-22 | 河南师范大学 | RhFGF-21(recombinant human fibroblast growth factor-21) fusion protein capable of treating metabolic diseases as well as preparation method and application of rhFGF-21 fusion protein |
| CN106397607A (en) * | 2016-09-13 | 2017-02-15 | 河南师范大学 | Recombinant human fibroblast growth factor 21 fusion protein and application thereof in preparation of medicine for treating metabolic diseases |
| CN106432509B (en) * | 2016-09-13 | 2019-05-21 | 河南师范大学 | A kind of 21 fusion protein of recombinant human fibroblast growth factor and its preparation method and application for treating metabolic disease |
| CN106220724B (en) * | 2016-09-13 | 2019-10-11 | 河南师范大学 | 21 recombinant protein of human fibroblastic growth factor and its preparation method and application |
| CN106220724A (en) * | 2016-09-13 | 2016-12-14 | 河南师范大学 | Human fibroblastic growth factor 21 recombiant protein and its preparation method and application |
| CN111484557A (en) * | 2019-01-25 | 2020-08-04 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying recombinant human serum albumin-epidermal growth factor fusion protein from genetically engineered rice seeds |
| CN111484557B (en) * | 2019-01-25 | 2023-07-18 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying recombinant human serum albumin-epidermal growth factor fusion protein from genetically engineered rice seeds |
| CN113087808A (en) * | 2021-04-01 | 2021-07-09 | 芜湖英特菲尔生物制品产业研究院有限公司 | Brewing yeast expression human rKGF-1-HSA fusion protein and preparation method of standard substance thereof |
| CN113105557A (en) * | 2021-04-01 | 2021-07-13 | 芜湖英特菲尔生物制品产业研究院有限公司 | Saccharomyces cerevisiae expression human rVEGF-HSA fusion protein and preparation method of standard substance thereof |
| CN113105558A (en) * | 2021-04-01 | 2021-07-13 | 芜湖英特菲尔生物制品产业研究院有限公司 | Saccharomyces cerevisiae expression human rPDGF-HSA fusion protein and preparation method of standard substance thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101121753B (en) | 2011-11-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101121753B (en) | Human serum albumin recombination fusion protein with continuous repairing function to multifarious skin cell | |
| WO2009043277A1 (en) | Skin care composition containing hsa fusion protein, method for preparation and use thereof | |
| CN102311503A (en) | Recombinant human serum albumin / FGF fusion protein with continuous effect on restoration of a plurality of skin cells | |
| CN110845603B (en) | Human collagen 17-type polypeptide, production method and use thereof | |
| CN114671941B (en) | Nerve growth factor mutant | |
| JPH07500968A (en) | Recombinant bone morphogenetic protein heterodimers, compositions and methods of use | |
| CN100582121C (en) | Thymosin beta 4 derivatives and use thereof | |
| CN113683679B (en) | Recombinant I-type humanized collagen C1L6T and preparation method and application thereof | |
| US5158935A (en) | Human epidermal growth factor having substitution at position 11 | |
| US6191106B1 (en) | Muteins of epidermal growth factor exhibiting enhanced binding at low pH | |
| CN113683680B (en) | Recombinant I-type humanized collagen C1L1T and preparation method and application thereof | |
| CN113637068B (en) | Recombinant I-type humanized collagen C1L5T and preparation method and application thereof | |
| WO2023035632A1 (en) | Active peptide and use thereof | |
| CN112409471B (en) | Low-pain nerve growth factor mutant | |
| CN104004097A (en) | Recombinant human serum albumin/insulin-like growth factor fusion protein | |
| KR100775958B1 (en) | Non-activated Polypeptides Having a Function of Tissue Regeneration and Method for Preparing the Same | |
| CN102803500A (en) | Immunoregulatory peptides and methods of use | |
| CN105884901B (en) | Tool persistently controls recombination human serum albumin/glicentin class peptide fusion protein of blood-sugar content function | |
| CN113683681B (en) | Recombinant I-type humanized collagen C1L3T and preparation method and application thereof | |
| CN104004096A (en) | Recombinant human serum albumin/platelet-derived growth factor fusion protein | |
| CN105777908B (en) | Recombinant human serum albumin/keratinocyte growth factor fusion protein | |
| CN108949730A (en) | A kind of preparation method and applications recombinating allosteric clostridiopetidase A | |
| CN101659700B (en) | Short peptide | |
| CN100535005C (en) | Long chain human insulin-like growth factor(LR3IGF-1) and its preparing process and application method | |
| CN113788891B (en) | Recombinant I-type humanized collagen C1L4T and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: TIANJIN LINDA BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: TIANJIN SINO BIOTECHNOLOGY LTD., FORTUNEROCK INC. Effective date: 20110915 Owner name: BEIJING MEIFUYUAN BIO-PHARM TECH. CO., LTD. TIANJI Free format text: FORMER OWNER: AMERICA FORD GLORY GROUP (FORTUNE ROCK INC.) |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20110915 Address after: Taihua road 300457 Tianjin TEDA No. 12 business center Applicant after: TIANJIN LINDA BIOTECHNOLOGY Co.,Ltd. Co-applicant after: BEIJING MEIFUYUAN BIOMEDICAL TECHNOLOGY CO.,LTD. Co-applicant after: TIANJIN SINOBIOTECH LTD. Address before: 300457, Tianjin TEDA Development Zone, 5 Avenue, international entrepreneurship center, 10 floor, Tianjin Applicant before: TIANJIN SINOBIOTECH LTD. Co-applicant before: Fortune Rock (Inc.) |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PP01 | Preservation of patent right | ||
| PP01 | Preservation of patent right |
Effective date of registration: 20220321 Granted publication date: 20111116 |
|
| PD01 | Discharge of preservation of patent | ||
| PD01 | Discharge of preservation of patent |
Date of cancellation: 20220926 Granted publication date: 20111116 |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231207 Address after: Building 15, 2nd Floor, 201, No. 50 Huatuo Road, Daxing Biomedical Industry Base, Zhongguancun Science and Technology Park, Daxing District, Beijing, 100000 RMB Patentee after: BEIJING MEIFUYUAN BIOMEDICAL TECHNOLOGY CO.,LTD. Patentee after: TIANJIN LINDA BIOTECHNOLOGY Co.,Ltd. Address before: 300457 Tianjin Teda Development Zone No.12 Taihua Road Entrepreneurship Center Patentee before: TIANJIN LINDA BIOTECHNOLOGY Co.,Ltd. Patentee before: BEIJING MEIFUYUAN BIOMEDICAL TECHNOLOGY CO.,LTD. Patentee before: TIANJIN SINOBIOTECH LTD. |