CN101870734B - 一种抑制新生血管生成的融合多肽及其编码基因与应用 - Google Patents
一种抑制新生血管生成的融合多肽及其编码基因与应用 Download PDFInfo
- Publication number
- CN101870734B CN101870734B CN2010101820014A CN201010182001A CN101870734B CN 101870734 B CN101870734 B CN 101870734B CN 2010101820014 A CN2010101820014 A CN 2010101820014A CN 201010182001 A CN201010182001 A CN 201010182001A CN 101870734 B CN101870734 B CN 101870734B
- Authority
- CN
- China
- Prior art keywords
- cell
- polypeptide
- mek
- growth
- fusogenic polypeptide
- 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.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 46
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 42
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 15
- 230000002401 inhibitory effect Effects 0.000 title abstract description 4
- 230000000799 fusogenic effect Effects 0.000 title abstract 5
- 206010029113 Neovascularisation Diseases 0.000 title abstract 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 6
- 201000010099 disease Diseases 0.000 claims abstract description 5
- 239000003814 drug Substances 0.000 claims abstract description 4
- 230000004927 fusion Effects 0.000 claims description 18
- 230000005764 inhibitory process Effects 0.000 claims description 15
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 7
- 102000040430 polynucleotide Human genes 0.000 claims description 4
- 108091033319 polynucleotide Proteins 0.000 claims description 4
- 239000002157 polynucleotide Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 abstract description 39
- 210000004027 cell Anatomy 0.000 abstract description 39
- 108010079709 Angiostatins Proteins 0.000 abstract description 14
- 102400001047 Endostatin Human genes 0.000 abstract description 14
- 108010079505 Endostatins Proteins 0.000 abstract description 14
- FZCSTZYAHCUGEM-UHFFFAOYSA-N aspergillomarasmine B Natural products OC(=O)CNC(C(O)=O)CNC(C(O)=O)CC(O)=O FZCSTZYAHCUGEM-UHFFFAOYSA-N 0.000 abstract description 14
- 210000002889 endothelial cell Anatomy 0.000 abstract description 12
- 238000012360 testing method Methods 0.000 abstract description 7
- 230000005012 migration Effects 0.000 abstract description 5
- 238000013508 migration Methods 0.000 abstract description 5
- 108010000684 Matrix Metalloproteinases Proteins 0.000 abstract description 3
- 102000002274 Matrix Metalloproteinases Human genes 0.000 abstract description 3
- 230000035755 proliferation Effects 0.000 abstract 2
- 102400000068 Angiostatin Human genes 0.000 abstract 1
- 238000002560 therapeutic procedure Methods 0.000 abstract 1
- 210000003725 endotheliocyte Anatomy 0.000 description 17
- 230000000694 effects Effects 0.000 description 14
- 102000012936 Angiostatins Human genes 0.000 description 13
- 201000011510 cancer Diseases 0.000 description 13
- 125000000539 amino acid group Chemical group 0.000 description 9
- 210000004204 blood vessel Anatomy 0.000 description 9
- 230000012010 growth Effects 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 210000004881 tumor cell Anatomy 0.000 description 8
- 230000002792 vascular Effects 0.000 description 8
- 239000011159 matrix material Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 108091005804 Peptidases Proteins 0.000 description 6
- 102000035195 Peptidases Human genes 0.000 description 6
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 6
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 108010082117 matrigel Proteins 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 101000605403 Homo sapiens Plasminogen Proteins 0.000 description 5
- 230000006907 apoptotic process Effects 0.000 description 5
- 206010020718 hyperplasia Diseases 0.000 description 5
- 239000002184 metal Substances 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 239000002516 radical scavenger Substances 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 4
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 4
- 230000000118 anti-neoplastic effect Effects 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 230000010595 endothelial cell migration Effects 0.000 description 4
- 239000003102 growth factor Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000004862 vasculogenesis Effects 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 101000627872 Homo sapiens 72 kDa type IV collagenase Proteins 0.000 description 3
- 102000013566 Plasminogen Human genes 0.000 description 3
- 108010051456 Plasminogen Proteins 0.000 description 3
- 230000033115 angiogenesis Effects 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000000452 restraining effect Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- 210000003954 umbilical cord Anatomy 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 102100026802 72 kDa type IV collagenase Human genes 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 102000009465 Growth Factor Receptors Human genes 0.000 description 2
- 108010009202 Growth Factor Receptors Proteins 0.000 description 2
- 208000032843 Hemorrhage Diseases 0.000 description 2
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- 229940095054 ammoniac Drugs 0.000 description 2
- 230000002491 angiogenic effect Effects 0.000 description 2
- 230000003527 anti-angiogenesis Effects 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000008034 disappearance Effects 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 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
- 239000003292 glue Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 210000001525 retina Anatomy 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 description 2
- 230000007998 vessel formation Effects 0.000 description 2
- ZODMADSIQZZBSQ-FXQIFTODSA-N Ala-Gln-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZODMADSIQZZBSQ-FXQIFTODSA-N 0.000 description 1
- HHRAXZAYZFFRAM-CIUDSAMLSA-N Ala-Leu-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O HHRAXZAYZFFRAM-CIUDSAMLSA-N 0.000 description 1
- KUFVXLQLDHJVOG-SHGPDSBTSA-N Ala-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C)N)O KUFVXLQLDHJVOG-SHGPDSBTSA-N 0.000 description 1
- KWKQGHSSNHPGOW-BQBZGAKWSA-N Arg-Ala-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)NCC(O)=O KWKQGHSSNHPGOW-BQBZGAKWSA-N 0.000 description 1
- CLICCYPMVFGUOF-IHRRRGAJSA-N Arg-Lys-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O CLICCYPMVFGUOF-IHRRRGAJSA-N 0.000 description 1
- OLVIPTLKNSAYRJ-YUMQZZPRSA-N Asn-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N OLVIPTLKNSAYRJ-YUMQZZPRSA-N 0.000 description 1
- YRTOMUMWSTUQAX-FXQIFTODSA-N Asn-Pro-Asp Chemical compound NC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O YRTOMUMWSTUQAX-FXQIFTODSA-N 0.000 description 1
- YNQMEIJEWSHOEO-SRVKXCTJSA-N Asn-Tyr-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O YNQMEIJEWSHOEO-SRVKXCTJSA-N 0.000 description 1
- XOASPVGNFAMYBD-WFBYXXMGSA-N Asp-Trp-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C)C(O)=O XOASPVGNFAMYBD-WFBYXXMGSA-N 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010081589 Becaplermin Proteins 0.000 description 1
- 108010051834 CTTHWGFTLC peptide Proteins 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- WXKWQSDHEXKKNC-ZKWXMUAHSA-N Cys-Asp-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CS)N WXKWQSDHEXKKNC-ZKWXMUAHSA-N 0.000 description 1
- WAJDEKCJRKGRPG-CIUDSAMLSA-N Cys-His-Ser Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CS)N WAJDEKCJRKGRPG-CIUDSAMLSA-N 0.000 description 1
- CNBIWHCVAZHRBI-IHRRRGAJSA-N Cys-Met-Phe Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CS)N CNBIWHCVAZHRBI-IHRRRGAJSA-N 0.000 description 1
- NAPULYCVEVVFRB-HEIBUPTGSA-N Cys-Thr-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)CS NAPULYCVEVVFRB-HEIBUPTGSA-N 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 201000008808 Fibrosarcoma Diseases 0.000 description 1
- 230000010190 G1 phase Effects 0.000 description 1
- YPFFHGRJCUBXPX-NHCYSSNCSA-N Gln-Pro-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCC(N)=O)C(O)=O YPFFHGRJCUBXPX-NHCYSSNCSA-N 0.000 description 1
- CGYDXNKRIMJMLV-GUBZILKMSA-N Glu-Arg-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O CGYDXNKRIMJMLV-GUBZILKMSA-N 0.000 description 1
- NKLRYVLERDYDBI-FXQIFTODSA-N Glu-Glu-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NKLRYVLERDYDBI-FXQIFTODSA-N 0.000 description 1
- TZOVVRJYUDETQG-RCOVLWMOSA-N Gly-Asp-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CN TZOVVRJYUDETQG-RCOVLWMOSA-N 0.000 description 1
- BUEFQXUHTUZXHR-LURJTMIESA-N Gly-Gly-Pro zwitterion Chemical compound NCC(=O)NCC(=O)N1CCC[C@H]1C(O)=O BUEFQXUHTUZXHR-LURJTMIESA-N 0.000 description 1
- CLNSYANKYVMZNM-UWVGGRQHSA-N Gly-Lys-Arg Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CCCN=C(N)N CLNSYANKYVMZNM-UWVGGRQHSA-N 0.000 description 1
- GWNIGUKSRJBIHX-STQMWFEESA-N Gly-Tyr-Arg Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)CN)O GWNIGUKSRJBIHX-STQMWFEESA-N 0.000 description 1
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 1
- SVHKVHBPTOMLTO-DCAQKATOSA-N His-Arg-Asp Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O SVHKVHBPTOMLTO-DCAQKATOSA-N 0.000 description 1
- CUEQQFOGARVNHU-VGDYDELISA-N His-Ser-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O CUEQQFOGARVNHU-VGDYDELISA-N 0.000 description 1
- JVEKQAYXFGIISZ-HOCLYGCPSA-N His-Trp-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(O)=O)C1=CN=CN1 JVEKQAYXFGIISZ-HOCLYGCPSA-N 0.000 description 1
- 101000577881 Homo sapiens Macrophage metalloelastase Proteins 0.000 description 1
- 206010020565 Hyperaemia Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- LHSGPCFBGJHPCY-UHFFFAOYSA-N L-leucine-L-tyrosine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 LHSGPCFBGJHPCY-UHFFFAOYSA-N 0.000 description 1
- HQUXQAMSWFIRET-AVGNSLFASA-N Leu-Glu-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HQUXQAMSWFIRET-AVGNSLFASA-N 0.000 description 1
- CSFVADKICPDRRF-KKUMJFAQSA-N Leu-His-Leu Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C([O-])=O)CC1=CN=CN1 CSFVADKICPDRRF-KKUMJFAQSA-N 0.000 description 1
- 102100027998 Macrophage metalloelastase Human genes 0.000 description 1
- 206010027336 Menstruation delayed Diseases 0.000 description 1
- DSWOTZCVCBEPOU-IUCAKERBSA-N Met-Arg-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CCCNC(N)=N DSWOTZCVCBEPOU-IUCAKERBSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- KLYYKKGCPOGDPE-OEAJRASXSA-N Phe-Thr-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O KLYYKKGCPOGDPE-OEAJRASXSA-N 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- DIZLUAZLNDFDPR-CIUDSAMLSA-N Pro-Cys-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H]1CCCN1 DIZLUAZLNDFDPR-CIUDSAMLSA-N 0.000 description 1
- SNIPWBQKOPCJRG-CIUDSAMLSA-N Pro-Gln-Cys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CS)C(=O)O SNIPWBQKOPCJRG-CIUDSAMLSA-N 0.000 description 1
- LCUOTSLIVGSGAU-AVGNSLFASA-N Pro-His-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O LCUOTSLIVGSGAU-AVGNSLFASA-N 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 1
- FKYWFUYPVKLJLP-DCAQKATOSA-N Ser-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO FKYWFUYPVKLJLP-DCAQKATOSA-N 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 210000000447 Th1 cell Anatomy 0.000 description 1
- LXWZOMSOUAMOIA-JIOCBJNQSA-N Thr-Asn-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N)O LXWZOMSOUAMOIA-JIOCBJNQSA-N 0.000 description 1
- XKWABWFMQXMUMT-HJGDQZAQSA-N Thr-Pro-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O XKWABWFMQXMUMT-HJGDQZAQSA-N 0.000 description 1
- KERCOYANYUPLHJ-XGEHTFHBSA-N Thr-Pro-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O KERCOYANYUPLHJ-XGEHTFHBSA-N 0.000 description 1
- AAZOYLQUEQRUMZ-GSSVUCPTSA-N Thr-Thr-Asn Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(N)=O AAZOYLQUEQRUMZ-GSSVUCPTSA-N 0.000 description 1
- HXMJXDNSFVNSEH-IHPCNDPISA-N Trp-Cys-Tyr Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)N HXMJXDNSFVNSEH-IHPCNDPISA-N 0.000 description 1
- TZXFLDNBYYGLKA-BZSNNMDCSA-N Tyr-Asp-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 TZXFLDNBYYGLKA-BZSNNMDCSA-N 0.000 description 1
- YQYFYUSYEDNLSD-YEPSODPASA-N Val-Thr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O YQYFYUSYEDNLSD-YEPSODPASA-N 0.000 description 1
- 241000405217 Viola <butterfly> Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 230000002634 anti-blastic effect Effects 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 1
- 210000004618 arterial endothelial cell Anatomy 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 108700041737 bcl-2 Genes Proteins 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000004718 centriole Anatomy 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000000994 depressogenic effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000004528 endothelial cell apoptotic process Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000008175 fetal development Effects 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 108010051307 glycyl-glycyl-proline Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 108010012058 leucyltyrosine Proteins 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 210000004924 lung microvascular endothelial cell Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000010046 negative regulation of endothelial cell proliferation Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000000683 nonmetastatic effect Effects 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 210000001147 pulmonary artery Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000002512 suppressor factor Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010408 sweeping Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 206010055031 vascular neoplasm Diseases 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- MECHNRXZTMCUDQ-RKHKHRCZSA-N vitamin D2 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)/C=C/[C@H](C)C(C)C)=C\C=C1\C[C@@H](O)CCC1=C MECHNRXZTMCUDQ-RKHKHRCZSA-N 0.000 description 1
- 235000001892 vitamin D2 Nutrition 0.000 description 1
- 239000011653 vitamin D2 Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/8146—Metalloprotease (E.C. 3.4.24) inhibitors, e.g. tissue inhibitor of metallo proteinase, TIMP
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/14—Decongestants or antiallergics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physical Education & Sports Medicine (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Rheumatology (AREA)
- Ophthalmology & Optometry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Pain & Pain Management (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种抑制新生血管生成的融合多肽及其编码基因与应用，该融合多肽是靶向性抑制金属基质蛋白酶的活性十肽、恩度的前25肽和人纤溶酶原的kringle 5结构域组成的三靶点多肽。细胞试验表明，该融合多肽可以有效的抑制内皮细胞的增殖、迁移，进而抑制新生血管的生成，从而可应用于制备治疗由新生血管增生异常产生的疾病的药物，并可用于肿瘤治疗。
Description
技术领域
本发明涉及一种血管新生的抑制肽(蛋白)，该多肽的编码基因，以及该多肽在治疗新生血管增生异常的疾病中的应用。
背景技术
一般血管生成(angiogenesis)分为病理的与正常的。正常的如胚胎发育期间除主动脉、主静脉以外的器官发育都涉及到血管形成，以维持形成器官的各类细胞的养分、氧气的供给，使之不因为养料不足或氧气不足而发生坏死。这样的情况还见诸于怀孕(pregnancy)、伤口愈合等。
另一类angiogenesis指的是病理的，比如动脉粥样硬化、实体肿瘤等。实体肿瘤从某种意义上也可以认为是一种器官发育，只是这种“器官发育”是有害的，失去控制的，无法逆转的。实体肿瘤在早期并不形成血管，在细胞发生癌变时，往往只有极少数可以顽强的存活下来，而大部分癌细胞早期都已被免疫系统或者因为自身的不稳定性以及代谢方面的障碍而很快被消灭或坏死。但癌细胞一旦存活下来，为了适应机体复杂的环境，需要对自身基因组进行调整。如癌细胞表面的凋亡受体缺失时，癌细胞将变得对营养条件不敏感，因其生长因子受体等已经可以自我激活，可以适应对苛刻条件的耐受等。这么一个过程在临床上被认为是潜伏期(dormancy)，甚至无法通过临床的手段进行监测。而癌细胞一旦经过这个过程，将变得无法抑制，表现为癌细胞群体的几何级数增长。肿瘤生长到1cm3时(这时大概有1012个癌细胞)，如果没有血管来供给养分与氧气，癌细胞最终会走向坏死。但大部分癌细胞都能突破这个极限！也就在这个数目(约1012个癌细胞)下，癌细胞群体开始分泌生长因子与各类蛋白酶，为血管的形成准备充分条件。
血管的主要成分中，单纯的血管腔主要是基质组成的复杂结构，起支架作用；在血管腔外部附着的细胞叫外皮细胞(mural cells)，而贴在内侧的叫内皮细胞(endothelial cells)。
血管的各个成分来源于骨髓里的干细胞，内皮细胞与外皮细胞都由同样的祖先分化而来(coomon precursors)，血管内皮生长因子VEGF主要使祖先细胞分化为内皮细胞，而血小板衍生生长因子PDGF则使祖先细胞分化为外皮细胞。内皮细胞还分为动脉内皮细胞与静脉内皮细胞，这种命运的决定是由Notch信号通路决定的。
形成血管还需要其他的辅助细胞，比如巨噬细胞、T细胞、单核细胞、树突状细胞、中心粒细胞、肥大细胞等免疫细胞，通过这些免疫细胞的参与来检测或抗衡生长因子的作用以及进行免疫监视，使得不正常的发育受到即时调控。但肿瘤组织作为失去平衡的一种器官发育，免疫细胞已经被肿瘤细胞分泌的各种因子教育过，而转而为肿瘤细胞服务了，不但表现为抗肿瘤免疫能力降低，反而还分泌很多金属基质蛋白酶来使肿瘤细胞周围的组织发生降解，为肿瘤的迁移做打扫道路的作用。同时，由于肿瘤细胞生长迅速，各方面调节来不及有效平衡，使得肿瘤组织的血管常常是没有足够多的外皮细胞来限制血管腔，血管腔通透性很差，经常发生出血。这种出血现象如果严重的话，常会在大小便中显示，表明已到癌的晚期，再施救已经来不及了。
肿瘤血管的形成中，内皮细胞扮演了很重要的角色。癌细胞会分泌VEGF、成纤维细胞生长因子FGF等生长因子，使得骨髓中内皮祖细胞分化为内皮细胞，而内皮细胞由于表面的各种黏附分子，常常可以一个一个黏附在一起，并可以牢牢的黏附在血管腔上。同时肿瘤组织里的巨噬细胞分泌各种蛋白酶，主要是金属基质蛋白酶(MMP)，使血管腔发生渗漏，为毛细血管的形成准备条件。毛细血管只有在现有血管的基础上才能形成。
鉴于大约一半的肿瘤都是实体肿瘤且都能形成血管，并且临床也显示抗血管增生类药物可以显著提高患者的生命时间或改善生活质量。抗肿瘤血管治疗因此而具有非常深刻而广泛的意义。因此，靶向性作用肿瘤血管的内皮细胞为肿瘤治疗提供了新的思路，具有看好的临床前景。
自Folkman提出抑制肿瘤血管可以治疗肿瘤的假说以来(N Engl J Med，1971，285：1182-1186.)，许多具有抗血管生成作用的物质相继被发现，其中尤以内源性血管生成因子备受人们的重视。
Angiostatin(AS，血管生长抑素)是纤溶酶原的一个蛋白片段，分子量38kD，是一个较强的血管生成和内皮细胞迁移、增殖抑制剂(Cell，1994，79：315-328)。人纤溶酶原含有5个kringles结构，Angiostatin包括纤溶酶原(本身含有5个kringles结构)的第1～4kringles结构，这4个kringles结构具有很高的同源性，而纤溶酶原的第5个kringle结构与前4个kringles的结构并无同源性，kringle5抑制bFGF刺激的血管内皮细胞生长的活性大于Angiostatin，可作为一个新颖的内皮细胞生长抑制剂，同时也可作为内皮细胞迁移的选择性抑制剂(J Bio Chem，1996，271：29461-29467)。Angiostatin的抑制血管生成活性主要通过kringles1～3来完成，在kringles1～3片段中kringle 1、3的活性大于kringle 2的活性。系统地运用Angiostatin能阻断裸鼠移植瘤的血管生成和肿瘤转移。研究表明不同类型的肿瘤以 相同的或完全不同的方法产生Angiostatin，一种类型肿瘤也可有多种机制产生AS。Lewis肺癌细胞依赖巨噬细胞产生的基质金属蛋白酶(MMP)产生Angiostatin。实验证明巨噬细胞MMP(或MMP12)是最有效的产生Angiostatin的MMP。研究表明，肿瘤细胞能直接产生Angiostatin或与肿瘤相关的巨噬细胞协同产生Angiostatin。机体在缺乏实体瘤的情况下也可产生Angiostatin。Angiostatin的抗血管生成活性可能具有种属特异性。用人AS治疗鼠血管瘤，发现AS也明显抑制了原发的非转移瘤的生长，这种抑制作用是通过诱发肿瘤细胞的凋亡而实现，对肿瘤细胞的增生并无抑制，AS通过多种形式诱导内皮细胞的凋亡。
Endostatin(恩度，内皮生长抑素)分子量约为18kD，由184个氨基酸残基构成。作为胶原蛋白18的球形羧基末端的一部分，Endostatin有着广泛的组织分布，其中肝细胞也是其来源之一。Endostatin最主要的生物学功能是抑制内皮细胞的生长、移动及生血管作用。Endostatin作为内源性的抗血管生成活性物质，可以抑制Lewis肺癌细胞、T241纤维肉瘤细胞及EOMA细胞在肿瘤移植动物体内的生长。其对实体肿瘤的抑制作用机理较复杂，已知Zn2+在Th1细胞的影响下可以激活内源性Endostatin抑制肿瘤的生长，并可明显抑制VEGF诱导的内皮细胞的生长和移动，对VEGF诱导的细胞移动产生多步骤的抑制。PhanabalM等报道Endostatin可使内皮细胞停留在细胞生长周期的G1期，并可引起人HUVEC及人HMVEC细胞的凋亡，对非内皮细胞无此作用(J Bio Chem.1999，274：11721-11726)。Endostatin可引起牛肺动脉内皮细胞凋亡并导致bcl-2基因及Bcl2-xl抗凋亡蛋白表达水平的明显下降但对Bax蛋白无显著影响，提示Endostatin可通过诱使内皮细胞发生凋亡来抑制新生血管生长。Endostatin可能通过结合参与细胞生长因子信号转导的细胞外生长因子受体而抑制细胞生长因子信号内传，从而抑制肿瘤细胞生长与移动，而发挥强效的抗肿瘤血管生成作用。
早年，通过筛选研究者发现一个人工十肽在抑制基质金属蛋白酶方面有效，并在小鼠肿瘤模型中显示出治疗效果。
基于国内外大量关于抗肿瘤血管形成和癌细胞迁移的理论与应用研究的基础上，我们曾设计和研制出既能抑制内皮细胞增殖，又能有效抑制内皮细胞迁移的双靶点抗肿瘤多肽(参见中国专利ZL 200410006549.8)。在此基础上，本发明设计出更强效的三靶点抗肿瘤多肽。
发明内容
本发明的目的是提供一种人工合成的融合多肽(蛋白)及其编码基因，该融合多肽具 有抑制新生血管生成的作用，进一步的，利用该融合多肽来治疗新生血管增生异常的疾病，如类风湿，二型糖尿病引发的视网膜充血，以及肿瘤等。
在本发明的第一方面，本发明所提供的抑制新生血管生成的多肽，命名为MEK，是具有序列表中SEQ ID No.2所示氨基酸序列的多肽。
SEQ ID No.2所示氨基酸序列多肽由125个氨基酸残基组成。本领域的技术人员应当理解，将SEQ ID No.2的氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加而衍生的多肽，在不涉及关键位点的情况下，可具有与SEQ ID No.2所示氨基酸序列的多肽相同或相近的活性。因此，本发明的抑制新生血管生成的多肽也包括此类衍生多肽，而不局限于SEQ ID No.2所示氨基酸序列的多肽。
本发明的抑制新生血管生成的多肽MEK是一个三靶点多肽，具有如下排列：
十肽-endo的前25肽-kringle 5
其中：十肽，是靶向性抑制金属基质蛋白酶(MMP2/MMP9)的活性肽；endo的前25肽，是来源于恩度(endostatin)基因编码的蛋白序列的前25个氨基酸残基，此25个氨基酸残基具有全长恩度的功效；kringle 5(简称k5)是来源于人纤溶酶原的第五个结构域，是人纤溶酶原最具活力的结构域。
在本发明的第二方面，本发明还提供了上述多肽的编码基因，可以是序列表中SEQ ID No.2所示的多核苷酸，也可以是其他的编码相同多肽的多核苷酸。
序列表中SEQ ID No.1所示的多核苷酸由375个碱基组成，编码SEQ ID No.2所示的融合多肽。
含有本发明多肽的编码基因的表达载体及细胞系均属于本发明的保护范围。
本发明的多肽MEK是将三个高活性的特异性作用内皮细胞的分子用基因工程手段整合在一起，细胞实验证明，相比于我们较早得到的双靶点基因工程重组蛋白RK5，，该多肽可以更有效的抑制内皮细胞的增殖、迁移，进而抑制新生血管的生成。因此，MEK可应用于制备治疗由新生血管增生异常产生的疾病的药物，如用于治疗类风湿、二型糖尿病引发的视网膜充血、肿瘤等。在肿瘤治疗中，可以更有效的抑制肿瘤的生长和转移，为肿瘤治疗增加了更高效的新药。
附图说明
图1是实施例1提纯的MEK融合多肽的电泳图谱。
图2是实施例2中MEK对细胞增殖抑制试验的结果。
图3是实施例3中MEK对细胞迁移抑制试验的结果。
图4是实施例4中在光学显微镜下观察到的未长微细血管的内皮细胞形态，其中A是未经染色的图像，B是结晶紫染色后的图像。
图5是实施例4中对生长出的毛细血管用不同蛋白(RK5和MEK)处理前后的对照图。
具体实施方式
下面通过实施例进一步详细描述本发明，但不以任何方式限制本发明的范围。
实施例1、融合多肽MEK的表达
1、融和多肽(蛋白)的设计
将一个抑制金属基质蛋白酶(MMP2或MMP9)十肽，endostatin的前25肽以及人纤溶酶原的k5序列，按照顺序十肽-25肽-K5连在一起，形成了MEK的融合序列，如序列表中SEQ ID No.2所示，其中：最前端的10个氨基酸残基(CTTHWGFTLC)组成的是抑制金属基质蛋白酶MMP2/9的活性小肽；接下来的25个氨基酸残基组成的是endostatin的前25肽；随后的9个氨基酸残基(ERETPSEED)是连接肽序列；最后的81个氨基酸残基组成的是人纤溶酶原的k5结构域。
2、融合多肽表达载体的构建
设计下述引物：
引物1：5’-CTCGAGAAAAGATGTACAACTCACTGGGGTTTCACACTTTGCCACAGC CACCGCGACTTC-3’(下划线部分是XhoI的酶切位点)；
引物2：5’-TTCGGAAGGAGTCTCTCTTTCGCCCCGCATGCCGCCTGACAG-3’；
引物3：5’-CTGTCAGGCGGCATGCGGGGCGAAAGAGAGACTCCTTCCGAA-3’；
引物4：5’-GAATTCTCAGGCACACTGAGGGACATC-3’(下划线部分是EcoRI的酶切位点)。
以引物1为上游引物，引物2为下游引物，人脐静脉内皮细胞(HUVEC)(北大医院肿瘤研究所赠与)的cDNA为模板PCR得到十肽-25肽融合片段的基因序列。以引物3为上游引物，引物4为下游引物，质粒pPIC9K-RK5(本实验室构建并保存，构建过程参见中国专利ZL200410006549.8说明书实施例1部分)为模板PCR得到连接肽和k5结构域融合片段的基因序列。将两个PCR的产物混合在一起作为模板，以引物1为上游引物，引物4为下游引物再次PCR获得编码全长三靶点融合多肽的DNA片段，将该DNA片段连入T载体(购自北京Genestar公司)并转化细胞进行扩增，然后提取质粒用XhoI和EcoRI双酶切将 编码三靶点融合多肽的核酸片段切下，与同样用XhoI和EcoRI酶切的高效表达质粒pPIC9K(购自美国Invitrogen公司)进行连接，得到重组质粒pPIC9K-MEK。
通过上述基因工程方法将编码十肽-25肽-k5核苷酸序列融合在一起，形成的融合核苷酸序列如序列表中SEQ ID No.1所示。所得到的重组质粒pPIC9K-MEK通过基因测序鉴定，确定其序列的正确性。
3、融合多肽的生产
将重组质粒pPIC9K-MEK转染酵母细胞GS115(毕赤酵母菌，购自Invitrogen公司)。转染后筛选能稳定表达融合蛋白MEK的克隆株。将这样的菌株收藏保存作为MEK的生产菌株。
将筛选得到的转化子分别接种于200mL BMGY(甘油培养基)液体培养基中，30℃振荡培养至OD600为2.0以上。无菌状态下收集菌液，6000r/min离心6min，去上清，用60mLBMMY(甲醇培养基)酵母培养基重悬细胞，30℃，振荡培养4天，每天在培养液中加入终浓度为1％的甲醇诱导表达。
发酵液5000g离心收集上清，加入硫酸氨至终浓度为85％进行硫酸氨沉淀，10000g离心收集沉淀。沉淀物重悬于50ml 0.05M PBS(pH 7.4，含1.7M硫酸氨)，上疏水柱Phenyl Sepharose 6 Fast Flow(15×60mm)，用0.05M PBS(pH 7.4)洗脱，收集。洗脱液在0.05M PBS(pH 8)中透析，然后上离子交换柱DEAE Sepharose Fast Flow column。用0.05M PBS(pH 8，with 1M NaCl)洗脱，收集，15％SDS-PAGE鉴定，考马斯亮蓝染色。鉴定结果如图1所示，其中最左侧的泳道是分子量标记物，每个泳道的蛋白染色代表相应收集管中蛋白的纯度。
实施例2、MEK对细胞增殖抑制的试验
HUVEC是一种血管内皮细胞，该细胞是公认的用于抑制血管生成的研究材料。本试验使用的材料：内皮细胞株(HUVEC)为新生儿脐带分离，10代以内，北大医院肿瘤研究所赠与；MTT购自sigma公司；细胞培养液来自GIBCO公司；小牛血清来自GIBCO公司
方法：内皮细胞HUVEC在M199，20％FBS培养基中培养。将培养好的细胞按1.2×105个均等转移至12孔细胞板中，每孔1ml培养基培养两天，加入不同浓度的k5、RK5、MEK(浓度分别为0.125μg/ml、0.25μg/ml、0.75μg/ml、1.5μg/ml)，处理72小时后每孔加入50ml MTT(5μg/ml)作用4小时。然后吸干培养基加入1.6ml无水乙醇，570nm检测紫外吸收。
试验结果如图2所示。从图2可以看出在0.25μg-1.5μg浓度下，MEK具有很好的抑制内皮细胞增殖的能力。
实施例3、MEK对细胞转移抑制的试验
材料：内皮细胞株(HUVEC)为新生儿脐带分离，24孔悬挂式Transwell迁移小室购自Millipore公司，matrigel基质胶购自BD公司，结晶紫购自Merk公司，bFGF、VEGF购自sigma公司。
方法：将matrigel基质胶4℃融化后，按照1∶3(matrigel基质胶∶无血清M199)的体积比用无血清的M199稀释，上室加入50μg稀释后的基质胶。过夜凝固，上室用3％FBS的M199接种2×105个细胞，下室加入含20％FBS的M199，60ng/ml VEGF，60ng/ml bFGF，并在下室加入蛋白，迁移72小时内，用4％甲醛固定20分钟后，用去离子水溶解的0.1％结晶紫染色，充分用PBS洗涤后，进行拍照或统计。
试验结果如图3所示，表明在浓度为1μg/ml的蛋白处理下，MEK对HUVEC的迁移抑制大于RK5。
实施例4、MEK对HUVEC形成毛细血管能力影响的试验
材料：内皮细胞株(HUVEC)为新生儿脐带分离，10代内可用。bFGF购自sigma公司。
方法：将matrigel胶按照1∶2体积比用无血清M199稀释后，加入60ng/ml bFGF，铺至24孔板，2小时凝固后，将HUVEC细胞接种在铺有matrigel胶的孔里。大约2天内形成毛细血管样形状。用蛋白处理48-72小时。处理完后拍照。
结果如下：在基质(matrix)中，生长因子会促进内皮细胞产生毛细血管样的细胞形态。可以在体外模拟血管形成的状态。双靶点RK5可以微弱抑制血管形成，而三靶点MEK可以强烈抑制血管形态的生成。图4是在光学显微镜下观察到的未长微细血管的内皮细胞形态，其中A是未经染色的图像，B是结晶紫染色后的图像。图5是在生长毛细血管时用不同蛋白处理前后的对照图，其中A和C分别是用RK5蛋白处理前和1微克RK5蛋白处理72h后的图像，B和D分别是用MEK蛋白处理前和1微克MEK蛋白处理72h后的图像，可以看出MEK可以较强抑制内皮细胞迁移能力。
SEQUENCE LISTING
<110>北京大学
<120>一种抑制新生血管生成的融合多肽及其编码基因与应用
<130>JSP100154
<160>2
<170>PatentIn version 3.1
<210>1
<211>125
<212>PRT
<213>人工序列
<400>1
Cys Thr Thr His Trp Gly Phe Thr Leu Cys His Ser His Arg Asp Phe
1 5 10 15
Gln Pro Val Leu His Leu Val Ala Leu Asn Ser Pro Leu Ser Gly Gly
20 25 30
Met Arg Gly Glu Arg Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly
35 40 45
Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr
50 55 60
Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe
65 70 75 80
Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg
85 90 95
Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro
100 105 110
Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala
115 120 125
<210>2
<211>375
<212>DNA
<213>人工序列
<400>2
tgtacaactc actggggttt cacactttgc cacagccacc gcgacttcca gccggtgctc 60
cacctggttg cgctcaacag ccccctgtca ggcggcatgc ggggcgaaag agagactccc 120
tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcaaccact 180
gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 240
actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 300
gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 360
gtccctcagt gtgcc 375
Claims (6)
1.一种融合多肽，其氨基酸序列如SEQ ID No.2所示。
2.一种融合多肽的编码基因，是编码SEQ ID No.2所示氨基酸序列的多核苷酸。
3.如权利要求2所述的编码基因，其特征在于，所述基因的碱基序列如SEQ ID No.1所示。
4.含权利要求2所述基因的表达载体。
5.含权利要求2所述基因的细胞系。
6.权利要求1所述融合多肽在制备治疗抑制新生血管异常生长引起的疾病的药物中的应用。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101820014A CN101870734B (zh) | 2010-05-25 | 2010-05-25 | 一种抑制新生血管生成的融合多肽及其编码基因与应用 |
| PCT/CN2010/001044 WO2011147057A1 (zh) | 2010-05-25 | 2010-07-13 | 抗血管生成的融合多肽，其基因和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101820014A CN101870734B (zh) | 2010-05-25 | 2010-05-25 | 一种抑制新生血管生成的融合多肽及其编码基因与应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101870734A CN101870734A (zh) | 2010-10-27 |
| CN101870734B true CN101870734B (zh) | 2012-06-20 |
Family
ID=42995831
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101820014A Active CN101870734B (zh) | 2010-05-25 | 2010-05-25 | 一种抑制新生血管生成的融合多肽及其编码基因与应用 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN101870734B (zh) |
| WO (1) | WO2011147057A1 (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102532326B (zh) * | 2011-11-18 | 2017-06-09 | 南京大学 | 一种肿瘤靶向的人纤溶酶原Kringle5变体及其应用 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998034634A1 (en) * | 1997-02-06 | 1998-08-13 | Entremed, Inc. | Compositions and methods for inhibiting cellular proliferation |
| CN1355196A (zh) * | 2000-12-01 | 2002-06-26 | 复旦大学 | 一种新的多肽——吉本猿白血病病毒受体2－14.63和编码这种多肽的多核苷酸 |
| CN1502625A (zh) * | 2002-11-20 | 2004-06-09 | 中国人民解放军军事医学科学院放射医 | 基质金属蛋白酶2的小肽抑制剂 |
| CN1666997A (zh) * | 2004-03-08 | 2005-09-14 | 北京大学 | 一种融合蛋白及其编码基因与应用 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7122181B2 (en) * | 2000-12-19 | 2006-10-17 | Research Development Foundation | Lentiviral vector-mediated gene transfer and uses thereof |
| KR20080080684A (ko) * | 2000-12-19 | 2008-09-04 | 리서치 디벨럽먼트 파운데이션 | 렌티바이러스 벡터-매개된 유전자 전달 및 이를 위한 조성물 |
| CN102264755A (zh) * | 2007-08-08 | 2011-11-30 | 得克萨斯大学体系董事会 | 靶向vegfr-1/nrp-1的肽 |
| JP2011525491A (ja) * | 2008-06-20 | 2011-09-22 | ザ ボード オブ リージェンツ オブ ザ ユニバーシティー オブ テキサス システム | Ｃｒｋｌ標的化ペプチド |
-
2010
- 2010-05-25 CN CN2010101820014A patent/CN101870734B/zh active Active
- 2010-07-13 WO PCT/CN2010/001044 patent/WO2011147057A1/zh active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998034634A1 (en) * | 1997-02-06 | 1998-08-13 | Entremed, Inc. | Compositions and methods for inhibiting cellular proliferation |
| CN1355196A (zh) * | 2000-12-01 | 2002-06-26 | 复旦大学 | 一种新的多肽——吉本猿白血病病毒受体2－14.63和编码这种多肽的多核苷酸 |
| CN1502625A (zh) * | 2002-11-20 | 2004-06-09 | 中国人民解放军军事医学科学院放射医 | 基质金属蛋白酶2的小肽抑制剂 |
| CN1666997A (zh) * | 2004-03-08 | 2005-09-14 | 北京大学 | 一种融合蛋白及其编码基因与应用 |
Non-Patent Citations (2)
| Title |
|---|
| 王勇等.核转录因子抑制剂对大鼠角膜新生血管的抑制作用.《中华眼科杂志》.2005,第41卷(第12期),1124-1128. * |
| 雷春涛等.奥曲肽抑制小鼠视网膜新生血管形成的研究.《眼科研究》.2004,第22卷(第06期),623-625. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101870734A (zh) | 2010-10-27 |
| WO2011147057A1 (zh) | 2011-12-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| De Larco et al. | A potential role for interleukin-8 in the metastatic phenotype of breast carcinoma cells | |
| ES2203668T3 (es) | Variantes de glicosilacion del activador de plasminogeno de tejido con propiedades terapeuticas mejoradas. | |
| JP5752033B2 (ja) | 遺伝子組み換えゼラチンを含む血管新生誘導剤 | |
| CN104045717A (zh) | 一种肿瘤血管阻断剂多肽、基因、表达载体及其应用 | |
| CN102311502A (zh) | 一种抑制血管新生或生长的融合蛋白及其医疗应用 | |
| Gyorffy et al. | Adenoviral vector expressing murine angiostatin inhibits a model of breast cancer metastatic growth in the lungs of mice | |
| Park et al. | Anti-proliferative effect of engineered neural stem cells expressing cytosine deaminase and interferon-beta against lymph node-derived metastatic colorectal adenocarcinoma in cellular and xenograft mouse models | |
| CN103319605B (zh) | 一种肝靶向肽与血管生成抑制因子融合蛋白及其制备方法与应用 | |
| CN101870734B (zh) | 一种抑制新生血管生成的融合多肽及其编码基因与应用 | |
| EP3041860A1 (en) | Semaphorin 3c variants, compositions comprising said variants and methods of use thereof | |
| CN102140473A (zh) | 抗肿瘤的核酸与多肽及其应用 | |
| CN102286074B (zh) | 一种cd13靶向肽ngr及其应用 | |
| CN100537765C (zh) | 重组人血管抑素k1-3的制备工艺及其制品在肿瘤治疗药物中的应用 | |
| Sun et al. | Blood vessel maturation in health and disease and its implications for vascularization of engineered tissues | |
| CN102085378B (zh) | hfgl2抑制剂在制备治疗肝癌的药物中的应用 | |
| JP2013517782A (ja) | 因子ｖｉｉ融合ポリペプチド | |
| US9850500B2 (en) | Recombinant adenoviruses capable of regulating angiogenesis | |
| CN102260352B (zh) | 靶向性白细胞介素融合蛋白及其制备方法与应用 | |
| CA2430625A1 (en) | A virus which can express tumor angiostatin factor with high efficiency in specific tumor cells and the use of it | |
| TWI541252B (zh) | 因子ｖｉｉ／ｖｉｉａ之量產方法 | |
| CN104479024A (zh) | 新型抗瘤融合肽及其用途 | |
| CN107098964A (zh) | 一种肿瘤特异性重组水蛭素及其制备方法和用途 | |
| KR101228668B1 (ko) | 혈관신생 억제 활성을 갖는 펩타이드 및 이의 용도 | |
| CN107216385A (zh) | 肿瘤特异性重组水蛭素及其制备方法和用途 | |
| CN102924605B (zh) | Telkp和lkp融合免疫毒素、原核表达载体及制备 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |