CN101851294B - Cell-penetrating peptide (Arg) 9 and lidamycin fusion protein (Arg) 9-LDP - Google Patents
Cell-penetrating peptide (Arg) 9 and lidamycin fusion protein (Arg) 9-LDP Download PDFInfo
- Publication number
- CN101851294B CN101851294B CN201010177517XA CN201010177517A CN101851294B CN 101851294 B CN101851294 B CN 101851294B CN 201010177517X A CN201010177517X A CN 201010177517XA CN 201010177517 A CN201010177517 A CN 201010177517A CN 101851294 B CN101851294 B CN 101851294B
- Authority
- CN
- China
- Prior art keywords
- arg
- ldp
- cell
- fusion protein
- fusion rotein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 62
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 62
- DGGZCXUXASNDAC-QQNGCVSVSA-N C-1027 chromophore Chemical compound COc1cc2OC(=C)C(=O)Nc2c(c1)C(=O)O[C@H]3COC(=O)C[C@H](N)c4cc(O)c(O[C@@H]5C#C\C=C\3/C#CC6=CC=C[C@]56O[C@@H]7OC(C)(C)[C@H]([C@@H](O)[C@H]7O)N(C)C)c(Cl)c4 DGGZCXUXASNDAC-QQNGCVSVSA-N 0.000 title claims abstract description 45
- 229960005535 lidamycin Drugs 0.000 title claims abstract description 44
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 title claims abstract description 17
- 102000020313 Cell-Penetrating Peptides Human genes 0.000 title claims abstract description 17
- XUNKPNYCNUKOAU-VXJRNSOOSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]a Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O XUNKPNYCNUKOAU-VXJRNSOOSA-N 0.000 title claims abstract description 4
- 229920002485 Peptide(-Arg) Polymers 0.000 title abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 31
- 150000001413 amino acids Chemical class 0.000 claims abstract description 10
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 3
- 230000004927 fusion Effects 0.000 claims description 68
- 238000000034 method Methods 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 10
- RZTAMFZIAATZDJ-UHFFFAOYSA-N felodipine Chemical compound CCOC(=O)C1=C(C)NC(C)=C(C(=O)OC)C1C1=CC=CC(Cl)=C1Cl RZTAMFZIAATZDJ-UHFFFAOYSA-N 0.000 claims description 6
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 2
- 239000002246 antineoplastic agent Substances 0.000 claims 2
- 229940041181 antineoplastic drug Drugs 0.000 claims 2
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 claims 1
- 238000003808 methanol extraction Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 15
- 102000006410 Apoproteins Human genes 0.000 abstract description 10
- 108010083590 Apoproteins Proteins 0.000 abstract description 10
- 208000032612 Glial tumor Diseases 0.000 abstract description 8
- 206010018338 Glioma Diseases 0.000 abstract description 8
- 239000003814 drug Substances 0.000 abstract description 5
- 241001465754 Metazoa Species 0.000 abstract description 3
- 238000001727 in vivo Methods 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 2
- 238000012404 In vitro experiment Methods 0.000 abstract 1
- 230000002147 killing effect Effects 0.000 abstract 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 117
- 210000004027 cell Anatomy 0.000 description 101
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 39
- 206010028980 Neoplasm Diseases 0.000 description 18
- 235000018102 proteins Nutrition 0.000 description 17
- 102000004169 proteins and genes Human genes 0.000 description 17
- 241000894006 Bacteria Species 0.000 description 15
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 14
- 210000004881 tumor cell Anatomy 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 9
- 230000006907 apoptotic process Effects 0.000 description 9
- 239000004202 carbamide Substances 0.000 description 9
- 210000003000 inclusion body Anatomy 0.000 description 9
- 108090000765 processed proteins & peptides Proteins 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- 241000699660 Mus musculus Species 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 8
- 210000000170 cell membrane Anatomy 0.000 description 8
- 238000011580 nude mouse model Methods 0.000 description 8
- 239000012466 permeate Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000012148 binding buffer Substances 0.000 description 7
- 230000003013 cytotoxicity Effects 0.000 description 7
- 231100000135 cytotoxicity Toxicity 0.000 description 7
- 201000007270 liver cancer Diseases 0.000 description 7
- 208000014018 liver neoplasm Diseases 0.000 description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000000502 dialysis Methods 0.000 description 5
- 230000009422 growth inhibiting effect Effects 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000003259 recombinant expression Methods 0.000 description 5
- 238000004153 renaturation Methods 0.000 description 5
- 241000193830 Bacillus <bacterium> Species 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 102100026679 Carboxypeptidase Q Human genes 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 210000001072 colon Anatomy 0.000 description 4
- 150000002460 imidazoles Chemical class 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 239000002504 physiological saline solution Substances 0.000 description 4
- 229920000724 poly(L-arginine) polymer Polymers 0.000 description 4
- 108010011110 polyarginine Proteins 0.000 description 4
- 239000011535 reaction buffer Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 3
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 3
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000022131 cell cycle Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 230000000149 penetrating effect Effects 0.000 description 3
- 230000035515 penetration Effects 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 230000000979 retarding effect Effects 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108010040476 FITC-annexin A5 Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 210000000633 nuclear envelope Anatomy 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 101710085003 Alpha-tubulin N-acetyltransferase Proteins 0.000 description 1
- 101710085461 Alpha-tubulin N-acetyltransferase 1 Proteins 0.000 description 1
- 101150019028 Antp gene Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 238000011729 BALB/c nude mouse Methods 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 208000013641 Cerebrofacial arteriovenous metameric syndrome Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 206010010254 Concussion Diseases 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000009331 Homeodomain Proteins Human genes 0.000 description 1
- 108010048671 Homeodomain Proteins Proteins 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- -1 MAP Proteins 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 230000018199 S phase Effects 0.000 description 1
- CWHJIJJSDGEHNS-MYLFLSLOSA-N Senegenin Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)C(CC[C@]4(CCC(C[C@H]44)(C)C)C(O)=O)=C4[C@@H](CCl)C[C@@H]3[C@]21C CWHJIJJSDGEHNS-MYLFLSLOSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 101710192266 Tegument protein VP22 Proteins 0.000 description 1
- 101710175714 Tyrosine aminotransferase Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000003277 amino acid sequence analysis Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 201000003959 cecum carcinoma Diseases 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 201000010989 colorectal carcinoma Diseases 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229940079938 nitrocellulose Drugs 0.000 description 1
- 108010043655 penetratin Proteins 0.000 description 1
- MCYTYTUNNNZWOK-LCLOTLQISA-N penetratin Chemical compound C([C@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(N)=O)C1=CC=CC=C1 MCYTYTUNNNZWOK-LCLOTLQISA-N 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000002831 pharmacologic agent Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 108010066925 sleep-promoting factor B Proteins 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000009871 tenuigenin Substances 0.000 description 1
- 108010062760 transportan Proteins 0.000 description 1
- PBKWZFANFUTEPS-CWUSWOHSSA-N transportan Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(N)=O)[C@@H](C)CC)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CN)[C@@H](C)O)C1=CC=C(O)C=C1 PBKWZFANFUTEPS-CWUSWOHSSA-N 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000004804 winding Methods 0.000 description 1
- FBTUMDXHSRTGRV-ALTNURHMSA-N zorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(\C)=N\NC(=O)C=1C=CC=CC=1)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 FBTUMDXHSRTGRV-ALTNURHMSA-N 0.000 description 1
- 229960000641 zorubicin Drugs 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to fusion protein (Arg)9-LDP and reinforced fusion protein (Arg)9-LDP-AE thereof. The fusion protein consists of cell-penetrating peptide (Arg)9, lidamycin apoprotein LDP and carboxyl terminal hexamerichistidine tail, wherein the full length of gene is 387bp, and 129 amino acids are encoded; the reinforced fusion protein is formed by fusion protein (Arg)9-LDP with molecular weight of about 14.5kDa and activated enediyne chromophore AE with molecular weight of 843Da; in-vivo and in-vitro experiment results prove that the fusion protein and reinforced fusion protein thereof have intense killing effect on glioma cells, and have remarkable curative effect in in-vivo animal experiments, and are expected to be developed into clinic efficient glioma treating medicament.
Description
Technical field:
The present invention relates to a kind of fusion rotein (Arg)
9-LDP, specifically, said fusion rotein contains cell-penetrating peptides (Arg)
9Aminoacid sequence with Lidamycin agon albumen LDP;
The invention still further relates to a kind of energized fusion protein that contains Enediyne chromophoric group (AE) (Arg) that can strengthen through molecule preparation
9-LDP-AE;
The invention still further relates to the application in oncotherapy of said fusion rotein and energized fusion protein thereof.
Background technology:
Lidamycin (LDM) (lidamycin, LDM, claim again C-1027), from Qianjiang county, China Hubei Province soil, to separate a strain styreptomyces globispotus strain Streptiomyces globisporus C-1027 who obtains (to be preserved in CGMCC on April 3rd, 2007, culture presevation number is: the Enediyne Antibiotic that CGMCC No.0704) produces, by enediyne chromophoric group (AE) and 110 apoprotein (LDP) two portions formations that amino-acid residue forms, both are by the hydrophobic interaction combination of chromophoric group core and apoprotein hydrophobic amino acid residue.Chromophoric group has strong activity; but unstable; apoprotein is folded into a hydrophobic pocket and protects chromophoric stability; detachable and the reconstruction of apoprotein and chromophoric group; molecule after the reconstruction and natural molecule are quite active; the detachable molecular structure characteristics of this uniqueness are convenient to DNA restructuring and molecule and are strengthened.
Lidamycin (LDM) has strong lethal effect to the tumour cell of vitro culture, presses IC
50Compare, stronger more than 1000 times than Zorubicin and mitomycin, experiment shows lidamycin (LDM) to Transplantable Murine colorectal carcinoma 26 and transplants in people's liver cancer Bel-7402 of nude mice and carcinoma of cecum Hee-8693 etc. in the animal body significant curative effect.Molecular pharmacology studies show that its main mechanism is to cause dna break, thus cell death inducing.Because the selective permeability of cytolemma and the existence of hemato encephalic barrier, lidamycin (LDM) there is not yet relevant report both domestic and external up to now to the lethal effect of cerebral tissue tumour.
Cell-penetrating peptides (cell penetrating peptides, CPPs) be that a class is directly passed the polypeptide that cytolemma enters cell with non-acceptor dependence mode, non-classical endocytosis mode, their length generally is no more than 30 amino acid and is rich in basic aminoacids, can guide the different kind organism active substance promptly pass cytolemma and (or) nuclear membrane.The CPPs that has been found that at present and synthesize has tens of kinds, is broadly divided into three classes according to its source, and (one) derives from the penetrating peptide of homeodomain, such as Penetratin; (2) derive from the natural albumen that exists of virus or microorganism, such as TAT, Antp, VP22 and PreS2 etc.; (3) the various small peptides of synthetic such as poly arginine, MAP, Transportan and various based on the synthetic peptide section of signal sequence are such as MTS etc.Research prompting so far, CPPs can mediate a series of bioactive moleculess in external or body such as protein, peptide class, oligonucleotide, DNAs, plasmid and liposome etc. enter various different tissues and cell, and these important discoveries will be applied to the research fields such as pharmacy, gene therapy, cytobiology new research direction is provided for hydrophilic protein, oligopeptides and oligonucleotide.
The amino acid sequence analysis of cell-penetrating peptides is found cell-penetrating peptides all is to be rich in arginicly, therefore, arginine may play an important role in cell-penetrating peptides permeates cell membranes or nuclear membrane process, poly arginine (Arg)
9Be exactly the cell-penetrating peptides of synthetic on this basis, can carry many kinds of substance and enter cell, have and well wear film activity.All be poly arginine (Arg) in the research in the past by chemical process
9With albumen coupling, by engineered method with poly arginine (Arg)
9Form fusion rotein with albumen, had not yet to see relevant report.
The present invention adopts engineered method express cell penetrating peptide (Arg)
9Fusion rotein (Arg) with Lidamycin agon albumen
9-LDP compares with chemical synthesis process, and the living albuminiferous cost of gene engineering method is lower, and output is higher, is easy to purifying and concentrated, without the impact of intermediate by-products.
One of purpose of the present invention is to make up cell-penetrating peptides (Arg)
9Fusion rotein (Arg) with Lidamycin agon albumen
9-LDP;
Two of purpose of the present invention is further to carry out the molecule reinforcement, structure energized fusion protein (Arg) with having highly active activated form enediyne (AE)
9-LDP-AE;
Summary of the invention:
Fusion rotein provided by the present invention (Arg)
9-LDP is by cell-penetrating peptides (Arg)
9Consist of full length gene 387bp, 129 amino acid of encoding with Lidamycin agon albumen LDP; Its energized fusion protein (Arg)
9-LDP-AE is assembled by fusion rotein and activated form enediyne chromophoric group AE, and its preparation method is:
The present invention utilizes engineered method express cell penetrating peptide (Arg)
9With fusion rotein and the energized fusion protein thereof of Lidamycin agon albumen, be used for gliomatous pharmacological agent.
Fusion rotein provided by the present invention (Arg)
9-LDP and energized fusion protein thereof (Arg)
9-LDP-AE, its preparation method may further comprise the steps:
Fusion rotein (Arg)
9The structure of-LDP recombinant expression plasmid;
Fusion rotein (Arg)
9-LDP is at colon bacillus BL21 (DE3) star
TMIn abduction delivering;
Fusion rotein (Arg)
9The affinitive layer purification of-LDP and renaturation;
Energized fusion protein (Arg)
9The preparation of-LDP-AE separates.
The present invention also provides fusion rotein (Arg)
9-LDP and energized fusion protein thereof (Arg)
9The biologic activity experimental study of-LDP-AE comprises:
Fusion rotein (Arg)
9The film ability of wearing of-LDP is measured;
Mtt assay detection enhancement fusion rotein (Arg)
9-LDP-AE is to the cytotoxicity of U87 cell, Bel-7402 cell, MCF-7 cell and A549 cell;
Energized fusion protein (Arg)
9-LDP-AE is on the impact of human glioma U87 cell cycle;
Energized fusion protein (Arg)
9-LDP-AE is to the apoptosis-induced effect of U87;
Fusion rotein (Arg)
9-LDP and energized fusion protein thereof (Arg)
9-LDP-AE is to the growth-inhibiting effect of rat liver cancer H22;
Fusion rotein (Arg)
9-LDP and energized fusion protein thereof (Arg)
9-LDP-AE is to the growth-inhibiting effect of human glioma's transplanted tumor in nude mice.
The invention effect:
Advantage of the present invention and positively effect are, the fusion rotein (Arg) that using gene engineering technique and molecule intensifying technology route prepare
9-LDP and energized fusion protein thereof (Arg)
9-LDP-AE is the fusion rotein medicine with penetration cell film activity, can guide the lidamycin (LDM) permeates cell membranes, promotes apoptosis of tumor cells, suppresses in vivo tumor growth, has good potential applicability in clinical practice.
Description of drawings:
Fig. 1: pET30-(arg)
9Ldp recombinant expression vector building process
Fig. 2: pcr amplification (arg)
9The ldp gene fragment
Wherein: 1-DNA molecular weight standard DL2000 (bp);
3-(arg)
9The ldp gene;
Fig. 3: pET30-(arg)
9The endonuclease analysis of ldp recombinant expression vector
Wherein: 1-DNA molecular weight standard DL2000 (bp);
2-recombinant plasmid pET30-(arg)
9Ldp;
3-recombinant plasmid pET30-(arg)
9Ldp+Nde I;
4-recombinant plasmid pET30-(arg)
9Ldp+Xho I;
5-recombinant plasmid pET30-(arg)
9Lap+Nde I/Xho I;
6-DNA molecular weight standard DL15000 (bp);
Fig. 4: fusion rotein (Arg)
9The SDS-PAGE of-LDP expression product analyzes
Wherein: 1,10-IPTG induces rear recombinant bacterium whole-cell protein;
2,9-IPTG induces rear recombinant bacterium inclusion body protein;
3-IPTG induces soluble proteins in the rear restructuring mycetocyte;
4-IPTG induces rear recombinant bacterium pericentral siphon chamber albumen;
5-IPTG induces white protein on the rear recombinant bacterium nutrient solution;
6-molecular weight of albumen standard (kDa);
7-IPTG induces front recombinant bacterium inclusion body protein;
8-IPTG induces front recombinant bacterium whole-cell protein;
Fig. 5: fusion rotein (Arg)
9The immunoblotting assay of-LDP expression product
Wherein: 1-IPTG induces rear recombinant bacterium bacterium liquid whole-cell protein;
2-IPTG induces rear recombinant bacterium inclusion body protein;
3-IPTG induces soluble proteins in the rear restructuring mycetocyte;
4-IPTG induces rear recombinant bacterium pericentral siphon chamber albumen;
5-IPTG induces white protein on the rear recombinant bacterium nutrient solution;
Fig. 6: fusion rotein (Arg)
9The SDS-PAGE of-LDP purified product analyzes
Wherein: 1-molecular weight of albumen standard (kDa);
2-IPTG induces rear recombinant bacterium whole-cell protein;
3-is through the recombinant bacterium inclusion body of urea dissolving;
The fusion rotein of 4～7-purifying (Arg)
9-LDp;
Fig. 7: fluoroscopic examination (Arg)
9-LDP fusion rotein permeates cell membranes ability
Wherein: the 1-Bel-7402 cell;
The 2-A549 cell;
The 3-H460 cell;
The 4-OVCAR3 cell;
The 5-U87 cell.
Fig. 8: fluoroscopic examination (Arg)
9The location of-LDP fusion rotein in tumour cell
Wherein: the 1-A549 cell;
The 2-H460 cell;
The 3-OVCAR3 cell.
Fig. 9: fluoroscopic examination different time (Arg)
9-LDP fusion rotein penetrates the tumour cell ability
Wherein: A1～A5 is that U87 cell and BSA-FITC are hatched 2h, 3h, 4h, 5h and 6h;
B1～B5 is that U87 cell and rLDP-FITC are hatched 2h, 3h, 4h, 5h and 6h;
C1～A5 be the U87 cell with (Arg)
9-LDP-FITC is hatched 2h, 3h, 4h, 5h and 6h.
Figure 10: flow cytometer detects (Arg)
9-LDP fusion rotein penetrates the tumour cell ability
Wherein: ◆-(Arg)
9-LDP-FITC;
■-rLDP-FITC；
▲-BSA-FITC。
Figure 11: the flow cytometer detected temperatures is to (Arg)
9-LDP fusion rotein penetrates the impact of U87 cell ability
Wherein: ◆-(Arg)
937 ℃ of-LDP-FITC;
■-(Arg)
9-LDP-FITC 4℃；
▲-rLDP-FITC 37℃；
●-rLDP-FITC 4℃。
Figure 12: the flow cytometer detected temperatures is to (Arg)
9-LDP fusion rotein penetrates the impact of A549 cell ability
Wherein: ◆-(Arg)
937 ℃ of-LDP-FITC;
■-(Arg)
9-LDP-FITC 4℃；
▲-rLDP-FITC 37℃；
●-rLDP-FITC 4℃。
Figure 13: energized fusion protein (Arg)
9-LDP-AE is to the cytotoxicity of Bel-7402 cell
Wherein: ■-LDM;
▲-(Arg)
9-LDP-AE。
Figure 14: energized fusion protein (Arg)
9-LDP-AE is to the cytotoxicity of MCF-7 cell
Wherein: ■-LDM;
▲-(Arg)
9-LDP-AE。
Figure 15: energized fusion protein (Arg)
9-LDP-AE is to the cytotoxicity of OVCAR3 cell
Wherein: ■-LDM;
▲-(Arg)
9-LDP-AE。
Figure 16: energized fusion protein (Arg)
9-LDP-AE is to the cytotoxicity of U87 cell
Wherein: ■-LDM;
▲-(Arg)
9-LDP-AE。
Figure 17: energized fusion protein (Arg)
9-LDP-AE is on the impact of U87 cell cycle
Wherein: 1-Control;
2-(Arg)
9-LDP-AE 0.1pM；
3-(Arg)
9-LDP-AE 1pM；
4-(Arg)
9-LDP-AE 0.01nM；
5-(Arg)
9-LDP-AE 0.1nM；
6-(Arg)
9-LDP-AE 1nM。
Figure 18: energized fusion protein (Arg)
9-LDP-AE is to the apoptosis-induced effect of U87 cell
Wherein: 1-Control;
2-(Arg)
9-LDP-AE 0.01nM；
3-(Arg)
9-LDP-AE 0.05nM；
4-(Arg)
9-LDP-AE 0.1nM；
5-(Arg)
9-LDP-AE 0.5nM；
6-(Arg)
9-LDP-AE 1nM。
Figure 19: energized fusion protein (Arg)
9-LDP-AE is to the apoptosis-induced effect of U87 cell
Wherein:
-apoptosis rate;
1-Control；
2-(Arg)
9-LDP-AE 0.01nM；
3-(Arg)
9-LDP-AE 0.05nM；
4-(Arg)
9-LDP-AE 0.1nM；
5-(Arg)
9-LDP-AE 0.5nM；
6-(Arg)
9-LDP-AE 1nM。
Figure 20: energized fusion protein (Arg)
9The impact that-LDP-AE is heavy on liver cancer H22 transplanted tumor mouse tumor
Wherein: 1-Control;
2-(Arg)
9-LDP 10mg/kg；
3-LDM 0.05mg/kg；
4-(Arg)
9-LDP-AE 0.3mg/kg；
5-(Arg)
9-LDP-AE 0.2mg/kg；
6-(Arg)
9-LDP-AE 0.1mg/kg。
Figure 21: energized fusion protein (Arg)
9-LDP-AE is on the impact of liver cancer H22 transplanted tumor Mouse Weight
Wherein: ◆-Control;
■-(Arg)
9-LDP 10mg/kg；
▲-LDM 0.05mg/kg；
●-(Arg)
9-LDP-AE 0.3mg/kg；
○-(Arg)
9-LDP-AE 0.2mg/kg；
△-(Arg)
9-LDP-AE 0.1mg/kg。
Figure 22: energized fusion protein (Arg)
9The growth curve of the human glioma U87 transplanted tumor in nude mice that-LDP-AE processes
Wherein: ◆-Control;
■-(Arg)
9-LDP 10mg/kg；
▲-LDM 0.05mg/kg；
□-(Arg)
9-LDP 10mg/kg+LDM 0.05mg/kg；
●-(Arg)
9-LDP-AE 0.3mg/kg；
○-(Arg)
9-LDP-AE 0.2mg/kg；
△-(Arg)
9-LDP-AE 0.1mg/kg。
Embodiment:
Following examples can make the present invention of those skilled in the art's comprehend, but not limit the present invention in any way.
＜embodiment 1 〉. fusion rotein (Arg)
9-LDP recombinant expression plasmid pET30-(arg)
9The structure of ldp
Recombinant plasmid pET30-vhldp (patent: CN200410034052.7) with the LDP gene.Introduce two restriction enzyme sites of NdeI, XhoI by PCR, (primer is synthetic by Invitrogen company).
Upstream primer: 5 ' GAATTC
CATATG 
(underscore partly is Nde I restriction enzyme site to GGCGCGCCCGCCTTCTCCGT 3 ', and italicized item is (arg)
9Gene)
Downstream primer: 5 ' GTTA
CTCGAGGCCGAAGGTCAGAGCCACGTG3 ' (underscore partly is Xho I restriction enzyme site)
Recombinant expression plasmid pET30-(arg)
9The building process of ldp as shown in Figure 1.Carry out pcr amplification take recombinant plasmid pET30-vhldp as template, obtain (arg)
9Ldp gene fragment (Fig. 2).The PCR reaction system is the amplified reaction that carries out 30 circulations behind 94 ℃ of sex change 5min: 94 ℃ of sex change 1min, and 58 ℃ of annealing 1min, 72 ℃ are extended 1min, are incubated 10min at 72 ℃ after last loop ends.PCR product utilization glass milk carries out double digestion with NdeI, Xho I after reclaiming the recovery of test kit (BioDev company product) purifying.Reclaim endonuclease bamhi, be cloned in the pET-30a of same double digestion (+) (available from Invitrogen company product), obtain recombinant plasmid pET30-(arg)
9Ldp.Carry out double digestion with NdeI and XhoI enzyme and identify (Fig. 3).Sequencing result shows: fusion rotein (Arg)
9-LDP full length gene 387bp, 129 amino acid of encoding, cell-penetrating peptides full length gene 30bp wherein, 10 amino acid of encoding, LDP full length gene 330bp, 110 amino acid of encoding, flexible peptide gene length 3bp between the two, 1 amino acid of encoding, the long 18bp of Histidine six aggressiveness coda genes of carboxyl terminal, 6 amino acid of encoding are introduced restriction enzyme site 6bp.
＜embodiment 2 〉. fusion rotein (Arg)
9-LDP is at colon bacillus BL21 (DE3) star
TMMiddle abduction delivering
PET30-after the evaluation (arg)
9The ldp recombinant plasmid transformed is to colon bacillus BL21 (DE3) star
TM(Invitrogen company product), picking recombinant conversion is inoculated in the LB liquid nutrient medium that 3ml contains 50 μ g/ml kantlex at random, and 37 ℃ of shaking culture are spent the night.Inoculate according to 5% ratio next day, and it is 0.8 that 37 ℃ of concussions are cultured to OD600, and adding IPTG is 0.2mM to final concentration, inducing culture 8h.Get an amount of bacterium liquid, full bacterium, substratum supernatant, cell pericentral siphon chamber component, solubility kytoplasm component and inclusion body component are carried out the expression product positioning analysis.15%SDS-PAGE electrophoresis and immunoblotting result show that the fusion protein molecule amount is about 14.5kDa with soluble formal representation in inclusion body (Fig. 4, Fig. 5).The gel imaging system quantitative analysis shows, accounts for bacterial protein about 8% with the fusion protein expression of top condition abduction delivering gained.Contain recombinant plasmid pET30a-arg9ldp, can expressed fusion protein (Arg)
9The conversion bacterial strain called after CAMS/ (Arg) of-LDP
9-LDP delivers in May, 2010 and to be positioned at the common micro-organisms center preservation of Pekinese China Committee for Culture Collection of Microorganisms, classification: colon bacillus Escherichia coli, deposit number: (CGMCC NO.3815).
＜embodiment 3 〉. fusion rotein (Arg)
9-LDP affinitive layer purification and separation preparation
3.1 the extraction of inclusion body protein
The centrifugal 10min of recombinant bacterium culture 10000g that the IPTG that learns from else's experience induces removes supernatant as far as possible, collects thalline.The thalline of every 1L volume of culture carries out resuspended with the 20mM Tris-HCl of 100ml.The ultrasonication smudge cells.4 ℃ of centrifugal 15min of 10000g collect inclusion body and cell debris albumen, supernatant discarded.Not urea-containing 1 * Binding Buffer (20mM Tris-HCl, 0.5M NaCl, 5mM imidazoles, pH8.0) resuspended precipitation with 100ml.4 ℃ of centrifugal 15min of 10000g, supernatant discarded, collecting precipitation.Repeat the aforesaid operations step, and usefulness 50ml 1 * Binding Buffer (20mM Tris-HCl, 0.5MNaCl, the 5mM imidazoles, 8M urea, pH8.0) resuspended precipitation, ice bath 1h dissolves inclusion body protein fully.4 ℃ of centrifugal 30min of 15000g remove insoluble material.Collect supernatant, behind 0.45 μ m membrane filtration, be stored in 4 ℃ to treat affinitive layer purification.
3.2 fusion rotein (Arg)
9-LDP affinitive layer purification
Adopt His-Trap
TMHP purification column (Amersham company product) purified protein samples.Behind the pre-treatment affinity column, contain 1 * Binding Buffer balance chromatography column with 6 times of column volumes, behind the soluble protein sample upper prop, respectively with 10 times of volume 1 * Binding Buffer and 6 times of volume 1 * Washing Buffer (20mM Tris-HCl, 0.5M NaCl, the 20mM imidazoles, 8M urea, pH8.0) washing chromatography column is at last with 6 times of volume 1 * Elute Buffer (20mM Tris-HCl, 0.5M NaCl, the 500mM imidazoles, 8M urea, pH8.0) elution of bound albumen, collect the wash-out component, the fusion rotein (Arg) behind the acquisition purifying
9-LDP (Fig. 6).
3.3 fusion rotein (Arg)
9The dialysis renaturation of-LDP
The dialysis tubing that will be immersed among the EDTA (disodium ethylene diamine tetraacetate) cleans up with distilled water, fills water and then discharges.The albumen of wash-out is diluted to 15 μ M with 1 * Binding Buffer, and adding 2 mercapto ethanol to final concentration is 10mM, and room temperature is placed 30min.With the albumen of reduction with the renaturation solution of at least 50 times of sample volumes (50mM Tris-HCl, 1mM EDTA, 200mM NaCl, 6M urea, pH8.0) 4 ℃ of dialysed overnight are to remove reductive agent.The renaturation solution (3M, 2M, 1M, 0.5M, 0M urea) that successively decreases successively with urea concentration carries out the substep dialysis with sample.Be the 1M stage at urea concentration, add the Sleep-promoting factor B of 750 μ M and the L-arginine of 400mM.Use again the PBS (pH7.4) of 50 times of volumes to dialyse in the final step gained sample of dialysing.Every 12h changes a dialyzate, totally 2 times.With the 4 ℃ of centrifugal 30min of 10000g of sample after the dialysis, collect supernatant, namely obtain renaturation fusion rotein (Arg)
9-LDP.
＜embodiment 4 〉. energized fusion protein (Arg)
9The preparation of-LDP-AE
4.1 the preparation of active chromophoric (AE)
Get highly active LDM dried frozen aquatic products (number of patent application: 001215272, publication number: 1284566) 10mg, add 5ml cold methanol jolting 5min, place 1h, middle jolting 1 time for-20 ℃; 0 ℃, the centrifugal 20min of 12000rpm is rich in chromophoric group AE in the supernatant, be precipitated as peptide chain, repeats to extract 2 times.4 ℃, lucifuge evaporation concentration chromophoric group ,-70 ℃ of preservations.
4.2 energized fusion protein (Arg)
9The preparation of-LDP-AE separates
Get the fusion rotein (Arg) of certain volume and concentration
9-LDP is dissolved among the PBS (pH7.4), adds the AE methanol solution of 3 times of mole numbers, mixes jolting, room temperature reaction 12h.Mixed solution with PD-10 post (business-like Sephadex G-25 post, Pharmacia product) chromatographic separation, behind 280nm, 343nm ultraviolet monitoring, is discarded excessive unreacted AE, collect energized fusion protein (Arg)
9-LDP-AE.
＜embodiment 5 〉. fluoroscopic examination fusion rotein (Arg)
9The permeates cell membranes ability of-LDP
5.1 FITC mark fusion rotein (Arg)
9-LDP, lidamycin (LDM) restructuring apoprotein rLDP and bovine serum albumin BSA
With fusion rotein (Arg)
9-LDP,, lidamycin (LDM) restructuring apoprotein rLDP and bovine serum albumin BSA be with carbonate buffer solution (Na
2CO
30.16mol/L, NaHCO
30.33mol/L pH 9.5) dialysed overnight.Take by weighing FITC (isothiocyanate), be dissolved among the DMSO (dimethyl sulfoxide (DMSO)), final concentration is 1mg/ml.The protein solution of dialysing is placed bottle, in the ratio of 150 μ gFITC/mg albumen, the mixing FITC solution while dripping.In 4 ℃ of lucifuge mixing 12-18h.Remove unconjugated FITC with the PBS dialysis.(A280-0.31 * A495), this value should be between 2.5-6.5 for the combining ratio F/P:F/P=3.1 * A495/ of calculating fluorescein and albumen.
5.2 fluoroscopic examination fusion rotein (Arg)
9-LDP penetrates the tumor cell membrane ability
Collect Bel-7402 cell, A549 cell, H460 cell, OVCAR3 cell and U87 cell and the counting of logarithmic phase, use the substratum re-suspended cell, with every hole 5 * 10
5The amount of cell joins in 6 well culture plates, 37 ℃ of overnight incubation.Discard original fluid, add new substratum, and the adding final concentration is the fusion rotein (Arg) of the FITC mark of 20 μ M
9-LDP-FITC, 37 ℃ of effect 2h.Supernatant discarded is washed 3 times with PBS, adds methyl alcohol, places shaking table to shake 10min, inhales and abandons methyl alcohol.Again add methyl alcohol, place-20 ℃ of fixedly 10min, wash 3 times with PBS, add the PBS mounting that 400 μ l contain 50% glycerine, observe under the inverted fluorescence microscope, take a picture.Result (Fig. 7) shows, with fusion rotein (Arg)
9After-LDP-FITC was hatched, all there was stronger green fluorescence reaction the inside of five kinds of tumour cells, illustrates that the fusion rotein with penetrating peptide can permeates cell membranes enter cell interior.
5.3 fluoroscopic examination fusion rotein (Arg)
9The location of-LDP in tumour cell
Collect A549 cell, H460 cell and OVCAR3 cell and the counting of logarithmic phase, use the substratum re-suspended cell, with every hole 5 * 10
5The amount of cell joins in 6 well culture plates, 37 ℃ of overnight incubation.Discard original fluid, add new substratum, and the adding final concentration is the fusion rotein (Arg) of the FITC mark of 20 μ M
9-LDP-FITC, 37 ℃ of effect 2h.Supernatant discarded is washed 3 times with PBS, adds methyl alcohol, places shaking table to shake 10min, inhales and abandons methyl alcohol.Again add methyl alcohol, place-20 ℃ of fixedly 10min, inhale and abandon methyl alcohol, wash 3 times with PBS, adding final concentration is that final concentration is the DAPI of 1 μ g/ml, room temperature lucifuge reaction 30min.Wash 3 times with PBS, add the PBS mounting that 400 μ l contain 50% glycerine, observe under the inverted fluorescence microscope, take a picture.Result (Fig. 8) shows, with fusion rotein (Arg)
9After-LDP-FITC is hatched, all there is stronger green fluorescence reaction three kinds of tumour cell inside, with DAPI (4 ', 6-diamidino-2-phenylindone) nucleus is redyed, find that the green fluorescence reaction mainly is present in the tenuigenin, weak green fluorescence reaction is also arranged in the nucleus simultaneously, illustrate that the fusion rotein with penetrating peptide can permeates cell membranes enter cell interior.
5.4 fluoroscopic examination different time fusion rotein (Arg)
9-LDP penetrates the tumour cell ability
Collect U87 cell and the counting of logarithmic phase, use the substratum re-suspended cell, with every hole 5 * 10
5The amount of cell joins in 6 well culture plates, 37 ℃ of overnight incubation.Discard original fluid, add new substratum, adding final concentration in different time is the fusion rotein (Arg) of the FITC mark of 20 μ M
9The restructuring apoprotein rLDP-FITC of-LDP-FITC, FITC mark and the bovine serum albumin BSA-FITC of FITC mark are hatched for 37 ℃.Supernatant discarded is washed 3 times with PBS, adds methyl alcohol, places shaking table to shake 10min, inhales and abandons methyl alcohol.Again add methyl alcohol, place-20 ℃ of fixedly 10min, inhale and abandon methyl alcohol, wash 3 times with PBS.Adding final concentration is the DAPI of 1 μ g/ml, room temperature lucifuge reaction 30min.Wash 3 times with PBS, add the PBS mounting that 400 μ l contain 50% glycerine, observe under the inverted fluorescence microscope, take a picture.Result (Fig. 9) demonstration, (Arg)
9-LDP-FITC was hatched 2 hours, can see obvious green fluorescence in the cell, prolongation along with the time, intracellular green fluorescence intensity strengthens gradually, in rLDP-FITC group and the BSA-FITC group cell a small amount of green fluorescence is arranged also, but along with the prolongation of time, green fluorescence intensity is without considerable change, with (Arg) in the born of the same parents
9-LDP-FITC group has been compared notable difference.
＜embodiment 6 〉. flow cytometer detects fusion rotein (Arg)
9-LDP permeates cell membranes ability
6.1 flow cytometer detects fusion rotein (Arg)
9-LDP penetration cell ability
The U87 cell of taking the logarithm vegetative period with counting after the trysinization, is adjusted cell 5 * 10
5Individual/ml, divide with every pipe 1ml to be filled in 19 centrifuge tubes.Centrifugal 5 minutes of 1000rpm, collecting cell is used PBS washed cell 3 times.Adding respectively final concentration is the fusion rotein (Arg) of the FITC mark of 20 μ M
9The restructuring apoprotein rLDP-FITC of-LDP-FITC, FITC mark and the bovine serum albumin BSA-FITC of FITC mark, supply volume to 100 μ l with reaction buffer (PBS+2%FBS), hatch for 37 ℃, respectively at 0.5,1,1.5,2,2.5, the 3h sampling, the negative control pipe that does not add albumen is set simultaneously.4 ℃ centrifugal after, with the resuspended cell mass of 4% trypan blue solution, room temperature 10min.Wash cell 3 times with reaction buffer (PBS+2%FBS); With FACS machine fluorescence intensity.Take fluorescence intensity as ordinate zou, make time response curve take sample time as X-coordinate.As shown in figure 10, in 0.5-3h, enter the fusion rotein (Arg) of cell
9-LDP-FITC is along with obviously increasing with the prolongation of time, and fluorescence intensity prolongation in time is without obvious increase, with (Arg) in the born of the same parents of rLDP-FITC group and BSA-FITC group
9-LDP-FITC group has been compared notable difference.
6.2 the flow cytometer detected temperatures is to fusion rotein (Arg)
9The impact of-LDP penetration cell ability
The U87 cell of taking the logarithm vegetative period and A549 cell with counting after the trysinization, are adjusted cell 5 * 10
5Individual/ml, divide with every pipe 1ml to be filled in 13 centrifuge tubes.Centrifugal 5 minutes of 1000rpm, collecting cell is used PBS washed cell 3 times.Adding respectively final concentration is the fusion rotein (Arg) of the FITC mark of 20 μ M
9The restructuring apoprotein rLDP-FITC of-LDP-FITC and FITC mark, supply volume to 100 μ l with reaction buffer (PBS+2%FBS), hatch respectively at 4 ℃ and 37 ℃, in 0.5,1,1.5,2,2.5, the 3h sampling, the negative control pipe that does not add albumen is set simultaneously.4 ℃ centrifugal after, with the resuspended cell mass of 4% trypan blue solution, room temperature 10min.Wash cell 3 times with reaction buffer (PBS+2%FBS); With FACS machine fluorescence intensity.Take fluorescence intensity as ordinate zou, make time response curve take sample time as X-coordinate.The result as shown in figure 11, (Arg)
9The interior fluorescence intensity of U87 cell prolongs obvious increase in time under 37 ℃ of conditions of-LDP-FITC group, under 4 ℃ of conditions, fluorescence intensity prolongs in time without considerable change in the U87 cell, the interior fluorescence intensity of U87 cell prolongs all in time under 4 ℃ and 37 ℃ conditions of rLDP-FITC group obviously increases without nothing, and (Arg) is described
9-LDP-FITC can permeates cell membranes enter cell interior, and it is relevant with temperature that it penetrates process, is an energy expenditure process.Also obtain same experimental result in the A549 cell, as shown in figure 12.
＜embodiment 7〉.MTT method detection enhancement fusion rotein (Arg)
9-LDP-AE is to the cytotoxicity of tumour cell
The MCF-7 cell of taking the logarithm vegetative period, Bel-7402 cell, U87 cell and OVCAR3 cell dissociation, counting, 4000/hole is laid on 96 orifice plates.37 ℃ of 5%CO
2After cultivating 24h, add the energized fusion protein/lidamycin (LDM) of different concns, each drug level is established 6 parallel holes.After cultivating 48h, every hole adds 20 μ l MTT (5mg/ml), and 37 ℃ are continued to cultivate 4h.Substratum is abandoned in suction, adds 150 μ l DMSO, shaking table vibration 10min, and microplate reader is measured the A570 value.Calculate cell survival rate and IC
50Value.Cell survival rate T/C (%)=(dosing group A570 value-acellular control group A 570 values)/(without medicine control group A 570 values-acellular control group A 570 values) * 100%.Then take cell survival rate as ordinate zou, drug level is that X-coordinate is made concentration-response curve (Figure 13, Figure 14, Figure 15, Figure 16).
The result shows (table 1), energized fusion protein and LDM have strong cytotoxicity to four kinds of cells, energized fusion protein and lidamycin (LDM) are close to the IC50 value of U87 cell and MCF-7 cell, and energized fusion protein is respectively 1/20 and 1/10 of lidamycin (LDM) to the IC50 value of OVCAR3 cell and Bel-7402 cell.
Table 1 energized fusion protein and lidamycin (LDM) are to the IC of tumour cell
50Value
＜embodiment 8 〉. energized fusion protein (Arg)
9-LDP-AE is on the impact of tumour cell cycle
With the U87 cell according to 1.5 * 10
5The density in individual/hole is seeded in 6 well culture plates, cultivates 24h for 37 ℃.When treating that cell grows to 50%-80%, replaced medium adds the energized fusion protein (Arg) of different concns simultaneously
9-LDP-AE.Set up simultaneously not dosing blank group.Behind the effect 48h, collecting cell is washed 2 times with PBS, adds 4 ℃ of 70% ethanol and fixedly spends the night, and PBS washes 2 times, and adding final concentration is the PI of 50 μ g/ml, carries out cells were tested by flow cytometry.The result as shown in figure 17, after the energized fusion protein of the different concns of the continuous 10 times of dilutions of 1nM to 0.1pM acted on respectively U87 cell 48h, singly dye flow cytometer sense cycle changes in distribution through PI, the result shows that the energized fusion protein of 0.1pM can cause the G of cell
2/ M the phase blocks, retarding degree increases and increases along with concentration, when concentration increases to 0.1nM and 1nM, retarding degree decreases, the energized fusion protein of 1pM can cause that the S phase of cell blocks, and retarding degree increases and increases along with concentration, as shown in figure 16, and along with the increase of concentration, the apoptotic cell number increases gradually.
＜embodiment 9〉.Annexin V-FITC/PI dyes in conjunction with flow cytometer detection enhancement fusion rotein (Arg)
9-LDP-AE is to the apoptosis-induced effect of tumour cell
According to 1.5 * 10
5The density in individual/hole is inoculated in the U87 cell in the 6 porocyte culture plates, 37 ℃ of environment 5%CO
2Cultivate 24h in the incubator of concentration.Replaced medium, the energized fusion protein (Arg) of adding different concns
9-LDP-AE sets up not dosing blank group simultaneously.Behind the effect 48h, collecting cell, PBS washes 2-3 time, 4 ℃ of centrifugal 10min of 1000rpm abandon supernatant, and cell is resuspended among the 150 μ l Binding Buffer, add gently mixing of 10 μ l Annexin V-FITC, lucifuge room temperature reaction 15min or 4 ℃ of reaction 30min add 150 μ l Binding Buffer and 5 μ l PI again, carry out cells were tested by flow cytometry.The result as shown in figure 18, with the energized fusion protein (Arg) of different concns
9After-LDP-AE (0.01nM, 0.05nM, 0.1nM, 0.5nM and 1nM) acted on human glioma U87 cell 48h, the apoptosis ratio raise with the concentration increase.Wherein, the energized fusion protein of 0.01nM, 0.05nM, 0.1nM and 0.5nM (Arg)
9-LDP-AE acts on human glioma U87 cell, and apoptosis rate is respectively 20.1%, 21.9%, 28.1 and 44.9%, and the apoptosis ratio raises with the concentration increase, compares with control group 2.1%, all has notable difference.The mortality ratio of cell is respectively 4.8%, 6.9%, 8.7% and 13.5%, compares with control group 0.9%, all has notable difference.The energized fusion protein of 1nM (Arg)
9The apoptosis rate that-LDP-AE acts on the U87 cell is 33.9%, than (Arg) of 0.5nM
9-LDP-AE slightly reduces, but the mortality ratio of cell is increased to 36%.The flow cytometer Analysis of test results as shown in Figure 19.
＜embodiment 10 〉. energized fusion protein (Arg)
9-LDP-AE is to the growth-inhibiting effect of mouse bearing liver cancer H22
Get the kunming mice that body weight is 18g-22g, random packet, 10 every group.Tested the 0th day, and got rat liver cancer H22 ascites, become cell count as 7.5 * 10 take normal saline dilution
5Individual/ml, it is subcutaneous to be inoculated in mouse armpit by every mouse 0.2ml.Inoculation awarded respectively physiological saline, lidamycin (LDM), fusion rotein (Arg) in rear 1,7 day
9The energized fusion protein of-LDP and various dose group (Arg)
9-LDP-AE, tail vein injection, 0.2ml/ are only.Per two days records of experimental session the weight of animals was put to death mouse on the 11st day, stripped the knurl piece and claimed knurl heavy, calculated tumour inhibiting rate according to tumour inhibiting rate (%)=(control group knurl weight-administration group knurl is heavy)/control group knurl heavy * 100%.
The result is shown in Figure 20, Figure 21 and table 2, (Arg)
9-LDP 10mg/kg group, the tumour inhibiting rate of LDM0.05mg/kg group is respectively 58.4% and 74.6%, (Arg)
9The tumour inhibiting rate of-LDP-AE 0.3mg/kg group and 0.2mg/kg group is respectively 89.2% and 85.3%, compares with the LDM0.05mg/kg group to be significantly improved, (Arg)
9The tumour inhibiting rate of-LDP-AE 0.1mg/kg group is 73.4%, and is near with the LDM0.05mg/kg winding.Each administration group is compared with control group all has significant difference, P value＜0.01.
Table 2 energized fusion protein (Arg)
9-LDP-AE is to the growth-inhibiting effect of rat liver cancer H22 transplanted tumor
*With control group ratio, P＜0.01
＜embodiment 11〉strengthen. fusion rotein (Arg)
9-LDP-AE is to the growth-inhibiting effect of human glioma U87 transplanted tumor in nude mice
Get the 6-8 female Balb/c nude mice in age in week, body weight 18～22g.After the U87 cell dissociation become single cell suspension, wash 3 times counting with stroke-physiological saline solution.With physiological saline resuspended 5 * 10
7Cell/ml, every mouse inoculation 0.2ml is subcutaneous in the right side armpit.Treat that the knurl block length when enough big or small, is cut into 2 * 2 * 2mm with it in stroke-physiological saline solution
3Fritter, with trochar with tumour transplatation to the right armpit of nude mice subcutaneous, with pyroxylin otch is clung.Behind inoculation knurl piece the 7th day, with the nude mice grouping, every group of 6 nude mices, each organizes body weight and knurl piece size homogeneous.Respectively the 7th day and tail vein injection administration in the 15th day behind inoculation knurl piece.Fusion rotein (Arg)
9-LDP group dosage is 10mg/kg, and LDM group dosage is 0.05mg/kg, fusion rotein (Arg)
9-LDP and LDM combination group, energized fusion protein (Arg)
9-LDP-AE 3 dosage groups are respectively 0.1mg/kg, 0.2mg/kg and 0.3mg/kg.Experimental session is every long and short footpath of tumour of measurement in 3 days and nude mice body weight, according to formula V=ab
2/ 2 calculate gross tumor volume.Experimental result (Figure 22) shows, (Arg)
9-LDP-AE has obvious restraining effect to the solid tumor that neuroglial cytoma forms, the tumour inhibiting rate of 0.2mg/kg and 0.3mg/kg dosage group is respectively 92.8% and 86.2% in the time of 19 days, specific force reaches mycin and has more significant result for the treatment of, and its significant difference is P＜0.01.
Sequence table
＜110〉Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences
＜120〉cell-penetrating peptides (Arg) 9 and white (Arg) 9-LDP of lidamycin fusion protein
<160>1
<210>1
<211>387
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
atgcgtcgtc gccgccgtcg tcgccgccgt ggcgcgcccg ccttctccgt cagtcccgcc 60
tcgggtctga gtgacggaca gagcgtgtcg gtgtcggtca gcggtgccgc cgccggcgag 120
acctactaca tcgcccagtg cgctccggtc ggtggccagg acgcgtgcaa cccggcgacc 180
gcgacgtcct tcaccacgga cgcgtccgga gcggcgtcgt tcagcttcgt cgtgcgcaag 240
tcgtacacgg gctccacgcc cgaaggcacg ccggtcggca gcgtcgactg cgccacggcc 300
gcctgtaacc tcggcgccgg caactccggg ctcgacctcg gccacgtggc tctgaccttc 360
ggcctcgagc accaccacca ccaccac 387
Claims (5)
1. a fusion rotein (Arg)
9-LDP is characterized in that said fusion rotein is by cell-penetrating peptides (Arg)
9, Lidamycin agon albumen LDP and carboxyl terminal Histidine six aggressiveness tails form, the full length gene 387bp of this fusion rotein of encoding, sequence shown in SEQ ID NO.1,129 amino acid of described genes encoding.
2. an energized fusion protein (Arg)
9-LDP-AE is characterized in that said energized fusion protein is the described fusion rotein of claim 1 (Arg) of 14.5kDa by molecular weight
9-LDP and molecular weight are that the activated form enediyne chromophoric group AE of 843Da consists of.
3. prepare the described energized fusion protein of claim 2 (Arg)
9The method of-LDP-AE is characterized in that the fusion rotein (Arg) that purifies and separates is obtained
9-LDP carries out molecule with the lidamycin (LDM) active form enediyne chromophoric group AE for preparing through methanol extraction and strengthens, and obtains energized fusion protein (Arg)
9-LDP-AE.
4. the described fusion rotein of claim 1 (Arg)
9The application of-LDP in the preparation antitumor drug.
5. energized fusion protein (Arg) as claimed in claim 2
9The application of-LDP-AE in the preparation antitumor drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010177517XA CN101851294B (en) | 2010-05-20 | 2010-05-20 | Cell-penetrating peptide (Arg) 9 and lidamycin fusion protein (Arg) 9-LDP |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010177517XA CN101851294B (en) | 2010-05-20 | 2010-05-20 | Cell-penetrating peptide (Arg) 9 and lidamycin fusion protein (Arg) 9-LDP |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101851294A CN101851294A (en) | 2010-10-06 |
| CN101851294B true CN101851294B (en) | 2013-02-13 |
Family
ID=42802991
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010177517XA Expired - Fee Related CN101851294B (en) | 2010-05-20 | 2010-05-20 | Cell-penetrating peptide (Arg) 9 and lidamycin fusion protein (Arg) 9-LDP |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101851294B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277364A (en) * | 2011-01-12 | 2011-12-14 | 四川大学 | Fusion protein LhFVII-LDP and reinforcement fusion protein LhFVII-LDP-AE, and application thereof |
| CN108721641B (en) * | 2017-04-14 | 2021-05-11 | 中国医学科学院医药生物技术研究所 | Antibody drug conjugate of anti-CD30antibody and lidamycin, preparation method and application thereof |
| CN112852853B (en) * | 2021-01-08 | 2023-06-02 | 重庆医科大学附属儿童医院 | High-strength focused ultrasound biological targeting synergist and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538331A (en) * | 2009-05-06 | 2009-09-23 | 中国医学科学院医药生物技术研究所 | Single chain antibody-lidamycin fusion protein ER (Fv-LDP) targeting EGFR |
-
2010
- 2010-05-20 CN CN201010177517XA patent/CN101851294B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538331A (en) * | 2009-05-06 | 2009-09-23 | 中国医学科学院医药生物技术研究所 | Single chain antibody-lidamycin fusion protein ER (Fv-LDP) targeting EGFR |
Non-Patent Citations (5)
| Title |
|---|
| HUANG YUN-HONG.Antitumor efficacy of lidamycin on hepatoma and active moiety of its molecule.《World J Gastroenterol》.2005,第11卷(第26期),第3980-3984页. * |
| 王薇等.细胞膜穿透肽促进脂质体包载的siRNA细胞内转运、定位及其功能.《药学学报》.2006,第41卷(第2期),第142-148页. * |
| 茹琴等.基因工程重组力达霉素的制备及抗肿瘤活性.《中国抗生素杂志》.2010,第35卷(第4期),第265-269页. * |
| 马迪等.新型烯二炔类抗肿瘤抗生素力达霉素的研究进展.《中国抗生素杂志》.2007,第32卷(第1期),第11-16页. * |
| 高瑞娟等.烯二炔类抗肿瘤抗生素力达霉素的作用机制研究进展.《中国新药杂志》.2006,第15卷(第13期),第1039-1043页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101851294A (en) | 2010-10-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101263212B1 (en) | A new cell-permeable peptide and its use | |
| CN104072581B (en) | D-configuration polypeptide with brain tumor targeting and tumor tissue penetrating capabilities and gene delivery system thereof | |
| CN103501818A (en) | Methods and reagents for efficient and targeted delivery of therapeutic molecules to cxcr4 cells | |
| CN105175526B (en) | Penetratin hPP8 and application thereof | |
| CN104140457A (en) | Tumor cell targeting penetrating peptide | |
| Liu et al. | Metabolically engineered bacteria as light-controlled living therapeutics for anti-angiogenesis tumor therapy | |
| CN103304637A (en) | Cell permeable peptide hPP3 and usage thereof | |
| CN104592395A (en) | Preparation method and application of VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) single-chain antibody and MICA fusion protein | |
| CN101851294B (en) | Cell-penetrating peptide (Arg) 9 and lidamycin fusion protein (Arg) 9-LDP | |
| CN105112383A (en) | Cell membrane penetrating peptide hPP5 and application thereof | |
| CN107987153A (en) | The soluble PD-1 molecules of high-affinity | |
| CN107417769A (en) | A kind of novel cell-penetrating peptide of mediate drug delivering and its application | |
| CN109608519B (en) | Cell-penetrating peptide and application thereof | |
| CN104628863B (en) | A kind of double bullet antineoplastic amalgamation proteins of double target spots, its encoding gene and purposes | |
| CN101538331A (en) | Single chain antibody-lidamycin fusion protein ER (Fv-LDP) targeting EGFR | |
| CN108864292A (en) | A kind of interferon recombination fusion albumen and its application | |
| CN115925988B (en) | Denatured collagen targeted antibacterial peptide and preparation method and application thereof | |
| CN104231050A (en) | Polypeptide and polypeptide complex for suppressing tumor metastasis and treating leukemia as well as preparation method for polypeptide complex and application of polypeptide and polypeptide complex | |
| CN104177502A (en) | Tachyplesin peptide-antibody fusion protein and preparation method thereof | |
| CN107226844B (en) | Structural molecule for enhancing integrin receptor affinity and target cell uptake capacity and application thereof | |
| CN104774246B (en) | NRP-1 specific tumours target polypeptide and its application | |
| CN112457370B (en) | Gene recombination cell-penetrating peptide RTP and preparation method and application thereof | |
| CN108727469B (en) | Novel cell-penetrating peptide for mediating drug delivery and application thereof | |
| CN105061562B (en) | A kind of oligopeptides and its application with effect of anti-lung cancer | |
| CN100352838C (en) | Interfusion protein of antineoplastic genetic engineering composed of target short peptide of receptor of epidermal growth factor and Lidamycin |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130213 Termination date: 20200520 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |