CN114853865B - 一种改造体抗菌肽dsNCM1及其应用 - Google Patents
一种改造体抗菌肽dsNCM1及其应用 Download PDFInfo
- Publication number
- CN114853865B CN114853865B CN202210467019.1A CN202210467019A CN114853865B CN 114853865 B CN114853865 B CN 114853865B CN 202210467019 A CN202210467019 A CN 202210467019A CN 114853865 B CN114853865 B CN 114853865B
- Authority
- CN
- China
- Prior art keywords
- dsncm1
- antibacterial peptide
- antibacterial
- engineered
- peptide
- 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
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 69
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 31
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 19
- 241000894006 Bacteria Species 0.000 claims abstract description 13
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 7
- 230000003214 anti-biofilm Effects 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 241000191967 Staphylococcus aureus Species 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 241000588626 Acinetobacter baumannii Species 0.000 claims description 9
- 230000002401 inhibitory effect Effects 0.000 claims description 9
- 241000588724 Escherichia coli Species 0.000 claims description 8
- 230000003115 biocidal effect Effects 0.000 claims description 8
- 241000192125 Firmicutes Species 0.000 claims description 4
- 108010093965 Polymyxin B Proteins 0.000 claims description 4
- 229960000723 ampicillin Drugs 0.000 claims description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 4
- 229960002260 meropenem Drugs 0.000 claims description 4
- DMJNNHOOLUXYBV-PQTSNVLCSA-N meropenem Chemical compound C=1([C@H](C)[C@@H]2[C@H](C(N2C=1C(O)=O)=O)[C@H](O)C)S[C@@H]1CN[C@H](C(=O)N(C)C)C1 DMJNNHOOLUXYBV-PQTSNVLCSA-N 0.000 claims description 4
- 229920000024 polymyxin B Polymers 0.000 claims description 4
- 229960005266 polymyxin b Drugs 0.000 claims description 4
- 239000002260 anti-inflammatory agent Substances 0.000 claims description 3
- 229940121363 anti-inflammatory agent Drugs 0.000 claims description 3
- 230000000845 anti-microbial effect Effects 0.000 claims description 3
- 241000193468 Clostridium perfringens Species 0.000 claims description 2
- 241000194032 Enterococcus faecalis Species 0.000 claims description 2
- 241000194031 Enterococcus faecium Species 0.000 claims description 2
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 claims description 2
- 241000607762 Shigella flexneri Species 0.000 claims description 2
- 241000191963 Staphylococcus epidermidis Species 0.000 claims description 2
- 241000607594 Vibrio alginolyticus Species 0.000 claims description 2
- 241000607618 Vibrio harveyi Species 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 229940032049 enterococcus faecalis Drugs 0.000 claims description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims 1
- MYPYJXKWCTUITO-KIIOPKALSA-N chembl3301825 Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)C(O)[C@H](C)O1 MYPYJXKWCTUITO-KIIOPKALSA-N 0.000 claims 1
- 229940088710 antibiotic agent Drugs 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 11
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 9
- 230000005764 inhibitory process Effects 0.000 abstract description 6
- 235000018417 cysteine Nutrition 0.000 abstract description 5
- 230000002195 synergetic effect Effects 0.000 abstract description 5
- 150000001945 cysteines Chemical class 0.000 abstract description 4
- 230000002757 inflammatory effect Effects 0.000 abstract description 2
- 238000001228 spectrum Methods 0.000 abstract description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 25
- 102000004196 processed proteins & peptides Human genes 0.000 description 24
- 229920001184 polypeptide Polymers 0.000 description 19
- 230000001580 bacterial effect Effects 0.000 description 17
- 239000000243 solution Substances 0.000 description 15
- 239000007788 liquid Substances 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 8
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 230000032770 biofilm formation Effects 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 229940079593 drug Drugs 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- 239000012071 phase Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 201000010099 disease Diseases 0.000 description 7
- 238000003556 assay Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 206010018910 Haemolysis Diseases 0.000 description 5
- 102000004889 Interleukin-6 Human genes 0.000 description 5
- 108090001005 Interleukin-6 Proteins 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 238000007865 diluting Methods 0.000 description 5
- 230000008588 hemolysis Effects 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 229940100601 interleukin-6 Drugs 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 4
- 241001232615 Acinetobacter baumannii ATCC 19606 = CIP 70.34 = JCM 6841 Species 0.000 description 4
- 241000270666 Testudines Species 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 208000035143 Bacterial infection Diseases 0.000 description 3
- 108010059993 Vancomycin Proteins 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 230000002949 hemolytic effect Effects 0.000 description 3
- 230000015784 hyperosmotic salinity response Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000010445 mica Substances 0.000 description 3
- 229910052618 mica group Inorganic materials 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000013112 stability test Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000010267 two-fold dilution method Methods 0.000 description 3
- 229960003165 vancomycin Drugs 0.000 description 3
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 3
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 3
- ITFICYZHWXDVMU-IPTZIORSSA-N (2S)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-6-amino-2-[[(2S,3S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-1-[(2S)-2-[[(2S,3R)-2-[[(2S,3S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-amino-4-carboxybutanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxybutanoyl]amino]-3-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-4-methylpentanoyl]pyrrolidine-2-carbonyl]amino]-3-sulfanylpropanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-3-methylpentanoyl]amino]hexanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-3-methylpentanoyl]amino]-4-oxobutanoyl]amino]-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-oxopentanoyl]amino]pentanedioic acid Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CS)NC(=O)[C@@H]4CCCN4C(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(=O)O)N ITFICYZHWXDVMU-IPTZIORSSA-N 0.000 description 2
- 241000270617 Cheloniidae Species 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 206010017533 Fungal infection Diseases 0.000 description 2
- 101000991061 Homo sapiens MHC class I polypeptide-related sequence B Proteins 0.000 description 2
- 102100030300 MHC class I polypeptide-related sequence B Human genes 0.000 description 2
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 2
- 208000031888 Mycoses Diseases 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 241000270708 Testudinidae Species 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000003674 animal food additive Substances 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 230000006866 deterioration Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000007598 dipping method Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229960003085 meticillin Drugs 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 108010050820 Antimicrobial Cationic Peptides Proteins 0.000 description 1
- 102000014133 Antimicrobial Cationic Peptides Human genes 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- IDZDFWJNPOOOHE-KKUMJFAQSA-N Cys-Phe-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CS)N IDZDFWJNPOOOHE-KKUMJFAQSA-N 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- WKJKBELXHCTHIJ-WPRPVWTQSA-N Gly-Arg-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N WKJKBELXHCTHIJ-WPRPVWTQSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010024642 Listless Diseases 0.000 description 1
- BRSGXFITDXFMFF-IHRRRGAJSA-N Lys-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)N BRSGXFITDXFMFF-IHRRRGAJSA-N 0.000 description 1
- RZHLIPMZXOEJTL-AVGNSLFASA-N Lys-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N RZHLIPMZXOEJTL-AVGNSLFASA-N 0.000 description 1
- OHXUUQDOBQKSNB-AVGNSLFASA-N Lys-Val-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O OHXUUQDOBQKSNB-AVGNSLFASA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- 239000000490 cosmetic additive Substances 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 208000017971 listlessness Diseases 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 208000019180 nutritional disease Diseases 0.000 description 1
- 210000003024 peritoneal macrophage Anatomy 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- 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
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Environmental Sciences (AREA)
- Immunology (AREA)
- Rheumatology (AREA)
- Birds (AREA)
- Dermatology (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Marine Sciences & Fisheries (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Pain & Pain Management (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
Abstract
本发明涉及一种改造体抗菌肽dsNCM1及其应用,该改造体抗菌肽dsNCM1的氨基酸序列如SEQ ID NO.1所示,其第1位和第16位半胱氨酸之间形成二硫键。本发明的改造体抗菌肽dsNCM1相比于未改造的绿海龟抗菌肽以及现有的绿海龟抗菌肽改造体,具有更广的抗菌谱、更强的抗炎活性和抗生物膜活性,对多种细菌、炎性因子都有较强的抑制作用。本发明还基于该改造体抗菌肽dsNCM1开发了一种药物组合物,与多种抗生素联用都具有协同抗菌作用。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种改造体抗菌肽dsNCM1及其应用。
背景技术
近年我国年上岸产卵绿海龟数量急剧下降,中国海龟保护行动计划鼓励通过海龟全人工繁育,探索野外种群补充新途径,以加快野生海龟资源恢复。但人工条件下亲龟活动空间受限,环境和食物的改变也会对子代的生长发育产生较大影响;水质变坏、体表外伤等容易导致龟苗染病,甚至大量死亡。目前,海龟疾病诊断和治疗困难,给人工繁育尤其龟苗规模化培育带来极大困扰。海水中的细菌会造成较为严重的细菌疾病,对大多数海水养殖对象包括鱼、虾、蟹和贝壳类等,均有不同程度危害。在人工养殖条件下,饵料和环境变差可导致海龟体质下降,易感染寄生类、消化呼吸类及营养类疾病。海龟常见疾病多由细菌、真菌和病毒感染引起,一般表现为肛门肿大、四肢及腹甲发炎或体表多处皮肤、甲壳糜烂等。如海龟腐甲病是环境恶化引起的一种常见的细菌、真菌混合感染疾病,若不加以控制,海龟会出现停食、精神萎靡和大面积糜烂,直至死亡。目前已有大量相关药敏实验来寻找绿海龟龟苗培育中常见疾病的治疗方法,虽然传统抗生素类药物在一定程度上减少了疾病的发生和提高动物生产性能,但抗生素等药物的残留和细菌耐药性等问题日渐严重,从而引发了人们对绿色养殖和食品安全的极大关注。
从上世纪末开始,越来越多的国家开始呼吁禁用抗生素,因此,无毒无公害的新型抗菌剂代替抗生素作为饲料添加剂已成为重要发展趋势。抗菌肽是核糖体合成的天然抗生素,是多细胞生物体免疫系统的一部分。抗菌肽的来源广泛,氨基酸的组成和结构差异很大,具有快速杀伤以及广谱的抗菌活性。抗菌肽是先天免疫反应的进化保守产物,可保护宿主免受昆虫、植物、细菌等产生的多种微生物的侵害。同抗生素相比,抗菌肽的优势在于具有广谱的抗菌活性、对宿主的选择性细胞毒性和不易诱导耐药性等。由于抗菌素耐药性的惊人增加,人们对抗菌肽作为替代抗菌素制剂的兴趣已愈来愈浓厚。这些具有天然抗生素之称的抗菌肽对革兰氏细菌有较强的杀伤作用,阳离子抗菌肽直接与带负电的细菌细胞膜结合,使其细胞膜通透性增加从而导致细菌死亡。抗菌肽功能特性应用也比较广泛,如抗菌活性、抗癌活性等。但目前关于绿海龟抗菌肽的研究尚不深入,因此本发明针对绿海龟抗菌肽进行改造获得一种新的抗菌肽。
发明内容
为解决上述技术问题,本发明提供了一种绿海龟(Cheloniamydas)抗菌肽Cm-CATH2的改造体抗菌肽dsNCM1,具有更广的抗菌谱以及更强的抗炎活性,对多种细菌和炎性因子都有较强的抑制作用。
本发明的第一个目的是提供一种改造体抗菌肽dsNCM1,所述改造体抗菌肽dsNCM1的氨基酸序列如SEQ ID NO.1所示,序列为:
Cys1Phe2Lys3Lys4Val5Arg6Lys7Gln8Leu9Gly10Arg11Val12Lys13Arg14His15Cys16Ser17Arg18Ile19Thr20Val21Gly22Gly23Arg24Met25Arg26Phe27。
进一步地,所述改造体抗菌肽dsNCM1的分子量为3230.96Da。
进一步地,所述改造体抗菌肽dsNCM1的等电点为12.01。
进一步地,上述改造体抗菌肽dsNCM1的制备方法包括以下步骤:根据上述改造体抗菌肽dsNCM1的氨基酸序列,采用多肽固相合成法进行化学合成,得到全序列,再利用HPLC反相柱层析脱盐,得到该改造体抗菌肽dsNCM1。
本发明的第二个目的是提供上述改造体抗菌肽dsNCM1在制备抗菌剂中的应用。
进一步地,所述的抗菌剂用于抑制革兰氏阳性细菌、革兰氏阴性细菌。
进一步地,所述的革兰氏阳性细菌包括但不限于金黄色葡萄球菌(如金黄色葡萄球菌CMCC26003、金黄色葡萄球菌ATCC43300、金黄色葡萄球菌31、金黄色葡萄球菌08032706)、粪肠球菌(如ATCC29212)、屎肠球菌、产气荚膜梭菌(如ATCC13124)和表皮葡萄球菌等。
进一步地,所述的革兰氏阴性细菌包括但不限于大肠杆菌(如大肠杆菌ATCC25922、大肠杆菌CMCC44102)、铜绿假单胞菌(如铜绿假单胞菌CMCC10104)、鲍曼不动杆菌(如鲍曼不动杆菌ATCC19606、鲍曼不动杆菌2、鲍曼不动杆菌6、鲍曼不动杆菌16)、福氏志贺氏菌(如ATCC12022)、鼠伤寒沙门氏菌(如ATCC14028)、溶藻弧菌和哈维氏弧菌等。
本发明的第三个目的是提供上述改造体抗菌肽dsNCM1在制备抗炎剂中的应用。
进一步地,所述的抗炎剂用于抑制肿瘤坏死因子(TNF-α)或白细胞介素6(IL-6)。
本发明的第四个目的是提供上述改造体抗菌肽dsNCM1在制备防腐剂、饲料添加剂或化妆品添加剂中的应用。
本发明的第五个目的是提供上述改造体抗菌肽dsNCM1在制备抗生物膜药物中的应用。
进一步地,抗生物膜药物用于清除生物膜或抑制生物膜的形成。
本发明的第六个目的是提供一种抗菌药物组合物,该抗菌药物组合物包括上述改造体抗菌肽dsNCM1和抗生素。
进一步地,所述抗生素可为美罗培南、多粘菌素B、氨苄青霉素和万古霉素等。
借由上述方案,本发明至少具有以下优点:
(1)本发明通过对绿海龟抗菌肽Cm-CATH2进行肽链缩短,然后在得到的最优抗菌肽的基础上,在第1位和第16位引入半胱氨酸,半胱氨酸之间形成二硫键,得到了进一步改造后的抗菌肽,其含有27个氨基酸残基,分子量3230.96Da,改造后的抗菌肽具有广谱高效的抗菌活性和极强的抗炎活性。同时,改造后的抗菌肽比母本Cm-CATH2序列短分子量小,所以其合成成本更低、免疫原性更低,具有结构简单、溶血活性低、稳定性高、制备方法简单等有益特点。
(2)本发明基于改造体抗菌肽dsNCM1开发了一种药物组合物,改造体抗菌肽dsNCM1与传统抗生素具有协同抗菌作用,在抗炎等方面也具有潜在应用。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明。
图1为改造体抗菌肽dsNCM1抗炎活性测定结果;
图2为改造体抗菌肽dsNCM1蛋白酶稳定性测定结果;
图3为改造体抗菌肽dsNCM1蛋白酶稳定性测试的峰面积变化图;
图4为改造体抗菌肽dsNCM1和NCM4对细菌生物膜的去除活性;其中,A为A.baumanniiATCC19606,B为S.aureusCMCC26003;*代表P<0.05,**代表P<0.01,***代表P<0.001;
图5为改造体抗菌肽dsNCM1和NCM4对细菌生物膜形成的抑制作用;其中,A为A.baumanniiATCC19606,B为S.aureusCMCC26003;*代表P<0.05,**代表P<0.01,***代表P<0.001。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1
改造体抗菌肽dsNCM1的化学合成
绿海龟抗菌肽Cm-CATH2是基因编码的一种多肽,含有33个氨基酸残基,分子量4089.9Da,等电点12.96。绿海龟抗菌肽Cm-CATH2全序列为:Arg1Arg2Ser3Arg4Phe5Gly6Arg7Phe8Phe9Lys10Lys11Val12Arg13Lys14Gln15Leu16Gly17Arg18Val19Lys20Arg21His22Ser23Arg24Ile2 5Thr26Val27Gly28Gly29Arg30Met31Arg32Phe33。
根据绿海龟抗菌肽Cm-CATH2的氨基酸序列,利用分子改造方法设计获得了中间改造体NCM4,全序列为:Phe1Lys2Lys3Val4Arg5Lys6Gln7Leu8Gly9Arg10Val11Lys12Arg13His14Ser1 5Arg16Ile17Thr18Val19Gly20Gly21Arg22Met23Arg24Phe25,然后进一步对中间改造体NCM4进行添加二硫键的结构改造,在第1位和第16位插入2个半胱氨酸而引入二硫键,得到抗菌肽dsNCM1,并利用多肽固相合成的方法对其进行了化学合成,具体制备方法如下:
Ⅰ、dsNCM1的制备方法:根据上述dsNCM1的氨基酸序列,用自动多肽合成仪(433A,Applied Biosystems)合成其全序列,利用HPLC反相柱层析脱盐。
Ⅱ、分子量测定采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)。
Ⅲ、纯化的dsNCM1用高效液相色谱HPLC方法鉴定其纯度,分子量测定采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF),等电聚焦电泳测定等电点。
测定结果为:
dsNCM1是绿海龟抗菌肽Cm-CATH2的一种改造体。dsNCM1是一种含一对分子内二硫键的抗菌肽,含有27个氨基酸残基,分子量3230.96Da,等电点为12.01。dsNCM1全序列为:Cys1Phe2Lys3Lys4Val5Arg6Lys7Gln8Leu9Gly10Arg11Val12Lys13Arg14His15Cys16Ser17Arg18Ile19Thr20Val21Gly22Gly23Arg24Met25Arg26Phe27,其中第1位和第16位的半胱氨酸之间形成二硫键。
实施例2
dsNCM1药理实验:
1.dsNCM1抗菌活性测定:
(1)分别挑取保存于斜面上的试验菌株均匀涂布于MH固体培养基(北京索莱宝科技有限公司)平板上,将经过灭菌的0.5cm直径的滤纸片置于培养基表面,滴加溶解于灭菌去离子水的2mg/ml的抗菌肽dsNCM1、NCM4和Cm-CATH2样品溶液10μl,于37℃倒置培养18-20小时,观察抑菌圈形成与否。若样品具有抗菌活性,则会在滤纸片周围形成清晰透明的抑菌圈,抑菌圈越大表明样品抗菌活性越强。
(2)抗菌肽dsNCM1最小抑菌浓度(Minimum Inhibitory Concentration)测定(2倍稀释法):
选择上步实验中具有抑菌圈的菌株进行MIC测定实验。试验菌株接种到MH液体培养基(北京索莱宝科技有限公司)中,37℃振荡培养到对数生长期,而后用新鲜MH液体培养基将培养至对数生长期的培养液稀释到2×105cfu/ml待用。
在无菌96孔板各孔中预先加入100μl MH液体培养基,然后在第一孔中加入100μl用MH液体培养基稀释到一定浓度的经0.22μm孔滤膜过滤的抗菌肽dsNCM1、NCM4和Cm-CATH2样品溶液,混匀后取100μl加入第2孔,依次倍比稀释(参见表1),自第9孔吸出100μl弃去,第10孔系对照管。
表1稀释方法
将上述各管混匀后放置37℃缓慢振荡培养18小时,于600nm波长处测定光吸收。最小抑菌浓度为看不见细菌生长的最低样品浓度。结果如表2所示。
表2改造体抗菌肽dsNCM1抗菌活性
MIC:最小抑菌浓度,以上结果为三次独立重复实验平均值。
由表2可见,抗菌肽dsNCM1对革兰氏阳性细菌、革兰氏阴性细菌均表现出很强的抗菌活性,其中包括部分临床分离致病菌,MIC值处于4.69-18.75μg/ml的范围。
2.dsNCM1溶血活性测定:
将采集的兔血与阿氏液混合抗凝,生理盐水洗涤2次并重悬成107-108cell/ml的悬浮液。上述稀释好的红细胞悬液与溶解于生理盐水的dsNCM1、NCM4或Cm-CATH2样品混合,37℃保温30min,再于1000rpm离心5min,上清液于540nm测吸收值。阴性对照使用生理盐水,阳性对照使用Triton X-100,溶血百分比按以下公式计算:溶血百分比H%=A样品-A阴性对照/A阳性对照×100%。
结果表明样品浓度为100μg/ml,dsNCM1的溶血百分比为1.82%,NCM4的溶血百分比为1.51%,而Cm-CATH2的溶血百分比为4.1%。说明dsNCM1具有很低的溶血活性,不易引起哺乳动物红细胞破裂溶解。尤其抗菌活性范围内,安全性高。
3.dsNCM1抗炎活性测定:
提取6-8周龄C57小鼠腹腔巨噬细胞,用含10%血清的1640培养基培养过夜,次日换成含2%血清的1640培养基,然后用终浓度为100ng/mL的大肠杆菌LPS(Sigma,美国)刺激细胞,同时给多肽dsNCM1或NCM4,终浓度为20μg/mL,设不给多肽和LPS的空白对照组与仅给LPS的阳性对照组,共孵育16h,取上清,用ELISA试剂盒(R&D,美国)检测上清液中促炎因子IL-6和TNF-α的含量。每个做三个平行。
结果如图1所示,dsNCM1能够显著抑制小鼠腹腔巨噬细胞中LPS诱导的促炎因子IL-6和TNF-α表达,表明dsNCM1具有极强的抗炎活性。与NCM4相比,dsNCM1对TNF-α的抗炎效果较弱,但是对IL-6的抗炎效果强于NCM4。
4.改造体抗菌肽dsNCM1稳定性实验研究:
(1)酶稳定性测试:将细胞消化用的0.25%胰酶与多肽样品,按照摩尔比1:200的比例混合,37℃下孵育,分别在0、6、12、24h时取样50μL,然后用多肽溶剂将所取样品稀释1倍,用0.22μm的滤膜过滤,取20μL用反向高效液相色谱测定多肽样品的残留量。其中A相用含0.1%三氟乙酸(TFA)的纯水,B相用含0.1%TFA的乙腈,进行梯度洗脱,得到多肽样品dsNCM1与胰酶混合后不同时间点的洗脱峰和积分面积,然后用软件Origin2018作图。
结果如图2和3所示,抗菌肽dsNCM1具有很强的酶稳定性,与酶作用24h后dsNCM1的峰高和峰面积仍然变化不大,而抗菌肽NCM4和Cm-CATH26h就显著降解,说明dsNCM1的稳定性显著优于NCM4和Cm-CATH2。
(2)盐耐受性、热耐受性和热稳定性测试
盐耐受性:大肠杆菌ATCC25922用MH液体培养基(青岛海博生物技术有限公司)于37℃培养12小时,然后分别用含0、50、100、150、200和400mM氯化钠的新鲜MH液体培养基稀释到106CFU/ml。用含相应氯化钠浓度的MH液体培养基制备不同浓度梯度的dsNCM1、NCM4和Cm-CATH2样品。利用2倍稀释法测定dsNCM1、NCM4和Cm-CATH2对大肠杆菌ATCC25922的MIC值,以此确定氯化钠对dsNCM1、NCM4和Cm-CATH2抗菌活性的影响。
热耐受性:dsNCM1、NCM4或Cm-CATH2溶解于灭菌的去离子水中(2mg/ml),在4、20、37、50、70和90℃孵育1小时,然后利用2倍稀释法测定样品对大肠杆菌ATCC25922的MIC值,以此确定不同温度加热对样品抗菌活性的影响。
热稳定性:dsNCM1、NCM4或Cm-CATH2溶解于灭菌的去离子水中(2mg/ml),在37℃孵育0-96小时。于0、6、12、24、48、72和96小时分别取样品检测对大肠杆菌ATCC25922的MIC值,以此确定样品的热稳定性。
如表3所示,dsNCM1、NCM4和Cm-CATH2具有很强的盐耐受性。在低于或等于人体生理盐浓度下(≤150mMNaCl),dsNCM1、NCM4和Cm-CATH2的抗菌活性保持不变。在盐浓度高于人体生理盐浓度后,dsNCM1、NCM4和Cm-CATH2的抗菌活性也只是随着盐浓度的升高而略有降低。
表3改造体抗菌肽dsNCM1盐耐受性
如表4所示,dsNCM1具有很强的热耐受性。dsNCM1溶液在90℃放置1小时之后,其抗菌活性不会改变。与之相比,NCM4和Cm-CATH2的热耐受性略差,在高温下加热1h后,其MIC值均有升高。
表4改造体抗菌肽dsNCM1热耐受性
许多传统的抗生素,如头孢类抗生素的溶液极不稳定,几个小时内就会失去活性,这大大限制了它们的使用。与之不同,dsNCM1溶液具有很好的热稳定性。dsNCM1溶液在37℃放置96小时后,其抗菌活性不会改变(如表5)。与之相比,NCM4和Cm-CATH2的热稳定性略差,在37℃放置96小时后,其MIC值均有升高。
表5改造体抗菌肽dsNCM1热稳定性
实施例3
改造体抗菌肽dNCM2和NCM4的生物膜清除和抑制活性测定。
1.生物膜清除活性测定
从-80℃冰箱中取出保存的菌株,37℃水浴快速融化,用接种环蘸取少许液体,在LB固体培养基上呈Z字形分四个区域划线,每次划线从上次末端开始,37℃恒温培养至长出单菌落;挑取单菌落于无菌液体MHB培养基中,37℃、180rpm振荡培养至对数生长期;检测菌液浓度,稀释成2×107CFU/mL。向无菌96孔板中加入上述菌液200μL,37℃培养48h使生物膜形成。吸出每孔的菌液,并用PBS洗涤三次。每孔加入200μL稀释后的多肽样品使多肽样品终浓度为0.5×MIC、1×MIC、2×MIC、4×MIC和8×MIC,37℃恒温培养24h。每孔加入结晶紫染液(0.1%),染色30min后吸出染液,无菌PBS洗涤三次,超净台中风干。每孔加入100μL无水乙醇,静置20min,溶解结晶紫。在紫外波长560nm下检测OD值,实验设置三个平行。用以下公式计算生物膜形成的百分比(Biofilm Retention%,BR%):BR(%)=100%-[100%×(F0-Fpeptide)/F0]该实验中选择PBS作为阴性对照,其测定值视为最大生物膜残留量。BiofilmRetention%,BR%为生物膜残存百分比,F0为PBS处理组的吸光值,Fpeptide为多肽处理组吸光值。
2.生物膜抑制活性测定
从-80℃冰箱中取出保存的菌株,37℃水浴快速融化,蘸取少许液体在LB固体培养基上呈Z字形划线,在37℃恒温培养至长出单菌落;挑取单菌落于无菌液体MHB培养基中,37℃,180rpm振荡培养至对数生长期;检测菌液浓度,稀释成2×107CFU/mL。向无菌96孔板中加入上述菌液190μL,每孔加入稀释后的多肽样品,每个浓度设置3个平行。使得多肽样品终浓度为0.5×MIC、1×MIC、2×MIC、4×MIC和8×MIC,37℃恒温培养48h。吸出每孔的菌液,并用PBS洗涤三次,把板置于超净台通风吹干。每孔加入结晶紫染液(0.1%),染色30min后吸出染液,无菌PBS洗涤三次,超净台中风干。每孔加入100μL无水乙醇,静置20min,溶解结晶紫。在紫外波长560nm下检测OD值。用以下公式计算生物膜形成的百分比(BiofilmFormation%,BF%):BF(%)=100%-[100%×(F0-Fpeptide)/F0]其中PBS(F0)为阴性对照,其测定值视为最大生物膜形成量。Biofilm Formation%,BF%为生物膜形成百分比,Fpeptide为多肽处理组吸光值。
如图4和5所示,改造体多肽dsNCM1和NCM4均能较有效的清除鲍曼不动杆菌(图4A)和金黄色葡萄球菌(图4B)产生的生物膜,存在浓度依赖性,在较高浓度下dsNCM1比NCM4更有效地清除金黄色葡萄球菌产生的生物膜,而在清除鲍曼不动杆菌产生的生物膜方面,dsNCM1效果略优于NCM4。改造体多肽dsNCM1和NCM4均能浓度依赖地抑制这两株菌产生的生物膜,并且在高浓度下dsNCM1较NCM4更有效地抑制这两株菌产生的生物膜,且差异显著。
实施例4
改造体抗菌肽dsNCM1和NCM4与抗生素协同抗菌作用测定
将多肽用无菌水配成20×8MIC的浓度,依次再倍比稀释直到20×1/64MIC,加上溶剂共11个浓度备用。称取抗生素(美罗培南、多粘菌素B、氨苄青霉素和万古霉素)药品,用无菌水溶成2mg/mL的溶液,依次倍比稀释得到4MIC-1/16MIC的浓度,加上溶剂共8个浓度备用。稀释菌液至5×105CFU/mL,备用。棋盘法测:在96孔板中加入90μL稀释好的菌液;将多肽加到菌中,每孔5μL,每一列一个浓度,共11列:将抗生素加到菌中,每孔5μL,每一行一个浓度,共8行;找到MICA,MICB,A,B计算FIC,FIC=FMICA+FMICB=A/MICA+B/MICB(A,B代表两药联用的最佳集合点处的浓度,MICA,MICB代表两药单用时的MIC)FIC<0.5时有协同作用,FIC>4时有拮抗作用,0.5<FIC<4时两药有相加作用。加药方式如下表所示。
选用鲍曼不动杆菌(A.baumanniiATCC19606)和耐甲氧西林金黄色葡萄球菌(S.aureusATCC43300)作为供试菌,检测了几种抗生素与改造体抗菌肽dsNCM1的协同抗菌作用。其中美罗培南和多粘菌素B均能与之协同发挥抗鲍曼不动杆菌的作用,FICI指分别为0.375和0.25。而氨苄青霉素和万古霉素能与改抗造体抗菌肽dsNCM1协同抗耐甲氧西林金黄色葡萄球菌的作用,FICI指数分别为0.375和0.375。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
序列表
<110> 苏州大学
<120> 一种改造体抗菌肽dsNCM1及其应用
<141> 2022-09-19
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> PRT
<213> (人工序列)
<400> 1
Cys Phe Lys Lys Val Arg Lys Gln Leu Gly Arg Val Lys Arg His Cys
1 5 10 15
Ser Arg Ile Thr Val Gly Gly Arg Met Arg Phe
20 25
Claims (10)
1.一种改造体抗菌肽dsNCM1,其特征在于:所述改造体抗菌肽dsNCM1的氨基酸序列如SEQ ID NO.1所示。
2.权利要求1所述的改造体抗菌肽dsNCM1在制备抗菌剂中的应用。
3.根据权利要求2所述的应用,其特征在于:所述抗菌剂用于抑制革兰氏阳性细菌或革兰氏阴性细菌。
4.根据权利要求3所述的应用,其特征在于:所述革兰氏阳性细菌包括金黄色葡萄球菌、粪肠球菌、屎肠球菌、产气荚膜梭菌或表皮葡萄球菌。
5.根据权利要求3所述的应用,其特征在于:所述的革兰氏阴性细菌包括大肠杆菌、铜绿假单胞菌、鲍曼不动杆菌、福氏志贺氏菌、鼠伤寒沙门氏菌、溶藻弧菌或哈维氏弧菌。
6.权利要求1所述的改造体抗菌肽dsNCM1在制备抗炎剂中的应用。
7.权利要求1所述的改造体抗菌肽dsNCM1在制备抗生物膜药物中的应用。
8.根据权利要求7所述的应用,其特征在于:所述抗生物膜药物用于清除生物膜或抑制生物膜的形成。
9.一种抗菌药物组合物,其特征在于:所述抗菌药物组合物包括权利要求1所述的改造体抗菌肽dsNCM1和抗生素。
10.根据权利要求9所述的抗菌药物组合物,其特征在于:所述的抗生素选自美罗培南、多粘菌素B、氨苄青霉素和万古霉素中的一种。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210467019.1A CN114853865B (zh) | 2022-04-29 | 2022-04-29 | 一种改造体抗菌肽dsNCM1及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210467019.1A CN114853865B (zh) | 2022-04-29 | 2022-04-29 | 一种改造体抗菌肽dsNCM1及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114853865A CN114853865A (zh) | 2022-08-05 |
CN114853865B true CN114853865B (zh) | 2024-03-15 |
Family
ID=82635330
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210467019.1A Active CN114853865B (zh) | 2022-04-29 | 2022-04-29 | 一种改造体抗菌肽dsNCM1及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114853865B (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116870132B (zh) * | 2023-07-31 | 2024-04-26 | 中国医学科学院医学生物学研究所 | 一种抗菌肽rh-16及其在制备抗耐药抑菌药物中的应用 |
CN116813713B (zh) * | 2023-07-31 | 2024-04-19 | 中国医学科学院医学生物学研究所 | 一种改造体抗菌肽ri-18及其应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111658761A (zh) * | 2020-06-19 | 2020-09-15 | 苏州大学 | 天然宿主防御肽Cm-CATH2的应用 |
CN112625108A (zh) * | 2020-11-30 | 2021-04-09 | 宜肌坊(厦门)生物科技有限公司 | 一种绿海龟抗菌肽的改造体抗菌肽c-cm5及其制备方法和应用 |
CN112625109A (zh) * | 2020-11-30 | 2021-04-09 | 宜肌坊(厦门)生物科技有限公司 | 一种绿海龟抗菌肽的改造体抗菌肽c-cm6及其制备方法和应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018039601A1 (en) * | 2016-08-26 | 2018-03-01 | Gangagen, Inc. | Staphtame activity on biofilms |
-
2022
- 2022-04-29 CN CN202210467019.1A patent/CN114853865B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111658761A (zh) * | 2020-06-19 | 2020-09-15 | 苏州大学 | 天然宿主防御肽Cm-CATH2的应用 |
CN112625108A (zh) * | 2020-11-30 | 2021-04-09 | 宜肌坊(厦门)生物科技有限公司 | 一种绿海龟抗菌肽的改造体抗菌肽c-cm5及其制备方法和应用 |
CN112625109A (zh) * | 2020-11-30 | 2021-04-09 | 宜肌坊(厦门)生物科技有限公司 | 一种绿海龟抗菌肽的改造体抗菌肽c-cm6及其制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN114853865A (zh) | 2022-08-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN114853865B (zh) | 一种改造体抗菌肽dsNCM1及其应用 | |
KR101896927B1 (ko) | 흰점박이꽃무지에서 유래한 항균 펩타이드 프로테티아마이신 1 및 그의 조성물 | |
CN111658761A (zh) | 天然宿主防御肽Cm-CATH2的应用 | |
KR100964136B1 (ko) | 울도하늘소유충으로부터 분리한 항균 및 항진균 펩타이드 유전자 및 항균 및 항진균 활성을 가지는 합성 펩타이드 | |
CN104497119A (zh) | 一种天然抗菌肽及其应用 | |
CN112625107A (zh) | 一种绿海龟抗菌肽的改造体抗菌肽c-cm8及其制备方法和应用 | |
CN112625108A (zh) | 一种绿海龟抗菌肽的改造体抗菌肽c-cm5及其制备方法和应用 | |
CN112625109A (zh) | 一种绿海龟抗菌肽的改造体抗菌肽c-cm6及其制备方法和应用 | |
CN111087460B (zh) | 一种广谱型抗菌肽及其应用 | |
KR20180052181A (ko) | 흰점박이꽃무지에서 유래한 항균 펩타이드 프로테티아마이신 2 및 그의 조성물 | |
CN110156875B (zh) | 抗菌肽H5-p5及其制备方法和应用 | |
CN113321708B (zh) | 一种人工设计抗菌肽的制备及其在水产上的应用 | |
KR101740551B1 (ko) | 벼메뚜기에서 유래한 항균 펩타이드 옥시야신-2 및 그의 조성물 | |
CN115043925B (zh) | 一种改造体抗菌肽oNCM及其应用 | |
CN111574619B (zh) | 脂肽Lin-Lf4NH2和Lin-Lf5NH2及其应用 | |
KR20170053879A (ko) | 벼메뚜기에서 유래한 항균 펩타이드 옥시야신-1 및 그의 조성물 | |
KR102146930B1 (ko) | 왕귀뚜라미에서 유래한 항균 펩타이드 테레오그릴루신 3 및 그의 조성물 | |
CN113999297B (zh) | 一种抗菌肽hrNCM及其制备方法与应用 | |
KR100459808B1 (ko) | 헬리코박터 파이로리균의 리보좀 단백질 l1 유래의새로운 항생 펩타이드 및 그의 용도 | |
KR101825952B1 (ko) | 벼메뚜기에서 유래한 항균 펩타이드 옥시야신-3 및 그의 조성물 | |
CN115043924B (zh) | 一种改造体抗菌肽及其应用 | |
CN113735956A (zh) | 一种抗菌肽ccm7wc及其制备方法与应用 | |
KR102146935B1 (ko) | 광대노린재로부터 유래된 포에실로코리신-1 펩타이드, 이를 유효성분으로 포함하는 항균, 항진균 또는 항염증용 조성물 | |
KR101465098B1 (ko) | 융합 항균펩타이드 paje 및 이를 합성하는 방법 | |
CN102796176B (zh) | 一种昆嵛林蛙抗菌肽Kunyuenin及其制备和应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |