CN106188264A - 一种抗微生物肽Cm‑CATH3及其基因、制备方法和应用 - Google Patents
一种抗微生物肽Cm‑CATH3及其基因、制备方法和应用 Download PDFInfo
- Publication number
- CN106188264A CN106188264A CN201610580361.7A CN201610580361A CN106188264A CN 106188264 A CN106188264 A CN 106188264A CN 201610580361 A CN201610580361 A CN 201610580361A CN 106188264 A CN106188264 A CN 106188264A
- Authority
- CN
- China
- Prior art keywords
- cath3
- antimicrobial peptide
- arg
- gene
- molecular weight
- 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.)
- Pending
Links
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 38
- 102000044503 Antimicrobial Peptides Human genes 0.000 title claims abstract description 19
- 108700042778 Antimicrobial Peptides Proteins 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 title claims description 20
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 16
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 15
- 230000003115 biocidal effect Effects 0.000 claims abstract description 13
- 230000001954 sterilising effect Effects 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 11
- 235000013305 food Nutrition 0.000 claims abstract description 11
- 230000036541 health Effects 0.000 claims abstract description 9
- 229920001184 polypeptide Polymers 0.000 claims abstract description 8
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 8
- 239000002537 cosmetic Substances 0.000 claims abstract description 7
- 206010059866 Drug resistance Diseases 0.000 claims abstract description 6
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 4
- 238000009364 mariculture Methods 0.000 claims abstract description 4
- 244000144972 livestock Species 0.000 claims abstract description 3
- 108060001132 cathelicidin Proteins 0.000 claims description 20
- 102000014509 cathelicidin Human genes 0.000 claims description 18
- POIUWJQBRNEFGX-XAMSXPGMSA-N cathelicidin Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C)C1=CC=CC=C1 POIUWJQBRNEFGX-XAMSXPGMSA-N 0.000 claims description 11
- 238000001228 spectrum Methods 0.000 claims description 7
- 230000000845 anti-microbial effect Effects 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 239000000654 additive Substances 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 4
- 238000001155 isoelectric focusing Methods 0.000 claims description 4
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 claims description 4
- 235000018102 proteins Nutrition 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 230000000996 additive effect Effects 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- 239000002243 precursor Substances 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 239000003242 anti bacterial agent Substances 0.000 claims description 2
- 230000002924 anti-infective effect Effects 0.000 claims description 2
- 229940088710 antibiotic agent Drugs 0.000 claims description 2
- 229960005475 antiinfective agent Drugs 0.000 claims description 2
- 238000010276 construction Methods 0.000 claims description 2
- 238000011033 desalting Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 239000002260 anti-inflammatory agent Substances 0.000 claims 1
- 229940124599 anti-inflammatory drug Drugs 0.000 claims 1
- 125000001151 peptidyl group Chemical group 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 8
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 6
- 208000015181 infectious disease Diseases 0.000 abstract description 5
- 238000012360 testing method Methods 0.000 abstract description 5
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 206010018910 Haemolysis Diseases 0.000 abstract description 2
- 230000008588 hemolysis Effects 0.000 abstract description 2
- 231100000167 toxic agent Toxicity 0.000 abstract description 2
- 239000003440 toxic substance Substances 0.000 abstract description 2
- 241000270607 Chelonia mydas Species 0.000 description 20
- 241000894006 Bacteria Species 0.000 description 18
- 239000004475 Arginine Substances 0.000 description 15
- 230000000694 effects Effects 0.000 description 13
- 239000000523 sample Substances 0.000 description 13
- 239000007788 liquid Substances 0.000 description 12
- 150000001413 amino acids Chemical class 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 239000004472 Lysine Substances 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 239000003755 preservative agent Substances 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- 239000004471 Glycine Substances 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 229960000310 isoleucine Drugs 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- 230000002335 preservative effect Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 229920005654 Sephadex Polymers 0.000 description 4
- 239000012507 Sephadex™ Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000007850 degeneration Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 230000001665 lethal effect Effects 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000004474 valine Substances 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000004007 reversed phase HPLC Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical class OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000000843 anti-fungal effect Effects 0.000 description 2
- 229940121375 antifungal agent Drugs 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000005611 electricity Effects 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 238000002270 exclusion chromatography Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000005227 gel permeation chromatography Methods 0.000 description 2
- 229960002989 glutamic acid Drugs 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 108010062940 pexiganan Proteins 0.000 description 2
- 238000009522 phase III clinical trial Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- WHNFPRLDDSXQCL-UAZQEYIDSA-N α-msh Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(N)=O)NC(=O)[C@H](CO)NC(C)=O)C1=CC=C(O)C=C1 WHNFPRLDDSXQCL-UAZQEYIDSA-N 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 229910014863 CaCl2—MgCl2 Inorganic materials 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 206010007134 Candida infections Diseases 0.000 description 1
- 102100038608 Cathelicidin antimicrobial peptide Human genes 0.000 description 1
- 101710140438 Cathelicidin antimicrobial peptide Proteins 0.000 description 1
- 241000938605 Crocodylia Species 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001360526 Escherichia coli ATCC 25922 Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 206010021531 Impetigo Diseases 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical group OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 241000270322 Lepidosauria Species 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 101800001751 Melanocyte-stimulating hormone alpha Proteins 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 241001105393 Mydas Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000007117 Oral Ulcer Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102100027467 Pro-opiomelanocortin Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 108010078256 antimicrobial peptide IB-367 Proteins 0.000 description 1
- 208000002399 aphthous stomatitis Diseases 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 201000003984 candidiasis Diseases 0.000 description 1
- 108010007004 cathelin Proteins 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- BQFCCCIRTOLPEF-UHFFFAOYSA-N chembl1976978 Chemical compound CC1=CC=CC=C1N=NC1=C(O)C=CC2=CC=CC=C12 BQFCCCIRTOLPEF-UHFFFAOYSA-N 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- -1 electrophoresis Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000004992 fast atom bombardment mass spectroscopy Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000005452 food preservative Substances 0.000 description 1
- 235000019249 food preservative Nutrition 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 244000038280 herbivores Species 0.000 description 1
- 230000007236 host immunity Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000013227 macrophage apoptotic process Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 229960002260 meropenem Drugs 0.000 description 1
- 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 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000031972 neutrophil apoptotic process Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- KGZGFSNZWHMDGZ-KAYYGGFYSA-N pexiganan Chemical compound C([C@H](NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(N)=O)C1=CC=CC=C1 KGZGFSNZWHMDGZ-KAYYGGFYSA-N 0.000 description 1
- 229950001731 pexiganan Drugs 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 238000001507 sample dispersion Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- 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
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
-
- 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
-
- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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)
- Dermatology (AREA)
- Organic Chemistry (AREA)
- Polymers & Plastics (AREA)
- Zoology (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Toxicology (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Peptides Or Proteins (AREA)
- Cosmetics (AREA)
Abstract
一种抗微生物肽Cm‑CATH3及其制备方法和应用,属于生物医学技术领域。Cm‑CATH3是一种直链多肽,理论等电点为12.23,分子量是4070.85Da,净电荷为+11,含有33个氨基酸残基。Cm‑CATH3对所有测试菌株,包括ATCC标准菌株和临床分离的耐药菌株,都有很强的广谱抗菌活性,最小抑菌浓度仅为0.2866μM;且杀菌效果迅速,无细菌恢复生长。Cm‑CATH3分子量小,低溶血、低细胞毒,可杀死耐药菌株,且不易产生耐药性,是理想的新型抗生素模板,可用于抗感染、抗炎、消毒等药物制备。也可应用于海产养殖,畜牧业饲料,日常化妆品、保健品和食品等。
Description
技术领域
本发明提供一种来源于绿海龟(C.mydas)的Cathelicidin家族广谱抗微生物肽Cm-CATH3及其制备方法,功能研究及其在抗感染、抗炎、消毒等药物制备,海产养殖,畜牧业饲料,日常化妆品、保健品和食品等添加剂的应用,属于生物医学技术领域。
背景技术
抗菌肽广泛分布于自然界各种生物体内,是生物体天然防御系统的一个重要组成部分。Cathelicidin是在哺乳动物体内发现的一类结构多变具有多功能的抗微生物肽家族,因在信号肽与成熟肽之间含有一段高度保守的cathelin而自成一个家族。目前在许多物种中都有发现,包括哺乳动物、鸟类、爬行动物、两栖动物、以及鱼类等。Cathelicidins家族抗菌肽在结构上存在较大差异,但是也存在一些共同点。Cathelicidins家族抗菌肽都富含带正电荷的赖氨酸和精氨酸残基,很少含或者不含带负电荷的天冬氨酸和谷氨酸残基,因此cathelicidins抗菌肽均带正电荷。Cathelicidins具有广谱的抗微生物活性,不但对普通革兰氏阳性细菌、革兰氏阴性细菌、真菌、霉菌、原虫和部分有包膜病毒有很强的活性,而且对许多临床耐药微生物同样具有作用。研究发现抗菌肽不仅能够直接抑制和杀灭病源微生物,同时具有多种免疫调节活性,在宿主的天然和适应性免疫中起着重要的作用。此外,cathelicidins具有许多其他生物学活性,如人类Cathelicidins LL-37和猪PR-39都具有血管原特性,参与伤口的治愈,诱导细胞趋化,结合内毒素;PR-39还可以抑制巨噬细胞的凋亡;而LL-37可以抑制嗜中性白细胞的凋亡,还具有抗肿瘤活性等。因而Cathelicidins具有广阔的应用前景。
由于传统抗生素的大量使用导致了耐药病原微生物的迅速增长,因此现在亟待开发出新型抗生素来对付世界范围内日益严重的病原微生物耐药性。抗菌肽在自然界分布广泛,是生物体产生的抵御外源性感染的内源性物质,历经数百万年而很少产生耐受性,具有作用迅速、强大、广谱等特点。抗菌肽显然已成为替代抗生素的理想模板。相比于普通抗生素,其抗菌谱广,相对分子质量小,热稳定性好,无免疫原性,不易引起耐药性。此外,抗菌肽通过其独特的作用机制和广泛的生物学功能,对真核细胞几乎没有作用,只对原核细胞和发生病变的真核细胞有选择性的杀伤作用,极有可能成为一种新的预防和治疗动物疾病的药物。目前抗菌肽的在医疗、医药方面应用研究有很多,并且已有许多新型药物逐步迈入医药市场。例如,Pexiganan又名MSI-78,为非洲爪蟾抗菌肽Maganin的类似物,是第一个进行商业开发的抗菌肽,由22个氨基酸残基组成,用来治疗脓疱病和糖尿病患者足底溃烂等,已经进入临床III期试验阶段;源自α-黑素细胞刺激素的CZEN-002,8氨基酸残基,用于治疗阴道念珠菌,处于临床II期;来自人BPI的Neuprex,用来治疗脑膜炎,已经III期成功;来源于猪protegrin的IB-367用于治疗肿瘤患者口腔溃疡,已进入临床III期试验;源于人的XMP.629,用于治疗痤疮,处于临床III期等。抗菌肽极有可能成为一种高效低毒且无残留的抗菌、抗病毒的新药物。
传统的防腐剂广泛应用于加入食品、药品、生物标本、化妆品、保健品等,是抑制物质腐败的药剂,即对以腐败物质为代谢底物的微生物的生长具有持续的抑制作用。以食品防腐剂为例,其作用是一把“双刃剑”,保证食物新鲜的同时,也有可能给人们的健康带来一定的麻烦。在我国,食品生产中使用的防腐剂绝大多数都是人工合成的,使用不当会有一定的副作用;有些防腐剂甚至含有微量毒素,长期过量摄入会对人体健康造成一定的损害,广泛使用的食品防腐剂苯甲酸为例,国际上对其使用一直存有争议。而抗菌肽具有广谱的抗菌作用,本身就是天然活性物质,是无毒、无害、无残留的绿色产品,其成分为易消化吸收的氨基酸,不会对人的身体造成伤害,是理想的传统防腐剂的替代品。不仅如此,抗菌肽还可以作为畜禽饲料添加剂取代或部分取代饲喂动物所用的抗生素,将会减少抗生素对动物体的危害,避免抗生素残留对人类的伤害。可见,Cathelicidin在生物制药、农业科学、食品化工和化妆品等领域具有很好的应用前景。
绿海龟(C.mydas)是体型最大的硬壳海龟之一,因其身上的脂肪为绿色而得名。其广泛分布在热带及亚热带海域中,为草食性动物。目前科研人员在很多两栖爬行类动物中得到了Cathelicidin抗菌肽的编码基因,并以两栖爬行动物Cathelicidin抗菌肽为模板进行了抗感染药物的开发研究,但是关于绿海龟cathelicidin抗菌肽的研究和应用还没有报道。
发明内容
本发明提供一种来源于绿海龟(C.mydas)的cathelicidin家族广谱抗微生物肽Cm-CATH3及其编码基因,制备方法,功能研究及其在抗感染、抗炎、消毒等药物制备,海产养殖,畜牧业饲料中的抗生素,以及日常化妆品、保健品和食品等中的防腐剂和抗氧化剂的替代中的应用。
为了实现本发明的目的,本发明提供了如下技术方案:
以绿海龟脾脏为原料,按照下述步骤分离纯化Cm-CATH3:
选取绿海龟脾脏组织,洗涤,匀浆,获得脾脏组织蛋白粗提物。随后,Sephadex G-50凝胶过滤层析,用自动部分收集器收集3mL/管,220nm检测并收集各峰检测抗菌活性,冻干备用。然后经反相高压液相层析(RP-HPLC)进行梯度洗脱,收集各多肽样品峰,用灭菌去离子水溶解,检测抗菌活性。最后对得到多肽样品进行一级结构的解析,包括采用电喷雾四级杆飞行时间串联质谱法(ESI-Q-TOF-MS,Biosystems/MDS Sciex多伦多,加拿大)测定分子量。利用等电聚焦电泳测定其等电点,用Edman降解法确定活性多肽氨基酸序列组成。其氨基酸序列为:苏氨酸1-精氨酸2-甘氨酸3-精氨酸4-色氨酸5-赖氨酸6-精氨酸7-苯丙氨酸8-色氨酸9-精氨酸10-甘氨酸11-丙氨酸12-甘氨酸13-精氨酸14-苯丙氨酸15-苯丙氨酸16-精氨酸17-精氨酸18-组氨酸19-赖氨酸20-谷氨酸21-赖氨酸22-异亮氨酸23-异亮氨酸24-精氨酸25-丙氨酸26-丙氨酸27-缬氨酸28-天冬氨酸29-异亮氨酸30-缬氨酸31-亮氨酸32-丝氨酸33。
绿海龟抗微生物肽Cm-CATH3基因的克隆包括:
绿海龟肝脏总RNA提取,mRNA纯化,mRNA反转录及cDNA文库构建,设计引物,利用PCR方法筛选绿海龟抗菌肽Cm-CATH3基因。获得阳性单克隆进行基因核苷酸序列测定。编码Cm-CATH3前体cathelicidin的基因由492个核苷酸组成,自5’端至3’端序列为:
其中391到489位是编码抗微生物肽Cm-CATH3的核苷酸序列。
抗微生物肽Cm-CATH3的化学制备方法:根据Cm-CATH3氨基酸序列,用全自动多肽合成仪合成其全序列;通过HPLC反相C18柱层析脱盐、纯化,然后用HPLC方法鉴定并确定其纯度大于97%;用基质辅助激光解析电离飞行时间质谱法(FAB-MS)测定其分子量;等电聚焦电泳测定等电点,最后用自动氨基酸测序仪测定氨基酸序列结构。
所述抗微生物肽Cm-CATH3可应用在抗感染、抗炎、消毒等药物制备,海产养殖,畜牧业饲料,日常化妆品、保健品和食品等添加剂中。
本发明的有益效果在于:基因克隆得到编码绿海龟cathelicidin抗微生物肽Cm-CATH3的基因,并利用化学合成方法得到抗微生物Cm-CATH3样品。体外活性实验发现,Cm-CATH3具有显著地抑制细菌和真菌的作用,包括对临床分离得到的耐药菌也有很好的抑制作用;其抗菌谱广,杀菌效果迅速;结构简单,人工合成方便;还具有低溶血、低细胞毒、不易产生耐药性、无免疫原性等有益特点。
具体实施方式
下面结合技术方案详细叙述本发明的具体实施例,但本发明的内容并不局限于此。
实施例1 Cm-CATH3的分离纯化:
(1)从动物园获取濒临死亡的绿海龟新鲜完整脾脏组织,用生理盐水少许洗涤脾脏组织表面。匀浆后,用少量生理盐水溶解,按PS液与正丁醇1:50(V/V)比例,把PS与正丁醇在室温下搅拌60min,13000r/min,20min离心两次,然后将沉淀冻干。随后,第一步SephadexG-50凝胶过滤层析:0.9g冻干粉用10ml 0.1M磷酸盐(Na2HPO4-NaH2PO4,pH 6.0)缓冲液溶解,12000rpm离心10min,取上清液上样于已平衡好的Sephadex G-50凝胶排阻色谱柱(1.6cm x 90cm,Amersham Bioscience),用同样缓冲液洗脱,流速3mL/10min,用自动部分收集器收集3mL/管,220nm检测并收集各峰检测抗菌活性,冻干备用。
(2)反相高压液相层析(RP-HPLC):将Sephadex G-50凝胶排阻色谱分离得到的活性成分的峰重新溶解于纯水中,4℃,12000rpm离心15min,取上清液,用0.45μm滤膜过滤,收集滤液上样于经含1‰三氟乙酸的超纯水充分平衡的C18反相柱(Hypersil BDS C18,30cmx 0.46cm)用乙腈(含1‰三氟乙酸)构成的洗脱系统进行梯度洗脱,215nm检测多肽浓度。收集得到的各峰,冻干浓缩,用灭菌的去离子水重新溶解并进行抗菌活性检测。
(3)活性多肽一级结构分析。纯化的Cm-CATH3采用电喷雾四级杆飞行时间串联质谱法(ESI-Q-TOF-MS,Biosystems/MDS Sciex多伦多,加拿大)测定分子量。利用等电聚焦电泳测定其等电点,用Edman降解法确定了其氨基酸序列组成为Cm-CATH3:TRGRWKRFWRGAGRFFRRHKEKIIRAAVDI VLS。
实施例2 Cm-CATH3前体基因的克隆及基因测序
第一步、绿海龟肝脏总RNA提取(以下实验所用器具和试剂均经过处理,无RNase):
A.从新鲜宰杀的绿海龟各种新鲜组织上分别剪下1g左右的小块,分别放入液氮预冷的细胞冻存管中,然后迅速放入液氮中保存;
B.将保存在液氮中的组织材料取出,放入预冷的研钵内,迅速充分研磨,期间不断向研钵内加入少许液氮;将大约30mg的组织粉末转移至预冷的1.5ml离心管中,向其中分别加入400μl Buffer R-I(裂解液,RNA Miniprep Kit提供),用21-25号针头的注射器反复抽吸8-10次,转入1.5ml离心管中,加入150μl Buffer R-П,涡旋振荡15-30s,4℃,12000rpm离心5min;
C.将离心后的上清转移至新的1.5ml离心管中,加入250ul异丙醇,迅速吸打混匀;将混合液分别转移至离心吸附柱,室温,6000rpm离心1min;弃废液,然后加入500μl BufferW1A,4℃,12000rpm离心1min;弃废液,加入700μl BufferW2A,4℃,12000rpm离心1min; 弃废液,加入700μl Buffer W2A,4℃,12000rpm离心1min;弃废液,空管12000rpm离心1min;将离心吸附柱转移至新的1.5ml离心管中,然后直接向吸附膜上滴加70-100μl Buffer TE,室温放置1min,12000rpm离心1min洗脱得到总RNA;
第二步、cDNA文库的构建
第一链的合成(mRNA反转录):
A.在新的0.2ml PCR管(无DNase和RNase)中配制下列混合液:
混合均匀,短暂离心;PCR仪中72℃保温5min后,然后42℃2min;短暂离心后,在上述管中配制如下反转录反应液:
混合均匀后,短暂离心;在PCR仪中完成以下程序:
42℃,90min;68℃,10min;4℃,保存。cDNA保存于-80℃。
第二链的合成:
第三步、采用半巢式PCR进行绿海龟cathelicidin的基因克隆筛选
引物使用前先12000rpm离心5min,然后根据标明的摩尔数加入相应体积的ddH2O溶解至20μM的浓度。以合成的肝脏cDNA为模板,以P1和In-Fusion SMARTer CDS为引物, 进行第一次PCR扩增。在0.2ml PCR管中加入下列试剂(总体积20μl):
混匀后,短暂离心。PCR条件为:94℃变性5min;25个循环:94℃变性30s,60℃退火30s,72℃延伸1min;72℃延伸10min;4℃保存。反应结束后,取5μl产物于1%琼脂糖凝胶电泳检测目的条带。
取上步PCR产物1μl加入99μl ddH2O稀释100倍作为模板,以P2和CDSⅢ为引物,进行第二次PCR扩增。在0.2ml PCR管中先后加入下列试剂(总体积20μl):
PCR条件为:94℃变性5min;25个循环:94℃变性30s,58℃退火30s,72℃延伸1min;72℃延伸10min;4℃保存。反应结束后,取5μl,1%琼脂糖凝胶电泳检测目的条带。
扩增完成后用胶回收试剂盒(天根生物)进行目的片段回收。将回收的目的DNA片段与测序载体pMD19-T Vecter连接,转化进CaCl2-MgCl2法制备好的DH5α感受态细胞。取100μl转化菌液均匀涂布在含有100μlg/ml氨苄青霉素(Amp)的LB琼脂培养基平板上;表面晾干后,放在37℃恒温培养箱中倒置培养12-16h。挑取单菌落用M13引物PCR检测插入片段大小。挑取阳性菌落,摇菌提取质粒,使用Applied Biosystems DNA sequencer,model ABIPRISM 377进行核苷酸测序。
第四步、绿海龟cathelicidin的基因序列测定和结果:
编码Cm-CATH3前体cathelicidin基因由492个核苷酸组成,自5’端至3’端序列为:
其中391到489是编码抗微生物肽Cm-CATH3的核苷酸序列,其氨基酸序列为:苏氨酸 1-精氨酸2-甘氨酸3-精氨酸4-色氨酸5-赖氨酸6-精氨酸7-苯丙氨酸8-色氨酸9-精氨酸10-甘氨酸 11-丙氨酸12-甘氨酸13-精氨酸14-苯丙氨酸15-苯丙氨酸16-精氨酸17-精氨酸18-组氨酸19-赖氨酸 20-谷氨酸21-赖氨酸22-异亮氨酸23-异亮氨酸24-精氨酸25-丙氨酸26-丙氨酸27-缬氨酸28-天冬氨酸29-异亮氨酸30-缬氨酸31-亮氨酸32-丝氨酸33。
实施例3 Cm-CATH3的化学制备方法:
(1)根据编码绿海龟cathelicidin基因推断的成熟肽Cm-CATH3氨基酸序列,用自动多肽合成仪(Applied Biosystems)合成其全序列。
(2)通过HPLC反相C18柱层析对合成多肽进行脱盐纯化:过程中使用柱子是4.6×250nm,Venusil XBP-C4;溶剂A是0.1%trifluoroacetic溶于100%乙腈,溶剂B是0.1%trifluoroacetic溶于100%水;梯度设为:0.01min(A 15%,B 85%),25min(A 40%,B60%),25.1min(A 100%,B 0%),30min(stop);流速为1.0ml/min,波长为220nm,体积为5μl,结果测得合成的多肽序列纯度大于97%。
(3)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):首先将Cm-CATH3样品分散在基质分子中并形成晶体,然后用激光照射晶体,基质从激光中吸收能量,样品解吸附,基质-样品之间发生电荷转移使得样品分子电离,电离的样品在电场作用下飞过真空的飞行管,根据到达检测器的飞行时间不同而被检测,即通过离子的质量电荷之比(M/Z)与离子的飞行时间成正比来分析离子,并测得样品分子的分子量,得到Cm-CATH3样品的分子量是4070.85Da。
(4)等电聚焦电泳测定等电点:配胶:胶液组成为载体两性电解质,凝胶贮液,蒸馏水,混合样品,染色物质等,灌胶,加电极液,电泳,样品收集进行检测,得到Cm-CATH3等电点。
(5)用自动氨基酸测序仪测定氨基酸序列结构。合成的Cm-CATH3抗菌肽可以溶于灭菌超纯水或PBS,用于药理活性检测。
实施例4 绿海龟抗微生物肽Cm-CATH3的药理实验:
(1)Cm-CATH3抗菌活性检测:
将化学合成的CM-CATH3以2mg/ml的浓度溶解在无菌超纯水中;用接种环挑取新活化 的微生物,然后均匀涂布在新的LB琼脂板上;将直径0.5cm的圆形无菌滤纸片放在上述琼脂板上,然后向纸片上滴加10μl CM-CATH3样品溶液;放入37℃恒温培养箱中培养12-24h;观察抑菌圈形成与否,将对Cm-CATH3敏感的菌株记录下来。
(2)Cm-CATH3对敏感菌株最小抑菌浓度(Minimum Inhibitory Concentration,MIC)的测定
该实验以无菌液体LB作阴性对照,最小抑菌浓度的定义为肉眼可以观察到的完全抑制微生物生长的最低多肽浓度,或者是光吸收值不高于阴性对照5%的最低浓度。挑取新活化的微生物,接种至无菌液体LB培养基,37℃恒温振荡器中200rpm培养10-16h至对数生长期;用紫外分光光度计测菌液600nm光波处的吸光值,吸光值为1时,浓度大约为109cfu/ml,将菌液用无菌液体LB培养基稀释至106cfu/ml,冰上待用;在96微孔板上配制浓度梯度为100μg/ml、50μg/ml、25μg/ml、12.5μg/ml、6.25μg/ml、3.13μg/ml、1.56μg/ml、0.78μg/ml、的经0.22μm孔径膜过滤的Cm-CATH3样品溶液(即24.4501、12.2250、6.1125、3.0563、1.5281、0.7641、0.3820、0.1910μM),每孔50μl;每孔加入50μl上述稀释菌液,充分混匀后,在37℃培养箱中培养10-16h;使用酶标仪测600nm吸光值或肉眼观察;上述实验重复3次,取平均值。
表1.Cm-CATH3的最小抑菌浓度(MIC)
*MIC:最小抑菌浓度,浓度值为3次重复实验的平均值;ND:2mg/ml剂量纸片抑菌试验无明显活性;IS:临床分离菌株。以上结果为三次独立重复实验平均值。
由表1可见,Cm-CATH3具有广谱的抗菌活性,革兰氏阴性细菌、革兰氏阳性菌和真菌都有很好的杀伤作用。其中,Cm-CATH3对大肠杆菌08040726、金黄色葡萄球菌08032712、金黄色葡萄球菌08032615和枯草杆菌08042313的最小抑菌浓度最小,仅为0.2866μM。Cm-CATH3不仅对标准菌株有抑制作用,对临床分离耐药菌也有很强的杀伤作用。其本身不易引起耐药性,又有显著地抑制耐药菌生长的作用,可作为抗微生物物质应用于制备抗微生物感染制剂,也可以替代水产养殖,动物饲料中的抗生素,也可以替代广泛使用于食品,化妆品等中的防腐剂和抗氧化剂。
(3)Cm-CATH3对大肠杆菌的杀菌动力学:
大肠杆菌ATCC25922接种到LB固体培养基平板上,37℃培养箱中倒置培养至菌落长出。用接种环挑取单菌落接种到LB液体培养基中,37℃振荡培养箱中培养到对数生长期。用新鲜的LB液体培养基将菌液稀释至1×106CFU/ml,将样品加入稀释好的菌液中,使其终浓度为5×MIC(阴性对照加入相应体积的灭菌超纯水)。加入样品的菌液迅速放入37℃振荡培养箱中,150rpm振荡培养,分别于0min、5min、10min、20min、30min、45min、60min、90min、120min取10μl菌液用灭菌的生理盐水稀释1×103倍,取50μl涂布LB固体平板。平板放入37℃培养箱中倒置培养16h,菌落计数。
表2.Cm-CATH3对大肠杆菌的杀菌动力学
表2杀菌动力学实验结果表明,Cm-CATH3杀菌作用迅速,在30min左右即可杀死所有细菌,且其杀菌作用是致死性的。与之相比,阳性对照抗生素美罗培南作用速度较慢,作用90分钟时仍有细菌存活。结果再次验证了Cm-CATH3的强烈抑菌效果,展现了其良好前景。
Claims (4)
1.一种抗微生物肽Cm-CATH3,其特征在于,Cm-CATH3是一种直链多肽,含有33个氨基酸残基,理论等电点为12.23,分子量是4070.85Da,净电荷为+11,其全序列一级结构为:Thr1-Arg2-Gly3-Arg4-Trp5-Lys6-Arg7-Phe8-Trp9-Arg10-Gly11-Ala12-Gly13-Arg14-Phe15-Phe16-Arg17-Arg18-His19-Lys20-Glu21-Lys22-Ile23-Ile24-Arg25-Ala26-Ala27-Val28-Asp29-Ile30-Val31-Leu32-Ser33(TRGRWKRFWRGAGRFFRRHKEKIIRAAVDI VLS);
编码Cm-CATH3前体cathelicidin的基因由492个核苷酸组成,自5’端至3’端序列为:
其中391到489位是编码抗微生物肽Cm-CATH3的核苷酸序列。
2.权利要求1所述一种抗微生物肽Cm-CATH3的制备方法,其特征在于根据编码Cm-CATH3抗微生物肽基因推断的成熟肽基酸序列,用自动多肽合成仪合成其全序列;通过HPLC反相柱层析脱盐纯化,并确定其纯度大于95%;用基质辅助激光解析电离飞行时间质谱测定其分子量;等电聚焦电泳测定等电点,用自动氨基酸测序仪测定氨基酸序列结构。
3.权利要求1所述一种抗微生物肽Cm-CATH3的应用,其特征在于:对标准菌株和临床分离的耐药菌株都有很强的广谱抗菌活性,杀菌效果迅速,相比于普通抗生素,其抗菌谱更广,不易引起耐药性,且分子量小,结构简单,不含有二硫键,生产成本低;热稳定性好,无免疫原性,视为理想的新型抗生素的开发模板,应用于抗感染药物制备、抗炎药物制备、消毒药物制备,海产养殖,畜牧业饲料,日常化妆品、保健品和食品中的添加剂。
4.根据权利要求3所述一种抗微生物肽Cm-CATH3基因的应用,其特征在于,据其编码合成的Cm-CATH3抗微生物肽具有很强的抗微生物作用,溶于灭菌超纯水,用于药理活性检测。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610580361.7A CN106188264A (zh) | 2016-07-22 | 2016-07-22 | 一种抗微生物肽Cm‑CATH3及其基因、制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610580361.7A CN106188264A (zh) | 2016-07-22 | 2016-07-22 | 一种抗微生物肽Cm‑CATH3及其基因、制备方法和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN106188264A true CN106188264A (zh) | 2016-12-07 |
Family
ID=57492303
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610580361.7A Pending CN106188264A (zh) | 2016-07-22 | 2016-07-22 | 一种抗微生物肽Cm‑CATH3及其基因、制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN106188264A (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109438556A (zh) * | 2018-10-31 | 2019-03-08 | 深圳凯联健康生物科技有限公司 | 活性肽、重组载体、重组细胞、抗炎组合物及其制备方法和应用 |
CN109517033A (zh) * | 2018-10-31 | 2019-03-26 | 深圳凯联健康生物科技有限公司 | 活性肽、重组载体、重组细胞、抗炎组合物及其制备方法和应用 |
CN111374317A (zh) * | 2019-12-18 | 2020-07-07 | 苏州大学 | 一种天然抗菌肽Hc-CATH在食品防腐保鲜中的应用 |
CN112625107A (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-cm8及其制备方法和应用 |
-
2016
- 2016-07-22 CN CN201610580361.7A patent/CN106188264A/zh active Pending
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109438556A (zh) * | 2018-10-31 | 2019-03-08 | 深圳凯联健康生物科技有限公司 | 活性肽、重组载体、重组细胞、抗炎组合物及其制备方法和应用 |
CN109517033A (zh) * | 2018-10-31 | 2019-03-26 | 深圳凯联健康生物科技有限公司 | 活性肽、重组载体、重组细胞、抗炎组合物及其制备方法和应用 |
CN109438556B (zh) * | 2018-10-31 | 2021-02-05 | 深圳凯联健康生物科技有限公司 | 活性肽、重组载体、重组细胞、抗炎组合物及其制备方法和应用 |
CN109517033B (zh) * | 2018-10-31 | 2021-02-05 | 深圳凯联健康生物科技有限公司 | 活性肽、重组载体、重组细胞、抗炎组合物及其制备方法和应用 |
CN111374317A (zh) * | 2019-12-18 | 2020-07-07 | 苏州大学 | 一种天然抗菌肽Hc-CATH在食品防腐保鲜中的应用 |
CN111374317B (zh) * | 2019-12-18 | 2022-08-30 | 苏州大学 | 一种天然抗菌肽Hc-CATH在食品防腐保鲜中的应用 |
CN112625107A (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-cm8及其制备方法和应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN106188265B (zh) | 一种抗微生物肽Cm-CATH2及其基因、制备方法和应用 | |
CN106188264A (zh) | 一种抗微生物肽Cm‑CATH3及其基因、制备方法和应用 | |
CN103665111B (zh) | 一种青环海蛇抗菌肽Hc-CATH的改造体HC-15及其制备方法和应用 | |
CN104151415A (zh) | 一种天然抗菌肽Alligatorin4及其应用 | |
CN106008677A (zh) | 一种抗菌肽se37及其应用 | |
KR101734331B1 (ko) | 한국산 해삼에서 분리한 신규 항균활성 펩타이드 및 그 용도 | |
CN108003223B (zh) | 一种抗菌肽fr-31及其应用 | |
CN107746429A (zh) | 一种末端对称抗菌肽pp及其制备方法和应用 | |
CN102924574A (zh) | 抗菌肽lz1和该抗菌肽在制备抗菌药物中的用途 | |
CN104292301A (zh) | 一种小分子合成抗菌肽及其制备方法与应用 | |
CN104761629B (zh) | 一种广谱高效抗微生物肽Pb‑CATH‑OH1及其基因、制备方法和应用 | |
CN104480059B (zh) | 一种表达菌丝霉素的重组菌及其应用 | |
CN110156875B (zh) | 抗菌肽H5-p5及其制备方法和应用 | |
CN104177485B (zh) | 一种扬子鳄抗菌肽Alligatorin6及其应用 | |
US20150218234A1 (en) | Extracting Hirudin from Leech in vivo | |
CN107903308B (zh) | 一种抗菌肽kk26及其应用 | |
CN101607992B (zh) | 奶牛血液中分离出的抗菌肽及其编码序列和用途 | |
CN104163861A (zh) | 一种爬行动物抗菌肽Alligatorin5及其应用 | |
CN103145833A (zh) | 一种单环刺螠血红蛋白及其分离纯化和应用 | |
CN103641903B (zh) | 一种牛蛙抗菌肽crc及其改造体、编码核酸和应用 | |
CN102516382B (zh) | 一种海南湍蛙抗微生物肽Hainanenin-5及其基因、应用 | |
WO2022104863A1 (zh) | 太湖白鱼来源的抗菌肽及其应用 | |
CN106008718A (zh) | 重组人源性抗菌肽c16ll-37及其对变异链球菌生物活性作用的应用方法 | |
CN102796176B (zh) | 一种昆嵛林蛙抗菌肽Kunyuenin及其制备和应用 | |
CN101058602A (zh) | 一种无指盘臭蛙抗菌肽及其制备方法和用途 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20161207 |
|
WD01 | Invention patent application deemed withdrawn after publication |