CN112194707B - 一种新型蜂毒肽及其应用 - Google Patents
一种新型蜂毒肽及其应用 Download PDFInfo
- Publication number
- CN112194707B CN112194707B CN202011100249.1A CN202011100249A CN112194707B CN 112194707 B CN112194707 B CN 112194707B CN 202011100249 A CN202011100249 A CN 202011100249A CN 112194707 B CN112194707 B CN 112194707B
- Authority
- CN
- China
- Prior art keywords
- melittin
- antibacterial
- solution
- medicines
- gram
- 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
- VDXZNPDIRNWWCW-JFTDCZMZSA-N melittin Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(N)=O)CC1=CNC2=CC=CC=C12 VDXZNPDIRNWWCW-JFTDCZMZSA-N 0.000 title claims abstract description 40
- 108010036176 Melitten Proteins 0.000 title claims abstract description 39
- 239000003814 drug Substances 0.000 claims abstract description 20
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 12
- 229940079593 drug Drugs 0.000 claims abstract description 12
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
- 238000002360 preparation method Methods 0.000 claims description 10
- 239000002220 antihypertensive agent Substances 0.000 claims description 4
- 229940127088 antihypertensive drug Drugs 0.000 claims description 4
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 claims description 4
- 230000002000 scavenging effect Effects 0.000 claims description 4
- ZTOJFFHGPLIVKC-CLFAGFIQSA-N abts Chemical compound S/1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C\1=N\N=C1/SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-CLFAGFIQSA-N 0.000 claims description 3
- 239000002417 nutraceutical Substances 0.000 claims 1
- 235000021436 nutraceutical agent Nutrition 0.000 claims 1
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 13
- 230000005764 inhibitory process Effects 0.000 abstract description 9
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 8
- 244000063299 Bacillus subtilis Species 0.000 abstract description 6
- 235000014469 Bacillus subtilis Nutrition 0.000 abstract description 6
- 241000588724 Escherichia coli Species 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 6
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 241000192125 Firmicutes Species 0.000 abstract description 5
- 239000002299 complementary DNA Substances 0.000 abstract description 5
- 201000010099 disease Diseases 0.000 abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
- 238000003757 reverse transcription PCR Methods 0.000 abstract description 5
- 230000002401 inhibitory effect Effects 0.000 abstract description 4
- 230000003276 anti-hypertensive effect Effects 0.000 abstract description 3
- 238000007254 oxidation reaction Methods 0.000 abstract description 3
- 238000001308 synthesis method Methods 0.000 abstract description 3
- 230000003647 oxidation Effects 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 36
- 239000000243 solution Substances 0.000 description 31
- 108090000765 processed proteins & peptides Proteins 0.000 description 17
- 229920001184 polypeptide Polymers 0.000 description 15
- 102000004196 processed proteins & peptides Human genes 0.000 description 15
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 10
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 9
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 description 9
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 9
- 150000003254 radicals Chemical class 0.000 description 9
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 8
- 235000006708 antioxidants Nutrition 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- 230000002292 Radical scavenging effect Effects 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000002904 solvent Substances 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 230000036772 blood pressure Effects 0.000 description 4
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 4
- 239000004327 boric acid Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 3
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 3
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 3
- 229930003268 Vitamin C Natural products 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 230000007760 free radical scavenging Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000001603 reducing effect Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 235000019154 vitamin C Nutrition 0.000 description 3
- 239000011718 vitamin C Substances 0.000 description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical group O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- -1 DPPH free radical Chemical class 0.000 description 2
- 206010034133 Pathogen resistance Diseases 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 229940124350 antibacterial drug Drugs 0.000 description 2
- 239000004599 antimicrobial Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical group [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- GAISRYMZAXLPHD-UHFFFAOYSA-N 2-hydroxybenzoic acid;methanol Chemical compound OC.OC(=O)C1=CC=CC=C1O GAISRYMZAXLPHD-UHFFFAOYSA-N 0.000 description 1
- 239000005541 ACE inhibitor Substances 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 229920002785 Croscarmellose sodium Polymers 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001360526 Escherichia coli ATCC 25922 Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 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 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 206010029803 Nosocomial infection Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 1
- 229960004977 anhydrous lactose Drugs 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 238000011482 antibacterial activity assay Methods 0.000 description 1
- 229960005475 antiinfective agent Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000003659 bee venom Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000001767 crosslinked sodium carboxy methyl cellulose Substances 0.000 description 1
- 235000010947 crosslinked sodium carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229960001375 lactose Drugs 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940031703 low substituted hydroxypropyl cellulose Drugs 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229960003085 meticillin Drugs 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 208000026775 severe diarrhea Diseases 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 229940032147 starch Drugs 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 239000002578 wasp venom Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- 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
-
- 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
Abstract
本发明公开了一种新型蜂毒肽及其应用,所述蜂毒肽的氨基酸序列为INWKGIAAMAKRLL;所述蜂毒肽的合成方法具体如下:提取凹纹胡蜂毒囊RNA,反转录PCR构建cDNA文库,利用特殊引物筛选出目标基因,并以此合成得到所述蜂毒肽。本发明蜂毒肽VV‑MP‑3对革兰氏阳性菌具有较好的抑制活性,试验证明,其对金黄色葡萄球菌和大肠杆菌有较好抑菌作用,抑菌率分别为0.998±0.003和1.000±0.001;蜂毒肽VV‑MP‑3对革兰氏阴性均枯草芽孢杆菌也具有抑制作用,可用来制备抗细菌药物或医用耗材;本发明蜂毒肽VV‑MP‑3具有显著的抗氧化性能,另外,对ACE活性具有一定的抑制作用,可用来制备抗氧化药物及降压药物。
Description
技术领域
本发明属于多肽药物技术领域,具体涉及一种具有良好抑菌、降血压、抗氧化效果的新型蜂毒肽及其应用。
背景技术
随着抗生素的滥用,细菌对于抗生素药物的耐药性已经成为了全球性的威胁,多种菌具有了耐药性,如耐甲氧西林葡萄球菌(MRSA)已经具备了光谱的耐药性。MRSA是一种常见的医院感染病原菌,其感染常发于外伤和手术中,2016年,国内主要医院MRSA的平均检出率为38.4,严重影响了病人康复,因此,开发新型抗菌药物极为重要。
天然抗菌肽(AMP)被认为是设计新型抗生素的优秀模板,与传统抗菌剂相比,AMP具有一些吸引人的特征:它们有广谱的、作用快的抗菌性,通常不诱导细菌耐药性,副作用少,当与传统抗生素结合表现出协同活性。因此,多肽抗菌剂将来可用作新型抗感染剂。
金黄色葡萄球菌属于葡萄球菌属,是革兰氏阳性菌的代表,可引起许多严重感染。大肠杆菌为革兰氏阴性短杆菌,一般多不致病,为人和动物肠道中的常居菌,在一定条件下可引起肠道外感染,某些血清型菌株的致病性强,引起腹泻,引起严重腹泻和败血症。
本发明在胡蜂蜂毒中发现了一种新型胡蜂肽,对革兰氏阳性菌和革兰氏阴性菌均有较好的杀伤作用,尤其是对金黄色葡萄球菌及大肠杆菌具有较好的抑菌活性。此外,本胡蜂肽还具有抗氧化和降血压的作用,具有开发成为抗氧化和降血压药物的潜力。
发明内容
本发明的第一目的在于提供一种新型蜂毒肽,本发明的第二目的在于所述提供蜂毒肽的合成方法,本发明的第三目的在于提供所述蜂毒肽在制备抗细菌产品或药物中的应用,本发明的第四目的在于提供所述蜂毒肽在制备抗氧化药物/保健品中的应用,本发明的第五目的在于提供所述蜂毒肽在制备降血压药物中的应用。
本发明的第一目的是这样实现的,一种蜂毒肽,所述蜂毒肽的氨基酸序列为INWKGIAAMAKRLL,命名为VV-MP-3。
本发明的第二目的是这样实现的,本发明蜂毒肽的合成方法,所述合成方法具体如下:提取凹纹胡蜂毒囊RNA,反转录PCR构建cDNA文库,利用特殊引物筛选出目标基因,并以此合成所述蜂毒肽。
本发明的第三目的是这样实现的,所述应用为在制备抗细菌产品或药物中的应用。
本发明的第四目的是这样实现的,所述应用为在制备抗氧化药物/保健品中的应用。
本发明的第五目的是这样实现的,所述应用为在制备降血压药物中的应用。
本发明的有益效果:
1、本发明首次通过提取凹纹胡蜂(Vespa velutina)毒囊RNA,反转录PCR构建cDNA文库,利用特殊引物5’-CATCATGAAGAACACGA-3’筛选出目标基因,并以此合成得到本新型蜂毒肽VV-MP-3;
2、蜂毒肽VV-MP-3对革兰氏阳性菌具有较好的抑制活性,试验证明,其对金黄色葡萄球菌和大肠杆菌有较好抑菌作用,抑菌率分别为0.998±0.003和1.000±0.001;蜂毒肽VV-MP-3对革兰氏阴性均枯草芽孢杆菌也具有抑制作用,在浓度为200μg/mL及100μg/mL时,抑菌率分别为0.707±0.001和0.533±0.005;可用来制备抗细菌药物或医用耗材。
3、蜂毒肽VV-MP-3具有显著的抗氧化性能,经试验检测,其对DPPH自由基、ABTS+自由基及·OH自由基均有显著的清除能力,可用来制备抗氧化药物或保健品;
4、蜂毒肽VV-MP-3对ACE活性具有一定的抑制作用,比化学合成的ACE抑制剂毒副作用小,具有作为抗血压药物的潜力,为抗血压药物的开发研究提供新的思路和途径。
附图说明
图1为本发明蜂毒肽VV-MP-3在不同浓度下对DPPH自由基的清除能力图;
图2为本发明蜂毒肽VV-MP-3在不同浓度下对ABTS+自由基的清除能力图;
图3为本发明蜂毒肽VV-MP-3在不同浓度下对·OH自由基的清除能力图。
具体实施方式
下面结合实施例对本发明作进一步的说明,但不以任何方式对本发明加以限制,基于本发明教导所作的任何变换或替换,均属于本发明的保护范围。
本发明一种蜂毒肽,所述蜂毒肽的氨基酸序列为INWKGIAAMAKRLL,如SEQ ID NO.1所示。
本发明蜂毒肽的合成方法,所述合成方法具体如下:提取凹纹胡蜂毒囊RNA,反转录PCR构建cDNA文库,利用特殊引物筛选出目标基因,并以此合成所述蜂毒肽。
所述特殊引物为5’-CATCATGAAGAACACGA-3’。
本发明一种蜂毒肽的应用,所述应用为在制备抗细菌产品或药物中的应用。
所述产品为医用耗材。
所述细菌为金黄色葡萄球菌或枯草芽孢杆菌或大肠杆菌。
所述药物使用的药辅料包括填充剂、粘合剂、崩解剂、润滑剂、吸收促进剂、香味剂、甜味剂、稀释剂、赋形剂、润湿剂、溶剂、增溶剂和着色剂。
所述填充剂为无水乳糖、淀粉、乳糖珠粒或葡萄糖,所述粘合剂为微晶纤维素,所述崩解剂为交联羧甲基淀粉钠、交联羧甲基纤维素钠、低取代羟丙基纤维素或交联PVP,所述润滑剂为硬脂酸镁。
所述药物的给药制剂为注射剂、霜剂、软膏剂、贴剂或喷雾剂。
本发明一种蜂毒肽的应用,所述应用为在制备抗氧化药物/保健品中的应用。
所述抗氧化药物/保健品可清除的自由基为DPPH自由基、ABTS+自由基或·OH自由基。
本发明一种蜂毒肽的应用,所述应用为在制备降血压药物中的应用。
实施例1多肽的合成及抗菌测试
1.多肽的合成
提取凹纹胡蜂(Vespa velutina)毒囊RNA,反转录PCR构建cDNA文库,利用特殊引物,筛选出目标基因,并以此合成本发明蜂毒多肽VV-MP-3,其中,
引物序列:5’-CATCATGAAGAACACGA-3’
蜂毒肽序列:INWKGIAAMAKRLL。
2.抗菌能力测试
采用液体法进行测定。
使用菌种:革兰氏阳性菌为金黄色葡萄球菌(Staphylococcus aureus ATCC25923)和枯草芽孢杆菌(Bacillus subtilis ATCC 1965),革兰氏阴性菌为大肠杆菌(Escherichia coli ATCC 25922),所有菌种来自昆明医科大学医学院。
上述细菌先培养至OD600=1,之后用LB培养基稀释3000倍。在96孔板中,先加入50μL稀释后的菌液,然后再加入50μL样品,37℃培养16-18h,之后检测OD600,确定在该浓度下样品能否抑制细菌生长。样品采用连续2倍稀释法,浓度分别为200μg/mL、100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL。
用酶标仪OD600测取96孔板中各孔洞吸光度值,绘制成图。
抑菌率(%)=(Ablank-Asample)×100/Ablank,不加样品的培养基作为空白生长对照。
3.实验结果
如表1可知,多肽VV-MP-3在浓度为200μg/mL时,对金黄色葡萄球菌和大肠杆菌有较好抑菌作用,抑菌率分别为0.998±0.003和1.000±0.001;浓度为200μg/mL及100μg/mL时,对枯草芽孢杆菌有抑制作用,抑菌率分别为0.707±0.001和0.533±0.005。
表1 VV-MP-3在不同浓度下对三种细菌的抑制率
(注:当抑菌剂的抑菌率>50%时,说明其有抑菌效果,当抑菌率>90%时,说明其有较好抑菌效果。)
实施例2抗氧化能力测试
1.清除DPPH自由基能力检测
1.1实验步骤
向96孔板的每个孔中加入190μL浓度为1×10﹣4M的DPPH甲醇溶液和10μL不同浓度的四种样品多肽甲醇溶液(5~200μg/mL),混合均匀。37℃孵化30分钟,517nm处测定对于空白的吸光度。
清除率(%)=(Ablank-Asample)×100/Ablank。
只加DPPH的孔为空白对照,2μg/mL的维生素C溶液做阳性对照,甲醇溶液做阴性对照。
1.2实验结果
如图1所示,VV-MP-3的甲醇溶液在5μg/mL、10μg/mL、20μg/mL、50μg/mL、100μg/mL和200μg/mL的浓度下均有极显著(P<0.001)的DPPH自由基清除能力,表现出优秀的抗氧化能力。
2.清除ABTS+自由基能力检测
2.1实验步骤
以甲醇为溶剂配置ABTS储备液(7.4mM)和K2S2O8储备液(2.6mM),将5mL的ABTS储备液和88μL的K2S2O8储备液混匀,静置12~16小时,用甲醇稀释40倍制成ABTS工作液备用。在96孔板中加入200μL的ABTS工作液和10μL不同浓度的四种样品多肽甲醇溶液(5~200μg/mL),静置6分钟,测定在734nm处的吸光度。
清除率(%)=(Ablank-Asample)×100/Ablank。
只加ABTS的孔为空白对照,2μg/mL的维生素C溶液做阳性对照,甲醇溶液做阴性对照。
2.2实验结果
如图2所示,VV-MP-3甲醇溶液在5μg/mL(P=0.033)的浓度下有ABTS+自由基清除能力;在10μg/mL(P=0.003)的浓度下有显著的ABTS+自由基清除能力;当浓度为20~200μg/mL(P<0.001)时,有极显著的ABTS+自由基清除能力。
3.清除·OH自由基能力检测
3.1实验步骤
向96孔板中加入10μL不同浓度的四种样品多肽甲醇溶液(5~200μg/mL)、70μL蒸馏水、10μL水杨酸-甲醇溶液(9.1mM)、10μL的FeSO4溶液(9mM),最后加入10μL 30%的H2O2溶液,混匀后在510nm处测定吸光度A1。用蒸馏水代替FeSO4溶液测定A2,用蒸馏水代替样品测定A3。
清除率(%)=[1-(A1-A2)/A3]*100。
2μg/mL的维生素C溶液做阳性对照,甲醇溶液做阴性对照。
3.2实验结果
如图3所示,VV-MP-3甲醇溶液在10μg/mL(P=0.050)的浓度下,有·OH自由基清除能力;在20μg/mL(P=0.009)和50μg/mL(P=0.002)的浓度下,有显著的·OH自由基清除能力;在100μg/mL(P<0.001)和200μg/mL(P<0.001)的浓度下,有极显著的·OH自由基清除能力。
实施例3降血压性测试
人体内ACE含量增高将会导致高血压疾病的发生,因此检测多肽对于ACE活性的抑制作用可表明此多肽在降血压活性,具体方法步骤如下:
1.溶液配制
血管紧张素转化酶(ACE)标准液:将0.1U的ACE溶于1mL的0.1mol/L硼酸缓冲液(PH8.3,含0.3mol/L NaCl)中。
马尿酰组氨酰亮氨酸(HHL)标准液:以0.1mol/L硼酸缓冲液(PH8.3,含0.3mol/LNaCl)为溶剂,配成5mmol/L的HHL溶液。
马尿酸标准液:用去离子水作为溶剂,配制成所需的马尿酸标准液。
多肽溶液:用0.1mol/L硼酸缓冲液(PH8.3,含0.3mol/L NaCl)溶解多肽,再用同种缓冲液配成所需浓度的溶液。
2.色谱条件
色谱柱:Symmetry C18分析型色谱柱(5μm 3.9×150mm);
检测波长:228nm;
流速:0.8mL/min;
流动相A:水(含0.05%三氟乙酸),流动相B:乙腈(含0.05%三氟乙酸);
进样量:10μL,手动进样;
柱温:25℃;
梯度洗脱条件:10%~60%B(10min),60%~10%B(2min)。
3.实验方法
在2.5mL离心管中加入120μL HHL溶液和20μL多肽溶液,混合均匀,在37℃恒温水浴锅中预热3~5min,然后向离心管中加入10μL ACE溶液,并充分混合,37℃下水浴60min后,最后加入150μL的1mol/L HCL溶液终止反应,得到反应液,该反应液用0.45μm滤膜过滤后直接在HPLC系统自动进样分析,同时用10μL pH8.3的硼酸缓冲液替代多肽溶液作为空白对照组,ACE抑制活性计算公式如下:
抑制活性(%)=(M-N)*100/M(式中:M为空白对照组中马尿酸的峰面积(mAU·s);N为添加抑制剂组中马尿酸的峰面积(mAU·s)
4.实验结果
VV-VP-3对ACE的抑制活性为(19.124±5.338)%。
SEQUENCE LISTING
<110> 中国科学院西双版纳热带植物园
<120> 一种新型蜂毒肽及其应用
<130> 20200625
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 14
<212> PRT
<213> 人工合成
<400> 1
Ile Asn Trp Lys Gly Ile Ala Ala Met Ala Lys Arg Leu Leu
1 5 10
Claims (3)
1.一种蜂毒肽在制备抗氧化药物/保健品中的应用,其特征在于,所述蜂毒肽的氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用,其特征在于,所述抗氧化药物/保健品可清除的自由基为DPPH自由基、ABTS+自由基或·OH自由基。
3.一种蜂毒肽在制备降血压药物中的应用,其特征在于,所述蜂毒肽的氨基酸序列如SEQ ID NO.1所示。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011100249.1A CN112194707B (zh) | 2020-10-15 | 2020-10-15 | 一种新型蜂毒肽及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011100249.1A CN112194707B (zh) | 2020-10-15 | 2020-10-15 | 一种新型蜂毒肽及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112194707A CN112194707A (zh) | 2021-01-08 |
CN112194707B true CN112194707B (zh) | 2023-08-29 |
Family
ID=74008757
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011100249.1A Active CN112194707B (zh) | 2020-10-15 | 2020-10-15 | 一种新型蜂毒肽及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112194707B (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20170031843A (ko) * | 2015-09-11 | 2017-03-22 | 대한민국(농촌진흥청장) | 멜리틴 항생펩타이드를 생산하는 형질전환 누에 |
CN110498848A (zh) * | 2019-09-10 | 2019-11-26 | 中国医学科学院基础医学研究所 | 一种新型蜂毒肽变体及其应用 |
CN111100190A (zh) * | 2020-01-10 | 2020-05-05 | 大理大学 | 一种胡蜂毒肽反序类似物wvd-ⅱ及其制备方法和应用 |
CN111494603A (zh) * | 2020-05-23 | 2020-08-07 | 河南科技学院 | 一种抗菌肽纳米软膏及其制备方法和用途 |
-
2020
- 2020-10-15 CN CN202011100249.1A patent/CN112194707B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20170031843A (ko) * | 2015-09-11 | 2017-03-22 | 대한민국(농촌진흥청장) | 멜리틴 항생펩타이드를 생산하는 형질전환 누에 |
CN110498848A (zh) * | 2019-09-10 | 2019-11-26 | 中国医学科学院基础医学研究所 | 一种新型蜂毒肽变体及其应用 |
CN111100190A (zh) * | 2020-01-10 | 2020-05-05 | 大理大学 | 一种胡蜂毒肽反序类似物wvd-ⅱ及其制备方法和应用 |
CN111494603A (zh) * | 2020-05-23 | 2020-08-07 | 河南科技学院 | 一种抗菌肽纳米软膏及其制备方法和用途 |
Non-Patent Citations (1)
Title |
---|
RecName: Full=Mastoparan-like peptide 12a;GenPept;《GenPept》;UniProtKB/Swiss-Prot: P0C1M4.1 * |
Also Published As
Publication number | Publication date |
---|---|
CN112194707A (zh) | 2021-01-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR102419616B1 (ko) | 항균 요법 | |
US6297215B1 (en) | Antimicrobial cationic peptides | |
US9820967B2 (en) | Methods and compositions for increasing antibiotic activity | |
HU180746B (en) | Process for preparing the antibiotic a-21978 | |
CN110498848B (zh) | 一种蜂毒肽变体及其应用 | |
KR20080064177A (ko) | 클로스트리듐 디피실리 관련 설사의 치료 방법 | |
Zhang et al. | Isolation, structure, and antibacterial activity of thiazomycin A, a potent thiazolyl peptide antibiotic from Amycolatopsis fastidiosa | |
AU2017375034A1 (en) | Antimicrobial peptides | |
CN112194707B (zh) | 一种新型蜂毒肽及其应用 | |
WO2024053836A1 (ko) | 항생제 내성균에 대한 항균 활성을 가지는 항균 펩타이드 h103b 및 이의 용도 | |
Giacometti et al. | In vitro activity of MSI-78 alone and in combination with antibiotics against bacteria responsible for bloodstream infections in neutropenic patients | |
US20020045574A1 (en) | Glycopeptide antibacterial compounds and methods of using same | |
US20040235745A1 (en) | Antimicrobial Peptides | |
Tehrani et al. | Synthesis of a peptide derivative of microcinJ25 and evaluation of antibacterial and biological activities | |
AU2002317071A1 (en) | Antimicrobial peptides | |
Conlon et al. | Antimicrobial and cytolytic properties of the frog skin peptide, kassinatuerin-1 and its L-and D-lysine-substituted derivatives | |
CN107827957B (zh) | 抗临床多重耐药菌小肽 | |
CN112110993B (zh) | 化学合成的具有抗细菌、真菌作用的二聚体多肽、制备方法及其应用 | |
CN110551191B (zh) | 一种低溶血活性的蜂毒肽及其应用 | |
KR20150038612A (ko) | 백본-고리형 펩타이드와의 조합물 | |
CN110467667B (zh) | 一种抗肿瘤活性肽及其应用 | |
KR101556414B1 (ko) | 생물학적 활성을 갖는 펩티드 화합물, 이의 제법 및 이의 용도 | |
NO329082B1 (no) | Memnopeptider, sammensetninger, fremgangsmate for deres fremstilling og anvendelse av disse. | |
CN101293821B (zh) | 一种抑制多药耐药金葡菌活性的化合物 | |
WO2018217880A1 (en) | Antimicrobial peptides and methods of treating gram-negative pathogen infections: polar and non-polar face analogs |
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 |