CN116041435B - 一种具有2019新型冠状病毒主蛋白酶抑制活性的榛仁蛋白衍生肽及其应用 - Google Patents
一种具有2019新型冠状病毒主蛋白酶抑制活性的榛仁蛋白衍生肽及其应用 Download PDFInfo
- Publication number
- CN116041435B CN116041435B CN202310145383.0A CN202310145383A CN116041435B CN 116041435 B CN116041435 B CN 116041435B CN 202310145383 A CN202310145383 A CN 202310145383A CN 116041435 B CN116041435 B CN 116041435B
- Authority
- CN
- China
- Prior art keywords
- wwnln
- ncov
- pro
- peptide
- derivative 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
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 78
- 241001678559 COVID-19 virus Species 0.000 title claims abstract description 67
- 235000001543 Corylus americana Nutrition 0.000 title claims abstract description 24
- 235000007466 Corylus avellana Nutrition 0.000 title claims abstract description 24
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 18
- 230000000694 effects Effects 0.000 title claims abstract description 12
- 101800000535 3C-like proteinase Proteins 0.000 title claims abstract description 11
- 101800002396 3C-like proteinase nsp5 Proteins 0.000 title claims abstract description 11
- 230000005764 inhibitory process Effects 0.000 title abstract description 23
- 240000007582 Corylus avellana Species 0.000 title 1
- 241000723382 Corylus Species 0.000 claims abstract description 23
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 23
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 239000003112 inhibitor Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000029087 digestion Effects 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 7
- 208000025721 COVID-19 Diseases 0.000 abstract description 6
- 230000002496 gastric effect Effects 0.000 abstract description 3
- 231100001083 no cytotoxicity Toxicity 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 239000012488 sample solution Substances 0.000 description 9
- 238000010532 solid phase synthesis reaction Methods 0.000 description 9
- 239000012452 mother liquor Substances 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 238000011282 treatment Methods 0.000 description 8
- 102000057297 Pepsin A Human genes 0.000 description 7
- 108090000284 Pepsin A Proteins 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000000502 dialysis Methods 0.000 description 7
- 238000010828 elution Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000003032 molecular docking Methods 0.000 description 6
- 229940111202 pepsin Drugs 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 241000711573 Coronaviridae Species 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004142 Trypsin Human genes 0.000 description 5
- 108090000631 Trypsin Proteins 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000012588 trypsin Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 235000012054 meals Nutrition 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 208000001528 Coronaviridae Infections Diseases 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 238000004007 reversed phase HPLC Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 208000000059 Dyspnea Diseases 0.000 description 2
- 206010013975 Dyspnoeas Diseases 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000005965 immune activity Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000010829 isocratic elution Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000000242 pagocytic effect Effects 0.000 description 2
- 210000003024 peritoneal macrophage Anatomy 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 108091005658 Basic proteases Proteins 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010003571 Nut Proteins Proteins 0.000 description 1
- 101710137908 Pepsin-2 Proteins 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 1
- 101710198474 Spike protein Proteins 0.000 description 1
- 102000005158 Subtilisins Human genes 0.000 description 1
- 108010056079 Subtilisins Proteins 0.000 description 1
- 108700010756 Viral Polyproteins Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012900 molecular simulation Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 230000006959 non-competitive inhibition Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 208000013220 shortness of breath Diseases 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229940126672 traditional medicines Drugs 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000014599 transmission of virus Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Images
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
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 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
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
- C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种具有2019新型冠状病毒主蛋白酶2019‑nCoV M‑pro抑制活性的榛仁蛋白衍生肽,其氨基酸序列为Trp‑Trp‑Asn‑Leu‑Asn(WWNLN)。本发明还公开了其在制备抑制2019新型冠状病毒感染的药品中的应用。本发明衍生肽WWNLN具有更好的2019‑nCoV M‑pro抑制活性,抑制率为74.13±1.93%,IC50值为6.695μM,属天然衍生肽,安全性高,具有热稳定性,抗酸碱稳定性、胃肠消化稳定性且无细胞毒性等优势,可用于制备抑制2019新型冠状病毒感染的药品,具有良好应用前景。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种具有2019新型冠状病毒主蛋白酶抑制活性的榛仁蛋白衍生肽及其应用。
背景技术
2019新型冠状病毒(2019-nCoV,亦称为SARS-CoV-2)是一种单链、正义RNA病毒,属于肉瘤病毒亚属的β冠状病毒,其主要通过呼吸道飞沫传播和接触传播。人感染了2019新型冠状病毒后,往往会导致新型冠状病毒肺炎(COVID-19),常见症状有呼吸道症状、发热、咳嗽、气促和呼吸困难等,部分感染患者会进展为重症,引起严重急性呼吸综合征、肾衰竭,甚至导致死亡。鉴于此,如何遏制病毒的传播,更好地治疗新型冠状病毒肺炎,成为全人类迫在眉睫的共同议题。据报道,2019新型冠状病毒的变异较快,目前至少有6种变异毒株被报道,而2021年11月报道的奥密克戎(Omicron)已取代德尔塔(Delta)成为主要的流行变异株。相对而言,Omicron变异株具有更强的免疫逃逸能力和传播能力,主要原因为Omicron在其刺突蛋白上发生了32种突变,从而使得疫苗和中和抗体的保护效力降低甚至失效。
2019新型冠状病毒主蛋白酶(2019-nCoV M-pro)是相对保守的蛋白酶,它负责催化病毒的多聚蛋白(ppla和pplab)水解,并产生多个功能性的非结构蛋白,组成复制-转录复合物,以完成后续遗传物质的复制和结构蛋白的合成。因此,通过抑制2019-nCoV M-pro的功能,可干扰病毒的复制过程,达到抗病毒的效果;值得注意的是,目前还没有已知的人源蛋白酶具有与2019-nCoV M-pro相同的切割特异性,这为针对2019-nCoV M-pro的药物发现提供了机会,且有望减少药物潜在的不良反应。
我国榛子资源丰富,榛仁蛋白含量可达16.2wt%~21.1wt%,作为植物源蛋白富含多种氨基酸(包含人体所需的全部8种必需氨基酸)。此外,研究还发现榛仁水解物具有多种生物活性,具有绿色、安全、易吸收等优势,具有极大的开发和应用价值。尤其是免疫活性,免疫活性肽能增强巨噬细胞吞噬能力和促进淋巴细胞增殖等,提高机体抵抗入侵病原体的能力,食源性免疫活性肽跟传统药物相比无毒副作用,通过食用食源性免疫活性肽来提高自身免疫力,日渐成为主流趋势。同时人体免疫力与新冠病毒感染息息相关,基于此,开发可抑制2019-nCoV M-pro的新型短肽可为2019新型冠状病毒的防治提供新的思路。
发明内容
本发明的目的在于提供一种具有2019新型冠状病毒主蛋白酶(2019-nCoV M-pro)抑制活性榛仁蛋白衍生肽及其在制备抑制2019新型冠状病毒感染的药品中的应用,以解决现有技术的不足。
本发明采用以下技术方案:
本发明第一方面提供了一种具有2019新型冠状病毒主蛋白酶2019-nCoV M-pro抑制活性的榛仁蛋白衍生肽,其氨基酸序列为Trp-Trp-Asn-Leu-Asn(WWNLN)。
本发明第二方面提供了上述具有2019新型冠状病毒主蛋白酶2019-nCoV M-pro抑制活性的榛仁蛋白衍生肽在制备抑制2019新型冠状病毒感染的药品中的应用。
进一步地,所述药品中还包括其他防治2019新型冠状病毒感染的成分和/或可接受的辅料。
本发明的有益效果:
本发明一种具有2019新型冠状病毒主蛋白酶2019-nCoV M-pro抑制活性的榛仁蛋白衍生肽,其氨基酸序列为Trp-Trp-Asn-Leu-Asn(WWNLN)。本发明以榛仁蛋白为原料,采用酶控制水解技术制备酶解物,经超滤得到分子量<3kDa组分并验证其对两种免疫细胞即小鼠腹腔巨噬细胞吞噬能力和脾脏淋巴细胞增殖能力具有积极影响,随后通过NANO-HPLC-MS/MS技术鉴定明确肽结构序列;筛选得到的活性肽WWNYN利用19种常见氨基酸进行替换,与2019-nCoV M-pro进行分子对接并筛选,获得2019-nCoV M-pro抑制活性更好的衍生肽WWNLN,抑制率为74.13±1.93%,IC50值为6.695μM;利用多肽合成仪进行固相化学合成,通过热稳定性,酸碱稳定性和体外胃肠道模拟实验以及对Vero-E6细胞毒性实验分析,确定其具有热稳定性、抗酸碱稳定性、胃肠消化稳定性且无细胞毒性。本发明是从坚果蛋白酶解物中获得的天然衍生肽,安全性高,可用于制备抑制2019新型冠状病毒感染的药品,具有良好应用前景。
附图说明
图1为衍生肽WWNLN的二级质谱图。
图2为衍生肽WWNLN与2019-nCoV M-pro分子对接2D图。
图3为衍生肽WWNLN与2019-nCoV M-pro酶促反应曲线图。
图4为衍生肽WWNLN对2019-nCoV M-pro IC50值图。
图5为衍生肽WWNLN对2019-nCoV M-pro抑制模式图。
图6为衍生肽WWNLN热稳定性图。
图7为衍生肽WWNLN酸碱稳定性图。
图8为衍生肽WWNLN体外胃肠道消化稳定性图。
图9为衍生肽WWNLN对Vero-E6细胞毒性图。
具体实施方式
下面结合实施例和附图对本发明做更进一步地解释。下列实施例仅用于说明本发明,但并不用来限定本发明的实施范围。
实施例1 2019-nCoV M-pro抑制活性肽WWNYN的筛选
本实施例提供的2019-nCoV M-pro抑制活性肽WWNYN,其于榛仁中提取筛选获得,具体提取筛选过程如下:
榛仁经挤压膨化去油后得到榛仁粕,将榛仁粕与蒸馏水以底物浓度10wt%搅拌混匀,在90~100℃水浴静置15min使蛋白变性,冷却至室温后逐滴加入1mol/L氢氧化钠调pH值到9.0,加入Alcalase 2.4L碱性蛋白酶(诺维信公司),加酶量14000U/g榛仁粕,45℃、10~40rpm下搅拌酶解2.5h,酶解过程中利用1mol/L氢氧化钠保持pH值为9.0不变,反应结束后迅速于90℃水浴静置15min以灭活酶活性,然后冷却至室温,用1mol/L盐酸调节pH至中性后4500r/min离心15min,取上清液,4℃保存备用。
将上述上清液即榛仁粕酶解得到的肽液分别使用膜孔径3000NMWC和10000NMWC的超滤***进行组分分离,分成3种不同分子量组分(<3kDa、3-10kDa、>10kDa),并将各组分肽液使用100Da透析袋透析除盐及浓缩:将各组分肽液分别转移至100Da透析袋中,并将透析袋置于盛有2L蒸馏水的烧杯中,在磁力搅拌器(10~40rpm)作用下进行透析,当透析袋已充满液体(大约30h),此时透析完成,随后使用旋转蒸发仪在45℃条件下对其浓缩,收集后在-50℃、10Kpa真空条件下冷冻干燥备用。利用小鼠腹腔巨噬细胞和脾脏淋巴细胞对4种不同组分(<3kDa、3-10kDa、>10kDa,及未经超滤的酶解物)进行免疫活性鉴定,随后将免疫活性最好的组分(<3kDa组分)进行NANO-HPLC-MS/MS分析,鉴定后序列经肽链结构特点和分子模拟筛选,得到目标序列Trp-Trp-Asn-Tyr-Asn(WWNYN)。经试验证明其能抑制2019-nCoVM-pro(表3)。
实施例2 2019-nCoV M-pro抑制衍生肽WWNLN的筛选
本实施例提供的2019-nCoV M-pro抑制衍生肽WWNLN筛选过程如下:以2019-nCoVM-pro抑制活性肽WWNYN(由江苏吉泰肽业科技有限公司固相合成得到)为模板,进行氨基酸替换,通过与2019-nCoV M-pro进行分子对接,以结合能为指标筛选获得2019-nCoV M-pro抑制活性更好的衍生肽Trp-Trp-Asn-Leu-Asn(WWNLN),其二级质谱图如图1所示。
2019-nCoV M-pro抑制衍生肽WWNLN与2019-nCoV M-pro进行分子对接,如图2和表1所示,结果显示,经与2019-nCoV M-pro分子对接,WWNLN与2019-nCoV M-pro稳定结合形成7条氢键,分子对接结合能为-8.1kcal/mol。
表1 2019-nCoV M-pro抑制衍生肽WWNLN与2019-nCoV M-pro结合的稳定氢键数量、名称及结合能
实施例3 2019-nCoV M-pro抑制活性肽WWNYN、2019-nCoV M-pro抑制衍生肽WWNLN对2019-nCoV M-pro抑制作用
(1)2019-nCoV M-pro抑制活性实验
使用检测缓冲液(500mM Tris,150mM NaCl,1mM EDTA,50v/v%甘油)配置2019-nCoV M-pro母液、活性肽WWNYN(由江苏吉泰肽业科技有限公司固相合成得到)母液、衍生肽WWNLN(由江苏吉泰肽业科技有限公司固相合成得到)母液,使用95v/v%DMSO配置荧光十肽底物(Mca-AVLQSGFR-K(Dnp)-K)母液。将实验组(含有不同浓度:1.0、2.5、5.0、10.0、20.0μM的WWNYN/WWNLN,20μM荧光十肽底物,2nM 2019-nCoV M-pro)及对照组(含20μM荧光十肽底物,2nM 2019-nCoV M-pro)溶液分别点入96孔黑色酶标板后放入30℃酶标仪中静置孵育30min。酶标仪程序设置:激发波长320nm,发射波长405nm,温度30℃,动力学循环150次,每10秒测一次,记录一段时间内的实验数据。根据实验检测的荧光强度和酶促反应时间绘制酶促反应过程曲线,曲线上点的斜率表示该时刻的反应速率,根据公式计算抑制率。
Vi:加入WWNYN/WWNLN的反应速率,
V0:不加WWNYN/WWNLN的反应速率。
(2)2019-nCoV M-pro IC50值计算
根据不同浓度的抑制率利用Graphpad Prrism8.0.1软件非线性拟合出IC50值。
(3)2019-nCoV M-pro抑制模式实验
表2抑制模式的12种实验组合
使用检测缓冲液(500mM Tris,150mM NaCl,1mM EDTA,50v/v%甘油)配置2019-nCoV M-pro母液、活性肽WWNYN(由江苏吉泰肽业科技有限公司固相合成得到)母液、衍生肽WWNLN(由江苏吉泰肽业科技有限公司固相合成得到)母液,使用95v/v%DMSO配置荧光十肽底物(Mca-AVLQSGFR-K(Dnp)-K)母液。如表2所示,4种浓度WWNYN/WWNLN(0、5、10、20μM)和3种浓度底物(2.5、5.0、10.0μM)分别设计12种浓度组合(各组合2019-nCoV M-pro浓度均为2nM),在激发波长320nm,发射波长405nm条件下测定荧光强度,根据Km和Vmax的趋势变化判断抑制剂的抑制模式。
表3活性肽WWNYN及其衍生肽WWNLN对2019-nCoV M-pro抑制作用
图3-图5和表3结果显示,活性肽WWNYN对2019-nCoV M-pro抑制率为59.36±1.05%,IC50值为19.925μM,而衍生肽WWNLN对2019-nCoV M-pro抑制率达74.13±1.93%,IC50值为6.695μM,抑制效果更好。活性肽WWNYN和衍生肽WWNLN在抑制剂浓度0-10μM时,Km增加,Vmax不变,为竞争性抑制,当浓度为20μM时,此时Vmax减小,为非竞争性抑制。
实施例4 2019-nCoV M-pro抑制衍生肽WWNLN热稳定性,酸碱稳定性和胃肠道消化稳定性实验
(1)2019-nCoV M-pro抑制衍生肽WWNLN热稳定性研究
使用蒸馏水将WWNLN(由江苏吉泰肽业科技有限公司固相合成得到)稀释成100μmol/L的溶液,取出未经不同温度处理的溶液用作空白对照。随后将稀释好的WWNLN样品溶液分别置于20、40、60、80、100℃水浴条件下静置2h。随后将空白对照和不同温度处理下的WWNLN样品溶液通过0.22μm孔径的滤膜注入RP-HPLC装置分析。检测波长:220nm;柱温:25℃。流动相A:1v/v%TFA+99v/v%乙腈;流动相B:1v/v%TFA+99v/v%水。洗脱条件:0-25min,72v/v%-47v/v%B(梯度洗脱);25min,47v/v%B(等度洗脱);25-25.1min,47v/v%-0v/v%B(梯度洗脱),以1.0mL/min的流速洗脱色谱柱。根据液相图谱的峰面积和出峰时间判定WWNLN在不同温度下是否降解。
(2)2019-nCoV M-pro抑制衍生肽WWNLN酸碱稳定性研究
使用蒸馏水将WWNLN(由江苏吉泰肽业科技有限公司固相合成得到)稀释成100μmol/L的溶液,取出未经不同酸碱度处理的溶液用作空白对照。随后利用1mol/L盐酸和1mol/L氢氧化钠将稀释好的WWNLN样品溶液pH分别调节至2、4、6、8、10。将空白对照和不同酸碱度处理下的WWNLN样品溶液于37℃水浴条件下静置2h。随后将空白对照和不同酸碱度处理下的WWNLN样品溶液通过0.22μm孔径的滤膜注入RP-HPLC装置分析。检测波长:220nm;柱温:25℃。流动相A:1v/v%TFA+99v/v%乙腈;流动相B:1v/v%TFA+99v/v%水。洗脱条件:0-25min,72v/v%-47v/v%B(梯度洗脱);25min,47v/v%B(等度洗脱);25-25.1min、47v/v%-0v/v%B(梯度洗脱),以1.0mL/min的流速洗脱色谱柱。根据液相图谱的峰面积和出峰时间判定WWNLN在不同pH条件下是否降解。
(3)2019-nCoV M-pro抑制衍生肽WWNLN模拟胃肠道消化稳定性分析
先用pH值为2的盐酸将WWNLN(由江苏吉泰肽业科技有限公司固相合成得到)稀释成100μmol/L的溶液。于1mL稀释好的WWNLN样品溶液中加入0.016g胃蛋白酶(北京索莱宝科技有限公司,P8390,胃蛋白酶活力1g:3000U),于37℃水浴静置2h,每隔1h取样,置于4℃备用。2h后取出WWNLN样品溶液,随后使用1mol/L氢氧化钠将胃蛋白酶酶解后的WWNLN样品溶液pH值调至7,加入0.010g胰蛋白酶(北京索莱宝科技有限公司,T8150,胰蛋白酶活力1g:250U),于37℃水浴静置2h,每隔1h取样,置于4℃备用。最后将取出的WWNLN样品溶液通过0.22μm孔径的滤膜注入RP-HPLC装置分析。洗脱条件同实施例4第(1)部分。根据液相图谱的峰面积和出峰时间判定WWNLN在经过胃蛋白酶和胰蛋白酶酶解后是否发生降解。
从图6中可以看出与空白对照相比,在不同温度处理下的WWNLN峰面积和出峰时间与空白对照均无明显差异,说明WWNLN未发生降解,热稳定性良好。从图7可以看出,不同pH处理后的WWNLN出峰时间基本一致,峰面积未发生显著变化,说明经不同酸碱环境的WWNLN未发生显著降解。从图8中可以看出,在胃蛋白酶消化1h(Pepsin-1)和2h(Pepsin-2)后,与空白对照相比,肽发生轻微降解,峰面积有所减少;经胰蛋白酶消化1h(Pepsin-Trypsin-1)和2h(Pepsin-Trypsin-2),同空白对照相比,峰面积变小,说明WWNLN发生降解。通过峰面积比较可知,经胃蛋白酶、胰蛋白酶先后消化2h后,此时WWNLN保留率达87.46%,稳定性良好。
实施例5 2019-nCoV M-pro抑制衍生肽WWNLN对Vero-E6细胞毒性实验
在96孔板中加入不同浓度WWNLN(由江苏吉泰肽业科技有限公司固相合成得到)溶液(0,25,50,100,200,400μM;DMEM细胞培养液配置,Gibco公司)和Vero-E6细胞(105个/mL),于培养箱中37℃静置培养24h后,从培养箱中取出96孔板,每孔加入MTT溶液,MTT溶液终浓度为0.5mg/mL,MTT溶液使用磷酸盐缓冲液(NaCl:137mM,KCl:2.7mM,Na2HPO4:10mM,KH2PO4:2mM,pH值7.2-7.4)配置。然后在CO2培养箱(CO2浓度为5v/v%)中37℃静置培养4h。然后1000rpm转速下离心5min,去除上清液,每孔添加150μL DMSO,置于摇床上70rpm震荡10min。使用酶标仪在490nm波长下读取各孔中的吸光度,使用Graphpad Prism 8.0.1计算出细胞存活率。本实施例从加入MTT溶液开始后注意避光处理。
表4衍生肽WWNLN对Vero-E6细胞毒性
图9和表4结果显示在0~400μM范围内,细胞存活率有下降趋势但经分析无显著性差异,表明衍生肽WWNLN对Vero-E6细胞无毒性。
Claims (2)
1.一种具有2019新型冠状病毒主蛋白酶2019-nCoV M-pro抑制活性的榛仁蛋白衍生肽,其特征在于,其氨基酸序列为Trp-Trp-Asn-Leu-Asn。
2.权利要求1所述的榛仁蛋白衍生肽在制备抑制2019新型冠状病毒主蛋白酶2019-nCoV M-pro活性的抑制剂中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310145383.0A CN116041435B (zh) | 2023-02-21 | 2023-02-21 | 一种具有2019新型冠状病毒主蛋白酶抑制活性的榛仁蛋白衍生肽及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310145383.0A CN116041435B (zh) | 2023-02-21 | 2023-02-21 | 一种具有2019新型冠状病毒主蛋白酶抑制活性的榛仁蛋白衍生肽及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116041435A CN116041435A (zh) | 2023-05-02 |
CN116041435B true CN116041435B (zh) | 2023-07-07 |
Family
ID=86113419
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310145383.0A Active CN116041435B (zh) | 2023-02-21 | 2023-02-21 | 一种具有2019新型冠状病毒主蛋白酶抑制活性的榛仁蛋白衍生肽及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116041435B (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103689215A (zh) * | 2013-12-25 | 2014-04-02 | 吉林农业大学 | 一种高活性坚果抗氧化肽及其制备方法 |
CN113677698A (zh) * | 2019-04-10 | 2021-11-19 | 纳维格蛋白质有限公司 | 用于癌症诊断和治疗的新的psma特异性结合蛋白 |
-
2023
- 2023-02-21 CN CN202310145383.0A patent/CN116041435B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103689215A (zh) * | 2013-12-25 | 2014-04-02 | 吉林农业大学 | 一种高活性坚果抗氧化肽及其制备方法 |
CN113677698A (zh) * | 2019-04-10 | 2021-11-19 | 纳维格蛋白质有限公司 | 用于癌症诊断和治疗的新的psma特异性结合蛋白 |
Non-Patent Citations (4)
Title |
---|
Identifying molecular insight of synergistic complexities for SARS-CoV-2 infection with pre-existing type 2 diabetes;M. Babul Islam等;Comput Biol Med;第136卷;104668 * |
SARS冠状病毒主蛋白酶抑制剂的筛选及抑制动力学研究;朱云鹏;王鹏;夏博然;唐延婷;王权;;中国生物工程杂志(04);42-49 * |
基于Mpro和PLP筛选潜在抗新型冠状病毒中药研究;马婧;霍晓乾;陈茜;朱卫星;姚美村;乔延江;张燕玲;;中国中药杂志(06);1219-1224 * |
榛仁免疫活性肽分离纯化及结构鉴定;王鹏等;食品科学;第39卷(第3期);200-205 * |
Also Published As
Publication number | Publication date |
---|---|
CN116041435A (zh) | 2023-05-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109234344B (zh) | 一种藜麦肽及其制备方法和应用 | |
CN104031967B (zh) | 一种沙丁鱼降血压肽及其制备方法与应用 | |
WO2021082294A1 (zh) | 一种免疫调节肽的制备及方法 | |
CN107849551A (zh) | 新型胰蛋白酶同功异型物和其用途 | |
CN116589533A (zh) | 南极磷虾小分子活性肽及其制备方法和应用 | |
CN111518164B (zh) | Ace抑制肽p2、其应用及其制备方法 | |
CN112457387A (zh) | 一种厚壳贻贝寡肽及其用途 | |
CN105463046B (zh) | 一种鳄鱼骨胶原蛋白肽粉的制备方法及其用途 | |
CN108796016B (zh) | 核桃肽及其酶解提取方法 | |
CA3203208A1 (en) | Multifunctional panax quinquefolius hydrolyzed peptide and preparation method and application thereof | |
WO2020108629A1 (zh) | 一种多肽rdp1及其提纯方法与应用 | |
CN108840909B (zh) | 一种紫菜降压肽和紫菜降压肽提取物及应用 | |
CN116041435B (zh) | 一种具有2019新型冠状病毒主蛋白酶抑制活性的榛仁蛋白衍生肽及其应用 | |
CN111378712A (zh) | 可食用酵母多肽及其制备方法和应用 | |
CN101126105A (zh) | 一种用大米分离蛋白制备降血压肽的方法 | |
CN103740797B (zh) | 一种利用高温花生粕制备高水解度功能性短肽的方法 | |
CN113555060A (zh) | 具有降血糖、降血压活性的山羊奶酪蛋白肽的制备方法 | |
CN106755230B (zh) | 一种双齿围沙蚕抗肺癌多肽的制备方法 | |
CN102643889A (zh) | 一种降血压菜籽活性肽及其制备方法和应用 | |
CN115960165B (zh) | 一种来源于辣木叶的富硒ace抑制肽及其应用 | |
CN113087773B (zh) | 一种具有降血糖和抗氧化功能的牦牛骨肽及其制备方法 | |
CN114032267B (zh) | 一种活性肽的制备方法和应用 | |
CN111499691B (zh) | Ace抑制肽p1、其应用及其制备方法 | |
CN109400687B (zh) | 一种西兰花蛋白来源的ace抑制肽及其制备方法和应用 | |
CN109402206B (zh) | 一种疣荔枝螺降压肽的制备方法 |
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 |