CN113072623A - 一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用 - Google Patents
一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用 Download PDFInfo
- Publication number
- CN113072623A CN113072623A CN202110466103.7A CN202110466103A CN113072623A CN 113072623 A CN113072623 A CN 113072623A CN 202110466103 A CN202110466103 A CN 202110466103A CN 113072623 A CN113072623 A CN 113072623A
- Authority
- CN
- China
- Prior art keywords
- cov
- sars
- protein
- nip
- amino acid
- 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.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 87
- 108091006197 SARS-CoV-2 Nucleocapsid Protein Proteins 0.000 title claims abstract description 41
- 230000008685 targeting Effects 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 150000001413 amino acids Chemical class 0.000 claims abstract description 25
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 13
- 230000002452 interceptive effect Effects 0.000 claims abstract description 9
- 230000002441 reversible effect Effects 0.000 claims abstract description 7
- 238000006471 dimerization reaction Methods 0.000 claims abstract description 5
- 239000002994 raw material Substances 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 21
- 208000025721 COVID-19 Diseases 0.000 claims description 13
- 208000037847 SARS-CoV-2-infection Diseases 0.000 claims description 13
- RAVVEEJGALCVIN-AGVBWZICSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-5-amino-2-[[(2s)-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-2-[[2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]acetyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]hexanoyl]amino]hexanoyl]amino]-5-(diamino Chemical group NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 RAVVEEJGALCVIN-AGVBWZICSA-N 0.000 claims description 3
- 108700000788 Human immunodeficiency virus 1 tat peptide (47-57) Proteins 0.000 claims description 3
- 241001678559 COVID-19 virus Species 0.000 abstract description 41
- 239000003814 drug Substances 0.000 abstract description 34
- 101710141454 Nucleoprotein Proteins 0.000 abstract description 12
- 238000006384 oligomerization reaction Methods 0.000 abstract description 11
- 230000005764 inhibitory process Effects 0.000 abstract description 7
- 230000015788 innate immune response Effects 0.000 abstract description 7
- 241001465754 Metazoa Species 0.000 abstract description 5
- 230000002401 inhibitory effect Effects 0.000 abstract description 5
- 230000029812 viral genome replication Effects 0.000 abstract 1
- 210000004072 lung Anatomy 0.000 description 31
- 238000011830 transgenic mouse model Methods 0.000 description 28
- 102100035765 Angiotensin-converting enzyme 2 Human genes 0.000 description 27
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 27
- 241000699660 Mus musculus Species 0.000 description 26
- 210000001519 tissue Anatomy 0.000 description 26
- 229940079593 drug Drugs 0.000 description 24
- 241000699670 Mus sp. Species 0.000 description 19
- 238000002474 experimental method Methods 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 230000014509 gene expression Effects 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 102100023727 Mitochondrial antiviral-signaling protein Human genes 0.000 description 11
- 101710142315 Mitochondrial antiviral-signaling protein Proteins 0.000 description 11
- 238000010586 diagram Methods 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 9
- 241001529936 Murinae Species 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 102100026720 Interferon beta Human genes 0.000 description 7
- 108090000467 Interferon-beta Proteins 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 102100031673 Corneodesmosin Human genes 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- 101710139375 Corneodesmosin Proteins 0.000 description 5
- 101150007193 IFNB1 gene Proteins 0.000 description 5
- 241000711408 Murine respirovirus Species 0.000 description 5
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 5
- 230000000840 anti-viral effect Effects 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- 241000711573 Coronaviridae Species 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 238000000540 analysis of variance Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000003902 lesion Effects 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 230000019491 signal transduction Effects 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000003364 immunohistochemistry Methods 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 210000004969 inflammatory cell Anatomy 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- 241000283707 Capra Species 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 102000004389 Ribonucleoproteins Human genes 0.000 description 2
- 108010081734 Ribonucleoproteins Proteins 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 230000035931 haemagglutination Effects 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 229940126586 small molecule drug Drugs 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 102100030988 Angiotensin-converting enzyme Human genes 0.000 description 1
- 101710185050 Angiotensin-converting enzyme Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229940022962 COVID-19 vaccine Drugs 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 101000929928 Homo sapiens Angiotensin-converting enzyme 2 Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- OFFWOVJBSQMVPI-RMLGOCCBSA-N Kaletra Chemical compound N1([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=2C=CC=CC=2)NC(=O)COC=2C(=CC=CC=2C)C)CC=2C=CC=CC=2)CCCNC1=O.N([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=1C=CC=CC=1)NC(=O)OCC=1SC=NC=1)CC=1C=CC=CC=1)C(=O)N(C)CC1=CSC(C(C)C)=N1 OFFWOVJBSQMVPI-RMLGOCCBSA-N 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 101001054328 Mus musculus Interferon beta Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 101000629313 Severe acute respiratory syndrome coronavirus Spike glycoprotein Proteins 0.000 description 1
- QTENRWWVYAAPBI-YZTFXSNBSA-N Streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@H]1[C@H](N=C(N)N)[C@@H](O)[C@H](N=C(N)N)[C@@H](O)[C@@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@H]1[C@H](N=C(N)N)[C@@H](O)[C@H](N=C(N)N)[C@@H](O)[C@@H]1O QTENRWWVYAAPBI-YZTFXSNBSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000008358 core component Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 102000048657 human ACE2 Human genes 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- XXSMGPRMXLTPCZ-UHFFFAOYSA-N hydroxychloroquine Chemical compound ClC1=CC=C2C(NC(C)CCCN(CCO)CC)=CC=NC2=C1 XXSMGPRMXLTPCZ-UHFFFAOYSA-N 0.000 description 1
- 229960004171 hydroxychloroquine Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000008088 immune pathway Effects 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000008629 immune suppression Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 229940113983 lopinavir / ritonavir Drugs 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108091005601 modified peptides Proteins 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 230000005727 virus proliferation Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16071—Demonstrated in vivo effect
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
- C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20051—Methods of production or purification of viral material
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20071—Demonstrated in vivo effect
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Veterinary Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pulmonology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种靶向SARS‑CoV‑2N蛋白的干扰肽的制备方法,该方法包括如下步骤:设计干扰肽段靶向位于SARS‑CoV‑2N蛋白的二聚化结构域内的氨基酸;将所述干扰肽段与HIV‑TAT融合;将与HIV‑TAT融合的干扰肽段修饰为逆向异构体,获得最终干扰肽NIP‑V的氨基酸序列;使用D‑型氨基酸为原料合成干扰肽NIP‑V。上述干扰肽药物NIP‑V能够与SARS‑CoV‑2N蛋白的二聚结构域相互作用,抑制N蛋白寡聚化,进而解除N蛋白对先天免疫的抑制,从而达到抑制SARS‑CoV‑2病毒在细胞和动物体内复制的目的。
Description
技术领域
本发明属于药物制备技术领域,具体涉及一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用。
背景技术
SARS-CoV-2属冠状病毒属(Coronavirus,CoV),其基本结构由刺突(spike,S)蛋白、包膜(envelope,E)蛋白、膜(membrane,M)蛋白、核衣壳(nucleocapsid,N)蛋白以及基因组单链RNA组成。N蛋白是病毒粒子的核心成分,SARS-CoV-2 N蛋白全长419个氨基酸,主要由N端RNA结合结构域、C端二聚结构域以及连接两个结构域的其他序列构成。SARS-CoV-2 N蛋白全序列含有多个较为保守的结合RNA的正电荷分布区域,其与病毒基因组RNA结合,将RNA包装成核糖核蛋白(ribonucleocapsid,RNP)复合体,此外其二聚结构域能够介导SARS-CoV-2 N蛋白形成同源寡聚体。目前在新型冠状病毒肺炎防治相关工作中,预防以全球逐渐开始接种的多类SARS-CoV-2疫苗为主流,但治疗则缺乏特效药。小分子药物一般研制周期很长,目前都是旧药新用,且药效饱受质疑,比如羟氯喹、洛匹那韦/利托那韦以及瑞德西韦等。除小分子药物外,血浆疗法虽好,但有一定风险。因为血浆因人而异,是一个复杂的混合物,而且血浆来源有限,不能大规模使用。
因此,针对上述问题,有必要提出进一步的解决方案。
发明内容
本发明目的是提供一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,以制备一种干扰肽药物(以下命名为NIP-V)用于抑制细胞和动物体内SARS-CoV-2的复制和增殖,治疗SARS-CoV-2感染相关的疾病。
本发明的技术方案是:一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,该方法包括如下步骤:
(a)设计干扰肽段靶向位于SARS-CoV-2 N蛋白二聚结构域内的氨基酸;
(b)将所述干扰肽段与HIV-TAT融合;
(c)将与HIV-TAT融合的干扰肽段修饰为逆向异构体,获得最终干扰肽NIP-V的氨基酸序列;
(d)使用D-型氨基酸为原料合成干扰肽NIP-V。
进一步的,在步骤(a)中,所述氨基酸为346-357位氨基酸。
进一步的,所述346-357位氨基酸的氨基酸序列为FKDQVILLNKHI。
进一步的,所述氨基酸为L-型天然氨基酸。
进一步的,在步骤(b)中,所述HIV-TAT的氨基酸序列为YGRKKRRQRRR。
进一步的,在步骤(c)中,所述最终干扰肽NIP-V的氨基酸序列为IHKNLLIVQDKFPPRRRQRRKKRG,分子量为3040.69。
本发明的另一技术方案是:一种靶向SARS-CoV-2N蛋白的干扰肽在抗SARS-CoV-2感染药物中的应用。
本发明提供了一种靶向SARS-CoV-2 N蛋白的干扰肽NIP-V的制备方法及应用,其优点是:
1、靶向SARS-CoV-2 N蛋白的干扰肽NIP-V有效解除了SARS-CoV-2 N蛋白介导的抗病毒免疫抑制,显著地阻止了SARS-CoV-2在表达人源血管紧张素转化酶2(angiotensin-converting enzyme 2,ACE2)转基因小鼠中的复制和增殖,提升了小鼠抗SARS-CoV-2的能力;
2、蛋白间的相互作用通常为面-面互作,相比于传统的小分子药物,本发明涉及的靶向SARS-CoV-2N蛋白的干扰肽NIP-V可有效阻断SARS-CoV-2N蛋白自身的相互作用;
3、靶向SARS-CoV-2N蛋白的干扰肽NIP-V包含HIV-TAT序列,可直接使肽段穿过细胞膜进入细胞质内发挥作用,不需要任何的载体,避免载体引起的毒副作用;
4、D-型氨基酸较天然L-型氨基酸在动物体内的降解较为缓慢,将干扰肽修饰为D-型氨基酸逆向(D-retroinverso,DRI)异构体在以往的临床试验中被证明具有良好的耐受性和治疗效果,所以靶向SARS-CoV-2N蛋白的DRI修饰干扰肽NIP-V具备进行临床试验的可行性;
5、靶向SARS-CoV-2N蛋白的干扰肽NIP-V长度仅为24氨基酸小肽,根据免疫学原理本身不具备免疫原性,可避免引起超敏反应;
6、靶向SARS-CoV-2 N蛋白的干扰肽NIP-V可直接通过现有成熟的多肽合成技术获得,具有高纯度,质量可控,成药潜力较大。
附图说明
图1的左图为SARS-CoV-2N蛋白的二聚结构域(dimerization domain,DD)二聚体的三级结构图,右图为干扰肽NIP-V的靶序列示意图;
图2为用质谱法(mass spectrometry,MS)检测合成的干扰肽NIP-V的分子量示意图;
图3为用高效液相色谱法(high performance liquid chromatography,HPLC)检测合成的干扰肽NIP-V的纯度示意图;
图4为核酸检测分析发现使用干扰肽NIP-V处理ACE2转基因小鼠可显著抑制SARS-CoV-2在肺组织中的增殖示意图;
图5为通过苏木精-伊红(hematoxylin-eosin,HE)染色发现使用干扰肽NIP-V处理ACE2转基因小鼠可抑制SARS-CoV-2感染导致的肺部的病变的示意图;
图6为通过免疫荧光实验发现使用干扰肽NIP-V处理ACE2转基因小鼠可抑制SARS-CoV-2感染后肺部SARS-CoV-2N蛋白的表达的示意图;
图7为通过免疫组化实验发现使用干扰肽NIP-V处理ACE2转基因小鼠可抑制SARS-CoV-2感染后肺部SARS-CoV-2 S蛋白的表达的示意图;
图8为干扰肽NIP-V可增强SARS-CoV-2感染ACE2转基因小鼠后血清中的IFN-β分泌的示意图;
图9为干扰肽NIP-V可增强SARS-CoV-2感染ACE2转基因小鼠后脾、肝、肺组织中的IFN-β、ISG56 mRNA表达,降低SARS-CoV-2基因组RNA的载量的示意图;
图10为干扰肽NIP-V可解除SARS-CoV-2 N蛋白对固有免疫信号通路关键接头蛋白MAVS的寡聚化的抑制的示意图。
具体实施方式
本发明所述的一种靶向SARS-CoV-2N蛋白的干扰肽的制备方法,介于蛋白质-蛋白质相互作用由于作用面积较大,单个小分子可能无法有效干扰,利用大分子药物比如拥有类似作用面的多肽(干扰肽)来干扰蛋白间的相互作用十分有效。根据SARS-CoV-2 N蛋白的基本功能以及抑制机体先天抗病毒免疫的机制,人工设计并合成靶向N蛋白的干扰肽。该干扰肽通过结合介导SARS-CoV-2N蛋白寡聚的二聚结构域的作用面,破坏疏水相互作用,进而解除N蛋白对先天免疫的抑制,达到抑制SARS-CoV-2病毒在细胞内复制的目的,从而实现临床病症的有效治疗。
本发明所述的一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法,首先设计干扰肽段靶向位于SARS-CoV-2 N蛋白二聚结构域内的346-357位氨基酸,该段氨基酸序列为FKDQVILLNKHI,天然氨基酸为L-型,设计短肽用于特异性破坏N蛋白间的互作;
其次,为了促进细胞对NIP-V的吸收,干扰肽段设计为与HIV-TAT融合。HIV-TAT是一段亲水的序列,氨基酸序列为YGRKKRRQRRR,能够通过不依赖能量的方式使肽段跨过细胞膜从而被细胞吸收;
然后,DRI修饰肽段可以提高肽段在细胞和动物体内试验的稳定性和有效性。将整条干扰肽段修饰为逆向异构体,最终干扰肽NIP-V的氨基酸序列为IHKNLLIVQDKFPPRRRQRRKKRG,分子量为3040.69;
最后,使用D-型氨基酸为原料合成干扰肽NIP-V,肽段的纯度在98%以上。
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合实施例进一步说明本发明的技术方案。但是本发明不限于所列出的实施例,还应包括在本发明所要求的权利范围内其他任何公知的改变。
此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例,也不是单独的或选择性的与其他实施例互相排斥的实施例。
实施例1
干扰肽药物NIP-V的合成和检测
本发明设计的干扰肽药物NIP-V氨基酸序列为IHKNLLIVQDKFPPRRRQRRKKRG,靶向序列如图1所示,使用D-型氨基酸为原料于吉尔生化(上海)有限公司合成。
如图2所示,合成的干扰肽药物NIP-V经Agilent-6125B液质联用***(AgilentTechnologies)鉴定分子量为3040.69,HPLC采用Inertsil ODS-SP液相色谱柱(岛津,4.6mm×250mm)为固定相,使用流动相A(100%腈,0.1%三氟乙酸)和流动相B(100%超纯水,0.1%三氟乙酸)进行梯度洗脱,如图3及表1所示,经HPLC鉴定,纯度大于98%。
保留时间 | 含量(%) | 峰面积 | 峰高度 |
11.891 | 98.03 | 6613214 | 514625 |
12.236 | 1.966 | 132591 | 20430 |
表1
实施例2
使用干扰肽药物NIP-V处理ACE2转基因小鼠可显著降低SARS-CoV-2在小鼠体内的增殖。
1实验材料
ACE2转基因小鼠,达安基因新型冠状病毒(2019-nCoV)核酸检测试剂盒(荧光PCR法),SARS-CoV-2,实施例1中制备的NIP-V干扰肽药物。
2实验方法
使用灭菌PBS溶解NIP-V干扰肽药物,使其浓度为1mg/mL。ACE2转基因小鼠分为4组,每组8只,第一三组注射0.5mL灭菌PBS作为对照,第二四组注射0.5mL(0.5mg)NIP-V药物。1小时后,所有四组小鼠麻醉后鼻内接种SARS-CoV-2,每只小鼠接种约1×105TCID50病毒。感染病毒16小时、24小时后,分别取第一二组、第三四组小鼠肺组织,通过达安基因公司的新型冠状病毒(2019-nCoV)核酸检测试剂盒对小鼠肺部SARS-CoV-2的核酸含量进行检测。结果的统计分析以“均值±标准偏差”(mean±SEM)表示,采用方差分析(ANOVA)进行比较,p<0.05为显著性差异,p<0.01为极显著性差异。
3实验结果
请参阅图4,如图4所示,干扰肽药物NIP-V可显著降低SARS-CoV-2在ACE2转基因小鼠肺组织中的载量。
核酸检测试剂盒是检测SARS-CoV-2病毒载量的常用手段之一,通过达安基因新型冠状病毒(2019-nCoV)核酸检测试剂盒通过绝对定量PCR方法检测组织中SARS-CoV-2基因组RNA拷贝数,结果见表2。
表2
表2结果表明在PBS处理组中(1、3组),随着SARS-CoV-2病毒载量随时间出现上升趋势,而在NIP-V处理组中(2、4组),SARS-CoV-2病毒载量大大降低,且多数小鼠肺组织中无法检测到SARS-CoV-2。由此可知,干扰肽药物NIP-V可显著降低SARS-CoV-2在ACE2转基因小鼠肺组织中的载量。
实施例3
使用干扰肽药物NIP-V处理ACE2转基因小鼠可显著抑制SARS-CoV-2感染导致的小鼠肺部的病变。
1实验材料
ACE2转基因小鼠,SARS-CoV-2,实施例1中制备的NIP-V干扰肽药物。组织固定、包埋以及HE染色相关材料试剂(生工)。
2实验方法
使用灭菌PBS溶解NIP-V干扰肽药物,使其浓度为1mg/mL。ACE2转基因小鼠分为3组,第一组不感染,第二组注射0.5mL灭菌PBS,1小时后鼻内接种1×105TCID50的SARS-CoV-2,第三组注射0.5mg的NIP-V药物,1小时后鼻内接种1×105TCID50的SARS-CoV-2。病毒感染24小时后,取小鼠肺组织,置于4%多聚甲醛/PBS中进行组织固定,制作肺组织的石蜡切片,通过HE染色检测小鼠肺部的病变。
3实验结果
请参阅图5,如图5所示,干扰肽药物NIP-V可显著降低SARS-CoV-2感染导致的ACE2转基因小鼠肺部病变。未感染病毒的小鼠的肺组织形态正常,肺泡清晰,间隔纤细。而PBS+SARS-CoV-2处理组肺泡间隔显著增厚,局部可见病毒感染引起的血细胞凝集以及炎性细胞浸润,证明病毒感染引起了明显的炎症反应。而NIP-V+SARS-CoV-2处理组肺泡间隔增厚不明显,且血细胞凝集和炎性细胞浸润现象显著低于PBS+SARS-CoV-2组。
实施例4
使用干扰肽药物NIP-V处理ACE2转基因小鼠可显著抑制小鼠肺组织中SARS-CoV-2的N蛋白、S蛋白的表达。
1实验材料
ACE2转基因小鼠,SARS-CoV-2,实施例1中制备的NIP-V干扰肽药物。多聚甲醛等组织固定、包埋相关材料试剂均为国产。兔抗SARS-CoV-2 N蛋白抗体(Abcam),鼠抗SARS-CoV-2 S蛋白抗体(Abcam),DAPI,FITC偶连的羊抗兔IgG,HRP偶连的羊抗鼠IgG(CST),DAB显色试剂盒(生工)
2实验方法
ACE2转基因小鼠的给药和病毒刺激同实施例3。SARS-CoV-2感染24小时后,取小鼠肺组织,使用4%多聚甲醛/PBS中进行组织固定,制作肺组织的石蜡切片。通过免疫组化检测小鼠肺部SARS-CoV-2 S蛋白的表达情况;通过免疫荧光实验检测小鼠肺部SARS-CoV-2 N蛋白的表达情况。
3实验结果
请参阅图6和图7,如图6中所示,我们通过偶连FITC的荧光二抗检测SARS-CoV-2 N蛋白的表达,在未感染病毒的小鼠的肺组织中几乎无荧光信号,而在PBS+SARS-CoV-2处理组小鼠的肺组织中具有较强的荧光信号,在NIP-V+SARS-CoV-2处理组小鼠的肺组织中只能检测到较弱的N蛋白的信号。同样地,如图7所示,通过免疫组化检测SARS-CoV-2 S蛋白在ACE2转基因小鼠肺部的表达,通过DAB显色法在存在S蛋白的部位出现棕红色染色。在未感染病毒的小鼠的肺组织中无S蛋白信号,在PBS+SARS-CoV-2处理组小鼠出现强S蛋白信号和炎性细胞浸润,而在NIP-V+SARS-CoV-2处理组小鼠的肺组织中的S蛋白信号远低于PBS+SARS-CoV-2处理组小鼠。由此可知,干扰肽药物NIP-V可明显降低SARS-CoV-2感染ACE2转基因小鼠后肺部的SARS-CoV-2 N蛋白和S蛋白表达。
实施例5
使用干扰肽药物NIP-V处理ACE2转基因小鼠可显著提升小鼠感染SARS-CoV-2的抗病毒固有免疫反应,降低组织中病毒增殖。
1实验材料
ACE2转基因小鼠,SARS-CoV-2,实施例一中制备的NIP-V干扰肽药物,鼠IFN-βELISA检测试剂盒,Trizol日本(TAKARA公司),反转录试剂盒,qPCR试剂盒。表3为进行qPCR所需要的引物(金唯智合成)
Primers | Sequence(5′-3′) |
Murine 18S forward | CGCGGTTCTATTTTGTTGGT |
Murine 18S reverse | AGTCGGCATCGTTTATGGTC |
Murine Ifnb1 forward | TCCTGCTGTGCTTCTCCACCACA |
Murine Ifnb1 reverse | AAGTCCGCCCTGTAGGTGAGGTT |
Murine Isg56forward | AAGACAAGGCAATCACCCTCTACT |
Murine Isg56reverse | GTCTTTCAGCCACTTTCTCCAAA |
SARS-CoV-2forward | CTTCTCGTTCCTCATCACGTAGTC |
SARS-CoV-2reverse | TTGCTCTCAAGCTGGTTCAATC |
表3
2实验方法
ACE2转基因小鼠的给药和病毒刺激同实施例1。SARS-CoV-2感染16、24小时后,从小鼠眼眶取血,将血液通过1200rpm 5min离心去除血细胞,保留血清。使用鼠IFN-β ELISA检测试剂盒检测血清中的IFN-β含量。取小鼠的脾、肝、肺组织,使用Trizol法提取总RNA,反转录后通过qPCR检测脾、肝、肺组织中的Ifnb1、Isg56mRNA的表达以及SARS-CoV-2基因组RNA的载量。结果的统计分析以“均值±标准偏差”(mean±SEM)表示,采用方差分析(ANOVA)进行比较,p<0.05为显著性差异,p<0.01为极显著性差异。
3实验结果
通过鼠IFN-β ELISA检测试剂盒检测血清中的IFN-β含量,结果见表4。
表4
如表4所示,干扰肽药物NIP-V显著提升SARS-CoV-2感染ACE2转基因小鼠血清中IFN-β的含量。请参阅图8,如图8所示,在PBS处理组中(1、3组),小鼠感染SARS-CoV-2后血清中IFN-β随时间出现下降趋势,而在NIP-V处理组中(2、4组),小鼠血清中IFN-β含量显著高于PBS处理组(图8)。由此可知,干扰肽药物NIP-V可明显提升SARS-CoV-2感染ACE2转基因小鼠后血清中的IFN-β含量。
如图9所示,使用干扰肽药物NIP-V预处理ACE2转基因小鼠后,感染SARS-CoV-2所诱导的Ifnb1、Isg56 mRNA的表达相对PBS处理组均有所提升,证明NIP-V处理增强了小鼠对SARS-CoV-2的抗病毒固有免疫反应;从而降低组织内SARS-CoV-2的增殖。相对于未刺激小鼠,mRNA增加倍数如下表5所示。
表5
如表5所示,qPCR结果表明NIP-V处理提升SARS-CoV-2感染ACE2转基因小鼠脾、肝、肺组织中Ifnb1、Isg56 mRNA的表达,抑制SARS-CoV-2增殖。
实施例6
使用干扰肽药物NIP-V处理细胞可解除SARS-CoV-2 N蛋白对MAVS寡聚化的抑制。
1实验材料
SARS-CoV-2 N蛋白表达质粒Myc-NP,按照实施例一中制备的NIP-V干扰肽药物,仙台病毒(SeV),胎牛血清、DMEM培养基、青霉素/链霉素溶液(Gibco);HEK 293T细胞系(ATCC来源),MAVS抗体、Myc抗体及相关二抗(CST公司)。
2实验方法
将HEK 293T细胞铺6孔板,待细胞密度达到70%时,在3个孔中转染Myc-NP质粒,24h后使用50μM、100μM NIP-V处理细胞,1h后使用SeV刺激细胞8h。8h后收集细胞,通过半变性电泳(SDD-PAGE)的方法检测MAVS寡聚化的情况。
3实验结果
MAVS是固有免疫信号通路中重要的接头蛋白,寡聚化是抗病毒固有免疫通路激活的重要标志之一。而SARS-CoV-2 N蛋白可通过作用于MAVS抑制抗病毒固有免疫。我们通过SDD-PAGE实验研究NIP-V是否可拯救被N蛋白抑制的固有免疫信号通路。如图10所示,在静息状态下,MAVS无寡聚化发生;SeV刺激激活固有免疫信号通路,可诱导MAVS发生寡聚化;在细胞中转染N蛋白可抑制SeV诱导的MAVS寡聚化;而使用NIP-V预先处理细胞,可呈剂量依赖性地恢复MAVS的寡聚化,证明NIP-V可解除SARS-CoV-2 N蛋白对MAVS的抑制,增强固有免疫信号传递。
综上所述,本发明提供的干扰肽药物NIP-V能够与SARS-CoV-2 N蛋白的二聚结构域相互作用,抑制N蛋白寡聚化,进而解除N蛋白对先天免疫的抑制,从而达到抑制SARS-CoV-2病毒在细胞和动物体内复制的目的。
应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (7)
1.一种靶向SARS-CoV-2N蛋白的干扰肽的制备方法,其特征在于,该方法包括如下步骤:
(a)设计干扰肽段靶向位于SARS-CoV-2N蛋白二聚结构域内的氨基酸;
(b)将所述干扰肽段与HIV-TAT融合;
(c)将与HIV-TAT融合的干扰肽段修饰为逆向异构体,获得最终干扰肽NIP-V的氨基酸序列;
(d)使用D-型氨基酸为原料合成干扰肽NIP-V。
2.根据权利要求1所述的一种靶向SARS-CoV-2N蛋白的干扰肽的制备方法,其特征在于:在步骤(a)中,所述氨基酸为346-357位氨基酸。
3.根据权利要求2所述的一种靶向SARS-CoV-2N蛋白的干扰肽的制备方法,其特征在于:所述346-357位氨基酸的氨基酸序列为FKDQVILLNKHI。
4.根据权利要求2所述的一种靶向SARS-CoV-2N蛋白的干扰肽的制备方法,其特征在于:所述氨基酸为L-型天然氨基酸。
5.根据权利要求1所述的一种靶向SARS-CoV-2N蛋白的干扰肽的制备方法,其特征在于:在步骤(b)中,所述HIV-TAT的氨基酸序列为YGRKKRRQRRR。
6.根据权利要求1所述的一种靶向SARS-CoV-2N蛋白的干扰肽的制备方法,其特征在于:在步骤(c)中,所述最终干扰肽NIP-V的氨基酸序列为IHKNLLIVQDKFPPRRRQRRKKRG,分子量为3040.69。
7.一种靶向SARS-CoV-2N蛋白的干扰肽在抗SARS-CoV-2感染中的应用。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110466103.7A CN113072623B (zh) | 2021-04-28 | 2021-04-28 | 一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用 |
US17/920,637 US20230212229A1 (en) | 2021-04-28 | 2022-01-11 | PREPARATION METHOD AND APPLICATION OF INTERFERING PEPTIDE TARGETING SARS-CoV-2 N PROTEIN |
PCT/CN2022/071356 WO2022227702A1 (zh) | 2021-04-28 | 2022-01-11 | 一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110466103.7A CN113072623B (zh) | 2021-04-28 | 2021-04-28 | 一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113072623A true CN113072623A (zh) | 2021-07-06 |
CN113072623B CN113072623B (zh) | 2022-10-14 |
Family
ID=76619112
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110466103.7A Active CN113072623B (zh) | 2021-04-28 | 2021-04-28 | 一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用 |
Country Status (3)
Country | Link |
---|---|
US (1) | US20230212229A1 (zh) |
CN (1) | CN113072623B (zh) |
WO (1) | WO2022227702A1 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113880947A (zh) * | 2021-07-26 | 2022-01-04 | 中国人民解放军军事科学院军事医学研究院 | 小分子抗体及其编码基因和制备方法及应用和药物组合物 |
WO2022227702A1 (zh) * | 2021-04-28 | 2022-11-03 | 苏州大学 | 一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111647048A (zh) * | 2020-06-22 | 2020-09-11 | 中国科学院昆明动物研究所 | 干扰多肽在制备抗SARS-CoV-2药物中的应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2522329A1 (en) * | 2003-04-14 | 2005-01-13 | Merck & Co., Inc. | Inhibitors of coronavirus |
CN113072623B (zh) * | 2021-04-28 | 2022-10-14 | 苏州大学 | 一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用 |
-
2021
- 2021-04-28 CN CN202110466103.7A patent/CN113072623B/zh active Active
-
2022
- 2022-01-11 US US17/920,637 patent/US20230212229A1/en active Pending
- 2022-01-11 WO PCT/CN2022/071356 patent/WO2022227702A1/zh active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111647048A (zh) * | 2020-06-22 | 2020-09-11 | 中国科学院昆明动物研究所 | 干扰多肽在制备抗SARS-CoV-2药物中的应用 |
Non-Patent Citations (1)
Title |
---|
DESIREE SCHÜTZ等: "Peptide and peptide-based inhibitors of SARS-CoV-2 entry", 《ADVANCED DRUG DELIVERY REVIEWS》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022227702A1 (zh) * | 2021-04-28 | 2022-11-03 | 苏州大学 | 一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用 |
CN113880947A (zh) * | 2021-07-26 | 2022-01-04 | 中国人民解放军军事科学院军事医学研究院 | 小分子抗体及其编码基因和制备方法及应用和药物组合物 |
Also Published As
Publication number | Publication date |
---|---|
WO2022227702A1 (zh) | 2022-11-03 |
CN113072623B (zh) | 2022-10-14 |
US20230212229A1 (en) | 2023-07-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20210322545A1 (en) | Smc combination therapy for the treatment of cancer | |
CN113072623B (zh) | 一种靶向SARS-CoV-2 N蛋白的干扰肽的制备方法及应用 | |
US10413586B2 (en) | Antiviral agent comprising recombinant mistletoe lectins | |
CN113769080B (zh) | 多肽免疫偶联物及其应用 | |
WO2021257781A1 (en) | Compositions and methods for treating covid-19 | |
US7399825B2 (en) | Synthetic peptide, inhibitor to DNA viruses | |
CN103372199B (zh) | 一种预防和治疗神经退行性疾病的新型脑部靶向制剂 | |
CN102584976A (zh) | 一种人血清淀粉样蛋白a1及其制备方法和应用 | |
WO2021195088A1 (en) | TGF-Bβ1 INHIBITORS FOR PREVENTING AND TREATING SARS-COV-2 | |
WO2023060483A1 (zh) | 多肽-rbd免疫偶联物及其用途 | |
JP4858803B2 (ja) | 抗mrd(微少残存白血病)薬 | |
CN114796233A (zh) | 积雪草酸在制备乙型肝炎治疗药物中的用途 | |
EP2981334A1 (en) | Compositions and methods for inhibiting viral activity | |
WO2021181279A1 (en) | Compositions and methods for treating covid-19 infections and/or symptoms thereof | |
WO2021118988A1 (en) | Compositions and methods for preventing recurrence of cancer | |
CN115850515A (zh) | 抑制SARS-CoV-2核衣壳蛋白SUMO化修饰的干扰肽的制备方法及应用 | |
CN110804100A (zh) | 融合多肽及其在增强干细胞归巢能力和抗凋亡能力中的应用和药物组合物 | |
US20220133837A1 (en) | Peptide inhibitors of nf kappa b and use thereof in treatment of covid-19 and inflammatory diseases | |
US20230190849A1 (en) | Use of huangqi extract for suppressing sars-cov-2 entry and treating covid-19 related cytokine storm | |
CN111991414B (zh) | 一种β-壳糖胺在制备预防和治疗新冠肺炎病毒感染的药物中的用途 | |
US20230167162A1 (en) | Homing peptide-guided decorin conjugates for use in treating epidermolysis bullosa | |
WO2022000167A1 (zh) | 转铁蛋白、转铁蛋白受体及其抗体在制备抗SARS-CoV-2病毒的药物中的应用 | |
US20230372468A1 (en) | Compositions and methods for targeting coronavirus using lipid vesicles including exosomes | |
KR20240086772A (ko) | 개선된 코로나 바이러스 억제용 펩타이드 및 그 용도 | |
CN1896100A (zh) | 肝再生因子及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |