CN117562981A - Snapin蛋白及其编码基因在抗流感病毒中的应用 - Google Patents
Snapin蛋白及其编码基因在抗流感病毒中的应用 Download PDFInfo
- Publication number
- CN117562981A CN117562981A CN202410023219.7A CN202410023219A CN117562981A CN 117562981 A CN117562981 A CN 117562981A CN 202410023219 A CN202410023219 A CN 202410023219A CN 117562981 A CN117562981 A CN 117562981A
- Authority
- CN
- China
- Prior art keywords
- snapin
- protein
- influenza virus
- cells
- replication
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101000633076 Homo sapiens SNARE-associated protein Snapin Proteins 0.000 title claims abstract description 70
- 102000008736 Snapin Human genes 0.000 title claims abstract description 70
- 241000712461 unidentified influenza virus Species 0.000 title claims abstract description 40
- 108090000623 proteins and genes Proteins 0.000 title claims description 20
- 230000014509 gene expression Effects 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 14
- 230000000091 immunopotentiator Effects 0.000 claims abstract description 5
- 238000000338 in vitro Methods 0.000 claims abstract description 5
- 230000010076 replication Effects 0.000 claims description 18
- 230000002452 interceptive effect Effects 0.000 claims description 11
- 230000002018 overexpression Effects 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 230000002401 inhibitory effect Effects 0.000 claims description 9
- 108020004414 DNA Proteins 0.000 claims description 5
- 102000053602 DNA Human genes 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- 101150002205 Snapin gene Proteins 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 230000029812 viral genome replication Effects 0.000 abstract description 18
- 230000000694 effects Effects 0.000 abstract description 15
- 108010050904 Interferons Proteins 0.000 abstract description 14
- 102000014150 Interferons Human genes 0.000 abstract description 14
- 229940079322 interferon Drugs 0.000 abstract description 14
- 241000700605 Viruses Species 0.000 abstract description 11
- 229940079593 drug Drugs 0.000 abstract description 7
- 206010022000 influenza Diseases 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 4
- 230000002708 enhancing effect Effects 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- 230000003042 antagnostic effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 76
- 230000003612 virological effect Effects 0.000 description 14
- 239000013612 plasmid Substances 0.000 description 12
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 12
- 239000013598 vector Substances 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 7
- 241000713666 Lentivirus Species 0.000 description 6
- 230000001939 inductive effect Effects 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 229950010131 puromycin Drugs 0.000 description 6
- 238000012216 screening Methods 0.000 description 5
- 101710154606 Hemagglutinin Proteins 0.000 description 4
- 108010006232 Neuraminidase Proteins 0.000 description 4
- 102000005348 Neuraminidase Human genes 0.000 description 4
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 4
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 4
- 241000156302 Porcine hemagglutinating encephalomyelitis virus Species 0.000 description 4
- 101710176177 Protein A56 Proteins 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 239000000185 hemagglutinin Substances 0.000 description 4
- 230000000670 limiting effect Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 102100035473 2'-5'-oligoadenylate synthase-like protein Human genes 0.000 description 3
- 101000597360 Homo sapiens 2'-5'-oligoadenylate synthase-like protein Proteins 0.000 description 3
- 101001128393 Homo sapiens Interferon-induced GTP-binding protein Mx1 Proteins 0.000 description 3
- 101001034846 Homo sapiens Interferon-induced transmembrane protein 3 Proteins 0.000 description 3
- 101001057508 Homo sapiens Ubiquitin-like protein ISG15 Proteins 0.000 description 3
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 3
- 101150002750 IFIT1 gene Proteins 0.000 description 3
- 101710099622 Interferon lambda-2 Proteins 0.000 description 3
- 102100020989 Interferon lambda-2 Human genes 0.000 description 3
- 101710099621 Interferon lambda-3 Proteins 0.000 description 3
- 102100020992 Interferon lambda-3 Human genes 0.000 description 3
- 102100027355 Interferon-induced protein with tetratricopeptide repeats 1 Human genes 0.000 description 3
- 102100040035 Interferon-induced transmembrane protein 3 Human genes 0.000 description 3
- 101150056567 UL70 gene Proteins 0.000 description 3
- 102100027266 Ubiquitin-like protein ISG15 Human genes 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 108010018844 interferon type III Proteins 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 210000003771 C cell Anatomy 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108091092584 GDNA Proteins 0.000 description 2
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 241000712431 Influenza A virus Species 0.000 description 2
- 108010014726 Interferon Type I Proteins 0.000 description 2
- 102000002227 Interferon Type I Human genes 0.000 description 2
- MQUQNUAYKLCRME-INIZCTEOSA-N N-tosyl-L-phenylalanyl chloromethyl ketone Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N[C@H](C(=O)CCl)CC1=CC=CC=C1 MQUQNUAYKLCRME-INIZCTEOSA-N 0.000 description 2
- 101710085061 Orsellinic acid synthase Proteins 0.000 description 2
- 101710110277 Orsellinic acid synthase armB Proteins 0.000 description 2
- 231100000645 Reed–Muench method Toxicity 0.000 description 2
- 102000004389 Ribonucleoproteins Human genes 0.000 description 2
- 108010081734 Ribonucleoproteins Proteins 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- 101150063416 add gene Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003443 antiviral agent Substances 0.000 description 2
- 230000004900 autophagic degradation Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000010805 cDNA synthesis kit Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000003197 gene knockdown Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 210000003712 lysosome Anatomy 0.000 description 2
- 230000001868 lysosomic effect Effects 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 210000002845 virion Anatomy 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- UBCHPRBFMUDMNC-UHFFFAOYSA-N 1-(1-adamantyl)ethanamine Chemical compound C1C(C2)CC3CC2CC1(C(N)C)C3 UBCHPRBFMUDMNC-UHFFFAOYSA-N 0.000 description 1
- 102100027769 2'-5'-oligoadenylate synthase 1 Human genes 0.000 description 1
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 101001008907 Homo sapiens 2'-5'-oligoadenylate synthase 1 Proteins 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102100031802 Interferon-induced GTP-binding protein Mx1 Human genes 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 101710085938 Matrix protein Proteins 0.000 description 1
- 101710127721 Membrane protein Proteins 0.000 description 1
- 108090000301 Membrane transport proteins Proteins 0.000 description 1
- 102000003939 Membrane transport proteins Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 101001028244 Onchocerca volvulus Fatty-acid and retinol-binding protein 1 Proteins 0.000 description 1
- 241000712464 Orthomyxoviridae Species 0.000 description 1
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- DKNWSYNQZKUICI-UHFFFAOYSA-N amantadine Chemical compound C1C(C2)CC3CC2CC1(N)C3 DKNWSYNQZKUICI-UHFFFAOYSA-N 0.000 description 1
- 229960003805 amantadine Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 229940124393 anti-influenza virus drug Drugs 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 108091092328 cellular RNA Proteins 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000007762 localization of cell Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000009061 membrane transport Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- VSZGPKBBMSAYNT-RRFJBIMHSA-N oseltamivir Chemical compound CCOC(=O)C1=C[C@@H](OC(CC)CC)[C@H](NC(C)=O)[C@@H](N)C1 VSZGPKBBMSAYNT-RRFJBIMHSA-N 0.000 description 1
- 229960003752 oseltamivir Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 229960000888 rimantadine Drugs 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000007442 viral DNA synthesis Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0688—Cells from the lungs or the respiratory tract
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/91—Cell lines ; Processes using cell lines
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Veterinary Medicine (AREA)
- Pulmonology (AREA)
- Animal Behavior & Ethology (AREA)
- Oncology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Communicable Diseases (AREA)
- Toxicology (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Marine Sciences & Fisheries (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种宿主蛋白SNAPIN，经体外研究发现SNAPIN蛋白具有拮抗流感病毒复制的作用，并且SNAPIN蛋白具有增强干扰素和干扰素诱导基因表达的作用，在抗流感病毒药物制备或免疫增强剂制备中具有应用价值。
Description
技术领域
本发明属于生物技术领域，具体来说，涉及一种SNAPIN蛋白及其编码基因在抗流感病毒中的应用。
背景技术
A型流感病毒（influenza A virus, IAV）可以感染人和动物，严重威胁养殖业的发展和公共卫生安全，对人类健康造成潜在威胁。当前H5N1、H6N1、H7N7、H7N9、H9N2和H10N8等多种亚型流感病毒跨越宿主屏障感染人的事件时有发生，该现象是病毒和宿主共同作用的结果。
目前抗流感病毒药物主要有金刚烷胺、金刚乙胺、奥司他韦、干扰素等，但药物在长期使用过程中易产生耐药毒株或病毒产生免疫逃逸现象，某些药物若使用不当会引起急性肾衰竭等严重不良反应。
IAV属于正黏病毒科，流感病毒属，其基因组是八个节段的单股负链RNA。流感病毒粒子表面有两种主要糖蛋白，分别是血凝素（Hemagglutinin，HA）和神经氨酸酶（Neuraminidase，NA），根据HA和NA抗原性不同，IAV可以分为18中HA亚型和11中NA亚型。病毒内部基因分别为PB2、PB1、PA、NP、M和NS，分别编码不同的蛋白质。病毒脂质双分子层内部为基质蛋白M1，病毒粒子内部为核糖核蛋白复合体（Viral ribonucleoprotein complex，vRNP）。
SNAPIN蛋白（突触小体相关蛋白）在脑、心脏、肝脏、脾脏、肾脏、肺、睾丸等器官均可表达，主要分布于细胞质、高尔基体、溶酶体和细胞核。SNAPIN蛋白N端包含20个氨基酸组成的疏水区，C端有两个α螺旋区形成卷曲结构，疏水区及α螺旋之间通过短肽连接，通常SNAPIN卷曲结构可与其他蛋白互作，进而发挥生物学功能。SNAPIN蛋白在囊泡膜运输、自噬以及调控病毒复制方面发挥重要作用。SNAPIN与人巨细胞病毒（HCMV）编码的UL70蛋白互作，在人星形胶质瘤细胞（U373细胞）过表达SNAPIN导致UL70入核减少，病毒滴度下降，而siRNA干扰下调SNAPIN后UL70的入核增加，病毒复制增强，结果表明SNAPIN蛋白通过调节HCMV病毒UL70的细胞定位，进而调节病毒DNA合成和子代病毒粒子产生。SNAPIN在PRRSV复制中发挥重要作用，siRNA干扰SNAPIN后抑制病毒复制，可能与SNAPIN与GP5和M蛋白互作相关。猪血凝性脑脊髓炎病毒（PHEV）通过S1蛋白与SNAPIN互作调节病毒复制，感染PHEV的神经细胞中，下调SNAPIN可以抑制病毒复制，降低其病毒感染性，使得PHEV诱导的自噬过程中溶酶体发生异常聚积。
流感病毒在宿主体内复制时需要宿主因子的参与，当前发现的宿主因子可发挥两种作用。其一，某些宿主因子可以促进流感病毒复制，其二，某些宿主因子则可以抑制流感病毒复制。根据宿主因子抑制流感病毒复制的理论基础进行药物设计，被称为宿主导向疗法。这些抑制流感病毒复制的宿主因子可以作为抗病毒药物设计的靶点，具备潜在的应用价值。当前研究中，以宿主为导向的抗病毒药物开发仍处于初始阶段。从宿主自身表达蛋白入手设计药物具有很多优点，比如耐药毒株不易产生、无明显的毒副作用以及可能具备广谱的抗病毒作用等。
目前研究尚未发现SNAPIN在流感病毒复制中作用的报道。本研究中通过4D蛋白质组学技术筛选到A549细胞感染IAV后，SNAPIN表达下调。利用pLVX-3×Flag载体构建稳定表达SNAPIN蛋白的慢病毒质粒并命名为pLVX-SNAPIN，对照质粒命名为pLVX-Vector。之后利用293T细胞包装慢病毒，将慢病毒感染A549细胞，利用嘌呤霉素筛选，筛选后的细胞利用有限稀释法获得单克隆细胞，扩大培养后获得稳定表达SNAPIN蛋白的A549细胞系。利用过表达SNAPIN的A549细胞系感染流感病毒，检测过表达SNAPIN蛋白对病毒复制的影响。
发明内容
本发明的目的在于提供一种宿主蛋白SNAPIN，经体外研究发现SNAPIN蛋白具有拮抗流感病毒复制的作用，并且SNAPIN蛋白具有增强干扰素和干扰素诱导基因表达的作用，在抗流感病毒药物制备或免疫增强剂制备中具有应用价值。
为实现上述目的，本发明采用如下技术方案：
本发明提供了一种SNAPIN蛋白在抗流感病毒中的应用，所述蛋白质由SEQ ID NO:1所示的氨基酸序列组成。SEQ ID NO:1序列为：
MAGAGSAAVSGAGTPVAGPTGRDLFAEGLLEFLRPAVQQLDSHVHAVRESQVELREQIDNLATELCRINEDQKVALDLDPYVKKLLNARRRVVLVNNILQNAQERLRRLNHSVAKETARRRAMLDSGIYPPGSPGK。
所述蛋白质可以有如下应用：
a.制备抑制流感病毒复制的药物;
b.制备抑制流感病毒复制的免疫增强剂;
c.制备抑制流感病毒在体外复制的药物；
d.制备治疗或预防流感病毒的药物。
所述蛋白质的编码基因是如SEQ ID NO:2所示的DNA分子。
SEQ ID NO:2序列为：
ATGGCGGGGGCTGGTTCCGCCGCTGTATCGGGGGCAGGGACCCCGGTGGCGGGGCCCACAGGCCGCGACCTTTTCGCCGAAGGGCTGCTGGAGTTCCTGCGACCCGCTGTGCAGCAGCTCGACTCTCACGTACACGCCGTCAGAGAGAGCCAGGTAGAGCTCCGGGAACAAATTGACAACCTAGCCACAGAACTGTGCCGCATAAATGAGGATCAGAAGGTGGCCCTGGATCTTGACCCCTATGTTAAGAAGCTACTTAATGCCCGGCGACGCGTTGTCTTGGTTAACAACATTCTACAGAATGCTCAGGAACGACTGAGACGGCTAAACCACAGTGTTGCCAAGGAAACAGCCCGCAGGAGAGCAATGCTGGATTCGGGAATTTACCCCCCTGGCTCCCCAGGCAAATAA。
本发明提供了种含有SEQ ID NO:2所示DNA分子的过表达载体、过表达细胞系、干扰SNAPIN蛋白表达载体或干扰SNAPIN基因表达细胞系。
本发明利用pLKO.1载体构建敲低SNAPIN蛋白的质粒，命名为pLKO-SNAPIN。之后利用293T细胞包装慢病毒，将慢病毒感染A549细胞，利用嘌呤霉素筛选，筛选的细胞利用有限稀释法获得单克隆细胞，扩大培养得到稳定敲低SNAPIN蛋白的A549细胞系。利用稳定敲低SNAPIN蛋白的A549细胞系感染流感病毒，检测敲低SNAPIN蛋白对流感病毒复制的影响。
在稳定过表达SNAPIN蛋白的A549细胞系上，利用qPCR技术检测过表达SNAPIN蛋白的A549细胞中I型干扰素、III型干扰素和干扰素诱导基因MxA、OASL、OAS1、IFITM3、ISG56和ISG15的表达情况。
现有技术相比，本发明具有以下有益效果:
本发明研究证明了SNAPIN蛋白具有诱导干扰素表达，刺激干扰素诱导基因表达作用，发挥抗流感病毒复制的作用，夯实了SNAPIN在流感病毒复制中发挥重要的调节功能，可以作为抗流感病毒的重要药物靶点或作为免疫增强剂。
附图说明
图1为过表达SNAPIN蛋白抑制流感病毒复制效果结果；其中图1A为过表达SNAPIN蛋白对WSN病毒复制的影响，图1B为过表达SNAPIN蛋白对PR8病毒复制的影响。
图2为干扰SNAPIN蛋白促进流感病毒复制的效果；其中图2A为干扰SNAPIN蛋白促进流感病毒WSN复制效果，图2B为干扰SNAPIN蛋白促进流感病毒PR8复制效果。
图3为 SNAPIN促进干扰素和干扰素诱导基因（ISG）表达效果。
具体实施方式
为使本发明实施方式的目的、技术方案和优点更加清楚，下面将结合本发明实施方式中的附图，对本发明实施方式中的技术方案进行清楚、完整地描述，显然，所描述的实施方式是本发明一部分实施方式，而不是全部的实施方式。基于本发明中的实施方式，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式，都属于本发明保护的范围。因此，以下对在附图中提供的本发明的实施方式的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施方式。基于本发明中的实施方式，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式，都属于本发明保护的范围。
实施材料：本实验研究中所使用的材料和耗材，如无特殊指明，均可从商业途径购买到。所用293T细胞(人胚肾细胞系)购自American Type Culture Collection, ATCC，编号为CRL-3216。A549细胞(人肺癌细胞系)购自American type culture collection，ATCC，编号为CCL-185。MDCK细胞(犬肾细胞系)购自American Type Culture Collection，ATCC，编号为CCL-34。质粒载体来自于Addgene或本实验室独立构建。流感病毒株H1N1（WSN）和PR8（H1N1）均由本实验保存。下述实验均为标准的分子生物学、细胞生物学或病毒学等操作方法，对本领域内的研究人员均可方便地理解和操作。
下面以具体的实施例对本发明进行详细的阐述，但不构成对本发明保护范围的限制。
实施例1.构建过表达SNAPIN质粒
利用Nucleozol提取A549细胞RNA，加入oligo（dT）和随机引物，利用HiScript II1st Strand cDNA Synthesis Kit (+gDNA wiper)（Vazyme，R212-01）进行反转录得到cDNA。利用引物pLVX-SNAPIN-F：CGCGGATCCATGGCGGGGGCTGGTTCCGCCG和pLVX-SNAPIN-R：CCGGAATTCTTATTTGCCTGGGGAGCCAGGG扩增SNAPIN全长，用BamHI和EcoRl分别酶切SNAPIN片段和pLVX-3×Flag载体，酶切后的产物胶回收，利用T4 DNA连接酶连接。连接产物转化大肠杆菌感受态细胞，挑取单克隆菌落，鉴定为阳性后扩大培养。提取阳性质粒，命名为pLVX-SNAPIN，送生工生物工程(上海)股份有限公司测序。
实施例2.构建过表达SNAPIN蛋白的A549细胞系
六孔板铺293T细胞，待细胞密度到达80%，利用Lipo8000™转染试剂（Beyotime，C0533）将pSPAX2、PVSV-G和pLVX-SNAPIN（或pLVX-Vector）质粒按（7.5μg:2.5μg:10μg）比例转染到细胞内。8 h后换成新鲜培养基。转染后48 h，收集293T细胞上清，用0.22μm滤膜过滤除去细胞碎片，获得慢病毒。A549细胞铺六孔板，每孔加入2 mL的慢病毒感染细胞，避光加入Polybrene（终浓度为3 μg/mL），置于水平离心机中离心2 h，之后将六孔板细胞放置37℃细胞培养箱培养。感染8 h后，将六孔板A549细胞中上清弃掉，换成10% FBS完全培养基。六孔板细胞长满后传代到直径10 cm平皿中，加入终浓度为4 μg/mL嘌呤霉素进行筛选，72h后用倒置荧光显微镜观察细胞荧光。持续加嘌呤霉素筛选2周，直至对照细胞全部死亡。构建的细胞系分别命名为A549-pLVX-Vector和A549-pLVX-SNAPIN。利用qPCR和Western Blot检测SNAPIN过表达水平。
实施例3.构建干扰SNAPIN质粒
利用pLKO.1载体构建干扰SNAPIN蛋白表达的质粒。pLKO.1载体利用AgeI和EcoRI进行双酶切，双酶切产物进行胶回收纯化。设计引物pLKO-SNAPIN-F: CCGGCAATGCTGGATTCGGGAATTTCTCGAGAAATTCCCGAATCCAGCATTGTTTTTG和pLKO- SNAPIN-R: AATTCAAAAACAATGCTGGATTCGGGAATTTCTCGAGAAATTCCCGAATCCAGCATTG。根据Annealing Buffer for DNA Oligos(5X)（Beyotime，D0251）说明书操作，将pLKO-SNAPIN-F和pLKO-SNAPIN-R退火形成双链结构。利用T4 DNA连接酶将双酶切的pLKO.1载体和片段连接，之后转化大肠杆菌感受态细胞，挑取单克隆菌落扩大培养，提质粒命名为pLKO-SNAPIN，送生工生物工程(上海)股份有限公司测序。对照质粒选用pLKO-GFP（Addgene，30323）。
实施例4.构建干扰SNAPIN基因的A549细胞系
六孔板铺293T细胞，待细胞密度到达80%，利用Lipo8000™转染试剂（Beyotime，C0533）将pSPAX2、PVSV-G和pLKO-SNAPIN（或pLKO-GFP）质粒按（7.5μg:2.5μg:10μg）比例转染到细胞内。8 h后换成新鲜培养基。转染后48 h，收集293T细胞上清，用0.22μm滤膜过滤除去细胞碎片，获得慢病毒。A549细胞铺六孔板，每孔加入2 mL的慢病毒感染细胞，避光加入Polybrene（终浓度为3 μg/mL），置于水平离心机中离心2 h，之后将六孔板细胞放置37 ℃细胞培养箱培养。感染8 h后，将六孔板A549细胞中上清弃掉，换成10% FBS完全培养基。六孔板细胞长满后传代到直径10 cm平皿中，加入终浓度为4 μg/mL嘌呤霉素进行筛选。持续加嘌呤霉素筛选2周，直至对照细胞全部死亡。构建的细胞系分别命名为A549-pLKO-GFP和A549-pLKO-SNAPIN。利用qPCR和Western Blot检测SNAPIN表达水平。
实施例5.过表达SNAPIN蛋白抑制流感病毒复制
利用A549-pLVX-Vector和A549-pLVX-SNAPIN细胞铺12孔板，将WSN和PR8毒株以MOI=1接种12孔板细胞，37℃孵育1 h，之后换成含TPCK（0.125μg/mL）的DMEM维持液，分别于感染后12 h、24 h、36 h和48 h收获细胞上清。利用MDCK细胞滴定收获细胞上清的病毒滴度，TCID50用Reed-Muench方法计算。过表达SNAPIN蛋白的A549细胞感染WSN病毒，24 h检测病毒滴度开始下降，48 h病毒滴度下降562倍（图1A）。过表达SNAPIN蛋白的A549细胞感染PR8病毒，36 h检测病毒滴度开始下降，36 h病毒滴度下降最多，为6.8倍（图1B）。结果表明SNAPIN蛋白可有效抑制流感病毒复制。
实施例6.干扰SNAPIN蛋白促进流感病毒复制
利用A549-pLKO-GFP和A549-pLKO-SNAPIN细胞铺12孔板，将WSN和PR8毒株以MOI=1接种12孔板细胞，37℃孵育1 h，之后换成含TPCK（0.125μg/mL）的DMEM维持液，分别于感染后12 h、24 h、36 h和48 h收获细胞上清。利用MDCK细胞滴定收获细胞上清的病毒滴度，TCID50用Reed-Muench方法计算。干扰SNAPIN蛋白表达的A549细胞感染WSN病毒，12 h检测病毒滴度即增加，36 h病毒滴度增大15.8倍（图2A）。干扰SNAPIN蛋白的A549细胞感染PR8病毒，36 h检测病毒滴度开始增加，与对照比，干扰SNAPIN蛋白后病毒增大39.5倍（图2B）。
实施例7. SNAPIN促进干扰素和干扰素诱导基因（ISG）表达
利用A549-pLVX-Vector和A549-pLVX-SNAPIN细胞铺六孔板，细胞密度长到90%，弃掉上清，收集细胞。利用Nucleozol提取细胞RNA，根据HiScript II 1st Strand cDNASynthesis Kit (+gDNA wiper)（Vazyme，R212-01）说明书反转录得到cDNA，利用qPCR检测A549-pLVX-Vector和A549-pLVX-SNAPIN细胞中I型干扰素（IFN-α，IFN-β）、III型干扰素（IL-28A和IL-28B）和干扰素诱导基因MxA、OASL、OAS1、IFITM3、ISG56和ISG15的表达情况（图3）。结果表明过表SNAPIN蛋白，IFN-β、IL-28A、IL-28B和MxA、OASL、OAS1、IFITM3、ISG56和ISG15表达均上调，表明SNAPIN蛋白促进I型干扰素、III型干扰素（IL-28A和IL-28B）和ISG表达。
试验证明，SNAPIN蛋白可以抑制流感病毒复制。A549细胞过表达SNAPIN蛋白后感染流感病毒，与对照相比，病毒滴度显著降低，表明SNAPIN蛋白可在体外细胞水平抑制流感病毒复制。利用shRNA干扰技术，A549细胞敲低SNAPIN后感染流感病毒，可显著增强病毒复制。进一步地，A549细胞过表达SNAPIN蛋白，可以促进I型和III型干扰素以及干扰素诱导基因（ISG）表达。综上，本发明鉴定了一种宿主蛋白SNAPIN，该蛋白具有抑制流感病毒复制的功能，为抗流感病毒药物的制备和设计提供了新的选择。
显然，上述实施例仅仅是为清楚地说明本发明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明的保护范围之中。
Claims (4)
1.一种SNAPIN蛋白在抗流感病毒中的应用，其特征在于，所述蛋白质由SEQ ID NO:1所示的氨基酸序列组成。
2.根据权利要求1所述的应用，其特征在于，所述蛋白质在如下a和/或b和/或c和/或d中的应用：
制备抑制流感病毒复制的药物;
制备抑制流感病毒复制的免疫增强剂;
制备抑制流感病毒在体外复制的药物；
制备治疗或预防流感病毒的药物。
3.根据权利要求1或2所述的应用，其特征在于，所述蛋白质的编码基因是如SEQ IDNO:2所示的DNA分子。
4.一种含有SEQ ID NO:2所示DNA分子的过表达载体、过表达细胞系、干扰SNAPIN蛋白表达载体或干扰SNAPIN基因表达细胞系。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410023219.7A CN117562981A (zh) | 2024-01-08 | 2024-01-08 | Snapin蛋白及其编码基因在抗流感病毒中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410023219.7A CN117562981A (zh) | 2024-01-08 | 2024-01-08 | Snapin蛋白及其编码基因在抗流感病毒中的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117562981A true CN117562981A (zh) | 2024-02-20 |
Family
ID=89862665
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410023219.7A Pending CN117562981A (zh) | 2024-01-08 | 2024-01-08 | Snapin蛋白及其编码基因在抗流感病毒中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117562981A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117925858A (zh) * | 2024-03-01 | 2024-04-26 | 华南农业大学 | Snap29在猪蓝耳病抗性鉴定、育种或疾病防治药物制备中的应用 |
-
2024
- 2024-01-08 CN CN202410023219.7A patent/CN117562981A/zh active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117925858A (zh) * | 2024-03-01 | 2024-04-26 | 华南农业大学 | Snap29在猪蓝耳病抗性鉴定、育种或疾病防治药物制备中的应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2021530985A (ja) | フソソームの組成物及びその使用 | |
| CA3120082A1 (en) | Fusosome compositions for cns delivery | |
| AU2019380517A1 (en) | Fusosome compositions for hematopoietic stem cell delivery | |
| Patel et al. | High efficiency gene transfer to airways of mice using influenza hemagglutinin pseudotyped lentiviral vectors | |
| CN117562981A (zh) | Snapin蛋白及其编码基因在抗流感病毒中的应用 | |
| KR20050084607A (ko) | 인플루엔자 치료제 | |
| KR20020086207A (ko) | 센다이바이러스 벡터를 사용한 ａｉｄｓ 바이러스 백신 | |
| Liang | Pathogenicity and virulence of influenza | |
| CN107208071B (zh) | 用于流感病毒的减弱活疫苗 | |
| US20170240898A1 (en) | Method for inhibiting ebola virus via mirna | |
| JP2023551982A (ja) | マルチシストロン性ｒｎａワクチン及びその使用 | |
| Xu et al. | Detection of viral components in exosomes derived from NDV-infected DF-1 cells and their promoting ability in virus replication | |
| CN110205321B (zh) | 一种dna片段及其在构建表达红色荧光蛋白基因的重组流感病毒中的应用 | |
| CN111373032A (zh) | 用于增强干扰素敏感性病毒的生产、生长、扩散或溶瘤和免疫治疗功效的组合物和方法 | |
| US10563178B2 (en) | PIV5-based amplifying virus-like particles | |
| CN104694576A (zh) | 一种沉默df-1细胞系中ifnar1基因的方法 | |
| WO2018168586A1 (ja) | ボルナウイルスベクター及びその利用 | |
| Chen et al. | A promising IFN-deficient system to manufacture IFN-sensitive influenza vaccine virus | |
| KR20240109241A (ko) | Piv5-기반 코로나바이러스 백신 및 이의 사용 방법 | |
| Yondola et al. | Un-“ESCRT”-ed budding | |
| CN113105537B (zh) | 一种促进a型流感病毒复制的宿主蛋白及其应用 | |
| CN102002489B (zh) | 抑制H1N1型流感病毒增殖的microRNA及其应用 | |
| Groenke | Molecular mechanism of virus attenuation by codon pair deoptimization | |
| Arora | Novel production system for influenza A virus-derived defective interfering particles and analysis of antiviral activity | |
| US20240238404A1 (en) | Influenza virus nucleic acid lipid particle vaccine |
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 |