CN113502298A - 非受体酪氨酸激酶tnk2蛋白在防治流感病毒感染中的应用 - Google Patents
非受体酪氨酸激酶tnk2蛋白在防治流感病毒感染中的应用 Download PDFInfo
- Publication number
- CN113502298A CN113502298A CN202110686648.9A CN202110686648A CN113502298A CN 113502298 A CN113502298 A CN 113502298A CN 202110686648 A CN202110686648 A CN 202110686648A CN 113502298 A CN113502298 A CN 113502298A
- Authority
- CN
- China
- Prior art keywords
- tnk2
- influenza virus
- tyrosine kinase
- receptor tyrosine
- protein
- 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
- 241000712461 unidentified influenza virus Species 0.000 title claims abstract description 41
- 230000009385 viral infection Effects 0.000 title claims abstract description 25
- 102000037979 non-receptor tyrosine kinases Human genes 0.000 title claims abstract description 19
- 108091008046 non-receptor tyrosine kinases Proteins 0.000 title claims abstract description 19
- 108091033409 CRISPR Proteins 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 15
- 238000005516 engineering process Methods 0.000 claims abstract description 11
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 9
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 7
- 229940079593 drug Drugs 0.000 claims abstract description 6
- 108020005004 Guide RNA Proteins 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 11
- 108020004414 DNA Proteins 0.000 claims description 10
- 230000008685 targeting Effects 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- 101150052413 TNK2 gene Proteins 0.000 claims description 7
- 230000000903 blocking effect Effects 0.000 claims description 7
- 108010019644 Oligodendrocyte Transcription Factor 2 Proteins 0.000 claims description 6
- 102100026058 Oligodendrocyte transcription factor 2 Human genes 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 102100026073 Oligodendrocyte transcription factor 1 Human genes 0.000 claims description 5
- 101710195940 Oligodendrocyte transcription factor 1 Proteins 0.000 claims description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 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 claims description 4
- 238000001890 transfection Methods 0.000 claims description 4
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000007480 sanger sequencing Methods 0.000 claims description 3
- 239000013598 vector Substances 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 108091034117 Oligonucleotide Proteins 0.000 claims description 2
- 239000012124 Opti-MEM Substances 0.000 claims description 2
- 239000006143 cell culture medium Substances 0.000 claims description 2
- 239000013599 cloning vector Substances 0.000 claims description 2
- 238000001976 enzyme digestion Methods 0.000 claims description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 229950010131 puromycin Drugs 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims 2
- 125000003729 nucleotide group Chemical group 0.000 claims 2
- 238000001816 cooling Methods 0.000 claims 1
- 239000001963 growth medium Substances 0.000 claims 1
- 101000928956 Homo sapiens Activated CDC42 kinase 1 Proteins 0.000 abstract description 41
- 102100036409 Activated CDC42 kinase 1 Human genes 0.000 abstract description 40
- 210000004027 cell Anatomy 0.000 abstract description 36
- 108090000623 proteins and genes Proteins 0.000 abstract description 12
- 241000282414 Homo sapiens Species 0.000 abstract description 6
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 5
- 210000002919 epithelial cell Anatomy 0.000 abstract description 3
- 238000010362 genome editing Methods 0.000 abstract description 3
- 210000004072 lung Anatomy 0.000 abstract description 3
- 241000700605 Viruses Species 0.000 description 15
- 239000000243 solution Substances 0.000 description 9
- 238000005406 washing Methods 0.000 description 8
- 238000003556 assay Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000010166 immunofluorescence Methods 0.000 description 5
- 206010022000 influenza Diseases 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 101100433980 Homo sapiens TNK2 gene Proteins 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 239000002356 single layer Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- 241000725681 Swine influenza virus Species 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- 108700010900 influenza virus proteins Proteins 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 229960005486 vaccine Drugs 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
- 241000203069 Archaea Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000011068 Cdc42 Human genes 0.000 description 1
- 108050001278 Cdc42 Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 231100000645 Reed–Muench method Toxicity 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000010448 genetic screening Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 102000054534 human TNK2 Human genes 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000021014 regulation of cell growth Effects 0.000 description 1
- 230000005903 regulation of histone modification Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000010023 transfer printing Methods 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- 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
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- 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
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
-
- 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
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/10—Protein-tyrosine kinases (2.7.10)
- C12Y207/10002—Non-specific protein-tyrosine kinase (2.7.10.2), i.e. spleen tyrosine kinase
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Cell Biology (AREA)
- Epidemiology (AREA)
- Pulmonology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Dermatology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及生物医药技术领域,是一种抗流感病毒感染的新靶点及应用。本发明以人肺上皮细胞(A549)作为靶细胞,采用CRISPR/Cas9基因编辑技术抑制或阻碍靶细胞宿主蛋白的表达,来挖掘可有效抑制流感病毒感染宿主的宿主因子。本发明提供了非受体酪氨酸激酶TNK2在防止流感病毒感染中的应用,本发明通过实验发现非受体酪氨酸激酶TNK2蛋白在流感病毒感染A549中发挥着重要作用。本发明提供了非受体酪氨酸激酶TNK2蛋白在制备预防或治疗流感病毒感染药物中的应用。
Description
技术领域
本发明涉及生物医药技术领域,是一种抗流感病毒感染的新靶点及应用。
背景技术
流感病毒引起严重的呼吸道疾病,危害公共环境和生命安全,90年代至今已经造成四次全球性流感大流行,危害性极大。尽管成功的疫苗研发,已经可以有效预防流感病毒感染,但是由于流感病毒具有高度变异性,导致疫苗效果达不到理想状态。根据流感病毒感染宿主的特性,我们发现宿主因子广泛的参与病毒复制周期中,并且宿主因子具有明显的广谱特性,因此,利用宿主因子作为药物靶点在抗流感病毒感染中具有重要的作用。非受体酪氨酸激酶TNK2蛋白属于非受体酪氨酸激酶家族成员之一,其含有多个结构域,包括一个SAM区、一个酪氨酸激酶催化区、一个Cdc42/Rac结合区以及一个泛素结合区等,并且其能够定位于细胞内多个组分,导致赋予其功能多样性的特性,例如调控细胞生长周期、调节EGFR内吞、泛素化以及细胞内信号转导等;此外,研究发现,TNK2也参与多种肿瘤的发生发展过程中,TNK2可以通过EGFR持续表达促进乳腺癌细胞的迁移,通过调控组蛋白修饰影响***癌等。因此,TNK2在癌症早期筛查、靶向治疗和预后评价中具有重要的作用。
CRISPR/Cas9 技术是基于广泛存在于细菌及古细菌中的一种获得性免疫防疫机制,后期经由人工改造开发而建立,通过导向 RNA序列在靶标基因特定位点进行切割引起DNA双链断裂,进而诱发细胞内的非同源末端修复,造成碱基缺失、***,进而实现基因功能敲除的目的。该技术使用方便,操作简单,成本低,为基因组定向改造、调控与应用等带来突破性革命。目前,CRISPR/Cas9 技术已广泛应用于特定基因敲除功能研究,以及敲除基因的动物模型构建等领域,对深入挖掘基因功能、精准治疗方面具有广阔的前景。
发明内容
本发明的目的在于提供一种抗流感病毒感染的新靶点。
本发明的另一目的在于提供非受体酪氨酸激酶TNK2蛋白的新用途,特别是在抗流感病毒感染中的应用。
本发明的第三目的在于提供抑制或阻碍非受体酪氨酸激酶TNK2蛋白分子表达的gRNA。
本发明的主要技术方案是:
本发明,以人肺上皮细胞(A549)作为靶细胞,采用CRISPR/Cas9基因编辑技术抑制或阻碍宿主蛋白的表达,来挖掘可有效抑制流感病毒感染的宿主因子,从而保护机体免受侵害的功能。本实验发现非受体酪氨酸激酶TNK2蛋白在流感病毒感染人肺上皮细胞(A549)中发挥着重要作用,利用设计的人和猪的TNK2 gRNA抑制或阻碍TNK2蛋白表达,并通过分子生物学和免疫实验,来观察对流感病毒感染的影响。结果发现抑制或阻碍TNK2蛋白表达能显著抑制流感病毒感染。
本发明的第一方面,提供了非受体酪氨酸激酶TNK2作为一种抗流感病毒感染的新靶点。
本发明的第二方面,提供了非受体酪氨酸激酶TNK2在制备预防或治疗流感病毒感染药物中的应用。
进一步地,本发明还提供非受体酪氨酸激酶TNK2在制备预防或治疗流感药物中的应用。
本发明所述的非受体酪氨酸激酶TNK2在制备预防或治疗流感病毒感染药物药物中的应用,该药物具体是指能够抑制或阻碍TNK2表达的试剂。
所述的抑制或阻碍TNK2表达的试剂可以是gRNA 或者是包含gRNA的重组载体等。
本发明的第三方面,本发明提供了抑制或阻碍TNK2表达的双gRNA在制备预防或治疗流感病毒感染药物中的应用,或TNK2在制备预防或治疗流感药物中的应用,所述的药物为TNK2 gRNA,其序列如下:
所述TNK2-gRNA1的靶向序列为: TGGCAGGAATAGGGGACGT
所述TNK2-gRNA2的靶向序列为: CTCATCATTCTGACTACCG。
本发明的有益技术效果:
本发明利用利用全基因遗传筛选技术鉴定到TNK2是流感病毒感染的关键宿主蛋白,并进一步通过构建靶向TNK2基因的特异性双gRNA,利用 CRISPR/Cas9基因编辑技术获得不表达TNK2蛋白的单克隆细胞系,然后利用免疫荧光和病毒滴度检测技术观察了抑制或阻碍TNK2分子表达,能够显著抑制流感病毒蛋白的表达,并且能显著抑制不同种属流感病毒(包括猪和禽流感病毒)的复制,表面抑制TNK2表达可能展现出广谱的抗病毒效果。本发明为抗流感病毒的治疗提供了新的候选靶点,扩展了传统抗病毒治疗的手段,为临床预防和治疗因流感病毒感染所导致的生理疾病提供了新的治疗方案。因此具有非常好的应用价值。
附图说明
图1 为本发明实施例1提供的pU6gRNA_PB_BbsI载体骨架图谱;
图2 为本发明实施例1提供的重组表达载体靶向切割检测(PCR检测);
图3 为本发明实施例1提供的重组表达载体靶向切割检测(Sanger测序检测);
图4 为本发明实施例2提供的单克隆细胞TNK2表达检测(Western Blooting检测结果);
图5 利用免疫荧光实验证实抑制或阻碍TNK2表达后人流感病毒蛋白表达下降;
图6 利用空斑实验证实抑制或阻碍TNK2表达后能降低人流感病毒(H1N1)的滴度;
图7 利用免疫荧光实验证实抑制或阻碍TNK2表达后禽流感病毒(H9N2)复制减少;
图8 利用TCID50实验证实抑制或阻碍TNK2表达后能降低猪流感病毒(H1N1)的滴度。
具体实施方式
以下将结合附图对本发明各实施例的技术方案进行清晰、完整的描述,显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例;基于本发明的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施例,都属于本发明所保护的范围。
实施例1 人TNK2基因被敲除所需的双gRNA质粒载体构建及检测
(1) 人TNK2基因双gRNA质粒载体构建
从Ensembl数据库获得人TNK2(ENSG00000061938)的基因序列,然后针对敲除TNK2第一个外显子来设计特异性的上下游敲除位点,综合软件***评分获得两条最佳的靶序列TNK2-sgRNA1(靶向序列为: TGGCAGGAATAGGGGACGT)和 TNK2-sgRNA2(靶向序列为:CTCATCATTCTGACTACCG)。根据BbsI限制性内切酶在sgRNA的两端设计酶切位点,在gRNA的5'端加上CACCG ,3' 端 加上GT;反向互补序列的5'端加上TAAAAC, 设计 2对sgRNA寡核苷酸链,对应的序列如下:TNK2-sgRNA1 Oligo1: 5'- CACCGTGGCAGGAATAGGGGACGTGT-3';TNK2-sgRNA1 Oligo2: 5'-TAAAAC ACGTCCCCTATTCCTGCCAC-3'; TNK2-sgRNA2 Oligo1:5'-CACC GCTCATCATTCTGACTACCG GT-3'; TNK2-sgRNA2 Oligo2: 5'-TAAAACCGGTAGTCAGAATGATGAGC-3'; 然后将Oligo1和Oligo2混合到10 mM Tris-HCl(pH8.0) 和 5 mM MgCl2的缓冲液中,在95°C 孵育5分钟,然后冰上冷却,形成双链;双链gRNAs再分别克隆至酶切后的gRNA克隆载体pKLV-flipedU6gRNA_CCDB_PB_BbsI_PGKpuro2ABFP中,并转化大肠杆菌TOP10, 挑单克隆菌株,提取质粒。
(2) 人TNK2基因双gRNA在靶细胞中的效果鉴定
本发明以人非小细胞肺癌细胞系A549作为靶细胞。将提前构建后的稳转Cas9的A549细胞(Cas9-A549)复苏,在转染前将Cas9-A549细胞接种到12孔板,然后将构建好的重组质粒TNK2-gRNA1和TNK2-gRNA2,各1μg与Lipofectamine 3000,4μl(按照比例1μg:2μL)加至50μl的Opti-MEM中,静止5min后将两者混合。将四种脂质体-质粒DNA混合物静置15min后直接加至细胞培养基中。将细胞重新置于37℃、5%CO2培养箱中培养48h。然后用含有嘌呤霉素的培养基继续培养细胞7-14天,获得阳性单克隆细胞。提取基因组DNA,利用PCR技术和Sanger测序鉴定是否敲除TNK2基因。
实例2 转染TNK2双gRNA后,获得的单克隆A549细胞中TNK2蛋白表达检测
将实例1中筛选获得的单克隆细胞系,接种至6孔板扩大培养, 将转染前Cas9-A549细胞设为对照,待细胞长满后,收取细胞放置冰上,加入适量SDS-PAGE上样缓冲液充分搅拌裂解,金属浴放置10min变性,离心后,取上清进行SDS-PAGE。电泳后通过湿转法转移至PVDF膜,转印后使用5%的脱脂奶粉封闭,然后孵育兔抗人TNK2抗体(1:1000,Abcam),羊抗兔IgG (IgG-HRP)为二抗,进行抗原抗体复合物检测,对TNK2基因敲除的A549单克隆细胞系在蛋白表达水平进行验证。实验结果如图4所示,在野生型的细胞中检测到清晰的TNK2蛋白条带,而转染TNK2的双gRNA的单克隆细胞系中没有检测到TNK2蛋白条带。
实例3 TNK2敲除后对流感病毒感染的影响
利用获得稳定敲除TNK2的单克隆细胞系,被分别感染人、猪和禽流感病毒,感染相应时间就,利用免疫荧光、病毒滴度检测技术检测抑制或阻碍TNK2表达对流感病毒感染的影响。
免疫荧光的具体方法如下:敲除TNK2的单克隆细胞系和野生型细胞感染流感病毒相应时间后,吸除上清液,以PBS快洗两次;用4%的多聚甲醛固定细胞10分钟后,弃去固定液, PBS洗3次;加入0 .2%Triton-X 100室温放置5分钟,增加细胞膜的通透性;弃去Triton-X 100,1×PBS洗3次;加汉10%BSA的PBS,放置于水平摇床上封闭30分钟;孵一抗:按抗体说明书推荐的浓度,用含10%BSA 的PBS稀释,4度孵育过夜;然后PBS洗3次,每次10分钟;孵二抗:按推荐浓度,用含10%BSA PBS稀释,孵二抗后所有过程开始需要避光,室温孵育一个小时;PBS洗3次,每次10分钟,避光;DAPI染核,10分钟,避光;PBS洗3次,每次10分钟,避光;用抗荧光衰减封片剂将盖玻片封于载玻片上;荧光显微镜下观察拍照。
病毒滴度的具体检测方法:
1. 空斑实验(plaque assay): MDCK细胞平铺到12孔板中,24小时后,病毒原液以汉克缓冲液 (Hank's solution) 做十倍连续稀释,由10-1稀释至10-7;把单层细胞的上清液吸掉后,加上病毒稀释液400ul,并放置在37 ℃温箱一小时,其间每十五分钟摇动一次,使病毒可充分与细胞接触;二倍的无血清DMEM液与2.5%的Avecil等量混合,每一孔内加1ml。放37 ℃温箱培养,并含二氧化碳5 %。48~96小时,去除上清液后,用PBS缓冲液洗2次,以1 % 结晶紫染液染色10分钟,再以自来水冲洗染液,直至无染料洗出为止;自然干燥后,便显出无色的不规则空洞。
2. 半数组织培养感染剂量(TCID50): 单层细胞制备, MDCK细胞于96孔中37℃培养24h,待生成单层后备用。病毒液稀释,病毒液作10倍稀释,先将稀释液分装于10个试管中,每管0.9ml,取0.1ml病毒悬液加入第一管中,摇匀后为10-1,换一支吸管,在10-1病毒液中吸放3~4次,取0.1ml放入第二管中摇匀后为10-2........,如此稀释至10-10。病毒接种,取细胞培养板,用多道加样器吸去96孔板中的培养液,用PBS缓冲液洗2次, 然后将稀释好的病毒液加到96孔板上,每孔100ul,37℃培养1小时后,取出培养板中的病毒液,加入含有0.2%bsa的培养液继续在37℃ CO2培养箱中培养。每天观察细胞病变情况,7天后统计观察结果,利用Reed-Muench方法,找出能引起半数细胞感染的病毒稀释倍数,并按照公式:[(高于50%病变率的百分数-50%)/(高于50%病变率的百分数-低于50%病变率的百分数)]x稀释度对数之间的差+高于50%病变率的稀释度的对数;计算出病毒液的TCID50.实验结果发现与对照组相比,TNK2敲除的细胞系有较强的抵抗病毒特性,提示敲除TNK2基因能够降低流感病毒感染。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
序列表
<110> 武汉轻工大学
<120> 非受体酪氨酸激酶TNK2蛋白在防治流感病毒感染中的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tggcaggaat aggggacgt 19
<210> 2
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctcatcattc tgactaccg 19
<210> 3
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
caccgtggca ggaatagggg acgtgt 26
<210> 4
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
taaaacacgt cccctattcc tgccac 26
<210> 5
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
caccgctcat cattctgact accggt 26
<210> 6
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
taaaaccggt agtcagaatg atgagc 26
Claims (6)
1.一种建立抗流感病毒感染的细胞系的方法,其特征在于,利用CRISPR/Cas9***敲除细胞中的TNK2基因,所述CRISPR/Cas9***使用靶向TNK2 基因的双gRNA序列,靶向TNK2 基因的双gRNA序列,包括TNK2-gRNA1和TNK2-gRNA2,
所述TNK2-gRNA1的靶向序列为: TGGCAGGAATAGGGGACGT,
所述TNK2-gRNA2的靶向序列为: CTCATCATTCTGACTACCG。
2.根据权利要求1所述的方法,其特征在于,
所述TNK2-gRNA1的核苷酸序列包括:
TNK2-gRNA1 Oligo1: 5'-CACCGTGGCAGGAATAGGGGACGTGT-3'
TNK2-gRNA1 Oligo2: 5'-TAAAACACGTCCCCTATTCCTGCCAC-3'
所述 TNK2-gRNA2的核苷酸序列包括:
TNK2-gRNA2 Oligo1: 5'-CACCGCTCATCATTCTGACTACCGGT-3'
TNK2-gRNA2 Oligo2: 5'-TAAAACCGGTAGTCAGAATGATGAGC-3'。
3.根据权利要求1所述的方法,其特征在于,
设计 2对sgRNA寡核苷酸链,然后将Oligo1和Oligo2混合到10 mM Tris-HCl (pH8.0)和 5 mM MgCl2的缓冲液中,在95°C 孵育5分钟,然后冰上冷却,形成双链;双链gRNAs再分别克隆至酶切后的gRNA克隆载体pKLV-flipedU6gRNA_CCDB_PB_BbsI_PGKpuro2ABFP中,并转化大肠杆菌TOP10, 挑单克隆菌株,提取质粒;
将提前构建后的稳转Cas9的A549细胞Cas9-A549复苏,在转染前将Cas9-A549细胞接种到12孔板,然后将构建好的重组质粒TNK2-gRNA1和TNK2-gRNA2,各1μg与Lipofectamine3000,4μl加至50μl的Opti-MEM中,静止5min后将两者混合,将四种脂质体-质粒DNA混合物静置15min后直接加至细胞培养基中,将细胞重新置于37℃、5%CO2培养箱中培养48h,然后用含有嘌呤霉素的培养基继续培养细胞7-14天,获得阳性单克隆细胞;
提取基因组DNA,利用PCR技术和Sanger测序鉴定是否敲除TNK2基因。
4.一种制备防治流感病毒感染的药物的方法,其特征在于,该药物能够抑制或阻碍非受体酪氨酸激酶TNK2蛋白表达的试剂,所述的药物是指能够抑制或阻碍非受体酪氨酸激酶TNK2蛋白表达的试剂是非受体酪氨酸激酶TNK2蛋白的gRNA或包含gRNA的重组载体。
5.根据权利要求4所述的一种制备防治流感病毒感染的药物的方法,其特征在于,所述的药物是抑制或阻碍非受体酪氨酸激酶TNK2的TNK2-gRNA1和TNK2-gRNA2:
所述TNK2-gRNA1的靶向序列为: TGGCAGGAATAGGGGACGT 、
所述TNK2-gRNA2的靶向序列为: CTCATCATTCTGACTACCG。
6.非受体酪氨酸激酶TNK2蛋白在制备预防或治疗流感病毒感染药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110686648.9A CN113502298A (zh) | 2021-06-21 | 2021-06-21 | 非受体酪氨酸激酶tnk2蛋白在防治流感病毒感染中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110686648.9A CN113502298A (zh) | 2021-06-21 | 2021-06-21 | 非受体酪氨酸激酶tnk2蛋白在防治流感病毒感染中的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113502298A true CN113502298A (zh) | 2021-10-15 |
Family
ID=78010552
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110686648.9A Pending CN113502298A (zh) | 2021-06-21 | 2021-06-21 | 非受体酪氨酸激酶tnk2蛋白在防治流感病毒感染中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113502298A (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20130090367A1 (en) * | 2009-12-11 | 2013-04-11 | Megan Shaw | Compositions and Methods for Inhibiting Human Host Factors Required for Influenza Virus Replication |
CN111849979A (zh) * | 2020-06-22 | 2020-10-30 | 中国农业科学院兰州兽医研究所 | 一种靶向敲除RPSA基因的sgRNA及RPSA基因敲除细胞系的构建方法 |
-
2021
- 2021-06-21 CN CN202110686648.9A patent/CN113502298A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20130090367A1 (en) * | 2009-12-11 | 2013-04-11 | Megan Shaw | Compositions and Methods for Inhibiting Human Host Factors Required for Influenza Virus Replication |
CN111849979A (zh) * | 2020-06-22 | 2020-10-30 | 中国农业科学院兰州兽医研究所 | 一种靶向敲除RPSA基因的sgRNA及RPSA基因敲除细胞系的构建方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Fehr et al. | The conserved coronavirus macrodomain promotes virulence and suppresses the innate immune response during severe acute respiratory syndrome coronavirus infection | |
CN106434752A (zh) | 敲除Wnt3a基因的过程及其验证方法 | |
CN111849979B (zh) | 一种靶向敲除RPSA基因的sgRNA及RPSA基因敲除细胞系的构建方法 | |
CN110607280A (zh) | Emc3基因的应用及其定点敲除方法 | |
CN114058619A (zh) | Riplet敲除细胞系的构建及作为小核糖核酸病毒科病毒疫苗生产细胞系的应用 | |
CN109929014A (zh) | 一种抑制鸡先天性免疫反应鸡马立克氏病病毒蛋白及其应用 | |
CN106244682B (zh) | Trem-2基因在鉴别猪对蓝耳病毒易感性中的应用 | |
CN109207577B (zh) | Marco在筛选抗蓝耳病猪中的应用 | |
CN113502298A (zh) | 非受体酪氨酸激酶tnk2蛋白在防治流感病毒感染中的应用 | |
CN116948975A (zh) | Ifnar1基因敲除的mdck细胞株及其构建方法和应用 | |
CN111705162A (zh) | 一种检测新型冠状病毒2019-nCOV的RNA探针及其制备方法与应用 | |
CN103215267B (zh) | 抑制流感病毒相关基因的siRNA及其应用 | |
CN113444726B (zh) | 一种与仔猪细菌性腹泻相关的lncRNA ALDB-898及其应用 | |
CN113637709B (zh) | Kif5b基因作为靶点在抑制***病毒复制中的应用 | |
CN107602674B (zh) | 一种筛选i型鸭肝炎病毒3c蛋白抑制剂的试剂盒 | |
CN111454908A (zh) | 一种Tpl2缺陷型MDCK细胞株及其构建方法和应用 | |
CN107475452B (zh) | 一种检测鸭瘟病毒icp4基因的引物以及rt-pcr和荧光定量pcr检测方法 | |
CN107475453B (zh) | 一种检测鸭瘟病毒ul48基因的引物以及rt-pcr和荧光定量pcr检测方法 | |
CN111228292B (zh) | 人tpt1/tctp基因在制备抗肿瘤药物中的应用 | |
CN109371140B (zh) | 一种基于ext1的表达量筛选抗蓝耳病猪的方法 | |
CN113564165B (zh) | 一种胞内编辑伪狂犬病毒关键基因的细胞株及其构建方法和应用 | |
CN117487009B (zh) | 抗鸡pml单克隆抗体及其应用 | |
CN111690688B (zh) | 表达靶向REV的CRISPR/Cas9的重组马立克氏病病毒及其应用 | |
CN110804662B (zh) | 一种基于sirt2的表达量筛选抗蓝耳病猪的方法 | |
CN103589753A (zh) | 特异性抑制人SIRT1 基因表达的shRNA慢病毒表达载体及其构建方法与应用 |
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 | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20211015 |
|
WD01 | Invention patent application deemed withdrawn after publication |