CN118105462A - 一种非洲猪瘟病毒蛋白qp383r的应用 - Google Patents
一种非洲猪瘟病毒蛋白qp383r的应用 Download PDFInfo
- Publication number
- CN118105462A CN118105462A CN202410006267.5A CN202410006267A CN118105462A CN 118105462 A CN118105462 A CN 118105462A CN 202410006267 A CN202410006267 A CN 202410006267A CN 118105462 A CN118105462 A CN 118105462A
- Authority
- CN
- China
- Prior art keywords
- qp383r
- seq
- swine fever
- fever virus
- african swine
- 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
- 101100187083 African swine fever virus (strain Badajoz 1971 Vero-adapted) Ba71V-124 gene Proteins 0.000 title claims abstract description 48
- 241000701386 African swine fever virus Species 0.000 title claims abstract description 23
- 230000002757 inflammatory effect Effects 0.000 claims abstract description 22
- 102000004127 Cytokines Human genes 0.000 claims abstract description 19
- 108090000695 Cytokines Proteins 0.000 claims abstract description 19
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- 230000002452 interceptive effect Effects 0.000 claims abstract description 12
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 11
- 239000003814 drug Substances 0.000 claims abstract description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 230000005764 inhibitory process Effects 0.000 claims abstract description 5
- 102000002689 Toll-like receptor Human genes 0.000 claims description 22
- 108020000411 Toll-like receptor Proteins 0.000 claims description 22
- 239000013598 vector Substances 0.000 claims description 10
- 230000034190 positive regulation of NF-kappaB transcription factor activity Effects 0.000 claims description 9
- 229940079593 drug Drugs 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 8
- 230000001404 mediated effect Effects 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
- 230000019491 signal transduction Effects 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 108090000978 Interleukin-4 Proteins 0.000 claims description 4
- 108090001007 Interleukin-8 Proteins 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000010276 construction Methods 0.000 claims description 4
- 102000012410 DNA Ligases Human genes 0.000 claims description 3
- 108010061982 DNA Ligases Proteins 0.000 claims description 3
- 102000010168 Myeloid Differentiation Factor 88 Human genes 0.000 claims description 3
- 108010077432 Myeloid Differentiation Factor 88 Proteins 0.000 claims description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 3
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 238000001976 enzyme digestion Methods 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 238000011084 recovery Methods 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 10
- 206010061218 Inflammation Diseases 0.000 abstract description 6
- 230000004054 inflammatory process Effects 0.000 abstract description 6
- 238000006243 chemical reaction Methods 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 231100000673 dose–response relationship Toxicity 0.000 abstract description 3
- 230000009471 action Effects 0.000 abstract description 2
- 230000008439 repair process Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 41
- 238000001890 transfection Methods 0.000 description 8
- 108060001084 Luciferase Proteins 0.000 description 7
- 239000005089 Luciferase Substances 0.000 description 7
- 230000000638 stimulation Effects 0.000 description 5
- 239000013613 expression plasmid Substances 0.000 description 4
- 102000007863 pattern recognition receptors Human genes 0.000 description 4
- 108010089193 pattern recognition receptors Proteins 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 208000035473 Communicable disease Diseases 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 102000004388 Interleukin-4 Human genes 0.000 description 3
- 102000004890 Interleukin-8 Human genes 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 2
- 208000007407 African swine fever Diseases 0.000 description 2
- 239000012103 Alexa Fluor 488 Substances 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 108010057466 NF-kappa B Proteins 0.000 description 2
- 102000003945 NF-kappa B Human genes 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000000556 agonist Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000015788 innate immune response Effects 0.000 description 2
- 210000005007 innate immune system Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 229940115272 polyinosinic:polycytidylic acid Drugs 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- OEDPHAKKZGDBEV-GFPBKZJXSA-N (2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-2-[[(2r)-3-[2,3-di(hexadecanoyloxy)propylsulfanyl]-2-(hexadecanoylamino)propanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]hexanoyl]amino]hexanoyl]amino]hexanoic acid Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)CCCCCCCCCCCCCCC)CSCC(COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC OEDPHAKKZGDBEV-GFPBKZJXSA-N 0.000 description 1
- 102100032814 ATP-dependent zinc metalloprotease YME1L1 Human genes 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- -1 IL-1β Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 101800000795 Proadrenomedullin N-20 terminal peptide Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010864 dual luciferase reporter gene assay Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 208000021760 high fever Diseases 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008741 proinflammatory signaling process Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000005253 yeast cell Anatomy 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
- A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- 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
- 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
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/12011—Asfarviridae
- C12N2710/12022—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
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Rheumatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Cell Biology (AREA)
- Pain & Pain Management (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种非洲猪瘟病毒蛋白的应用。所述应用为制备干扰TLR信号通路和/或抑制炎性细胞因子药物，所述非洲猪瘟病毒蛋白QP383R的氨基酸序列如SEQ ID NO.2所示。QP383R作为一种小分子蛋白，不会产生过度的免疫排斥反应；对TLR信号通路的干扰作用存在剂量依赖关系，通过降低作用浓度使其产生适度的抑制，但却不损害宿主的防御能力，以避免过度抑炎反应造成的副作用。可通过多条途径同时抑制多种因子，利于损伤组织的修复。
Description
技术领域
本发明涉及一种非洲猪瘟病毒蛋白QP383R的应用，具体涉及一种非洲猪瘟病毒蛋白QP383R在制备干扰TLR信号通路和/或抑制炎性细胞因子药物中的应用，属于生物技术领域。
背景技术
非洲猪瘟(African swine fever)是非洲猪瘟病毒(African swine fevervirus，ASFV)感染猪而引起的一种传染病，以病程短、高热和出血性病变为特征。ASFV含有数量众多具有免疫逃逸功能的编码蛋白，这些蛋白逃逸宿主免疫功能的机制多样且复杂。ASFV传播能力较强，急性感染死亡率高达100％，只能通过在疫区实施严格的卫生措施，依靠预防或宰杀患病动物以进行控制，是世界动物卫生组织(OIE)规定的必须报告的疫病，在我国也被划分为一类动物传染病，严重威胁养猪业的发展。
细胞通过模式识别受体(PRRs)识别病原体相关分子模式(Pathogen-associatedmolecular patterns，PAMPs)，激活宿主天然免疫，分泌细胞因子/趋化因子，通过吞噬作用呈现抗原和清除病原体。Toll样受体(TLRs)属于模式识别受体(PRRs)超家族，在哺乳动物中，有13个确定的TLRs在天然免疫系统的调节中发挥着重要作用。这些受体是高度保守的蛋白质，它们构成了必不可少的第一道防线。在收到外界刺激后，TLR激活核转录因子κB(Nuclear factorκB,NF-κB)信号通路，诱导炎性基因的表达，产生抗菌和抗病毒作用。
NF-κB信号通路被认为是一种典型的促炎信号通路，与肿瘤、炎症及自身免疫性疾病、感染性休克、病毒感染、免疫组织增生等有关。这些天然免疫反应的变化与炎症的发病机制有关。TLR启动细胞内信号转导，激活NF-κB，触发肠道炎症介质的释放，导致效应细胞分泌细胞因子，如IL-1β、IL-6和TNF-α。最终，机体免疫稳态被破坏，导致炎症发展。许多感染性疾病、自身免疫病和恶性肿瘤都会引发机体产生过度炎症反应，因而靶向天然免疫应答和信号传递的药物具有潜在的医疗前景。
ASFV通过自身蛋白采用多种免疫逃逸机制以确保病毒的繁殖与扩散，其逃逸TLRs-NF-κB信号通路的机制为未来抗炎性药物的研究提供了理论基础。病毒通过进化出相应的策略来抑制机体的炎性应答，这些病毒效应蛋白分子本身或经修饰后或许会具有治疗药物的潜能。
发明内容
发明目的：本发明的第一目的是提供了一种非洲猪瘟病毒蛋白QP383R在制备干扰TLR信号通路和/或抑制炎性细胞因子药物中的应用。本发明的第二目的是提供了一种含有QP383R基因的重组质粒、表达盒或重组细胞在制备干扰TLR信号通路和/或抑制炎性细胞因子药物中的应用。本发明的第三目的是提供了一种干扰TLR信号通路和/或抑制炎性细胞因子药物组合物。
技术方案：本发明所述的一种非洲猪瘟病毒蛋白QP383R在制备干扰TLR信号通路和/或抑制炎性细胞因子药物中的应用，所述非洲猪瘟病毒蛋白QP383R的氨基酸序列如SEQID NO.2所示。
其中，编码所述非洲猪瘟病毒蛋白QP383R的基因的核苷酸序列如SEQ ID NO.1所示。
其中，所述干扰TLR信号通路为抑制MyD88介导的NF-κB的活化。
其中，所述炎性细胞因子包括IL-4、IL-8、IFN-γ或TNF-α中的一种或几种。
本发明所述的一种含有QP383R基因的重组质粒、表达盒或重组细胞在制备干扰TLR信号通路和/或抑制炎性细胞因子药物中的应用，所述QP383R基因的核苷酸序列如SEQID NO.1所示。
本发明所述的一种构建上述重组质粒的方法，包括以下步骤：
(1)以QP383R基因为模板，进行PCR扩增，琼脂糖凝胶电泳回收，得到QP383R产物，所述QP383R基因的核苷酸序列如SEQ ID NO.1所示；
(2)将步骤(1)所述QP383R产物和pCMV载体进行双酶切，使用C115 DNA连接酶连接，转化至感受态细胞中，筛选后得到重组质粒pCMV-Myc-QP383R。
构建重组细胞过程中的宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌、链霉菌属、鼠伤寒沙门菌的细菌细胞；真菌细胞如酵母；植物细胞；果蝇S2或Sf9的昆虫细胞；CHO、COS、293细胞或Bowes黑素瘤细胞的动物细胞等。
其中，宿主细胞为HEK293T和HeLa细胞。
其中，所述PCR扩增的引物对的序列如SEQ ID NO.3和SEQ ID NO.4所示。
其中，所述感受态细胞为大肠杆菌DH5α。
本发明所述的一种干扰TLR信号通路和/或抑制炎性细胞因子药物组合物，所述药物组合物中的活性成分为QP383R蛋白，所述QP383R蛋白的氨基酸序列如SEQ ID NO.2所示。
其中，所述药物还包括药学上可接受的载体或赋形剂。
有益效果：与现有技术相比，本发明具有如下突出的显著优点：QP383R作为一种小分子蛋白，不会产生过度的免疫排斥反应；对TLR信号通路的干扰作用存在剂量依赖关系，通过降低作用浓度使其产生适度的抑制，但却不损害宿主的防御能力，以避免过度抑炎反应造成的副作用。可通过多条途径同时抑制多种因子，利于损伤组织的修复。
附图说明
图1：为Western blotting鉴定真核表达质粒pQP383R在细胞中的表达。其中，Mock表示HEK293T(pCMV-Myc)，QP383R表示HEK293T(pCMV-Myc-QP383R)。一抗为Myc标签单抗，1:10000稀释。
图2：为间接免疫荧光鉴定真核表达质粒pQP383R在细胞中的表达。其中，Mock表示HEK293T(pCMV-Myc)，QP383R表示HEK293T(pCMV-Myc-QP383R)。一抗为Myc标签单抗，1:10000稀释，二抗为山羊抗小鼠IgG H&L(Alexa Fluor488)二抗。
图3：为QP383R抑制MyD88介导的NF-κB的活化。
图4：为QP383R在不同浓度下抑制MyD88介导的NF-κB的活化。
图5：为在HeLa细胞中QP383R可抑制不同TLRLs诱导的NF-κB的活化。
图6：为QP383R抑制炎性细胞因子的产生。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例1Western blotting技术鉴定HEK293T细胞表达并分泌QP383R
(1)构建真核表达质粒pCMV-Myc-QP383R
首先，设计带有EcoR I和Sal I双酶切位点的引物(由北京擎科生物科技股份有限公司合成)：
正向引物：5’-tggccatggaggcccgaattcggATGGGATGTGACGGTAACGAGT-3’(SEQ IDNO.3)
反向引物：5’-ccgcggccgcggtacctcgagTTAAGAAAAAGAAGAAGAGTGGCTCG-3’(SEQ IDNO.4)
然后，通过查找获得ASFV Wuhan 2019-1(genbank登录号MN393476.1)株的基因序列，从中查找到QP383R，并通过合成获得QP383R的序列作为模板，以SEQ ID NO：3和SEQ IDNO：4的序列为扩增引物PCR扩增QP383R基因(SEQ ID NO.2：ATGGGATGTGACGGTAACGAGTACCATGTTCTTTTTACCAGCAGCTCCGAGGAAGCAAATACTCATATGATCATGGCCGCCGTGCGTCGCCATTTGCTGCGGACGCAGCAAAGGCCTCATGTCATTATCGGAGCAGCCGAGCCCCCTAGCGTCACCGAATGTGTGAAGGCATTGGCGCAGGAAAAACGCTGCGTATACACCATCATCCCCCTAAAAAATTTTGAAATAGATCCTGTTGCGGTATACGATGCCATACAAAGCAATACCTGCTTAGCGTGCATTTCAGGCACTAATGCTGTTGTCAAAACGTTCAACAAACTCCAGGACATCAGCAACGTGTTAAAAGGTATTCCCCTGCACTCAGAAGTGAGTGATCTTGTTTATCAAGGATGTATTCAACAAAATCCGCCCGCTGATAGTTTTTCAATAAATAGTCTCTACGGCTTCCTGGGAGTCGGTGTTTTGGGAATGAAGAAAAAGGTCATGCAAGGATTGGGGCCGCTCATTTTTGGAGGAGGGCTGAGAGGCGGAAGCCCTAATATACCCGGAATTCATGCCATGTATAAAACGCTAACCCAGCAAAGGCCTTCTATGAAAAAAATAAATACAATACATACGCTGTTCATGAAAACTTTAAAAAAACATCAGCATGTATATCTACCCATAGGGGGCGTGTCTGCAGAGGACACGTCTGCAGAAAACATATCTACAAAAGACATGCCTGTTGAAGGCCCGAAGGGACTCCCGGGCTATATTTTATTTAGCGTTGGCCGTCGCGCCGAGGAGCTACAAAAAAAAATTTTCACTAAATTTAATATAAAGGTTGGCCGTGTTGTTGACTTACAAGAGATACTGTTTCGTATCAAAATACCCCAAAAATACTGGGAGACATTATTGTTCATCCAATTAAGAGATAATTTGACCAAAGAGGACATAAAAAGAGTTATGGTTGTTTTGATGCATTTAGATACCATCACTCCTCGTGGCTCTCTTCCTCCTCCGAGCCACTCTTCTTCTTTTTCTTAA)后进行琼脂糖凝胶电泳回收，将QP383R产物和pCMV-Myc载体(QYV0529，Qualityard公司)分别进行双酶切并回收，分别加入1μg载体+1μl EcoR I+1μl Sal I，并用灭菌水定容至20μl的体系，双酶切37℃ 3h并回收，使用C115 DNA连接酶(C115-02，诺唯赞)连接，转化至大肠杆菌DH5α，并涂布于有抗性的LB固体培养基进行筛选。挑取阳性克隆进行鉴定，测序(由北京擎科生物科技股份有限公司测序)正确后得到真核表达质粒pCMV-Myc-QP383R。其中，QP383R产物的蛋白的氨基酸序列如SEQ ID NO.1所示(MGCDGNEYHVLFTSSSEEANTHMIMAAVRRHLLRTQQRPHVIIGAAEPPSVTECVK ALAQEKRCVYTIIPLKNFEIDPVAVYDAIQSNTCLACISGTNAVVKTFNKLQDISNVLKGIPLHSEVSDLVYQGCIQQNPPADSFSINSLYGFLGVGVLGMKKKVMQGLGPLIFGGGLRGGSPNIPGIHAMYKTLTQQRPSMKKINTIHTLFMKTLKKHQHVYLPIGGVSAEDTSAENISTKDMPVEGPKGLPGYILFSVGRRAEELQKKIFTKFNIKVGRVVDLQEILFRIKIPQKYWETLLFIQLRDNLTKEDIKRVMVVLMHLDTITPRGSLPPPSHSSSFS)。
(2)Western blotting技术鉴定HEK293T细胞表达并分泌Myc-QP383R
在HeLa细胞铺板2×105/孔18h后，转染pCMV-Myc-QP383R 500ng/ml，转染后24h后，弃去上清，收集细胞，并用WB裂解液裂解5min。使用Myc单抗(AM926，碧云天)鉴定Myc-QP383R的表达情况，Western blotting结果显示转染后可检测到Myc-QP383R，表明HEK293T是可表达Myc-QP383R蛋白的(结果如图1所示)，表明pCMV-Myc-QP383R具有编码蛋白功能的基因。
(3)间接免疫荧光技术鉴定HEK293T细胞表达Myc-QP383R
在HeLa细胞铺板2×105/孔18h后，转染pCMV-Myc-QP383R 500ng/ml，转染24h后，弃去上清，收集细胞，并用甲醛固定10min。使用Myc单抗(AM926，碧云天)鉴定Myc-QP383R的表达情况，间接免疫荧光结果显示转染后可检测到Myc-QP383R，表明HEK 293T是可表达并分泌Myc-QP383R蛋白的(结果如图2所示)，表明pCMV-Myc-QP383R具有编码蛋白功能的基因。一抗为Myc标签单抗(AM926，碧云天)，1:10000稀释，二抗为山羊抗小鼠IgG H&L(AlexaFluor488)(AB150113，Abcam)。
实施例2QP383R可干扰TLR信号通路并抑制炎性细胞因子分泌
(1)QP383R可抑制MyD88介导的NF-κB的活化
pGL 4.32-NF-κB luciferase reporter、pcDNA3.1-Flag-MyD88、p-EMPTY为实验室构建保存质粒，已有相关文章发表(Xiong D,Song L,Geng S,Jiao Y,Zhou X,Song H,Kang X,Zhou Y,Xu X,Sun J,Pan Z,Jiao X.Salmonella Coiled-Coil-and TIR-Containing TcpS Evades the Innate Immune System and Subdues Inflammation.CellRep,2019,28(3):804-818.)。
HEK293T细胞为实验室保存。
共转染NF-κB luciferase reporter(200ng/mL)、pcDNA3.1-Flag-MyD88(30ng/mL，实验室保藏)和pQP383R(实施例1构建的pCMV-Myc-QP383R，浓度分别为100ng/mL、200ng/mL和500ng/mL)到HEK293T细胞中作为实验组(pQP383R)；转染NF-κBluciferasereporter(200ng/mL)、pcDNA3.1-Flag-MyD88(30ng/mL，实验室保藏)和pCMV-Myc载体(100ng/mL)到HEK293T细胞中作为对照组(p-EMPTY，MyD88)；转染NF-κB luciferasereporter(200ng/mL)、pCMV-Myc载体(100ng/mL)到HEK293T细胞中作为空白组(p-EMPTY)；24h后检测荧光素酶活性(Dual Luciferase Reporter Assay Kit，诺唯赞)。
结果表明QP383R在HEK293T细胞中可以显著抑制MyD88(79％)介导的NF-κB的活化(结果如图3所示)，且这种抑制作用存在剂量依赖关系(结果如图4所示)。
(2)在HeLa细胞中QP383R可抑制不同TLRLs诱导的NF-κB的活化
共转染NF-κB luciferase reporter和pQP383R(实施例1构建的pCMV-Myc-QP383R)到HeLa细胞中作为实验组pQP383R；转染NF-κB luciferase reporter和pCMV-Myc载体到HeLa细胞中作为对照组(p-EMPTY，Pam3CSK4、Poly(I:C)、LPS)；转染pCMV-Myc载体到HeLa细胞中作为空白组(p-EMPTY)；转染24h后分别用1μg/mL Pam3CSK4(TLR2激动剂)(tlrl-pms，Invivogen)，2.5μg/mL Poly(I:C)(TLR3激动剂)(tlrl-picwlv，Invivogen)，100ng/mL LPS(TLR4激活剂)(L4391，SIGMA)刺激。刺激5h后检测荧光素酶活性。结果表明相对空白组，转染pQP383R组在不同配体的刺激下均显著抑制了NF-κB的活化(抑制率分别为52％、54％、58％)(如图5所示)。
(3)QP383R可抑制炎性细胞因子的表达
为直接检测QP383R对炎性细胞因子的影响，转染pQP383R(实施例1构建的pCMV-Myc-QP383R)到HeLa细胞中作为实验组(pQP383R)；转染pCMV-Myc载体到HeLa细胞中作为对照组(p-EMPTY)；转染pCMV-Myc载体到HeLa细胞中作为空白组(p-EMPTY，不用LPS刺激)；转染24h后100ng/mL LPS进行刺激(实验组和对照组)，刺激5h后裂解细胞提取RNA，去DNA后反转录成cDNA，荧光定量PCR(qRT-PCR)检测炎性因子IL-4、IL-8、IFN-γ、TNF-α的转录水平。
结果(图6)表明同NF-κB活性检测结果一致，QP383R可显著抑制炎性细胞因子IL-4(98％)、IL-8(59％)、IFN-γ(70％)和TNF-α(39％)的产生(内参为GAPDH)。
Claims (10)
1.一种非洲猪瘟病毒蛋白QP383R在制备干扰TLR信号通路和/或抑制炎性细胞因子药物中的应用，其特征在于，所述非洲猪瘟病毒蛋白QP383R的氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的非洲猪瘟病毒蛋白QP383R在制备抑制炎性细胞因子药物中的应用，其特征在于，编码所述非洲猪瘟病毒蛋白QP383R的基因的核苷酸序列如SEQ ID NO.1所示。
3.根据权利要求1所述的非洲猪瘟病毒蛋白QP383R在制备抑制炎性细胞因子药物中的应用，其特征在于，所述干扰TLR信号通路为抑制MyD88介导的NF-κB的活化。
4.根据权利要求1所述的非洲猪瘟病毒蛋白QP383R在制备抑制炎性细胞因子药物中的应用，其特征在于，所述炎性细胞因子包括IL-4、IL-8、IFN-γ或TNF-α中的一种或几种。
5.一种含有QP383R基因的重组质粒、表达盒或重组细胞在制备干扰TLR信号通路和/或抑制炎性细胞因子药物中的应用，所述QP383R基因的核苷酸序列如SEQ ID NO.1所示。
6.根据权利要求5所述的应用，其特征在于，所述重组质粒的构建包括以下步骤：
(1)以QP383R基因为模板，进行PCR扩增，琼脂糖凝胶电泳回收，得到QP383R产物，所述QP383R基因的核苷酸序列如SEQ ID NO.1所示；
(2)将步骤(1)所述QP383R产物和pCMV载体进行双酶切，使用C115 DNA连接酶连接，转化至感受态细胞中，筛选后得到重组质粒pCMV-Myc-QP383R。
7.根据权利要求6所述的应用，其特征在于，所述PCR扩增的引物对的序列如SEQ IDNO.3和SEQ ID NO.4所示。
8.根据权利要求6所述的应用，其特征在于，所述感受态细胞为大肠杆菌DH5α。
9.一种干扰TLR信号通路和/或抑制炎性细胞因子药物组合物，其特征在于，所述药物组合物中的活性成分为QP383R蛋白，所述QP383R蛋白的氨基酸序列如SEQ ID NO.2所示。
10.根据权利要求9所述的干扰TLR信号通路和/或抑制炎性细胞因子药物组合物，其特征在于，所述药物还包括药学上可接受的载体或赋形剂。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410006267.5A CN118105462A (zh) | 2024-01-03 | 2024-01-03 | 一种非洲猪瘟病毒蛋白qp383r的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410006267.5A CN118105462A (zh) | 2024-01-03 | 2024-01-03 | 一种非洲猪瘟病毒蛋白qp383r的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118105462A true CN118105462A (zh) | 2024-05-31 |
Family
ID=91215215
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410006267.5A Pending CN118105462A (zh) | 2024-01-03 | 2024-01-03 | 一种非洲猪瘟病毒蛋白qp383r的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118105462A (zh) |
-
2024
- 2024-01-03 CN CN202410006267.5A patent/CN118105462A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Orzalli et al. | An antiviral branch of the IL-1 signaling pathway restricts immune-evasive virus replication | |
| Sang et al. | Differential expression and activity of the porcine type I interferon family | |
| WO2016011906A1 (zh) | 泛素化途径相关因子在调控辅助性t细胞功能中的应用 | |
| Tanaka-Kataoka et al. | In vivo antiviral effect of interleukin 18 in a mouse model of vaccinia virus infection | |
| CN101918440B (zh) | 禽类细胞因子及编码其的遗传序列的应用 | |
| Xia et al. | Hypoxia/ischemia promotes CXCL10 expression in cardiac microvascular endothelial cells by NFkB activation | |
| CN101460634A (zh) | 用于靶向c-rel的方法和组合物 | |
| KR20010015711A (ko) | 폴리펩티드, 그 폴리펩티드를 암호화하는 ｃＤＮＡ 및그들의 용도 | |
| WO2009054996A2 (en) | Systems and methods for viral therapy | |
| JPH06504785A (ja) | インターロイキン−１０による腫瘍性疾患の処置 | |
| Grayfer et al. | Cytokine regulation of teleost inflammatory responses | |
| Ciechomska et al. | Targeting interferons as a strategy for systemic sclerosis treatment | |
| WO2023087823A1 (zh) | 一种线粒体定位多肽、定位系统及其应用 | |
| CN118105462A (zh) | 一种非洲猪瘟病毒蛋白qp383r的应用 | |
| CN110716035B (zh) | 基于棘球蚴微管蛋白为靶点的抗棘球蚴病高通量药物筛选方法 | |
| Zhu et al. | Immune responses to SARS-CoV-2 infection in Humans and ACE2 humanized mice | |
| RU2563989C1 (ru) | Композиция для подавления экспрессии гена цитокина интерлейкина-4 | |
| CN111848780A (zh) | 一种IL-36的可溶性受体sIL-36R及其应用 | |
| CN116693626A (zh) | 订书肽及其用途和在体外扩增干细胞的方法 | |
| Klostergaard et al. | Tumoricidal effector mechanisms of murine BCG-activated macrophages: role of TNF in conjugation-dependent and conjugation-independent pathways | |
| KR101433794B1 (ko) | 프로그래뉼린 억제제를 유효성분으로 함유하는 골다공증 예방 또는 치료용 조성물 | |
| CN110294808B (zh) | 一种具有抗炎作用的多肽sr4及其应用 | |
| KR100692226B1 (ko) | 신규 폴리펩티드, 그 폴리펩티드를 암호화하는 ｃＤＮＡ 및 그 용도 | |
| CN117965568A (zh) | 一种编码asfv蛋白mgf 505-3r的基因及其应用 | |
| Jaekal et al. | Cloning and characterization of bovine interleukin-32 beta isoform |
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 |