LU102855B1 - Method for establishing anti-new coronavirus drug screening and evaluation model and application thereof - Google Patents

Method for establishing anti-new coronavirus drug screening and evaluation model and application thereof Download PDF

Info

Publication number
LU102855B1
LU102855B1 LU102855A LU102855A LU102855B1 LU 102855 B1 LU102855 B1 LU 102855B1 LU 102855 A LU102855 A LU 102855A LU 102855 A LU102855 A LU 102855A LU 102855 B1 LU102855 B1 LU 102855B1
Authority
LU
Luxembourg
Prior art keywords
spike
cells
drug screening
evaluation model
novel coronavirus
Prior art date
Application number
LU102855A
Other languages
English (en)
Inventor
Yihang Wu
Yanzhu Zhou
Zihong Ye
Runtong Lian
Xiaoping Yu
Original Assignee
China Jilian Univ
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by China Jilian Univ filed Critical China Jilian Univ
Application granted granted Critical
Publication of LU102855B1 publication Critical patent/LU102855B1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Food Science & Technology (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Vascular Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Claims (6)

1. Verfahren zur Bildung eines Drogenscreening -und Auswertungsmodells gegen das neuartige Coronavirus, dadurch gekennzeichnet, dass das Drogenscreening -und Auswertungsmodell in vitro unter Verwendung der Wechselwirkung des Spike-Proteins des neuartigen Coronavirus mit dem ACF2-Rezeptor als Ziel aufgebaut wird.
2. Verfahren zur Bildung eines Drogenscreening -und Auswertungsmodells gegen das neuartige Coronavirus nach Anspruch 1, dadurch gekennzeichnet, dass als Verfahren zum Aufbau eines Trägers eine bimolekulare Fluoreszenzkomplementierungstechnik verwendet wird.
3. Verfahren zur Bildung eines Drogenscreening -und Auswertungsmodells gegen das neuartige Coronavirus nach Anspruch 2, dadurch gekennzeichnet, dass das Spike-Protein-Gen des neuartigen Coronavirus, das menschliche ACE2-Gen und zwei nicht-fluoreszierende komplementäre Fragmente des fluoreszierenden Proteins Venus auf einzelnen Vektor oder auf verschiedene Vektoren zur Zelltransfektion aufgebaut werden.
4. Verfahren zur Bildung eines Drogenscreening -und Auswertungsmodells gegen das neuartige Coronavirus nach Anspruch 3, dadurch gekennzeichnet, dass: 1) unter Verwendung des pcDNA4-TO-Plasmids, der selbstspaltenden Figenschaft des 2A-Polypeptids und mRFP als Expressionskontrolle, das menschliche ACE2-Gen, das Spike-Protein-Gen des neuartigen Coronavirus und zwei nicht-fluoreszierende komplementäre Fragmente des fluoreszierenden Proteins Venus beziehungsweise Fragmente VN155 und VC155 auf einzelnen Vektor aufgebaut werden, wobei der Vektor pcDNA-ACF2-ACFE2-VN155-P2A-mRFP-T2A-Spike-VC155 aufgebaut wird, und wobei einer Gensequenz wie SEQ ID NO. 1 dargestellt wird; 1108008 2) Nach der Zellhaftung der Vero-E6-Zellen die Zellen mit den Wildtyp- und Mutantenvektoren transfiziert werden und die Zellen entweder mit einer prophylaktischen Verabreichung vor der Transfektion oder einer Medikamentenintervention nach der Transfektion behandelt werden, je nach der Eigenschaft und Wirkmechanismus des zu testenden Medikaments, und wobei eine leere Vektor-Kontrolle, eine Lôsungsmittel-Kontrolle und eine unbehandelte Zellkontrolle eingerichtet werden; 3) Die Fluoreszenzsignalintensität und die subzelluläre Lokalisierung der Proteininteraktionen mittels Fluoreszenzmikroskopie für jede Gruppe von Zellen zu verschiedenen Zeitpunkten nach der Behandlung analysiert werden.
4) Nach Abschluss der Analyse der Fluoreszenzsignalintensität und der subzellulären Lokalisierung der Proteininteraktionen die transfizierten Zellen gesammelt werden, um die Gen- und Proteinexpressionsniveaus von ACF2 und Spike zu bestimmen, wobei der Effekt der Medikamentenintervention auf die Expression von ACF2 und Spike in den transfizierten Zellen analysiert wird.
5. Verfahren zur Bildung eines Drogenscreening -und Auswertungsmodells gegen das neuartige Coronavirus nach Anspruch 4, dadurch gekennzeichnet, dass die Expressionsniveaus von ACF2 und Spike mRNA durch RT-PCR nachgewiesen werden; wobei die Expressionsniveaus von ACF2 und Spike-Protein durch Western Blotting nachgewiesen werden.
6. Verwendung des Verfahrens zur Bildung eines Drogenscreening -und Auswertungsmodells gegen das neuartige Coronavirus nach Anspruch 1-5 beim Drogenscreening und Auswertung gegen das neuartige Coronavirus.
LU102855A 2021-07-19 2021-08-13 Method for establishing anti-new coronavirus drug screening and evaluation model and application thereof LU102855B1 (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110812726.5A CN113549651A (zh) 2021-07-19 2021-07-19 一种抗新型冠状病毒药物筛选与评价模型的建立方法及应用

Publications (1)

Publication Number Publication Date
LU102855B1 true LU102855B1 (en) 2022-03-14

Family

ID=78132013

Family Applications (1)

Application Number Title Priority Date Filing Date
LU102855A LU102855B1 (en) 2021-07-19 2021-08-13 Method for establishing anti-new coronavirus drug screening and evaluation model and application thereof

Country Status (2)

Country Link
CN (1) CN113549651A (de)
LU (1) LU102855B1 (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115414364A (zh) * 2022-09-23 2022-12-02 四川大学 一种以rna g-四链体为靶点的抗新冠病毒药物及其筛选方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108265078A (zh) * 2016-12-29 2018-07-10 中国计量大学 一种基于宿主细胞靶点的抗乙肝模型建立及应用
CN111662884B (zh) * 2020-06-18 2022-05-03 中吉当康(北京)基因技术有限公司 一种假病毒及其包装方法和药物评估系统

Also Published As

Publication number Publication date
CN113549651A (zh) 2021-10-26

Similar Documents

Publication Publication Date Title
Lippé Flow virometry: a powerful tool to functionally characterize viruses
González-Jara et al. The multiplicity of infection of a plant virus varies during colonization of its eukaryotic host
Fischer et al. Insectivorous bats carry host specific astroviruses and coronaviruses across different regions in Germany
Fang et al. Real-time monitoring of flavivirus induced cytopathogenesis using cell electric impedance technology
Banerjee et al. Tools to study pathogen-host interactions in bats
Perdikaris et al. Development of a portable, high throughput biosensor system for rapid plant virus detection
Frohns et al. Potassium ion channels of chlorella viruses cause rapid depolarization of host cells during infection
Trottier et al. High numbers of viral RNA copies in the central nervous system of mice during persistent infection with Theiler's virus
LU102855B1 (en) Method for establishing anti-new coronavirus drug screening and evaluation model and application thereof
Brussel et al. Use of a new RNA next generation sequencing approach for the specific detection of virus infection in cells
Gee et al. Pseudovirus mimics cell entry and trafficking of the human polyomavirus JCPyV
Scallan et al. Defective RNAs in mosquito cells persistently infected with Bunyamwera virus
Chen et al. The intraviral protein-protein interaction of SARS-CoV-2 reveals the key role of N protein in virus-like particle assembly
Vera et al. A common pattern of influenza A virus single cell gene expression heterogeneity governs the innate antiviral response to infection
Edwards et al. Evaluation of henipavirus chemical inactivation methods for the safe removal of samples from the high-containment PC4 laboratory
CN102439458A (zh) 组织分析
US20220065869A1 (en) Methods, Compositions, and Systems for Detecting Coronavirus Neutralizing Antibodies
Kooi et al. Early events of importance in determining host cell permissiveness to mouse hepatitis virus infection
US9523132B2 (en) Rapid and highly fieldable viral diagnostic
CN106754982A (zh) 表达绿色荧光蛋白的限制性复制西尼罗病毒系统及其应用
Warren et al. Quantification of virus-infected cells using RNA FISH-Flow
Li et al. Pseudotyped virus for henipavirus
Dangler et al. Limitations of in situ hybridization for the detection of bluetongue virus in blood mononuclear cells
Rother et al. Discovery of Zika virus host dependency factors in trophoblasts using CRISPR/Cas9 screening
CN115219726B (zh) 一种SARS-CoV-2假病毒检测血清中中和抗体滴度的方法

Legal Events

Date Code Title Description
FG Patent granted

Effective date: 20220314