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
German (de)
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

Abstract

The present invention discloses a method for establishing an anti-new coronavirus drug screening and evaluation model and an application thereof. Two non-fluorescent complementary fragments VN155 and VC155 based on a fluorescent protein Venus are recombined to form a fluorescent protein to emit a fluorescent signal, and simultaneous expression of multiple genes is implemented by using a self-cleavage property of a 2A polypeptide, to detect a binding effect between a Spike protein in the new coronavirus and an ACE2 receptor, which can be used for screening and preclinical efficacy evaluation of anti-SARS-CoV-2 drugs capable of cutting off adsorption to and infection of human cells by the new coronavirus. The present invention has advantages of simplicity, rapidity, trace amount and intuition, with good reproducibility of results, strong specificity, high sensitivity, less interference factors, less false positive and false negative, and is suitable for research and development of new anti-SARS-CoV-2 drugs.

Description

METHOD FOR ESTABLISHING ANTI-NEW CORONAVIRUS DRUG SCREENING AND EVALUATION MODEL AND APPLICATION THEREOF
TECHNICAL FIELD The present invention relates to the field of biomedicine technologies, and in particular, to a method for establishing an anti-new coronavirus drug screening and evaluation model and an application thereof.
BACKGROUND New coronavirus pneumonia is an infectious disease caused by new coronavirus (SARS-CoV-2) infection. Since 2019, the global spread of the disease has intensified, and up to now, no clinically verified specific therapeutic drug has been approved for marketing. There is an urgent need to develop a safe and effective innovative drug against SARS-CoV-2, but there is a lack of reliable cell models for a specific mechanism of virus infection. Currently, a commonly used screening and evaluation method for an anti-SARS-CoV-2 drug in vitro mainly includes evaluating an antiviral effect of a drug by establishing an in-vitro virus infection cell system, that is, by isolating live SARS-CoV-2 viruses and infecting cultured Vero-E6 cells in vitro. Firstly, the Vero-E6 cells are cultured, SARS-CoV-2 are isolated and adsorbed into the cells by using a virus diluent, cytopathic changes are observed, and a 50% tissue culture infective dose (TCID50) of the viruses is calculated. Then viruses of a corresponding dose (TCIDs0) are used to infect the cells, and then an investigational drug at a maximum amount of non-toxic concentration is added for intervention. Since SARS-CoV-2 infected Vero-E6 cells may form plaque lesions, finally the 1 anti-SARS-CoV-2 effect of the drug is evaluated by calculating a drug-to-virus 02005 median inhibitory concentration (ICs) and therapeutic index (TI). In view of the strong infectious ability of the SARS-CoV-2, to protect researchers, China stipulates that live SARS-CoV-2 viruses can only be used in a high-level biosafety laboratory (P3 laboratory). However, the construction and operation cost of a P3 laboratory is extremely high, and most medical and research institutions do not have a P3 laboratory. Therefore, the institutions cannot carry out related research work such as isolation, culture and preservation of live SARS-CoV-2 viruses, which brings great obstacles and risks to drug activity screening by directly using live viruses to infect cells. Therefore, it is of great significance to establish a cell model for screening anti-SARS-CoV-2 drugs in a common laboratory.
Although it has been reported that SARS-CoV-2 pseudoviruses were used in researches related to SARS-CoV-2, the infection and replication process of the pseudoviruses cannot fully reflect the properties of live viruses, and therefore false positive is more likely to appear when using a pseudovirus system for drug screening, and active compounds screened by using the pseudoviruses still need to be confirmed by using live viruses. In view of the shortcomings of the pseudovirus technology, it is still necessary to establish a new in-vitro screening model for a key molecular mechanism of SARS-CoV-2 infection.
Theoretically, key proteins and links in the process of transmembrane entering of the SARS-CoV-2 into host cells, and transcription and translation, and assembly and release of new viruses in the host cells can be used as targets of anti-SARS-CoV-2 drugs, but the link that prevents viruses from entering host cells is an optimal target for anti-virus. ACE2 receptors from the host and the Spike proteins from the SARS CoV-2 are the main drug targets involved in blocking the process of entering of the SARS CoV-2 into host cells through endocytosis.
2
According to the interaction between Spike proteins and ACE2 receptor proteins. 02005 which is necessary for the SARS-CoV-2 to enter host cells, it is of great significance to establish an evaluation model of the interaction between viruses and host cells for screening and verifying anti-SARS-CoV-2 drugs, which accelerates the research and development of new anti-SARS-CoV-2 drugs.
SUMMARY In view of the defects in the prior art, the present invention establishes a novel model suitable for screening and evaluation of anti-SARS-CoV-2 drugs in a common laboratory by using a recombination property of fluorescent proteins and based on that interaction between the Spike proteins of the new coronavirus (SARS-CoV-2) and ACE2 receptors of host cells is a key link through which viruses infect cells and a key target of anti-SARS-CoV-2 drugs. Therefore, an objective of the present invention is to provide a method for establishing an anti-new coronavirus drug screening and evaluation model and an application thereof, thereby resolving the obstacles and risks brought about by carrying out drug activity screening by directly using live viruses to infect cells.
To achieve the above-mentioned objective, the present invention adopts the following technical solution: A method for establishing an anti-new coronavirus drug screening and evaluation model is provided, which is an in-vitro drug screening and evaluation model constructed by using interaction between Spike proteins of the new coronavirus and ACE2 receptors as a target.
Further, the vector construction method used is a bimolecular fluorescence complementation technique.
3
Further, a Spike protein gene of the new coronavirus, a human ACF2 gene, and 02058 two non-fluorescent complementary fragments of the fluorescent protein Venus are constructed on a single vector or different vectors to be transfected into cells.
Further, 1) by using a pcDNA4-TO plasmid, utilizing a self-cleavage property of a 2A polypeptide, and using an mRFP as expression control, the human ACE2 gene, the Spike protein gene of the new coronavirus, and the two non-fluorescent complementary fragments VN155 and VC155 of the fluorescent protein Venus are constructed on a single vector to construct the vector pcDNA-ACF2-VN155-P2A-mRFP-T2A-Spike-VC155 with a gene sequence as shown in SEQ ID NO.1.
2) After the cultured Vero-E6 cells adhere to the wall, the constructed wild-type and mutant vectors are separately transfected into the cells, and the cells are treated by preventive administration before transfection or drug intervention after transfection according to properties and action mechanisms of the drugs to be tested, and meanwhile, an empty vector control, a solvent control, and an untreated cell control are set up.
3) At different time after treatment, intensities of fluorescent signals and localization of subcells in which proteins interact in each group of cells are analyzed by using a fluorescence microscope.
4) After the analyses of the intensities of fluorescent signals and localization of subcells in which proteins interact, transfected cells are collected to measure expression levels of the ACE2 and Spike genes and proteins, so as to analyze the effect of drug intervention on the expression of ACE2 and Spike in transfected cells.
4
Further, the expression levels of ACE2 and Spike mRNA are measured by 02005 using the RT-PCR method and the expression levels of ACF2 and Spike proteins are measured by using the Western Blotting method.
An application of any one of the above-mentioned methods for establishing an anti-new coronavirus drug screening and evaluation model in screening and evaluation of anti-new coronavirus drugs.
The outstanding effects of the present invention are: The present invention, based on that two non-fluorescent complementary fragments VN155 and VC155 based on a fluorescent protein Venus are recombined to form a fluorescent protein to emit a fluorescent signal, and that simultaneous expression of multiple genes is implemented by using a self-cleavage property of a 2A polypeptide, to detect a binding effect between a Spike protein in the new coronavirus and an ACF2 receptor, can be used for screening and preclinical efficacy evaluation of anti-SARS-CoV-2 drugs capable of cutting off adsorption to and infection of human cells by the new coronavirus. The present invention has advantages of simplicity, rapidity, trace amount and intuition, with good reproducibility of results, strong specificity, high sensitivity, less interference factors, less false positive and false negative, and is suitable for research and development of new anti-SARS-CoV-2 drugs.
BRIEF OF THE ACCOMPANYING DRAWINGS FIG. 1 1s a schematic diagram of interaction between Spike proteins of the new coronavirus (SARS-CoV-2) and ACE2 receptors of host cells and fluorescent protein recombination of the present invention; and 5
FIG. 2 is a flowchart of a method according to an embodiment of the present 02005 invention.
DETAILED DESCRIPTION The following clearly and completely describes the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Apparently, the described embodiments are merely some embodiments, other than all embodiments, of the present invention.
Example: Main materials:
1. Vero-E6 cells, and competent cells DHSa.
2. DMEM medium (Gibco), pcDNA4-TO plasmid, DNA extraction kit (Omega), PCR purification kit (Vazyme), Plasmid Maxi Kit (OMEGA), dNTPs, high-fidelity pfu polymerase, Lipofectamine® 2000 transfection reagent (Sigma), T7 ligase (Thermo Fisher), RNase (Invitrogen), nucleic acid reference Marker (Thermo Fisher), and protein Marker (Amresco); Hind III, BamH I, Xba I, and Neh I endonuclease (Invitrogen™); and ammonium persulfate (APS) (Sigma), tetramethyl ethylenediamine (TEMED) (Sigma), B-mercaptoethanol (Gibco), and ACE2 and Spike expression detection reagents.
3. Instruments: 1) Inverted fluorescence microscope. 2) Other conventional molecular biology research devices. The flowchart of operations of this example 1s shown in FIG. 2, including: I. Verification of binding effect between Spike proteins of SARS-CoV-2 and human ACF2 receptors
1. Vero-E6 cell culture: 6
A DMEM medium containing 10% fetal bovine serum was cultured in an 02005 incubator containing 5% carbon dioxide at 37°C, and the cells can be used after growing into a monolayer.
2. pcDNA-ACF2-VN155-P2A-mRFP-T2A-Spike-VC155 vector construction: By using a pcDNA4-TO plasmid, utilizing a self-cleavage property of a 2A polypeptide, and using an mRFP as expression control, the human ACE2 gene, the Spike gene, and the two non-fluorescent complementary fragments VN155 and VC155 of the fluorescent protein Venus were constructed on a single vector simultaneously expressing multiple genes, that IS, pcDNA-ACF2-VN155-P2A-mRFP-T2A-Spike-VC155 (with a gene sequence as shown in SEQ ID NO.1). Meanwhile, on the basis of the vector, a vector expressing a Spike protein mutant gene was constructed and transfected into Vero-E6 cells as negative control.
3. Verification of interaction between Spike proteins and ACE2 receptors: After the Vero-E6 cells cultured in a 96-well plate adhered to the wall, the constructed wild-type and mutant vectors were separately transfected into the cells, and meanwhile, an empty vector control and an untreated cell control were set up.
In 24 and 48 hours after transfection, the intensity and location of fluorescence of each group of cells were analyzed by using the fluorescence microscope and software thereof. By observing the intensity and location of fluorescence, the interaction between human ACF2 receptors and Spike proteins of the new coronavirus and localization of subcells thereof can be learned and verified.
4. Result determination: As shown in FIG. 1, if the ACE2 receptor interacted with the Spike protein, two non-fluorescent complementary fragments VN155 and VCI155 of the 7 fluorescent protein Venus were caused to recombine to form a fluorescent protein, 02058 and emitted fluorescence which can be detected by the fluorescence microscope. The fluorescence observed by microscope can prove the interaction between ACF2 receptor and Spike protein. By observing the location of fluorescence in the cells (such as nucleus, cytoplasm, and cell membrane),the expression region of the ACF2 receptor in cells can be determined, thus implementing localization of subcells.
IL. Screening and evaluation methods for anti-SARS-CoV-2 drug activities
1. Vero-E6 cell culture: A DMEM medium containing 10% fetal bovine serum was cultured in an incubator containing 5% carbon dioxide at 37°C, and the cells can be used after growing into a monolayer.
2. pcDNA-ACE2-VN155-P2A-mRFP-T2A-Spike-VCI155 vector construction: By using a pcDNA4-TO plasmid, utilizing a self-cleavage property of a 2A polypeptide, and using an mRFP as expression control, the human ACE2 gene, the Spike gene, and the two non-fluorescent complementary fragments VN155 and VC155 of the fluorescent protein Venus were constructed on a single vector simultaneously expressing multiple genes, that IS, pcDNA-ACF2-VN155-P2A-mRFP-T2A-Spike-VC155 (with a gene sequence as shown in SEQ ID NO.1). Meanwhile, on the basis of the vector, a vector expressing a Spike protein mutant gene was constructed and transfected into Vero-E6 cells as negative control.
3. Screening of anti-SARS-CoV-2 drug activities: 1) According to properties and action mechanisms of the drugs to be tested, different administration methods (a) or (b) may be adopted: (a) Preventive administration before transfection: After the Vero-E6 cells 8 cultured in the DMEM medium adhered to the wall, the cells were pretreated with 02005 different concentrations of investigational drugs for 24 hours, and then the constructed wild-type and mutant vectors were separately transfected into the cells, and meanwhile, an empty vector control, a solvent control, and an untreated cell control were set up. In 24, 48, and 72 hours after transfection, the intensities of fluorescent signals and subcellular locations of each group of cells were analyzed by using the fluorescence microscope and software thereof.
(b) Drug intervention after transfection: After the Vero-E6 cells cultured in the DMEM medium adhered to the wall, the constructed wild-type and mutant vectors were separately transfected into the cells, and after transfection, the cells were treated with different concentrations of investigational drugs for 24 hours. In 24, 48, and 72 hours after the treatment with drugs, the intensities of fluorescent signals and subcellular locations of each group of cells were analyzed by using the fluorescence microscope and software thereof. Meanwhile, an empty vector control, a solvent control, and an untreated cell control were set up.
2) After the analyses of the intensities of fluorescent signals and localization of subcells in which proteins interact, transfected cells were collected to measure expression levels of the ACE2 and Spike genes by using the RT-PCR method and expression levels of the ACE2 and Spike proteins by using the Western Blotting method, so as to analyze the effect of intervention by the investigational drugs on the expression of ACE2 and Spike in transfected cells.
4. Result determination: The treatment of investigational drugs on cells will affect the interaction between ACE2 and Spike protein, the intervention by the investigational drugs on the binding of ACE2 and Spike proteins by detecting the presence, intensity, and 9 location of fluorescence can be learned. The anti-SARS-CoV-2 activity of he investigational drugs was evaluated comprehensively in combination with the expression levels of the ACF2 and Spike genes and proteins.
The foregoing are only preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent replacement or changes made by a person skilled in the art within the scope of protection of the technologies described in the present invention according to the technical solutions and inventive concept of the present invention shall fall within the scope of protection described in the present invention. 10

Claims (6)

CLAIMS 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.A method of forming a drug screening and evaluation model against the novel coronavirus, characterized in that the drug screening and evaluation model is constructed in vitro using the interaction of the spike protein of the novel coronavirus with the ACF2 receptor as a target. 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.2. Method for creating a drug screening and evaluation model against the novel coronavirus according to claim 1, characterized in that a bimolecular fluorescence complementation technique is used as a method for constructing a carrier. 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.3. A method for forming a drug screening and evaluation model against the novel coronavirus according to claim 2, characterized in that the spike protein gene of the novel coronavirus, the human ACE2 gene and two non-fluorescent complementary fragments of the fluorescent protein Venus on individual Vector or be built on different vectors for cell transfection. 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. A method for creating a drug screening and evaluation model against the novel coronavirus according to claim 3, characterized in that: 1) using the pcDNA4-TO plasmid, the self-cleaving DNA of the 2A polypeptide and mRFP as an expression control, the human ACE2- gene, the novel coronavirus spike protein gene and two non-fluorescent complementary fragments of the fluorescent protein Venus, respectively fragments VN155 and VC155, are constructed on a single vector, the vector being pcDNA-ACF2-ACFE2-VN155-P2A-mRFP-T2A- Spike-VC155 is constructed, and wherein a gene sequence such as SEQ ID NO. 1; 1108008 2) After cell attachment of the Vero-E6 cells, the cells are transfected with the wild-type and mutant vectors and the cells are treated with either prophylactic administration before transfection or drug intervention after transfection, depending on the property and mechanism of action of the to drug being tested, and establishing an empty vector control, a solvent control, and an untreated cell control; 3) Fluorescence signal intensity and subcellular localization of protein interactions are analyzed by fluorescence microscopy for each group of cells at different time points after treatment. 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.4) After completing the analysis of fluorescence signal intensity and subcellular localization of protein interactions, the transfected cells are collected to determine the gene and protein expression levels of ACF2 and Spike, analyzing the effect of drug intervention on the expression of ACF2 and Spike in the transfected cells will. 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.5. A method for creating a drug screening and evaluation model against the novel coronavirus according to claim 4, characterized in that the expression levels of ACF2 and Spike mRNA are detected by RT-PCR; whereby the expression levels of ACF2 and spike protein are detected by Western blotting. 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.6. Use of the method for forming a drug screening and evaluation model against the novel coronavirus according to claims 1-5 in drug screening and evaluation against the novel 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 (en) 2021-07-19 2021-07-19 Method for establishing anti-novel coronavirus drug screening and evaluating model and application

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 (en)
LU (1) LU102855B1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115414364A (en) * 2022-09-23 2022-12-02 四川大学 Anti-neocoronavirus drug taking RNA G-quadruplex as target spot and screening method thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108265078A (en) * 2016-12-29 2018-07-10 中国计量大学 It is a kind of based on the anti-hepatitis B model foundation of host cell target spot and application
CN111662884B (en) * 2020-06-18 2022-05-03 中吉当康(北京)基因技术有限公司 Pseudovirus, packaging method thereof and drug evaluation system

Also Published As

Publication number Publication date
CN113549651A (en) 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
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
Cheung et al. Virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq) defines projection neurons involved in sensorimotor integration
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
US20120183950A1 (en) Tissue analysis
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
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 (en) The restricted duplication west nile virus system of expressing green fluorescent protein and its application
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
Yang et al. SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

Legal Events

Date Code Title Description
FG Patent granted

Effective date: 20220314