WO2021217696A1 - Method for screening inhibitor for 3c protease activity of sars-cov-2 coronavirus and screening kit - Google Patents

Method for screening inhibitor for 3c protease activity of sars-cov-2 coronavirus and screening kit Download PDF

Info

Publication number
WO2021217696A1
WO2021217696A1 PCT/CN2020/089144 CN2020089144W WO2021217696A1 WO 2021217696 A1 WO2021217696 A1 WO 2021217696A1 CN 2020089144 W CN2020089144 W CN 2020089144W WO 2021217696 A1 WO2021217696 A1 WO 2021217696A1
Authority
WO
WIPO (PCT)
Prior art keywords
protease
cov
coronavirus
sars
expression vector
Prior art date
Application number
PCT/CN2020/089144
Other languages
French (fr)
Chinese (zh)
Inventor
刘青松
胡晨
王文超
任涛
王伟
王黎
陈先涛
Original Assignee
中国科学院合肥物质科学研究院
合肥中科普瑞昇生物医药科技有限公司
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 中国科学院合肥物质科学研究院, 合肥中科普瑞昇生物医药科技有限公司 filed Critical 中国科学院合肥物质科学研究院
Publication of WO2021217696A1 publication Critical patent/WO2021217696A1/en

Links

Images

Classifications

    • 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
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • 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)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • 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"
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of molecular and cell biology, and specifically relates to a screening method and a screening kit for inhibitors of virus 3C protease activity, in particular to a screening method and screening reagents for SARS-CoV-2 coronavirus 3C protease activity inhibitors box.
  • the new coronavirus SARS-CoV-2 is the seventh coronavirus discovered in 2019 that can infect humans. It was named by the World Health Organization on January 12, 2020. It can cause a new type of coronavirus pneumonia COVID-19, which is harmful to humans. Physical health poses a serious threat.
  • the SARS-COV-2 coronavirus belongs to the same single positive-stranded RNA virus as the 2003 SARS coronavirus (SARS-CoV) and the "Middle East Respiratory Syndrome" MERS coronavirus (MERS-CoV). Through gene sequence alignment, SARS-COV-2 has about 80% similarity with SARS-CoV and 40% similarity with MERS-CoV.
  • SARS-COV-2 coronavirus is highly contagious and is a major potential threat to human health. Therefore, it is imperative to find anti-SARS-COV-2 coronavirus drugs.
  • 3C protease also known as 3CL protease or M protease, plays an important role in the protein maturation process of single plus-strand RNA viruses. It can recognize specific enzyme cleavage sites, cut the virus's polymer protein, and cut it into Multiple active proteins eventually assemble into new virus particles. Therefore, inhibiting the catalytic function of 3C protease can effectively inhibit the cleavage of the viral precursor protein, block the replication of the virus, and play a role in resisting single positive-strand RNA viruses. Screening inhibitors that can inhibit the activity of 3C protease has become an important way to develop drugs against single plus-strand RNA viruses.
  • the present invention aims to provide a screening method and screening kit for inhibitors of virus 3C protease activity.
  • a method for screening virus 3C protease activity inhibitors which includes:
  • the expression vector expressing the active viral 3C protease, the expression vector expressing the substrate of the viral 3C protease and the candidate reagent are incubated in the cells as the experimental group, and a negative control without adding the candidate reagent is set according to the experimental group relative to the negative control Changes in the intensity of the fluorescent signal to screen for inhibitors of viral 3C protease activity;
  • the substrate of the virus 3C protease is a fusion protein formed by connecting IL1 ⁇ protein and Gussia luciferase through the restriction site of the virus 3C protease.
  • the viral 3C protease is SARS-CoV-2 coronavirus 3C protease.
  • the method for constructing the expression vector for expressing the active viral 3C protease includes:
  • step B) Cloning the 3C protease recombinant expression sequence of step A) into a eukaryotic expression vector to obtain an expression vector for expressing active viral 3C protease.
  • the method for constructing the expression vector expressing the substrate of the viral 3C protease includes:
  • step b) Cloning the fusion expression sequence of step a) into a eukaryotic expression vector to obtain an expression vector expressing the substrate of the viral 3C protease.
  • the change in the intensity of the fluorescent signal is a decrease in the intensity of the fluorescent signal.
  • an expression vector expressing active SARS-COV-2 coronavirus 3C protease which is constructed and obtained by the above method; optionally, the expression of active SARS-COV -2
  • the nucleotide sequence of the expression vector of coronavirus 3C protease is shown in SEQ ID NO: 7.
  • an expression vector expressing the substrate of SARS-COV-2 coronavirus 3C protease, which is constructed and obtained by the above method; optionally, the expression vector of SARS-COV-2 coronavirus
  • the nucleotide sequence of the expression vector of the viral 3C protease substrate is shown in SEQ ID NO: 3.
  • kits for screening SARS-CoV-2 coronavirus 3C protease activity inhibitors which includes the above-mentioned expression of the active SARS-CoV-2 coronavirus 3C protease Vector, and the above-mentioned expression vector for expressing the substrate of SARS-CoV-2 coronavirus 3C protease.
  • a method for measuring virus 3C protease activity which includes the following steps:
  • step 2) Clone the fusion expression sequence of step 1) into a eukaryotic expression vector to obtain an expression vector;
  • step 2) Co-express the expression vector of step 2) and the expression system of the viral 3C protease in cells, and characterize the activity of the viral 3C protease by measuring the fluorescence signal intensity of the co-expression system;
  • the expression system of the viral 3C protease in step 3) is an expression vector of the viral 3C protease constructed by cloning the expression sequence of the viral 3C protease into a eukaryotic expression vector.
  • the above-mentioned virus 3C protease is the 3C protease of a single positive-strand RNA virus, preferably SARS-CoV-2 coronavirus 3C protease.
  • kits for measuring the activity of the SARS-CoV-2 coronavirus 3C protease which includes the aforementioned expression vector for expressing the substrate of the SARS-CoV-2 coronavirus 3C protease; optionally; Specifically, the nucleotide sequence of the expression vector for expressing the substrate of the SARS-CoV-2 coronavirus 3C protease is shown in SEQ ID NO: 3.
  • the method for screening SARS-COV-2 coronavirus 3C protease activity inhibitors provided by the above technical solutions is to establish a system for expressing active SARS-COV-2 coronavirus 3C protease, and construct a system based on 3C protease digestion to activate fluorescence
  • the reporting system of the protease activity can evaluate the SARS-COV-2 coronavirus 3C protease activity at the cellular level, and this evaluation system can then be used to screen inhibitors of the SARS-COV-2 coronavirus 3C protease activity.
  • the screening method provides a kit for screening SARS-COV-2 coronavirus 3C protease activity inhibitors, which can quickly screen out inhibitors that can inhibit the SARS-COV-2 coronavirus 3C protease activity, which can be effectively used For the development of anti-SARS-COV-2 coronavirus drugs.
  • Figure 1 is a schematic diagram of the construction of the SARS-CoV-2 coronavirus 3C protease activity assay system
  • Figure 2 is a plasmid map of the expression vector S1;
  • Figure 3 is a plasmid map of the expression vector C1;
  • Figure 4 is a histogram of the activity comparison of 3C protease expressed by expression vector C1 and expression vector C2;
  • Figure 5 is a graph showing the inhibitory effects of candidate compounds and positive drugs on 3C protease activity
  • Figure 6 shows the inhibition curve of hydroxychloroquine on SARS-CoV-2 coronavirus 3C protease activity.
  • the present invention aims to provide a method for screening virus 3C protease activity inhibitors, in particular, to provide a method for screening SARS-COV-2 coronavirus 3C protease activity inhibitors, and to provide a rapid screening method for SARS-COV-2 based on the screening method.
  • COV-2 Coronavirus 3C protease activity inhibitor kit COV-2 Coronavirus 3C protease activity inhibitor kit.
  • IL1 ⁇ protein precursor will aggregate and precipitate in cells, and it must be cleaved by the protease CASPASE1 to release the C-terminal soluble IL1, constructing an IL1 ⁇ protein precursor and The fusion vector expressing Gussia luciferase (Gluc) in tandem, as shown in Figure 1, the two expressed proteins are connected with the 3C protease cleavage site.
  • the fusion protein When there is no 3C protease in the cell, the fusion protein will aggregate Precipitation, and when 3C protease cleaves the restriction site between IL1 ⁇ and Gussia luciferase, it will release activated and soluble Gussia luciferase, so it can be characterized by the intensity of the fluorescence (chemiluminescence) signal
  • the activity of 3C protease, and the system constructed by the present invention that can characterize the activity of 3C protease also belongs to the content of the present invention. In order to screen the inhibitor of SARS-COV-2 coronavirus 3C protease activity, it is the key to obtain an expression vector that expresses 3C protease with higher activity.
  • the inventors used the two sequences of SARS-COV-2 coronavirus, NSP7 and The length of the protein encoded by NSP8 is small, and the restriction site on the border has high homology with the restriction site of 3C protease.
  • An expression vector that can express high 3C protease activity is constructed and used for the analysis of 3C protease. Screening for inhibitors of protease activity.
  • the method for screening SARS-COV-2 coronavirus 3C protease activity inhibitors is taken as an example to further illustrate the present invention. It should be understood that the specific embodiments are only used to further illustrate the present invention, rather than to limit the content of the present invention.
  • the sequence used was synthesized by Shanghai Shenggong Co., Ltd.
  • a fusion protein containing the SARS-CoV-2 coronavirus 3C protease cleavage site was constructed as a substrate for the SARS-CoV-2 coronavirus 3C protease.
  • the substrate contains IL1 ⁇ protein.
  • -3C restriction site (the amino acid sequence of the 3C restriction site is shown in SEQ ID NO:1 in the sequence list) -Gussia luciferase (Gluc), that is, IL1 ⁇ protein and Gussia luciferase pass through the 3C restriction site
  • the amino acid sequence is shown in SEQ ID NO: 2 in the sequence table.
  • the specific construction method is:
  • the expression vector of the substrate is named expression vector S1.
  • the nucleotide sequence of the expression vector S1 is shown in SEQ ID NO: 3, and the plasmid map is shown in Figure 2.
  • the expression vector S1 was transfected into HEK 293T cells, and the fusion protein obtained by expression can be used as a substrate for the SARS-CoV-2 coronavirus 3C protease, and will be used in the following examples to determine the 3C protease activity.
  • the expression vector S1 obtained based on this example can provide a kit for measuring the SARS-CoV-2 coronavirus 3C protease activity.
  • the kit includes the expression vector S1.
  • the expression vector S1 can be used. It is co-transfected with the expression vector of SARS-CoV-2 coronavirus 3C protease in HEK 293T cells for co-expression, and the activity of 3C protease is characterized by reading the fluorescence signal intensity in the co-expression system.
  • the SARS-CoV-2 coronavirus After the SARS-CoV-2 coronavirus invades the human body, it uses its own RNA as a template to translate and synthesize a multimeric protein body containing multiple proteins. Only after the active 3C protease digestion can it be cleaved into independent active proteins. Subsequent copying and assembly.
  • the SARS-CoV-2 coronavirus 3C protease expressed in the prior art usually has very low protease activity or even no activity. Therefore, it is necessary to design the expression structure specifically to enable the expressed 3C protease to be self-scissing. Cleavage and dimerization to obtain its biological function and improve the activity of protease.
  • the 5'and 3'ends of the 3C protease expression sequence (the nucleotide sequence is shown in SEQ ID NO: 4) are respectively connected to SARS- COV-2 coronavirus NSP7 (its nucleotide sequence is shown in SEQ ID NO: 5) and NSP8 (its nucleotide sequence is shown in SEQ ID NO: 6) sequence, and the 3C protease is inserted at the junction
  • the expression sequence of the restriction site is constructed to obtain a recombinant expression sequence, which can promote 3C protease to complete self-cleavage and dimerization after expression, and then become an activated protein with biological functions.
  • an expression vector expressing the SARS-COV-2 coronavirus 3C protease with higher biological activity was obtained, which was named expression vector C1, and its nucleotide sequence was as sequenced
  • the SEQ ID NO: 7 in the list is shown, and as shown in Figure 3, the plasmid map of the expression vector C1 is shown.
  • a separate 3C protease expression sequence was synthesized and cloned into the eukaryotic expression vector pCDNA3.1 as a negative control, named as expression vector C2, and its nucleotide sequence is shown in SEQ ID NO: 8 in the sequence list.
  • HEK 293T cells were resuspended to 1 ⁇ 10 5 cells/ml, and 100 ⁇ l per well were plated in a 96-well plate for 8 hours Then DNA transfection was carried out; each well was transferred with 150ng DNA, which contained 50ng of plasmid from expression vector S1 (S1 plasmid) and gradient dilution of plasmids from expression vector C1 and C2 (0ng, 25ng, 50ng, 100ng, C1 /C2 plasmid), the insufficient DNA is filled with pCDNA3.1 empty vector.
  • the specific transfection operation is as follows: prepare 10 ⁇ l of Opti-MEM (Gibco) medium and add the corresponding DNA, mix well, add lipofiter transfection reagent (Hanbio, Shanghai) with 3 times the DNA quality, let stand at room temperature for 10 minutes, drop Add to a 96-well plate, incubate in a 5% CO 2 incubator for 24 hours, add 15 ⁇ l of Renilla-glo reagent (Promega) to each well, and read the fluorescence value after 5 minutes of reaction.
  • This example uses the expression vector S1 constructed in Example 1 and the expression vector C1 constructed in Example 2 to screen for inhibitors of the SARS-COV-2 coronavirus 3C protease activity, which specifically includes the following operations:
  • narfinavir did not show the inhibition of SARS-COV-2 coronavirus 3C protease activity
  • hydroxychloroquine showed the ability to inhibit SARS-COV-2 coronavirus 3C protease activity, and the same two Like positive drugs, they are dose-dependent. Therefore, hydroxychloroquine can be selected as a candidate compound for the preparation of anti-SARS-COV-2 coronavirus infection drugs.
  • the present invention constructs a system capable of measuring 3C protease activity and constructs a vector for expressing 3C protease with higher activity.
  • the two can be combined to screen SARS-COV-2 coronavirus 3C protease
  • An activity inhibitor kit that is, the kit includes an S1 vector expressing a 3C protease substrate and a C1 vector expressing an active 3C protease, both of which can be used to quickly screen out the SARS-COV-2 coronavirus 3C protease Active inhibitors provide important data support for the development of drugs against SARS-COV-2 coronavirus infection.
  • the invention provides a screening method and a screening kit for inhibitors of virus 3C protease activity, which can screen the inhibitors of virus 3C protease activity at the cell level, can be used for the development of antiviral drugs, and is suitable for industrial applications.

Landscapes

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

Abstract

A method for screening an inhibitor for 3C protease activity of a SARS-CoV-2 coronavirus and a screening kit. The method can screen an inhibitor for 3C protease activity of a virus at a cellular level by constructing a system capable of determining the 3C protease activity and a carrier for expressing 3C protease having a high activity, and can be used for developing antiviral drugs.

Description

SARS-CoV-2冠状病毒3C蛋白酶活性抑制剂的筛选方法及筛选试剂盒Method for screening SARS-CoV-2 coronavirus 3C protease activity inhibitor and screening kit 技术领域Technical field
本发明属于分子和细胞生物学领域，具体涉及一种病毒3C蛋白酶活性抑制剂的筛选方法及筛选试剂盒，尤其涉及一种SARS-CoV-2冠状病毒3C蛋白酶活性抑制剂的筛选方法及筛选试剂盒。The invention belongs to the field of molecular and cell biology, and specifically relates to a screening method and a screening kit for inhibitors of virus 3C protease activity, in particular to a screening method and screening reagents for SARS-CoV-2 coronavirus 3C protease activity inhibitors box.
背景技术Background technique
新型冠状病毒SARS-CoV-2是2019年发现的第7种可以感染人的冠状病毒，于2020年1月12日被世界卫生组织命名，其可以引发新型冠状病毒肺炎COVID-19，对人类的身体健康产生严重的威胁。The new coronavirus SARS-CoV-2 is the seventh coronavirus discovered in 2019 that can infect humans. It was named by the World Health Organization on January 12, 2020. It can cause a new type of coronavirus pneumonia COVID-19, which is harmful to humans. Physical health poses a serious threat.
SARS-COV-2冠状病毒与2003年“非典”SARS冠状病毒(SARS-CoV)和“中东呼吸综合征”MERS冠状病毒(MERS-CoV)一样，同属于单正链RNA病毒。通过基因序列比对，SARS-COV-2与SARS-CoV有约80％相似性，与MERS-CoV有40％的相似性。SARS-COV-2冠状病毒传染性强，对人类健康是一个重大的潜在威胁，因此寻找抗SARS-COV-2冠状病毒的药物势在必行。The SARS-COV-2 coronavirus belongs to the same single positive-stranded RNA virus as the 2003 SARS coronavirus (SARS-CoV) and the "Middle East Respiratory Syndrome" MERS coronavirus (MERS-CoV). Through gene sequence alignment, SARS-COV-2 has about 80% similarity with SARS-CoV and 40% similarity with MERS-CoV. The SARS-COV-2 coronavirus is highly contagious and is a major potential threat to human health. Therefore, it is imperative to find anti-SARS-COV-2 coronavirus drugs.
3C蛋白酶又称为3CL蛋白酶或M蛋白酶，在单正链RNA病毒的蛋白成熟过程中发挥着重要作用，其能够识别特异的酶切位点，剪切病毒的多聚蛋白，将其剪切为多个有活性的蛋白，最终组装为新的病毒颗粒。因此，抑制3C蛋白酶的催化功能可有效抑制病毒前体蛋白的切割、阻断病毒的复制，起到抗单正链RNA病毒的作用。通过筛选能够抑制3C蛋白酶活性的抑制剂已经成为当前开发抗单正链RNA病毒的药物的重要途径，因此，针对一种新发现的单正链RNA病毒-SARS-COV-2冠状病毒，在体外建立用于筛选其3C蛋白酶活性的抑制剂的方法对于抗SARS-COV-2冠状病毒药物的开发具有重要的临床意义。3C protease, also known as 3CL protease or M protease, plays an important role in the protein maturation process of single plus-strand RNA viruses. It can recognize specific enzyme cleavage sites, cut the virus's polymer protein, and cut it into Multiple active proteins eventually assemble into new virus particles. Therefore, inhibiting the catalytic function of 3C protease can effectively inhibit the cleavage of the viral precursor protein, block the replication of the virus, and play a role in resisting single positive-strand RNA viruses. Screening inhibitors that can inhibit the activity of 3C protease has become an important way to develop drugs against single plus-strand RNA viruses. Therefore, it is aimed at a newly discovered single plus-strand RNA virus-SARS-COV-2 coronavirus in vitro. The establishment of a method for screening inhibitors of its 3C protease activity has important clinical significance for the development of anti-SARS-COV-2 coronavirus drugs.
发明内容Summary of the invention
本发明旨在提供一种病毒3C蛋白酶活性抑制剂的筛选方法及筛选试剂盒。The present invention aims to provide a screening method and screening kit for inhibitors of virus 3C protease activity.
在本发明的第一个方面，提供了一种病毒3C蛋白酶活性抑制剂的筛选方法，其包括：In the first aspect of the present invention, a method for screening virus 3C protease activity inhibitors is provided, which includes:
将表达具有活性的病毒3C蛋白酶的表达载体、表达病毒3C蛋白酶的底物的表达载 体与候选试剂在细胞中共孵育作为实验组，同时设置不添加候选试剂的阴性对照，根据实验组相对于阴性对照的荧光信号强度的变化来筛选病毒3C蛋白酶活性抑制剂；The expression vector expressing the active viral 3C protease, the expression vector expressing the substrate of the viral 3C protease and the candidate reagent are incubated in the cells as the experimental group, and a negative control without adding the candidate reagent is set according to the experimental group relative to the negative control Changes in the intensity of the fluorescent signal to screen for inhibitors of viral 3C protease activity;
其中所述病毒3C蛋白酶的底物为IL1β蛋白和Gussia荧光素酶通过病毒3C蛋白酶的酶切位点连接而成的融合蛋白。The substrate of the virus 3C protease is a fusion protein formed by connecting IL1β protein and Gussia luciferase through the restriction site of the virus 3C protease.
在一个实施方案中，所述病毒3C蛋白酶为SARS-CoV-2冠状病毒3C蛋白酶。In one embodiment, the viral 3C protease is SARS-CoV-2 coronavirus 3C protease.
在一个实施方案中，所述表达具有活性的病毒3C蛋白酶的表达载体的构建方法包括：In one embodiment, the method for constructing the expression vector for expressing the active viral 3C protease includes:
A)在SARS-COV-2冠状病毒3C蛋白酶的核苷酸序列的5’和3’端分别连接SARS-COV-2冠状病毒的NSP7和NSP8序列，且在各连接处插入SARS-COV-2冠状病毒3C蛋白酶的酶切位点，获得SARS-COV-2冠状病毒3C蛋白酶重组表达序列；A) Connect the NSP7 and NSP8 sequences of SARS-COV-2 coronavirus to the 5'and 3'ends of the nucleotide sequence of the SARS-COV-2 coronavirus 3C protease respectively, and insert SARS-COV-2 at each connection The restriction site of the coronavirus 3C protease, the recombinant expression sequence of SARS-COV-2 coronavirus 3C protease was obtained;
B)将步骤A)的3C蛋白酶重组表达序列克隆至真核表达载体，获得表达具有活性的病毒3C蛋白酶的表达载体。B) Cloning the 3C protease recombinant expression sequence of step A) into a eukaryotic expression vector to obtain an expression vector for expressing active viral 3C protease.
在一个实施方案中，所述表达病毒3C蛋白酶的底物的表达载体的构建方法包括：In one embodiment, the method for constructing the expression vector expressing the substrate of the viral 3C protease includes:
a)将IL1β蛋白表达序列和Gussia荧光素酶表达序列通过SARS-COV-2冠状病毒3C蛋白酶的酶切位点连接，获得融合表达序列；a) Connect IL1β protein expression sequence and Gussia luciferase expression sequence through the restriction site of SARS-COV-2 coronavirus 3C protease to obtain fusion expression sequence;
b)将步骤a)的融合表达序列克隆至真核表达载体，获得表达病毒3C蛋白酶的底物的表达载体。b) Cloning the fusion expression sequence of step a) into a eukaryotic expression vector to obtain an expression vector expressing the substrate of the viral 3C protease.
在一个实施方案中，所述荧光信号强度的变化为荧光信号强度的降低。In one embodiment, the change in the intensity of the fluorescent signal is a decrease in the intensity of the fluorescent signal.
在本发明的第二方面，提供了一种表达具有活性的SARS-COV-2冠状病毒3C蛋白酶的表达载体，其由上述的方法构建获得；任选地，所述表达具有活性的SARS-COV-2冠状病毒3C蛋白酶的表达载体的核苷酸序列如SEQ ID NO:7所示。In the second aspect of the present invention, there is provided an expression vector expressing active SARS-COV-2 coronavirus 3C protease, which is constructed and obtained by the above method; optionally, the expression of active SARS-COV -2 The nucleotide sequence of the expression vector of coronavirus 3C protease is shown in SEQ ID NO: 7.
在本发明的第三方面，提供了一种表达SARS-COV-2冠状病毒3C蛋白酶的底物的表达载体，其由上述的方法构建获得；任选地，所述表达SARS-COV-2冠状病毒3C蛋白酶的底物的表达载体的核苷酸序列如SEQ ID NO:3所示。In the third aspect of the present invention, there is provided an expression vector expressing the substrate of SARS-COV-2 coronavirus 3C protease, which is constructed and obtained by the above method; optionally, the expression vector of SARS-COV-2 coronavirus The nucleotide sequence of the expression vector of the viral 3C protease substrate is shown in SEQ ID NO: 3.
在本发明的第四方面，提供了一种用于筛选SARS-CoV-2冠状病毒3C蛋白酶活性抑制剂的试剂盒，其包括上述的表达具有活性的SARS-CoV-2冠状病毒3C蛋白酶的表达载体，和上述的表达SARS-CoV-2冠状病毒3C蛋白酶的底物的表达载体。In the fourth aspect of the present invention, a kit for screening SARS-CoV-2 coronavirus 3C protease activity inhibitors is provided, which includes the above-mentioned expression of the active SARS-CoV-2 coronavirus 3C protease Vector, and the above-mentioned expression vector for expressing the substrate of SARS-CoV-2 coronavirus 3C protease.
在本发明的第五方面，还提供一种病毒3C蛋白酶活性的测定方法，其包括以下步骤：In the fifth aspect of the present invention, there is also provided a method for measuring virus 3C protease activity, which includes the following steps:
1)将IL1β蛋白表达序列和Gussia荧光素酶表达序列通过病毒3C蛋白酶的酶切位点连接，获得融合表达序列；1) Connect IL1β protein expression sequence and Gussia luciferase expression sequence through the restriction site of virus 3C protease to obtain fusion expression sequence;
2)将步骤1)的融合表达序列克隆至真核表达载体，获得表达载体；2) Clone the fusion expression sequence of step 1) into a eukaryotic expression vector to obtain an expression vector;
3)将步骤2)的表达载体与病毒3C蛋白酶的表达体系在细胞中进行共表达，通过 测定共表达体系的荧光信号强度来表征病毒3C蛋白酶的活性；3) Co-express the expression vector of step 2) and the expression system of the viral 3C protease in cells, and characterize the activity of the viral 3C protease by measuring the fluorescence signal intensity of the co-expression system;
任选地，步骤3)中所述病毒3C蛋白酶的表达体系为将病毒3C蛋白酶的表达序列克隆至真核表达载体所构建的病毒3C蛋白酶的表达载体。Optionally, the expression system of the viral 3C protease in step 3) is an expression vector of the viral 3C protease constructed by cloning the expression sequence of the viral 3C protease into a eukaryotic expression vector.
上述病毒3C蛋白酶为单正链RNA病毒的3C蛋白酶，优选为SARS-CoV-2冠状病毒3C蛋白酶。The above-mentioned virus 3C protease is the 3C protease of a single positive-strand RNA virus, preferably SARS-CoV-2 coronavirus 3C protease.
在本发明的第六方面，还提供一种测定SARS-CoV-2冠状病毒3C蛋白酶活性的试剂盒，其包括上述的表达SARS-CoV-2冠状病毒3C蛋白酶的底物的表达载体；任选地，所述表达SARS-CoV-2冠状病毒3C蛋白酶的底物的表达载体的核苷酸序列如SEQ ID NO:3所示。In the sixth aspect of the present invention, there is also provided a kit for measuring the activity of the SARS-CoV-2 coronavirus 3C protease, which includes the aforementioned expression vector for expressing the substrate of the SARS-CoV-2 coronavirus 3C protease; optionally; Specifically, the nucleotide sequence of the expression vector for expressing the substrate of the SARS-CoV-2 coronavirus 3C protease is shown in SEQ ID NO: 3.
基于以上技术方案提供的SARS-COV-2冠状病毒3C蛋白酶活性抑制剂的筛选方法通过建立表达具有活性的SARS-COV-2冠状病毒3C蛋白酶的体系，并构建一个基于3C蛋白酶酶切进而激活荧光素酶活性的报告体系，能够在细胞水平对SARS-COV-2冠状病毒3C蛋白酶活性进行评价，进而可以利用该评价体系对SARS-COV-2冠状病毒3C蛋白酶活性的抑制剂进行筛选。基于提供的筛选方法还提供了用于筛选SARS-COV-2冠状病毒3C蛋白酶活性抑制剂的试剂盒，可以快速筛选出能够抑制SARS-COV-2冠状病毒3C蛋白酶活性的抑制剂，能够有效用于抗SARS-COV-2冠状病毒的药物的开发。The method for screening SARS-COV-2 coronavirus 3C protease activity inhibitors provided by the above technical solutions is to establish a system for expressing active SARS-COV-2 coronavirus 3C protease, and construct a system based on 3C protease digestion to activate fluorescence The reporting system of the protease activity can evaluate the SARS-COV-2 coronavirus 3C protease activity at the cellular level, and this evaluation system can then be used to screen inhibitors of the SARS-COV-2 coronavirus 3C protease activity. Based on the screening method provided, it also provides a kit for screening SARS-COV-2 coronavirus 3C protease activity inhibitors, which can quickly screen out inhibitors that can inhibit the SARS-COV-2 coronavirus 3C protease activity, which can be effectively used For the development of anti-SARS-COV-2 coronavirus drugs.
附图说明Description of the drawings
图1为SARS-CoV-2冠状病毒3C蛋白酶活性测定体系的构建原理图；Figure 1 is a schematic diagram of the construction of the SARS-CoV-2 coronavirus 3C protease activity assay system;
图2为表达载体S1的质粒图谱；Figure 2 is a plasmid map of the expression vector S1;
图3为表达载体C1的质粒图谱；Figure 3 is a plasmid map of the expression vector C1;
图4为表达载体C1和表达载体C2表达的3C蛋白酶的活性对比柱状图；Figure 4 is a histogram of the activity comparison of 3C protease expressed by expression vector C1 and expression vector C2;
图5为候选化合物和阳性药物对3C蛋白酶活性的抑制效果图；Figure 5 is a graph showing the inhibitory effects of candidate compounds and positive drugs on 3C protease activity;
图6为羟氯喹对SARS-CoV-2冠状病毒3C蛋白酶活性的抑制曲线。Figure 6 shows the inhibition curve of hydroxychloroquine on SARS-CoV-2 coronavirus 3C protease activity.
具体实施方式Detailed ways
本发明旨在提供一种病毒3C蛋白酶活性抑制剂的筛选方法，尤其是提供一种SARS-COV-2冠状病毒3C蛋白酶活性抑制剂的筛选方法，并基于该筛选方法提供一种快速筛选SARS-COV-2冠状病毒3C蛋白酶活性抑制剂的试剂盒。The present invention aims to provide a method for screening virus 3C protease activity inhibitors, in particular, to provide a method for screening SARS-COV-2 coronavirus 3C protease activity inhibitors, and to provide a rapid screening method for SARS-COV-2 based on the screening method. COV-2 Coronavirus 3C protease activity inhibitor kit.
为了实现上述目的，本发明人参考以下原理：IL1β蛋白前体在细胞中会发生聚集沉淀，必须经过蛋白酶CASPASE1的剪切，才能释放出C端可溶的IL1，构建了一个IL1β蛋白前体和Gussia荧光素酶(Gluc)串联表达的融合载体，如图1所示，表达的两个蛋 白之间以3C蛋白酶的酶切位点相连接，当细胞中没有3C蛋白酶时，融合蛋白会发生聚集沉淀，而当3C蛋白酶剪切IL1β与Gussia荧光素酶之间的酶切位点后，将释放出活化可溶的Gussia荧光素酶，因此通过荧光(化学发光)信号强度的高低，就可以表征3C蛋白酶的活性，本发明构建的可以表征3C蛋白酶活性的体系也属于本发明的内容。为了筛选SARS-COV-2冠状病毒3C蛋白酶活性的抑制剂，获得一种表达具有较高活性的3C蛋白酶的表达载体是关键，本发明人利用SARS-COV-2冠状病毒的两个序列NSP7和NSP8编码的蛋白长度较小，且边界的酶切位点与3C蛋白酶的酶切位点同源性高的特点，构建获得一种可以表达具有较高3C蛋白酶活性的表达载体，用于对3C蛋白酶活性的抑制剂进行筛选。In order to achieve the above purpose, the inventors referred to the following principle: IL1β protein precursor will aggregate and precipitate in cells, and it must be cleaved by the protease CASPASE1 to release the C-terminal soluble IL1, constructing an IL1β protein precursor and The fusion vector expressing Gussia luciferase (Gluc) in tandem, as shown in Figure 1, the two expressed proteins are connected with the 3C protease cleavage site. When there is no 3C protease in the cell, the fusion protein will aggregate Precipitation, and when 3C protease cleaves the restriction site between IL1β and Gussia luciferase, it will release activated and soluble Gussia luciferase, so it can be characterized by the intensity of the fluorescence (chemiluminescence) signal The activity of 3C protease, and the system constructed by the present invention that can characterize the activity of 3C protease also belongs to the content of the present invention. In order to screen the inhibitor of SARS-COV-2 coronavirus 3C protease activity, it is the key to obtain an expression vector that expresses 3C protease with higher activity. The inventors used the two sequences of SARS-COV-2 coronavirus, NSP7 and The length of the protein encoded by NSP8 is small, and the restriction site on the border has high homology with the restriction site of 3C protease. An expression vector that can express high 3C protease activity is constructed and used for the analysis of 3C protease. Screening for inhibitors of protease activity.
以下以筛选SARS-COV-2冠状病毒3C蛋白酶活性的抑制剂的方法为例，对本发明进一步阐述。应当理解的是，具体实施例仅用于进一步说明本发明，而不是用于限制本发明的内容。The method for screening SARS-COV-2 coronavirus 3C protease activity inhibitors is taken as an example to further illustrate the present invention. It should be understood that the specific embodiments are only used to further illustrate the present invention, rather than to limit the content of the present invention.
下述实施例中所用方法如无特别说明均为常规方法，具体步骤可参见：《分子克隆实验指南》(《Molecular Cloning:A Laboratory Manual》Sambrook，J.，Russell，David W.，Molecular Cloning:A Laboratory Manual，3rd edition，2001，NY，Cold Spring Harbor)。The methods used in the following examples are conventional methods unless otherwise specified. For specific steps, please refer to: "Molecular Cloning: A Laboratory Manual" Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。The ways to obtain various biological materials described in the examples are only to provide an experimental way to achieve specific disclosure purposes, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and ethics can be replaced and used according to the prompts in the examples.
所用序列由上海生工有限公司合成。The sequence used was synthesized by Shanghai Shenggong Co., Ltd.
实施例1：SARS-CoV-2冠状病毒3C蛋白酶活性测定体系的构建Example 1: Construction of SARS-CoV-2 Coronavirus 3C Protease Activity Assay System
该实施例构建了一个含有SARS-CoV-2冠状病毒3C蛋白酶酶切位点的融合蛋白作为SARS-CoV-2冠状病毒3C蛋白酶的底物，如图1所示，该底物包含了IL1β蛋白-3C酶切位点(3C酶切位点的氨基酸序列如序列表中SEQ ID NO:1所示)-Gussia荧光素酶(Gluc)，即IL1β蛋白和Gussia荧光素酶通过3C酶切位点连接，其氨基酸序列如序列表中SEQ ID NO:2所示，具体的构建方法为：In this example, a fusion protein containing the SARS-CoV-2 coronavirus 3C protease cleavage site was constructed as a substrate for the SARS-CoV-2 coronavirus 3C protease. As shown in Figure 1, the substrate contains IL1β protein. -3C restriction site (the amino acid sequence of the 3C restriction site is shown in SEQ ID NO:1 in the sequence list) -Gussia luciferase (Gluc), that is, IL1β protein and Gussia luciferase pass through the 3C restriction site The amino acid sequence is shown in SEQ ID NO: 2 in the sequence table. The specific construction method is:
合成包含表达IL1β蛋白-SARS-CoV-2冠状病毒3C酶切位点-Gussia荧光素酶的基因序列，并将该基因序列克隆至载体pCDNA3.1(Thermo Fisher Scientific)上，获得表达3C蛋白酶的底物的表达载体，命名为表达载体S1，该表达载体S1的核苷酸序列如SEQ ID NO:3所示，质粒图谱如图2所示。将该表达载体S1转染HEK 293T细胞，表达获得的融合蛋白可以作为SARS-CoV-2冠状病毒3C蛋白酶的底物，将用于以下实施例中测定3C蛋白酶的活性。Synthesize the gene sequence containing IL1β protein-SARS-CoV-2 coronavirus 3C restriction site-Gussia luciferase, and clone the gene sequence into the vector pCDNA3.1 (Thermo Fisher Scientific) to obtain the 3C protease expression The expression vector of the substrate is named expression vector S1. The nucleotide sequence of the expression vector S1 is shown in SEQ ID NO: 3, and the plasmid map is shown in Figure 2. The expression vector S1 was transfected into HEK 293T cells, and the fusion protein obtained by expression can be used as a substrate for the SARS-CoV-2 coronavirus 3C protease, and will be used in the following examples to determine the 3C protease activity.
如果在表达载体S1进行表达时，体系中没有3C蛋白酶剪切表达的融合蛋白，那么 融合蛋白将发生聚集沉淀而失活；当存在3C蛋白酶对表达的融合蛋白进行剪切时，荧光素酶得以活化，因此可以通过读取荧光信号的强度变化表征3C蛋白酶的活性，进而根据3C蛋白酶的活性筛选抑制3C蛋白酶活性的抑制剂。因此可以基于该实施例获得的表达载体S1提供一种测定SARS-CoV-2冠状病毒3C蛋白酶活性的试剂盒，该试剂盒中包括表达载体S1，当使用该试剂盒时，可以将表达载体S1与SARS-CoV-2冠状病毒3C蛋白酶的表达载体共转染HEK 293T细胞共表达，通过读取共表达体系中荧光信号强度来表征3C蛋白酶的活性。If the expressed fusion protein is not cleaved by 3C protease when the expression vector S1 is expressed in the system, the fusion protein will aggregate and precipitate and become inactivated; when the expressed fusion protein is cleaved by 3C protease, luciferase will be able to cleave the expressed fusion protein. Therefore, the activity of the 3C protease can be characterized by reading the intensity change of the fluorescent signal, and then the inhibitors that inhibit the activity of the 3C protease can be screened according to the activity of the 3C protease. Therefore, the expression vector S1 obtained based on this example can provide a kit for measuring the SARS-CoV-2 coronavirus 3C protease activity. The kit includes the expression vector S1. When the kit is used, the expression vector S1 can be used. It is co-transfected with the expression vector of SARS-CoV-2 coronavirus 3C protease in HEK 293T cells for co-expression, and the activity of 3C protease is characterized by reading the fluorescence signal intensity in the co-expression system.
实施例2：在体外具有活性的SARS-CoV-2冠状病毒3C蛋白酶的表达Example 2: Expression of SARS-CoV-2 coronavirus 3C protease with activity in vitro
SARS-CoV-2冠状病毒入侵人体之后，以自身RNA为模板翻译合成含有多个蛋白的多聚蛋白体，只有在具有活性的3C蛋白酶酶切后，才能剪切为独立的有活性的蛋白进行后续的复制和组装。然而，现有技术中表达的SARS-CoV-2冠状病毒3C蛋白酶，通常其蛋白酶活性非常低，甚至没有活性，因此就需要对其表达结构进行针对性设计，使得表达的3C蛋白酶能够发生自剪切和二聚化，以获得其生物功能，提高蛋白酶的活性。After the SARS-CoV-2 coronavirus invades the human body, it uses its own RNA as a template to translate and synthesize a multimeric protein body containing multiple proteins. Only after the active 3C protease digestion can it be cleaved into independent active proteins. Subsequent copying and assembly. However, the SARS-CoV-2 coronavirus 3C protease expressed in the prior art usually has very low protease activity or even no activity. Therefore, it is necessary to design the expression structure specifically to enable the expressed 3C protease to be self-scissing. Cleavage and dimerization to obtain its biological function and improve the activity of protease.
该实施例为了提高表达的SARS-COV-2冠状病毒3C蛋白酶的活性，在3C蛋白酶表达序列(其核苷酸序列如SEQ ID NO:4所示)的5’和3’端分别连接SARS-COV-2冠状病毒的NSP7(其核苷酸序列如SEQ ID NO:5所示)和NSP8(其核苷酸序列如SEQ ID NO:6所示)序列，且在连接处插入了3C蛋白酶的酶切位点的表达序列，构建获得一重组表达序列，该重组表达序列可以促进3C蛋白酶在表达后完成自剪切和二聚化，进而成为具有生物功能的活化蛋白。将该重组表达序列克隆至真核表达载体pCDNA3.1后，获得表达具有较高生物活性的SARS-COV-2冠状病毒3C蛋白酶的表达载体，命名为表达载体C1，其核苷酸序列如序列表中SEQ ID NO:7所示，如图3所示，示出了该表达载体C1的质粒图谱。同时合成单独的3C蛋白酶表达序列，并克隆到真核表达载体pCDNA3.1作为阴性对照，命名为表达载体C2，其核苷酸序列如序列表中SEQ ID NO:8所示。In this example, in order to improve the activity of the expressed SARS-COV-2 coronavirus 3C protease, the 5'and 3'ends of the 3C protease expression sequence (the nucleotide sequence is shown in SEQ ID NO: 4) are respectively connected to SARS- COV-2 coronavirus NSP7 (its nucleotide sequence is shown in SEQ ID NO: 5) and NSP8 (its nucleotide sequence is shown in SEQ ID NO: 6) sequence, and the 3C protease is inserted at the junction The expression sequence of the restriction site is constructed to obtain a recombinant expression sequence, which can promote 3C protease to complete self-cleavage and dimerization after expression, and then become an activated protein with biological functions. After cloning the recombinant expression sequence into the eukaryotic expression vector pCDNA3.1, an expression vector expressing the SARS-COV-2 coronavirus 3C protease with higher biological activity was obtained, which was named expression vector C1, and its nucleotide sequence was as sequenced The SEQ ID NO: 7 in the list is shown, and as shown in Figure 3, the plasmid map of the expression vector C1 is shown. At the same time, a separate 3C protease expression sequence was synthesized and cloned into the eukaryotic expression vector pCDNA3.1 as a negative control, named as expression vector C2, and its nucleotide sequence is shown in SEQ ID NO: 8 in the sequence list.
为了验证该实施例获得的表达载体C1确实能够获得具有较高活性的3C蛋白酶，将HEK 293T细胞重悬至1×10 5个细胞/ml，以100μl每孔铺板于96孔板中，8小时后进行DNA转染；每孔转入150ng DNA，其中包含了50ng的来自表达载体S1的质粒(S1质粒)和梯度稀释的来自表达载体C1和C2的质粒(0ng、25ng、50ng、100ng，C1/C2质粒)，不足的DNA用pCDNA3.1空载体补齐。具体转染操作为：准备10μl的Opti-MEM(Gibco)培养基加入相应的DNA后，混匀，加入DNA质量3倍的lipofiter转染试剂(Hanbio，上海)，室温静置10分钟后，滴加入96孔板中，于5％CO 2培养箱中培养24小时后，每孔加入15μl的Renilla-glo试剂(Promega)，反应5分钟后，读取荧光值。 In order to verify that the expression vector C1 obtained in this example can indeed obtain 3C protease with higher activity, HEK 293T cells were resuspended to 1×10 5 cells/ml, and 100 μl per well were plated in a 96-well plate for 8 hours Then DNA transfection was carried out; each well was transferred with 150ng DNA, which contained 50ng of plasmid from expression vector S1 (S1 plasmid) and gradient dilution of plasmids from expression vector C1 and C2 (0ng, 25ng, 50ng, 100ng, C1 /C2 plasmid), the insufficient DNA is filled with pCDNA3.1 empty vector. The specific transfection operation is as follows: prepare 10 μl of Opti-MEM (Gibco) medium and add the corresponding DNA, mix well, add lipofiter transfection reagent (Hanbio, Shanghai) with 3 times the DNA quality, let stand at room temperature for 10 minutes, drop Add to a 96-well plate, incubate in a 5% CO 2 incubator for 24 hours, add 15 μl of Renilla-glo reagent (Promega) to each well, and read the fluorescence value after 5 minutes of reaction.
结果如图4所示，可见，将该实施例构建的表达载体C1转染HEK 293T细胞后表达 的3C蛋白酶能够呈质量依赖性的剪切由表达载体S1表达的底物，即加入的表达载体C1越多，剪切的底物越多，荧光信号强度越高(荧光值越大)，显著高于同等转染量的C2质粒剪切底物后的荧光值，证明表达载体C1表达的3C蛋白酶的活性远远高于表达载体C2表达的3C蛋白酶的活性，经计算，由C1表达的3C蛋白酶的酶切活性约为由C2表达的3C蛋白酶的酶切活性的1000倍。The results are shown in Figure 4, it can be seen that the 3C protease expressed by the expression vector C1 constructed in this example after transfection of HEK 293T cells can cleave the substrate expressed by the expression vector S1 in a quality-dependent manner, that is, the added expression vector The more C1, the more the sheared substrate, and the higher the fluorescence signal intensity (the greater the fluorescence value), which is significantly higher than the fluorescence value of the C2 plasmid with the same amount of transfection after shearing the substrate, which proves that the expression vector C1 expresses 3C The activity of the protease is much higher than that of the 3C protease expressed by the expression vector C2. According to calculations, the cleavage activity of the 3C protease expressed by C1 is about 1000 times that of the 3C protease expressed by C2.
实施例3：SARS-COV-2冠状病毒3C蛋白酶活性抑制剂的筛选Example 3: Screening of SARS-COV-2 Coronavirus 3C Protease Activity Inhibitors
该实施例利用上述实施例1构建的表达载体S1和实施例2构建的表达载体C1对SARS-COV-2冠状病毒3C蛋白酶活性的抑制剂进行筛选，具体包括以下操作：This example uses the expression vector S1 constructed in Example 1 and the expression vector C1 constructed in Example 2 to screen for inhibitors of the SARS-COV-2 coronavirus 3C protease activity, which specifically includes the following operations:
3.1、将HEK 293T细胞铺于10厘米的细胞培养皿内，密度达到75％时进行转染。3.1. Pour HEK 293T cells in a 10 cm cell culture dish and transfect when the density reaches 75%.
3.2、将表达3C蛋白酶的C1载体3μg和表达3C蛋白酶的底物的S1载体6μg混合，加入1ml的Opti-MEM培养基中，混匀，之后加入27μl转染试剂lipofiter，吹吸混匀，室温静置10分钟，滴加入HEK 293T细胞进行蛋白表达。转染8小时后，利用0.25％胰酶对细胞进行消化，并离心重悬，稀释至1×10 5个细胞/ml。 3.2. Mix 3μg of C1 vector expressing 3C protease and 6μg of S1 vector expressing 3C protease substrate, add 1ml of Opti-MEM medium, mix well, then add 27μl transfection reagent lipofiter, blow and suck and mix, room temperature Let stand for 10 minutes and add HEK 293T cells dropwise for protein expression. After 8 hours of transfection, the cells were digested with 0.25% trypsin, centrifuged to resuspend, and diluted to 1×10 5 cells/ml.
3.3、分别将梯度稀释(0.0、3.0、10.0、30.0μM)的溶于DMSO的化合物(阿比朵尔(Arbidol)、洛匹那韦(lopinavir)、那非那韦(nelfinavir)和羟氯喹(Hydroxychloroquine)(均购自MedChemExpress)，其中阿比朵尔和洛匹那韦作为阳性药物(Zhen Zhu等，Arbidol monotherapy is superior to lopinavir/ritonavir in treating COVID-19，Journal of Infection，doi:10.1016/j.jinf.2020.03.060；Bodee Nutho等，Why Are Lopinavir and Ritonavir Effective against the Newly Emerged Coronavirus 2019？Atomistic Insights into the Inhibitory Mechanisms，Biochemistry，2020.04.15，doi:10.1021/acs.biochem.0c00160)，那非那韦和羟氯喹作为候选化合物)按体积比为1:1000加入细胞重悬液，随后每孔100μl铺至96孔培养板，继续于5％CO 2的37℃培养箱中培养16个小时；将96孔培养板取出，加入15μl Renilla Glo reagent(Promega)，室温孵育5分钟，读取荧光值。同时设置不加细胞的空白培养基的空白对照和不加候选化合物或阳性药物的阴性对照，在扣除空白对照孔的读值后，分别通过各自的荧光值计算每种候选化合物或阳性药物在梯度浓度下的3C蛋白酶的相对活性。结果如图5所示，为上述两种候选化合物和两种阳性药物在不同浓度下的3C蛋白酶的相对活性，可见两种阳性药物均表现出对SARS-COV-2冠状病毒3C蛋白酶活性的抑制效果，并且呈现剂量依赖性，即在一定浓度范围内，阳性药物的浓度越高，对3C蛋白酶的活性抑制作用越强。另外两种候选化合物中那非那韦未表现出对SARS-COV-2冠状病毒3C蛋白酶活性的抑制，而羟氯喹表现出能够抑制SARS-COV-2冠状病毒3C蛋白酶的活性，并且同两种阳性药物一样，呈现出剂量依赖性，因此，可以选择羟氯喹作为制备抗SARS-COV-2冠状病毒感染的药物的候选化合物。 3.3. The compounds (Arbidol, lopinavir, nelfinavir and hydroxychloroquine (Arbidol), lopinavir, nelfinavir and hydroxychloroquine ( Hydroxychloroquine) (both purchased from MedChemExpress), in which Arbidol and Lopinavir are used as positive drugs (Zhen Zhu et al., Arbidol monotherapy is superior to lopinavir/ritonavir in treating COVID-19, Journal of Infection, doi: 10.1016/j .jinf.2020.03.060; Bodee Nutho et al., Why Are Lopinavir and Ritonavir Effective against the Newly Emerged Coronavirus 2019? Atomistic Insights into the Inhibitory Mechanisms, Biochemistry, 2020.04.15, doi: 10.1021/acs.biochem.0c00160), Nafei Navir and hydroxychloroquine as candidate compounds) Add cell resuspension at a volume ratio of 1:1000, then spread 100μl per well to a 96-well culture plate, and continue to incubate in a 5% CO 2 37°C incubator for 16 hours; Take out the 96-well culture plate, add 15μl Renilla Glo reagent (Promega), incubate at room temperature for 5 minutes, and read the fluorescence value. At the same time, set the blank control of the blank medium without cells and the negative control without the candidate compound or positive drug. After subtracting the reading value of the blank control well, calculate the gradient of each candidate compound or positive drug by their respective fluorescence values. The relative activity of 3C protease at different concentrations. The results are shown in Figure 5, showing the relative activity of the 3C protease of the above two candidate compounds and the two positive drugs at different concentrations. It can be seen that the two positive drugs both exhibited inhibition of the SARS-COV-2 coronavirus 3C protease activity. The effect is dose-dependent, that is, within a certain concentration range, the higher the concentration of the positive drug, the stronger the inhibitory effect on 3C protease activity. Among the other two candidate compounds, narfinavir did not show the inhibition of SARS-COV-2 coronavirus 3C protease activity, while hydroxychloroquine showed the ability to inhibit SARS-COV-2 coronavirus 3C protease activity, and the same two Like positive drugs, they are dose-dependent. Therefore, hydroxychloroquine can be selected as a candidate compound for the preparation of anti-SARS-COV-2 coronavirus infection drugs.
3.4、按照以上操作，使用梯度浓度为0、0.01、0.03、0.1、0.3、1、3、10、30、100 μM的羟氯喹对C1载体和S1载体在HEK 293T细胞中的共表达体系进行处理，同时设置不加细胞的空白培养基的空白对照和不加羟氯喹的阴性对照，在扣除空白对照孔的读值后，分别通过各自的荧光值计算每种浓度下对3C蛋白酶的抑制率。结果如图6所示，为梯度稀释浓度的羟氯喹对SARS-COV-2冠状病毒3C蛋白酶活性的抑制曲线，经计算，羟氯喹抑制SARS-COV-2冠状病毒3C蛋白酶活性的半数抑制浓度IC 50约为30nM。 3.4. According to the above operation, use hydroxychloroquine with gradient concentrations of 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 μM to process the co-expression system of C1 vector and S1 vector in HEK 293T cells , Set a blank control without cells and a negative control without hydroxychloroquine at the same time. After subtracting the reading value of the blank control well, calculate the inhibition rate of 3C protease at each concentration by the respective fluorescence values. The results are shown in Figure 6, which is the inhibition curve of hydroxychloroquine with gradient dilution concentration on SARS-COV-2 coronavirus 3C protease activity. After calculation, the half inhibitory concentration IC of hydroxychloroquine inhibiting SARS-COV-2 coronavirus 3C protease activity 50 is about 30nM.
综上实施例的描述，本发明构建一种能够测定3C蛋白酶活性的体系，并构建一种表达具有较高活性的3C蛋白酶的载体，两者可以组合作为筛选SARS-COV-2冠状病毒3C蛋白酶活性抑制剂的试剂盒，即该试剂盒中包括表达3C蛋白酶的底物的S1载体和表达具有活性的3C蛋白酶的C1载体，两者可以用于快速筛选出SARS-COV-2冠状病毒3C蛋白酶活性的抑制剂，对于抗SARS-COV-2冠状病毒感染的药物的开发提供了重要的数据支撑。In summary, the present invention constructs a system capable of measuring 3C protease activity and constructs a vector for expressing 3C protease with higher activity. The two can be combined to screen SARS-COV-2 coronavirus 3C protease An activity inhibitor kit, that is, the kit includes an S1 vector expressing a 3C protease substrate and a C1 vector expressing an active 3C protease, both of which can be used to quickly screen out the SARS-COV-2 coronavirus 3C protease Active inhibitors provide important data support for the development of drugs against SARS-COV-2 coronavirus infection.
以上实施例仅仅描述了SARS-COV-2冠状病毒3C蛋白酶活性抑制剂的筛选方法，由于在单正链RNA病毒中均存在3C蛋白酶，并且各自能够识别特异的酶切位点，因此可以根据上述实施例的描述构建单正链RNA病毒各自的测定3C蛋白酶活性的体系以及表达各自的具有活性的3C蛋白酶的表达载体，均可以用于筛选各自病毒的3C蛋白酶活性的抑制剂，用于抗病毒药物的开发。The above examples only describe the method for screening SARS-COV-2 coronavirus 3C protease activity inhibitors. Since 3C protease exists in single positive-strand RNA viruses, and each can recognize specific enzyme cleavage sites, it can be based on the above Description of Examples The construction of a system for measuring the 3C protease activity of each single positive-strand RNA virus and an expression vector expressing the respective active 3C protease can be used to screen the inhibitors of the 3C protease activity of the respective viruses for anti-virus Drug development.
此处描述的实施例只用于说明(作为例证)，技术人员所做的各种修改或变更也应包括在专利申请的实质范围内。The embodiments described here are only for illustration (as an illustration), and various modifications or changes made by technical personnel should also be included in the essential scope of the patent application.
工业应用性Industrial applicability
本发明提供了一种病毒3C蛋白酶活性抑制剂的筛选方法及筛选试剂盒，能够在细胞水平对病毒3C蛋白酶活性的抑制剂进行筛选，可以用于抗病毒的药物的开发，适于工业应用。The invention provides a screening method and a screening kit for inhibitors of virus 3C protease activity, which can screen the inhibitors of virus 3C protease activity at the cell level, can be used for the development of antiviral drugs, and is suitable for industrial applications.

Claims (11)

  1. 病毒3C蛋白酶活性抑制剂的筛选方法，其包括：The screening method of virus 3C protease activity inhibitor includes:
    将表达具有活性的病毒3C蛋白酶的表达载体、表达病毒3C蛋白酶的底物的表达载体与候选试剂在细胞中共孵育作为实验组，同时设置不添加候选试剂的阴性对照，根据实验组相对于阴性对照的荧光信号强度的变化来筛选病毒3C蛋白酶活性抑制剂；The expression vector expressing the active viral 3C protease, the expression vector expressing the substrate of the viral 3C protease and the candidate reagent are incubated in the cells as the experimental group, and a negative control without adding the candidate reagent is set according to the experimental group relative to the negative control Changes in the intensity of the fluorescent signal to screen for inhibitors of viral 3C protease activity;
    其中所述病毒3C蛋白酶的底物为IL1β蛋白和Gussia荧光素酶通过病毒3C蛋白酶的酶切位点连接而成的融合蛋白。The substrate of the virus 3C protease is a fusion protein formed by connecting IL1β protein and Gussia luciferase through the restriction site of the virus 3C protease.
  2. 根据权利要求1所述的方法，其中所述病毒3C蛋白酶为SARS-CoV-2冠状病毒3C蛋白酶。The method according to claim 1, wherein the viral 3C protease is SARS-CoV-2 coronavirus 3C protease.
  3. 根据权利要求2所述的方法，其中所述表达具有活性的病毒3C蛋白酶的表达载体的构建方法包括：The method according to claim 2, wherein the method for constructing the expression vector for expressing the active viral 3C protease comprises:
    A)在SARS-COV-2冠状病毒3C蛋白酶的核苷酸序列的5’和3’端分别连接SARS-COV-2冠状病毒的NSP7和NSP8序列，且在各连接处插入SARS-COV-2冠状病毒3C蛋白酶的酶切位点，获得SARS-COV-2冠状病毒3C蛋白酶重组表达序列；A) Connect the NSP7 and NSP8 sequences of SARS-COV-2 coronavirus to the 5'and 3'ends of the nucleotide sequence of the SARS-COV-2 coronavirus 3C protease respectively, and insert SARS-COV-2 at each connection The restriction site of the coronavirus 3C protease, the recombinant expression sequence of SARS-COV-2 coronavirus 3C protease was obtained;
    B)将步骤A)的3C蛋白酶重组表达序列克隆至真核表达载体，获得表达具有活性的病毒3C蛋白酶的表达载体。B) Cloning the 3C protease recombinant expression sequence of step A) into a eukaryotic expression vector to obtain an expression vector for expressing active viral 3C protease.
  4. 根据权利要求2所述的方法，其中所述表达病毒3C蛋白酶的底物的表达载体的构建方法包括：The method according to claim 2, wherein the method for constructing the expression vector expressing the substrate of the viral 3C protease comprises:
    a)将IL1β蛋白表达序列和Gussia荧光素酶表达序列通过SARS-COV-2冠状病毒3C蛋白酶的酶切位点连接，获得融合表达序列；a) Connect IL1β protein expression sequence and Gussia luciferase expression sequence through the restriction site of SARS-COV-2 coronavirus 3C protease to obtain fusion expression sequence;
    b)将步骤a)的融合表达序列克隆至真核表达载体，获得表达病毒3C蛋白酶的底物的表达载体。b) Cloning the fusion expression sequence of step a) into a eukaryotic expression vector to obtain an expression vector expressing the substrate of the viral 3C protease.
  5. 根据权利要求1-4中任一项所述的方法，其中所述荧光信号强度的变化为荧光信号强度的降低。The method according to any one of claims 1 to 4, wherein the change in the intensity of the fluorescent signal is a decrease in the intensity of the fluorescent signal.
  6. 用于权利要求2-5中任一项所述的方法的表达具有活性的SARS-COV-2冠状病毒3C蛋白酶的表达载体，任选地，所述表达具有活性的SARS-COV-2冠状病毒3C蛋白酶的表达载体的核苷酸序列如SEQ ID NO:7所示。The expression vector for expressing the active SARS-COV-2 coronavirus 3C protease used in the method of any one of claims 2-5, optionally, the expression of the active SARS-COV-2 coronavirus The nucleotide sequence of the expression vector of 3C protease is shown in SEQ ID NO:7.
  7. 用于权利要求2-5中任一项所述的方法的表达SARS-COV-2冠状病毒3C蛋白酶的底物的表达载体，任选地，所述表达SARS-COV-2冠状病毒3C蛋白酶的底物的 表达载体的核苷酸序列如SEQ ID NO:3所示。The expression vector for expressing the substrate of SARS-COV-2 coronavirus 3C protease used in the method of any one of claims 2-5, optionally, the expression vector for expressing SARS-COV-2 coronavirus 3C protease The nucleotide sequence of the expression vector of the substrate is shown in SEQ ID NO: 3.
  8. 用于筛选SARS-CoV-2冠状病毒3C蛋白酶活性抑制剂的试剂盒，其包括权利要求6所述的表达具有活性的SARS-CoV-2冠状病毒3C蛋白酶的表达载体，和权利要求7所述的表达SARS-CoV-2冠状病毒3C蛋白酶的底物的表达载体。A kit for screening SARS-CoV-2 coronavirus 3C protease activity inhibitor, which comprises the expression vector of claim 6 for expressing the active SARS-CoV-2 coronavirus 3C protease, and the claim 7 An expression vector that expresses the substrate of the SARS-CoV-2 coronavirus 3C protease.
  9. 病毒3C蛋白酶活性的测定方法，其包括以下步骤：The method for measuring virus 3C protease activity includes the following steps:
    1)将IL1β蛋白表达序列和Gussia荧光素酶表达序列通过病毒3C蛋白酶的酶切位点连接，获得融合表达序列；1) Connect IL1β protein expression sequence and Gussia luciferase expression sequence through the restriction site of virus 3C protease to obtain fusion expression sequence;
    2)将步骤1)的融合表达序列克隆至真核表达载体，获得表达载体；2) Clone the fusion expression sequence of step 1) into a eukaryotic expression vector to obtain an expression vector;
    3)将步骤2)的表达载体与病毒3C蛋白酶的表达体系在细胞中进行共表达，通过测定共表达体系的荧光信号强度来表征病毒3C蛋白酶的活性；3) Co-express the expression vector of step 2) and the expression system of the viral 3C protease in cells, and characterize the activity of the viral 3C protease by measuring the fluorescence signal intensity of the co-expression system;
    任选地，步骤3)中所述病毒3C蛋白酶的表达体系为将病毒3C蛋白酶的表达序列克隆至真核表达载体所构建的病毒3C蛋白酶的表达载体。Optionally, the expression system of the viral 3C protease in step 3) is an expression vector of the viral 3C protease constructed by cloning the expression sequence of the viral 3C protease into a eukaryotic expression vector.
  10. 根据权利要求9所述的方法，其中所述病毒3C蛋白酶为单正链RNA病毒的3C蛋白酶，优选为SARS-CoV-2冠状病毒3C蛋白酶。The method according to claim 9, wherein the viral 3C protease is the 3C protease of a single positive-strand RNA virus, preferably SARS-CoV-2 coronavirus 3C protease.
  11. 测定SARS-CoV-2冠状病毒3C蛋白酶活性的试剂盒，其包括权利要求7所述的表达SARS-CoV-2冠状病毒3C蛋白酶的底物的表达载体，任选地，所述表达SARS-CoV-2冠状病毒3C蛋白酶的底物的表达载体的核苷酸序列如SEQ ID NO:3所示。A kit for measuring the activity of SARS-CoV-2 coronavirus 3C protease, which includes the expression vector of claim 7 for expressing the substrate of SARS-CoV-2 coronavirus 3C protease, optionally, the expression of SARS-CoV -2 The nucleotide sequence of the expression vector of the substrate of the coronavirus 3C protease is shown in SEQ ID NO: 3.
PCT/CN2020/089144 2020-04-27 2020-05-08 Method for screening inhibitor for 3c protease activity of sars-cov-2 coronavirus and screening kit WO2021217696A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010343025.7 2020-04-27
CN202010343025.7A CN113637724B (en) 2020-04-27 2020-04-27 Screening method and screening kit for SARS-CoV-2 coronavirus 3C protease activity inhibitor

Publications (1)

Publication Number Publication Date
WO2021217696A1 true WO2021217696A1 (en) 2021-11-04

Family

ID=78331496

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/089144 WO2021217696A1 (en) 2020-04-27 2020-05-08 Method for screening inhibitor for 3c protease activity of sars-cov-2 coronavirus and screening kit

Country Status (2)

Country Link
CN (1) CN113637724B (en)
WO (1) WO2021217696A1 (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117088990B (en) * 2023-10-20 2024-02-20 浙江迪福润丝生物科技有限公司 Fluorescent reporting system for detecting activity of coronavirus protease inhibitor
CN117088988B (en) * 2023-10-20 2024-02-06 浙江迪福润丝生物科技有限公司 Fusion protein, plasmid and application thereof in HIV protease inhibitor screening and drug effect evaluation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105067574A (en) * 2015-06-18 2015-11-18 山东省医学科学院基础医学研究所 EV71 3C protease action target point detection kit and EV71 3C protease action target point detection method

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101921823B (en) * 2010-05-05 2013-01-02 中国科学院生物物理研究所 Method for screening SARS corona virus major protease inhibitor from traditional Chinese medicine and screened SARS corona virus major protease inhibitor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105067574A (en) * 2015-06-18 2015-11-18 山东省医学科学院基础医学研究所 EV71 3C protease action target point detection kit and EV71 3C protease action target point detection method

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BARTOK EVA, BAUERNFEIND FRANZ, KHAMINETS MARIA G, JAKOBS CHRISTOPHER, MONKS BRIAN, FITZGERALD KATHERINE A, LATZ EICKE, HORNUNG VEI: "iGLuc: a luciferase-based inflammasome and protease activity reporter", NATURE METHODS, NATURE PUBLISHING GROUP, UNITED STATES, 1 February 2013 (2013-02-01), United States , pages 147 - 154, XP055814592, [retrieved on 20210616], DOI: 10.1038/nmeth.2327 *
FENG YUFEI, XU QINGYUAN, YANG TAO, SUN ENCHENG, LI JUNPING, SHI DONGFANG, WU DONGLAI: "A novel self-cleavage system for production of soluble recombinant protein in Escherichia coli", PROTEIN EXPRESSION AND PURIFICATION, ACADEMIC PRESS, SAN DIEGO, CA., vol. 99, 1 July 2014 (2014-07-01), SAN DIEGO, CA. , pages 64 - 69, XP055777247, ISSN: 1046-5928, DOI: 10.1016/j.pep.2014.04.001 *
HEFEI HIGH-TECH INCUBATOR: "[Star Enterprise] The first new coronavirus 3C protease drug high-throughput detection cell system is launched!", 23 April 2020 (2020-04-23), CN, pages 1 - 2, XP009531691, Retrieved from the Internet <URL:https://www.sohu.com/a/390552824_99956155> *
KRICHEL BORIS, FALKE SVEN, HILGENFELD ROLF, REDECKE LARS, UETRECHT CHARLOTTE: "Processing of the SARS-CoV pp1a/ab nsp7–10 region", BIOCHEMICAL JOURNAL, PUBLISHED BY PORTLAND PRESS ON BEHALF OF THE BIOCHEMICAL SOCIETY., GB, vol. 477, no. 5, 13 March 2020 (2020-03-13), GB , pages 1009 - 1019, XP055862450, ISSN: 0264-6021, DOI: 10.1042/BCJ20200029 *
TE VELTHUIS AARTJAN J.W., VAN DEN WORM SJOERD H. E., SNIJDER ERIC J.: "The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 40, no. 4, 1 February 2012 (2012-02-01), GB , pages 1737 - 1747, XP055862453, ISSN: 0305-1048, DOI: 10.1093/nar/gkr893 *

Also Published As

Publication number Publication date
CN113637724B (en) 2023-07-21
CN113637724A (en) 2021-11-12

Similar Documents

Publication Publication Date Title
Perera et al. Cellular protein modification by poliovirus: the two faces of poly (rC)-binding protein
Li et al. DNA polymerase α interacts with H3-H4 and facilitates the transfer of parental histones to lagging strands
Xiang et al. Complete protein linkage map of poliovirus P3 proteins: interaction of polymerase 3Dpol with VPg and with genetic variants of 3AB
Verma et al. Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system
Noble et al. Conformational flexibility of the Dengue virus RNA-dependent RNA polymerase revealed by a complex with an inhibitor
Banerjee et al. Transcriptional repression of C4 complement by hepatitis C virus proteins
Zhong et al. Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis
Huber et al. Cleavage of RasGAP and phosphorylation of mitogen-activated protein kinase in the course of coxsackievirus B3 replication
Latha et al. Maspin mediates increased tumor cell apoptosis upon induction of the mitochondrial permeability transition
Poole et al. Apoptosis of liver-derived cells induced by parvovirus B19 nonstructural protein
Lackner et al. Persistence of bovine viral diarrhea virus is determined by a cellular cofactor of a viral autoprotease
Varjak et al. Magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of Semliki Forest virus
Adedeji et al. Biochemical characterization of Middle East respiratory syndrome coronavirus helicase
Yang et al. Formation and phosphorylation of the PINCH-1–integrin linked kinase–α-parvin complex are important for regulation of renal glomerular podocyte adhesion, architecture, and survival
Beran et al. The NS4A protein of hepatitis C virus promotes RNA-coupled ATP hydrolysis by the NS3 helicase
WO2021217696A1 (en) Method for screening inhibitor for 3c protease activity of sars-cov-2 coronavirus and screening kit
US7655419B2 (en) Methods and compositions for identifying anti-HCV agents
Ohbayashi et al. Enhancement of the structural stability of full-length clostridial collagenase by calcium ions
Ponia et al. Mitophagy antagonism by ZIKV reveals Ajuba as a regulator of PINK1 signaling, PKR-dependent inflammation, and viral invasion of tissues
Peters et al. Tethering soluble meprin α in an enzyme complex to the cell surface affects IBD-associated genes
Chen et al. N-terminal phosphorylation of phosphoprotein of vesicular stomatitis virus is required for preventing nucleoprotein from binding to cellular RNAs and for functional template formation
Gandikota et al. Mitochondrial import of dengue virus NS3 protease and cleavage of GrpEL1, a cochaperone of mitochondrial Hsp70
Niehues et al. The collectrin‐like part of the SARS‐CoV‐1 and‐2 receptor ACE2 is shed by the metalloproteinases ADAM10 and ADAM17
Ding et al. Klebsiella pneumoniae carbapenemase variants: the new threat to global public health
Bai et al. Identifying small-molecule inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase by establishing a fluorometric assay

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20932975

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20932975

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 06/06/2023)

122 Ep: pct application non-entry in european phase

Ref document number: 20932975

Country of ref document: EP

Kind code of ref document: A1