CN113637724B - Screening method and screening kit for SARS-CoV-2 coronavirus 3C protease activity inhibitor - Google Patents
Screening method and screening kit for SARS-CoV-2 coronavirus 3C protease activity inhibitor Download PDFInfo
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Abstract
The invention discloses a screening method and a screening kit of SARS-CoV-2 coronavirus 3C protease activity inhibitor. The screening method of the 3C protease activity inhibitor can screen the inhibitor of the virus 3C protease activity at the cellular level by constructing a system capable of measuring the 3C protease activity and a carrier for expressing the 3C protease with higher activity, and can be used for developing antiviral drugs.
Description
Technical Field
The invention belongs to the field of molecular and cell biology, in particular to a screening method and a screening kit of a virus 3C protease activity inhibitor, and particularly relates to a screening method and a screening kit of a SARS-CoV-2 coronavirus 3C protease activity inhibitor.
Background
The novel coronavirus SARS-CoV-2 is the 7 th coronavirus discovered in 2019 and capable of infecting human, and is named by the world health organization in 1/12 of 2020, and can cause novel coronavirus pneumonia COVID-19, which can cause serious threat to human health.
SARS-COV-2 coronavirus is the same as SARS coronavirus (SARS-CoV) in 2003 and MERS coronavirus (MERS-CoV) in "middle east respiratory syndrome", and belongs to single positive strand RNA virus. By gene sequence alignment, SARS-COV-2 has about 80% similarity to SARS-CoV and 40% similarity to MERS-CoV. The SARS-COV-2 coronavirus has strong infectivity and is a great potential threat to human health, so that the search for drugs against SARS-COV-2 coronavirus is imperative.
The 3C protease, also known as 3CL protease or M protease, plays an important role in the protein maturation process of single positive strand RNA viruses, and is capable of recognizing specific cleavage sites, cleaving the multimeric proteins of the virus, cleaving them into a plurality of active proteins, and finally assembling into new viral particles. Therefore, the catalytic function of inhibiting 3C protease can effectively inhibit the cleavage of virus precursor protein, block the replication of virus and play a role in resisting single positive strand RNA virus. The screening of inhibitors capable of inhibiting 3C protease activity has become an important way to currently develop drugs against single positive strand RNA virus, and thus, the establishment of a method for screening inhibitors of 3C protease activity in vitro against a newly discovered single positive strand RNA virus-SARS-COV-2 coronavirus has important clinical significance for the development of drugs against SARS-COV-2 coronavirus.
Disclosure of Invention
In view of one or more of the problems of the prior art, one aspect of the present invention provides a method for screening inhibitors of viral 3C protease activity, comprising:
co-incubating an expression vector expressing active viral 3C protease, an expression vector expressing a substrate for the viral 3C protease, and a candidate reagent in cells to form an experimental group, setting a negative control without the candidate reagent, and screening a viral 3C protease activity inhibitor according to the change of the fluorescence signal intensity of the experimental group relative to the negative control;
wherein the substrate of the virus 3C protease is fusion protein formed by connecting IL1 beta protein and Gussia luciferase through enzyme cleavage sites of the virus 3C protease.
The virus 3C protease is SARS-CoV-2 coronavirus 3C protease.
The construction method of the expression vector for expressing the virus 3C protease with activity comprises the following steps:
a) Connecting the 5 'and 3' ends of the nucleotide sequence of SARS-COV-2 coronavirus 3C protease with NSP7 and NSP8 sequences of SARS-COV-2 coronavirus respectively, and inserting enzyme cutting sites of SARS-COV-2 coronavirus 3C protease at each connecting place to obtain recombinant expression sequence of SARS-COV-2 coronavirus 3C protease;
b) Cloning the 3C protease recombinant expression sequence of the step A) to a eukaryotic expression vector to obtain an expression vector for expressing the virus 3C protease with activity.
The construction method of the expression vector for expressing the substrate of the viral 3C protease comprises the following steps:
a) Connecting an IL1 beta protein expression sequence and a Gussia luciferase expression sequence through an enzyme cleavage site of SARS-COV-2 coronavirus 3C protease to obtain a fusion expression sequence;
b) Cloning the fusion expression sequence of the step a) to a eukaryotic expression vector to obtain an expression vector for expressing a substrate of the viral 3C protease.
The change in fluorescence signal intensity is a decrease in fluorescence signal intensity.
Expression vectors for expressing active SARS-COV-2 coronavirus 3C protease for use in the above methods are also within the scope of the present invention; optionally, the nucleotide sequence of the expression vector for expressing active SARS-COV-2 coronavirus 3C protease is shown in SEQ ID NO. 7.
Expression vectors for the above methods for expressing a substrate for SARS-COV-2 coronavirus 3C protease are also within the scope of the present invention; optionally, the nucleotide sequence of the expression vector for expressing the substrate of SARS-COV-2 coronavirus 3C protease is shown as SEQ ID NO. 3.
In another aspect, the invention provides a kit for screening SARS-CoV-2 coronavirus 3C protease activity inhibitor, comprising the above expression vector for expressing active SARS-CoV-2 coronavirus 3C protease, and the above expression vector for expressing a substrate of SARS-CoV-2 coronavirus 3C protease.
In another aspect, the present invention provides a method for determining the activity of a viral 3C protease, comprising the steps of:
1) Connecting an IL1 beta protein expression sequence and a Gussia luciferase expression sequence through an enzyme cleavage site of a virus 3C protease to obtain a fusion expression sequence;
2) Cloning the fusion expression sequence of the step 1) to a eukaryotic expression vector to obtain an expression vector;
3) Co-expressing the expression vector of step 2) and the expression system of the viral 3C protease in cells, and characterizing the activity of the viral 3C protease by measuring the fluorescence signal intensity of the co-expression system;
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.
The viral 3C protease is a 3C protease of a single positive strand RNA virus, preferably a SARS-CoV-2 coronavirus 3C protease.
In yet another aspect, the present invention provides a kit for determining the activity of SARS-CoV-2 coronavirus 3C protease, comprising the above-mentioned expression vector for expressing a substrate for SARS-CoV-2 coronavirus 3C protease; optionally, the nucleotide sequence of the expression vector for expressing the substrate of SARS-CoV-2 coronavirus 3C protease is shown as SEQ ID NO. 3.
The screening method of SARS-COV-2 coronavirus 3C protease activity inhibitor based on the above technical scheme can evaluate SARS-COV-2 coronavirus 3C protease activity at cell level by establishing a system for expressing active SARS-COV-2 coronavirus 3C protease and constructing a report system for activating luciferase activity based on 3C protease cleavage, and can further screen SARS-COV-2 coronavirus 3C protease activity inhibitor by using the evaluation system. The screening method also provides a kit for screening the SARS-COV-2 coronavirus 3C protease activity inhibitor, so that the inhibitor capable of inhibiting the SARS-COV-2 coronavirus 3C protease activity can be rapidly screened out, and the method can be effectively used for developing medicaments for resisting the SARS-COV-2 coronavirus.
Drawings
FIG. 1 is a schematic diagram of the construction of a system for determining the activity of SARS-CoV-2 coronavirus 3C protease;
FIG. 2 is a plasmid map of expression vector S1;
FIG. 3 is a plasmid map of expression vector C1;
FIG. 4 is a graph showing comparison of activities of 3C protease expressed by expression vector C1 and expression vector C2;
FIG. 5 is a graph showing the inhibitory effect of candidate compounds and positive drugs on 3C protease activity;
FIG. 6 is a graph showing the inhibition of SARS-CoV-2 coronavirus 3C protease activity by hydroxychloroquine.
Detailed Description
The invention aims to provide a screening method of a virus 3C protease activity inhibitor, in particular to a screening method of a SARS-COV-2 coronavirus 3C protease activity inhibitor, and provides a kit for rapidly screening the SARS-COV-2 coronavirus 3C protease activity inhibitor based on the screening method.
To achieve the above object, the present inventors refer to the following principle: the IL1 beta protein precursor can be aggregated and precipitated in cells, and can release C-terminal soluble IL1 through the cleavage of protease CASPASE1, so that a fusion carrier for the serial expression of the IL1 beta protein precursor and Gussia luciferase (Gluc) is constructed, as shown in figure 1, the two expressed proteins are connected by a cleavage site of the 3C protease, when the 3C protease is not present in the cells, the fusion protein can be aggregated and precipitated, and when the 3C protease cleaves the cleavage site between the IL1 beta and Gussia luciferase, the activated soluble Gussia luciferase is released, therefore, the activity of the 3C protease can be represented through the high or low fluorescence (chemiluminescence) signal intensity, and the system capable of representing the activity of the 3C protease is also included in the invention. In order to screen an inhibitor of SARS-COV-2 coronavirus 3C protease activity, it is critical to obtain an expression vector for expressing 3C protease with higher activity, and the inventor constructs an expression vector capable of expressing 3C protease activity by utilizing the characteristics that two sequences NSP7 and NSP8 of SARS-COV-2 coronavirus encode proteins with smaller lengths and the homology of a boundary cleavage site and a cleavage site of 3C protease is high, so as to screen the inhibitor of 3C protease activity.
The present invention will be further described with reference to the following method for screening for inhibitors of SARS-COV-2 coronavirus 3C protease activity. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The methods used in the examples below are conventional methods unless otherwise specified, and specific steps can be found in: molecular cloning guidelines (Molecular Cloning: A Laboratory Manual) Sambrook, j., russell, david w., molecular Cloning: A Laboratory Manual,3rd edition,2001,NY,Cold Spring Harbor).
The various biomaterials described in the examples were obtained by merely providing an experimental route for achieving the objectives of the specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.
The sequences used were synthesized by Shanghai Biotechnology Inc.
Example 1: construction of SARS-CoV-2 coronavirus 3C protease activity determination system
The embodiment constructs a fusion protein containing a 3C protease cleavage site of SARS-CoV-2 coronavirus as a substrate of the 3C protease of SARS-CoV-2 coronavirus, as shown in figure 1, the substrate comprises an IL1 beta protein-3C cleavage site (the amino acid sequence of the 3C cleavage site is shown as SEQ ID NO:1 in a sequence table) -Gussia luciferase (Gluc), namely the IL1 beta protein and Gussia luciferase are connected through the 3C cleavage site, the amino acid sequence of the fusion protein is shown as SEQ ID NO:2 in the sequence table, and the specific construction method comprises the following steps:
the gene sequence of 3C restriction site-Gussia luciferase of 3C restriction enzyme site of 3C coronavirus for expressing IL1 beta protein-SARS-CoV-2 is synthesized, and cloned to a vector pCDNA3.1 (Thermo Fisher Scientific) to obtain an expression vector for expressing substrate of 3C protease, named as expression vector S1, the nucleotide sequence of the expression vector S1 is shown as SEQ ID NO. 3, and the plasmid map is shown as figure 2. The expression vector S1 is transfected into HEK293T cells, and fusion protein obtained by expression can be used as a substrate of SARS-CoV-2 coronavirus 3C protease, and the activity of the 3C protease can be measured in the following examples.
If the 3C protease is not used for shearing the expressed fusion protein in the system when the expression vector S1 is expressed, the fusion protein is subjected to aggregation precipitation to be inactivated; when the expressed fusion protein is sheared by the presence of the 3C protease, the luciferase is activated, so that the activity of the 3C protease can be characterized by reading the change in the intensity of the fluorescent signal, thereby screening inhibitors that inhibit the activity of the 3C protease according to the activity of the 3C protease. Thus, a kit for determining the activity of SARS-CoV-2 coronavirus 3C protease can be provided based on the expression vector S1 obtained in this example, wherein the kit comprises the expression vector S1, and when the kit is used, the expression vector S1 and the expression vector of SARS-CoV-2 coronavirus 3C protease can be co-transfected into HEK293T cells for co-expression, and the activity of 3C protease can be characterized by reading the fluorescence signal intensity in the co-expression system.
Example 2: expression of SARS-CoV-2 coronavirus 3C protease active in vitro
After SARS-CoV-2 coronavirus invades human body, it uses self-RNA as template to make translation synthesis of multimeric protein body containing several proteins, only after the 3C proteinase with activity is cut, it can be cut into independent active proteins for subsequent replication and assembly. However, the expressed SARS-CoV-2 coronavirus 3C protease in the prior art has very low protease activity or even no activity, so that the expression structure of the expressed 3C protease needs to be specifically designed, so that the expressed 3C protease can be subjected to self-shearing and dimerization to obtain the biological function of the expressed 3C protease, and the activity of the protease is improved.
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 of which is shown as SEQ ID NO: 4) are respectively connected with the NSP7 (the nucleotide sequence of which is shown as SEQ ID NO: 5) and NSP8 (the nucleotide sequence of which is shown as SEQ ID NO: 6) sequences of the SARS-COV-2 coronavirus, and the expression sequence of the enzyme cleavage site of the 3C protease is inserted at the connection part, so that a recombinant expression sequence is constructed, which can promote the 3C protease to complete self-shearing and dimerization after expression, thereby becoming an activated protein with biological functions. After cloning the recombinant expression sequence to eukaryotic expression vector pCDNA3.1, an expression vector for expressing SARS-COV-2 coronavirus 3C protease with higher biological activity is obtained, which is named as expression vector C1, the nucleotide sequence of which is shown as SEQ ID NO. 7 in a sequence table, as shown in FIG. 3, and the plasmid map of the expression vector C1 is shown. Meanwhile, an independent 3C protease expression sequence is synthesized and cloned to a eukaryotic expression vector pCDNA3.1 as a negative control, and the eukaryotic expression vector is named as an expression vector C2, and the nucleotide sequence of the eukaryotic expression vector is shown as SEQ ID NO. 8 in a sequence table.
To verify that the expression vector C1 obtained in this example was indeed able to obtain a 3C protease with higher activity, HEK293T cells were resuspended to 1X 10 5 Individual cells/ml, 100 μl per well was plated in 96-well plates, and DNA transfection was performed after 8 hours; 150ng of DNA was transferred per well, which contained 50ng of plasmid from expression vector S1 (S1 plasmid) and a gradient of diluted plasmids from expression vectors C1 and C2 (0 ng, 25ng, 50ng, 100ng, C1/C2 plasmid), and the deficient DNA was complemented with pCDNA3.1 empty vector. The specific transfection procedure was: preparation of 10. Mu.l of Opti-MEM (Gibco) medium was added to the corresponding DNA, mixed well, added with lipofiter transfection reagent (Hanbio, shanghai) 3 times the mass of DNA, allowed to stand at room temperature for 10 minutes, then added dropwise to a 96-well plate at 5% CO 2 After 24 hours of incubation in the incubator, 15. Mu.l of Renilla-glo reagent (Promega) was added to each well, and after 5 minutes of reaction, the fluorescence value was read.
As shown in FIG. 4, it can be seen that the 3C protease expressed by the HEK293T cell transfected by the expression vector C1 constructed in the example can cut the substrate expressed by the expression vector S1 in a quality-dependent manner, namely, the more the expression vector C1 is added, the more the substrate is cut, the higher the fluorescence signal intensity (the larger the fluorescence value) is, the fluorescence value after the substrate is cut by the C2 plasmid with the same transfection amount is significantly higher, the activity of the 3C protease expressed by the expression vector C1 is proved to be far higher than the activity of the 3C protease expressed by the expression vector C2, and the calculated enzyme cleavage activity of the 3C protease expressed by the C1 is about 1000 times of the enzyme cleavage activity of the 3C protease expressed by the C2.
Example 3: screening of inhibitors of SARS-COV-2 coronavirus 3C protease activity
This example uses the expression vector S1 constructed in example 1 and the expression vector C1 constructed in example 2 described above to screen for inhibitors of SARS-COV-2 coronavirus 3C protease activity, and specifically comprises the following procedures:
3.1, HEK293T cells were plated in 10 cm cell culture dishes and transfected when the density reached 75%.
3.2 mixing 3 mug of C1 vector for expressing 3C protease and 6 mug of S1 vector for expressing 3C protease substrate, adding into 1ml of Opti-MEM culture medium, mixing uniformly, then adding 27 mug of transfection reagent lipofiter, blowing and sucking uniformly, standing at room temperature for 10 minutes, and dripping HEK293T cells for protein expression. After 8 hours of transfection, the cells were digested with 0.25% pancreatin and resuspended by centrifugation and diluted to 1X 10 5 Individual cells/ml.
3.3 gradient dilutions (0.0, 3.0, 10.0, 30.0. Mu.M) of DMSO-dissolved compounds (Abidol (Arbidol), lopinavir (lopinavir), nelfinavir (nelfinavir) and Hydroxychloroquine (Hydroxychloroquinone) (all available from MedChemexpress) were added 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 2019Atomistic Insights into the Inhibitory Mechanisms,Biochemistry,2020.04.15,doi:10.1021/acs.0c00160) as candidate compounds) in a volume ratio of 1:1000 to a cell resuspension followed by 100. Mu.l of each well to 96Well plates continued at 5% CO 2 Is cultured in an incubator at 37 ℃ for 16 hours; the 96-well plate was removed, 15 μ l Renilla Glo reagent (Promega) was added, incubated at room temperature for 5 minutes, and fluorescence values were read. And simultaneously setting a blank control of a blank culture medium without adding cells and a negative control without adding candidate compounds or positive drugs, and respectively calculating the relative activity of 3C protease of each candidate compound or positive drug under the gradient concentration through respective fluorescence values after deducting the read value of the blank control hole. As a result, as shown in fig. 5, it was found that both of the above two candidate compounds and the two positive drugs showed inhibitory effects on the 3C protease activity of SARS-COV-2 coronavirus, and showed dose dependency, i.e., the higher the concentration of the positive drug, the stronger the inhibitory effect on the 3C protease activity, in a certain concentration range, for the relative activities of the 3C protease at different concentrations of the two candidate compounds and the two positive drugs. The nelfinavir of the other two candidate compounds does not show inhibition of SARS-COV-2 coronavirus 3C protease activity, while hydroxychloroquine shows inhibition of SARS-COV-2 coronavirus 3C protease activity and shows dose dependency as two positive drugs, so hydroxychloroquine can be selected as a candidate compound for preparing a drug for resisting SARS-COV-2 coronavirus infection.
3.4, following the above procedure, the co-expression system of C1 vector and S1 vector in HEK293T cells was treated with hydroxychloroquine at gradient concentrations of 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100. Mu.M, while a blank control without cell addition of blank medium and a negative control without hydroxychloroquine addition were set, and after subtraction of the read values from the blank control wells, the inhibition rate of 3C protease at each concentration was calculated by the respective fluorescence values. As shown in FIG. 6, the results of the curve of the gradient dilution concentration of hydroxychloroquine on the inhibition of SARS-COV-2 coronavirus 3C protease activity are shown, and the calculated half inhibition concentration IC of hydroxychloroquine on the inhibition of SARS-COV-2 coronavirus 3C protease activity 50 About 30nM.
In summary, the present invention constructs a system capable of measuring 3C protease activity and constructs a vector expressing 3C protease with higher activity, which can be combined as a kit for screening 3C protease activity inhibitors of SARS-COV-2 coronavirus, i.e. the kit comprises S1 vector expressing 3C protease substrate and C1 vector expressing 3C protease with activity, which can be used for rapidly screening out 3C protease activity inhibitors of SARS-COV-2 coronavirus, and provides important data support for the development of anti-SARS-COV-2 coronavirus infection drugs.
The above examples merely describe a screening method for inhibitors of 3C protease activity of SARS-COV-2 coronavirus, and since 3C protease is present in each single positive strand RNA virus and each is capable of recognizing a specific cleavage site, a system for measuring 3C protease activity of each single positive strand RNA virus and an expression vector expressing each active 3C protease can be constructed according to the description of the above examples, and can be used for screening inhibitors of 3C protease activity of each virus for development of antiviral drugs.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
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Ala Leu His Leu Asn Gly Gln Asn Ile Asn Gln Gln Val Ile Phe Ser
145 150 155 160
Met Ser Phe Val Gln Gly Glu Pro Ser Asn Asp Lys Ile Pro Val Ala
165 170 175
Leu Gly Leu Lys Gly Lys Asn Leu Tyr Leu Ser Cys Val Met Lys Asp
180 185 190
Gly Thr Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Gln Tyr Pro
195 200 205
Lys Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Val Lys
210 215 220
Ser Lys Val Glu Phe Glu Ser Ala Glu Phe Pro Asn Trp Tyr Ile Ser
225 230 235 240
Thr Ser Gln Ala Glu His Lys Pro Val Phe Leu Gly Asn Asn Ser Gly
245 250 255
Gln Asp Ile Ile Asp Phe Thr Met Glu Ser Val Ser Ser Ser Ala Val
260 265 270
Leu Gln Ser Gly Phe Arg Glu Ala Lys Pro Thr Glu Asn Asn Glu Asp
275 280 285
Phe Asn Ile Val Ala Val Ala Ser Asn Phe Ala Thr Thr Asp Leu Asp
290 295 300
Ala Asp Arg Gly Lys Leu Pro Gly Lys Lys Leu Pro Leu Glu Val Leu
305 310 315 320
Lys Glu Met Glu Ala Asn Ala Arg Lys Ala Gly Cys Thr Arg Gly Cys
325 330 335
Leu Ile Cys Leu Ser His Ile Lys Cys Thr Pro Lys Met Lys Lys Phe
340 345 350
Ile Pro Gly Arg Cys His Thr Tyr Glu Gly Asp Lys Glu Ser Ala Gln
355 360 365
Gly Gly Ile Gly Glu Ala Ile Val Asp Ile Pro Glu Ile Pro Gly Phe
370 375 380
Lys Asp Leu Glu Pro Met Glu Gln Phe Ile Ala Gln Val Asp Leu Cys
385 390 395 400
Val Asp Cys Thr Thr Gly Cys Leu Lys Gly Leu Ala Asn Val Gln Cys
405 410 415
Ser Asp Leu Leu Lys Lys Trp Leu Pro Gln Arg Cys Ala Thr Phe Ala
420 425 430
Ser Lys Ile Gln Gly Gln Val Asp Lys Ile Lys Gly Ala Gly Gly Asp
435 440 445
Asp Tyr Lys Asp Asp Asp Asp Lys
450 455
<210> 3
<211> 6761
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttggta ccgagctcgg atccgaattc gccaccatgg caactgttcc 960
tgaactcaac tgtgaaatgc caccttttga cagtgatgag aatgacctgt tctttgaagt 1020
tgacggaccc caaaagatga agggctgctt ccaaaccttt gacctgggct gtcctgatga 1080
gagcatccag cttcaaatct cgcagcagca catcaacaag agcttcaggc aggcagtatc 1140
actcattgtg gctgtggaga agctgtggca gctacctgtg tctttcccgt ggaccttcca 1200
ggatgaggac atgagcacct tcttttcctt catctttgaa gaagagccca tcctctgtga 1260
ctcatgggat gatgatgata acctgttagt ctgcgatgta cccattagac aactgcacta 1320
caggctccga gatgaacaac aaaaaagcct cgtgctgtcg gacccatatg agctgaaagc 1380
tctccacctc aatggacaga atatcaacca acaagtgata ttctccatga gctttgtaca 1440
aggagaacca agcaacgaca aaatacctgt ggccttgggc ctcaaaggaa agaatctata 1500
cctgtcctgt gtaatgaaag acggcacacc caccctgcag ctggagagtg tggatcccaa 1560
gcaataccca aagaagaaga tggaaaaacg gtttgtcttc aacaagatag aagtcaagag 1620
caaagtggag tttgagtctg cagagttccc caactggtac atcagcacct cacaagcaga 1680
gcacaagcct gtcttcctgg gaaacaacag tggtcaggac ataattgact tcaccatgga 1740
atccgtgtct tcctcagctg ttttgcagag tggttttaga gaggccaagc ccaccgagaa 1800
caacgaagac ttcaacatcg tggccgtggc cagcaacttc gcgaccacgg atctcgatgc 1860
tgaccgcggg aagttgcccg gcaagaagct gccgctcgag gtgctcaaag agatggaagc 1920
caatgcccgg aaagctggct gcaccagggg ctgtctgatc tgcctgtccc acatcaagtg 1980
cacgcccaag atgaagaagt tcatcccagg acgctgccac acctacgaag gcgacaaaga 2040
gtccgcacag ggcggcatag gcgaggcgat cgtcgacatt cctgagattc ctgggttcaa 2100
ggacttggag cccatggagc agttcatcgc acaggtcgat ctgtgtgtgg actgcacaac 2160
tggctgcctc aaagggcttg ccaacgtgca gtgttctgac ctgctcaaga agtggctgcc 2220
gcaacgctgt gcgacctttg ccagcaagat ccagggccag gtggacaaga tcaagggggc 2280
cggtggtgac gattacaagg atgacgacga taagtgagtt aactctagag ggcccgttta 2340
aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 2400
ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 2460
ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 2520
ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc 2580
tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg 2640
tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 2700
cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 2760
ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 2820
gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg 2880
atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 2940
ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt 3000
gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta 3060
attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga 3120
agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc 3180
ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc 3240
ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc 3300
tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag 3360
aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt 3420
atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa 3480
gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg 3540
gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc 3600
ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca 3660
gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc 3720
actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca 3780
tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat 3840
acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca 3900
cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg 3960
ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc 4020
gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct 4080
ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct 4140
acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac 4200
ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc 4260
tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag 4320
atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg 4380
ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact 4440
tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 4500
aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 4560
atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc 4620
ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 4680
gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 4740
ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 4800
ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 4860
cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 4920
cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 4980
accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 5040
acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 5100
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 5160
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 5220
atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 5280
agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 5340
acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 5400
gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 5460
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 5520
gcaaacaaac caccgctggt agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa 5580
aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 5640
aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 5700
ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 5760
acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 5820
ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 5880
gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 5940
taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 6000
tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 6060
gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 6120
cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 6180
aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 6240
cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 6300
tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 6360
gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 6420
tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 6480
gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 6540
ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 6600
cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 6660
agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 6720
gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c 6761
<210> 4
<211> 918
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
agtggtttta gaaaaatggc attcccatct ggtaaagttg agggttgtat ggtacaagta 60
acttgtggta caactacact taacggtctt tggcttgatg acgtagttta ctgtccaaga 120
catgtgatct gcacctctga agacatgctt aaccctaatt atgaagattt actcattcgt 180
aagtctaatc ataatttctt ggtacaggct ggtaatgttc aactcagggt tattggacat 240
tctatgcaaa attgtgtact taagcttaag gttgatacag ccaatcctaa gacacctaag 300
tataagtttg ttcgcattca accaggacag actttttcag tgttagcttg ttacaatggt 360
tcaccatctg gtgtttacca atgtgctatg aggcccaatt tcactattaa gggttcattc 420
cttaatggtt catgtggtag tgttggtttt aacatagatt atgactgtgt ctctttttgt 480
tacatgcacc atatggaatt accaactgga gttcatgctg gcacagactt agaaggtaac 540
ttttatggac cttttgttga caggcaaaca gcacaagcag ctggtacgga cacaactatt 600
acagttaatg ttttagcttg gttgtacgct gctgttataa atggagacag gtggtttctc 660
aatcgattta ccacaactct taatgacttt aaccttgtgg ctatgaagta caattatgaa 720
cctctaacac aagaccatgt tgacatacta ggacctcttt ctgctcaaac tggaattgcc 780
gttttagata tgtgtgcttc attaaaagaa ttactgcaaa atggtatgaa tggacgtacc 840
atattgggta gtgctttatt agaagatgaa tttacacctt ttgatgttgt tagacaatgc 900
tcaggtgtta ctttccaa 918
<210> 5
<211> 237
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
atgtctaaaa tgtcagatgt aaagtgcaca tcagtagtct tactctcagt tttgcaacaa 60
ctcagagtag aatcatcatc taaattgtgg gctcaatgtg tccagttaca caatgacatt 120
ctcttagcta aagatactac tgaagccttt gaaaaaatgg tttcactact ttctgttttg 180
ctttccatgc agggtgctgt agacataaac aagctttgtg aagaaatgct ggacaac 237
<210> 6
<211> 582
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gagtttagtt cccttccatc atatgcagct tttgctactg ctcaagaagc ttatgagcag 60
gctgttgcta atggtgattc tgaagttgtt cttaaaaagt tgaagaagtc tttgaatgtg 120
gctaaatctg aatttgaccg tgatgcagcc atgcaacgta agttggaaaa gatggctgat 180
caagctatga cccaaatgta taaacaggct agatctgagg acaagagggc aaaagttact 240
agtgctatgc agacaatgct tttcactatg cttagaaagt tggataatga tgcactcaac 300
aacattatca acaatgcaag agatggttgt gttcccttga acataatacc tcttacaaca 360
gcagccaaac taatggttgt cataccagac tataacacat ataaaaatac gtgtgatggt 420
acaacattta cttatgcatc agcattgtgg gaaatccaac aggttgtaga tgcagatagt 480
aaaattgttc aacttagtga aattagtatg gacaattcac ctaatttagc atggcctctt 540
attgtaacag ctttaagggc caattctgct gtcaaattac ag 582
<210> 7
<211> 1770
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atgtctaaaa tgtcagatgt aaagtgcaca tcagtagtct tactctcagt tttgcaacaa 60
ctcagagtag aatcatcatc taaattgtgg gctcaatgtg tccagttaca caatgacatt 120
ctcttagcta aagatactac tgaagccttt gaaaaaatgg tttcactact ttctgttttg 180
ctttccatgc agggtgctgt agacataaac aagctttgtg aagaaatgct ggacaactca 240
gctgttttgc agagtggttt tagaaaaatg gcattcccat ctggtaaagt tgagggttgt 300
atggtacaag taacttgtgg tacaactaca cttaacggtc tttggcttga tgacgtagtt 360
tactgtccaa gacatgtgat ctgcacctct gaagacatgc ttaaccctaa ttatgaagat 420
ttactcattc gtaagtctaa tcataatttc ttggtacagg ctggtaatgt tcaactcagg 480
gttattggac attctatgca aaattgtgta cttaagctta aggttgatac agccaatcct 540
aagacaccta agtataagtt tgttcgcatt caaccaggac agactttttc agtgttagct 600
tgttacaatg gttcaccatc tggtgtttac caatgtgcta tgaggcccaa tttcactatt 660
aagggttcat tccttaatgg ttcatgtggt agtgttggtt ttaacataga ttatgactgt 720
gtctcttttt gttacatgca ccatatggaa ttaccaactg gagttcatgc tggcacagac 780
ttagaaggta acttttatgg accttttgtt gacaggcaaa cagcacaagc agctggtacg 840
gacacaacta ttacagttaa tgttttagct tggttgtacg ctgctgttat aaatggagac 900
aggtggtttc tcaatcgatt taccacaact cttaatgact ttaaccttgt ggctatgaag 960
tacaattatg aacctctaac acaagaccat gttgacatac taggacctct ttctgctcaa 1020
actggaattg ccgttttaga tatgtgtgct tcattaaaag aattactgca aaatggtatg 1080
aatggacgta ccatattggg tagtgcttta ttagaagatg aatttacacc ttttgatgtt 1140
gttagacaat gctcaggtgt tactttccaa agtgcagtga aaagagagtt tagttccctt 1200
ccatcatatg cagcttttgc tactgctcaa gaagcttatg agcaggctgt tgctaatggt 1260
gattctgaag ttgttcttaa aaagttgaag aagtctttga atgtggctaa atctgaattt 1320
gaccgtgatg cagccatgca acgtaagttg gaaaagatgg ctgatcaagc tatgacccaa 1380
atgtataaac aggctagatc tgaggacaag agggcaaaag ttactagtgc tatgcagaca 1440
atgcttttca ctatgcttag aaagttggat aatgatgcac tcaacaacat tatcaacaat 1500
gcaagagatg gttgtgttcc cttgaacata atacctctta caacagcagc caaactaatg 1560
gttgtcatac cagactataa cacatataaa aatacgtgtg atggtacaac atttacttat 1620
gcatcagcat tgtgggaaat ccaacaggtt gtagatgcag atagtaaaat tgttcaactt 1680
agtgaaatta gtatggacaa ttcacctaat ttagcatggc ctcttattgt aacagcttta 1740
agggccaatt ctgctgtcaa attacagtag 1770
<210> 8
<211> 921
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
atgagtggtt ttagaaaaat ggcattccca tctggtaaag ttgagggttg tatggtacaa 60
gtaacttgtg gtacaactac acttaacggt ctttggcttg atgacgtagt ttactgtcca 120
agacatgtga tctgcacctc tgaagacatg cttaacccta attatgaaga tttactcatt 180
cgtaagtcta atcataattt cttggtacag gctggtaatg ttcaactcag ggttattgga 240
cattctatgc aaaattgtgt acttaagctt aaggttgata cagccaatcc taagacacct 300
aagtataagt ttgttcgcat tcaaccagga cagacttttt cagtgttagc ttgttacaat 360
ggttcaccat ctggtgttta ccaatgtgct atgaggccca atttcactat taagggttca 420
ttccttaatg gttcatgtgg tagtgttggt tttaacatag attatgactg tgtctctttt 480
tgttacatgc accatatgga attaccaact ggagttcatg ctggcacaga cttagaaggt 540
aacttttatg gaccttttgt tgacaggcaa acagcacaag cagctggtac ggacacaact 600
attacagtta atgttttagc ttggttgtac gctgctgtta taaatggaga caggtggttt 660
ctcaatcgat ttaccacaac tcttaatgac tttaaccttg tggctatgaa gtacaattat 720
gaacctctaa cacaagacca tgttgacata ctaggacctc tttctgctca aactggaatt 780
gccgttttag atatgtgtgc ttcattaaaa gaattactgc aaaatggtat gaatggacgt 840
accatattgg gtagtgcttt attagaagat gaatttacac cttttgatgt tgttagacaa 900
tgctcaggtg ttactttcca a 921
Claims (6)
1. A method of screening for inhibitors of viral 3C protease activity comprising:
co-incubating an expression vector expressing active viral 3C protease, an expression vector expressing a substrate for the viral 3C protease, and a candidate reagent in cells to form an experimental group, setting a negative control without the candidate reagent, and screening a viral 3C protease activity inhibitor according to the change of the fluorescence signal intensity of the experimental group relative to the negative control;
wherein the substrate of the virus 3C protease is fusion protein formed by connecting IL1 beta protein and Gussia luciferase through enzyme cleavage sites of the virus 3C protease;
the nucleotide sequence of the expression vector for expressing active SARS-COV-2 coronavirus 3C protease is shown as SEQ ID NO. 7;
the virus 3C protease is SARS-CoV-2 coronavirus 3C protease.
2. The method according to claim 1, wherein the construction method of the expression vector for expressing the active viral 3C protease comprises:
a) Connecting the 5 'and 3' ends of the nucleotide sequence of SARS-COV-2 coronavirus 3C protease with NSP7 and NSP8 sequences of SARS-COV-2 coronavirus respectively, and inserting enzyme cutting sites of SARS-COV-2 coronavirus 3C protease at each connecting place to obtain recombinant expression sequence of SARS-COV-2 coronavirus 3C protease;
b) Cloning the 3C protease recombinant expression sequence of the step A) to a eukaryotic expression vector to obtain an expression vector for expressing the virus 3C protease with activity.
3. The method according to claim 1, wherein the method for constructing the expression vector for expressing the substrate of the viral 3C protease comprises:
a) Connecting an IL1 beta protein expression sequence and a Gussia luciferase expression sequence through an enzyme cleavage site of SARS-COV-2 coronavirus 3C protease to obtain a fusion expression sequence;
b) Cloning the fusion expression sequence of the step a) to a eukaryotic expression vector to obtain an expression vector for expressing a substrate of the viral 3C protease.
4. A method according to any one of claims 1-3, wherein the change in fluorescence signal intensity is a decrease in fluorescence signal intensity.
5. An expression vector for expressing active SARS-COV-2 coronavirus 3C protease for use in the method of any one of claims 1-4, said expression vector expressing active SARS-COV-2 coronavirus 3C protease having a nucleotide sequence as set forth in SEQ ID NO. 7.
6. A kit for screening an inhibitor of SARS-CoV-2 coronavirus 3C protease activity comprising the expression vector of claim 5 for expressing active SARS-CoV-2 coronavirus 3C protease and the expression vector for expressing a substrate for SARS-CoV-2 coronavirus 3C protease, wherein the substrate for the viral 3C protease is a fusion protein of il1β protein and Gussia luciferase linked by a cleavage site for the viral 3C protease.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010343025.7A CN113637724B (en) | 2020-04-27 | 2020-04-27 | Screening method and screening kit for SARS-CoV-2 coronavirus 3C protease activity inhibitor |
| 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 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN101921823A (en) * | 2010-05-05 | 2010-12-22 | 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 |
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| 【明星企业】首个新冠病毒3C蛋白酶药物高通量检测细胞体系推出;中科普瑞昇;《搜狐》;20200423;全文 * |
| iGLuc: a luciferase-based inflammasome and protease activity reporter;Eva Bartok等;《Nat Methods》;20140811;摘要 * |
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