Screening method and screening kit for SARS-CoV-2 coronavirus 3C protease activity inhibitor
阅读说明：本技术 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 (Screening method and screening kit for SARS-CoV-2 coronavirus 3C protease activity inhibitor ) 是由 刘青松 胡晨 王文超 任涛 王伟 王黎 陈先涛 于 2020-04-27 设计创作，主要内容包括：本发明公开一种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。提供的3C蛋白酶活性抑制剂的筛选方法通过构建一个可以测定3C蛋白酶活性的体系以及表达具有较高活性的3C蛋白酶的载体,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。(The invention discloses a screening method and a screening kit for a SARS-CoV-2 coronavirus 3C protease activity inhibitor. The screening method of the 3C protease activity inhibitor can screen the viral 3C protease activity inhibitor at a cellular level by constructing a system capable of measuring the 3C protease activity and a carrier expressing the 3C protease with higher activity, and can be used for developing antiviral drugs.)
1. A method of screening for inhibitors of viral 3C protease activity comprising:
incubating an expression vector for expressing active virus 3C protease, an expression vector for expressing a substrate of the virus 3C protease and a candidate reagent in cells to serve as an experimental group, simultaneously setting a negative control without adding the candidate reagent, and screening the virus 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 a fusion protein formed by connecting IL1 beta protein and Gussia luciferase through the enzyme cutting site of the virus 3C protease.
2. The method of claim 1, wherein the viral 3C protease is SARS-CoV-2 coronavirus 3C protease.
3. The method of claim 2, wherein the expression vector for expressing the active viral 3C protease is constructed by a method comprising:
A) respectively connecting the NSP7 and NSP8 sequences of SARS-COV-2 coronavirus 3C protease at the 5 'end and the 3' end of the nucleotide sequence of SARS-COV-2 coronavirus 3C protease, and inserting the restriction enzyme cutting sites of SARS-COV-2 coronavirus 3C protease at each connection position to obtain the SARS-COV-2 coronavirus 3C protease recombinant expression sequence;
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.
4. The method of claim 2, wherein the expression vector for expressing a substrate of viral 3C protease is constructed by a method comprising:
a) connecting the IL1 beta protein expression sequence with the Gussia luciferase expression sequence through the restriction enzyme cutting 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.
5. The method of any one of claims 1 to 4, wherein the change in fluorescence signal intensity is a decrease in fluorescence signal intensity.
6. The expression vector for expressing active SARS-COV-2 coronavirus 3C protease for use in the method of any one of claims 2-5, optionally, the nucleotide sequence of the expression vector for expressing active SARS-COV-2 coronavirus 3C protease is set forth in SEQ ID No. 7.
7. An expression vector for expressing a substrate of SARS-COV-2 coronavirus 3C protease for use in the method of any one of claims 2-5, optionally the nucleotide sequence of the expression vector for expressing a substrate of SARS-COV-2 coronavirus 3C protease is as shown in SEQ ID NO 3.
8. A kit for screening inhibitors of SARS-CoV-2 coronavirus 3C protease activity, comprising the expression vector of claim 6 expressing an active SARS-CoV-2 coronavirus 3C protease, and the expression vector of claim 7 expressing a substrate for SARS-CoV-2 coronavirus 3C protease.
9. A method for determining viral 3C protease activity comprising the steps of:
1) connecting an IL1 beta protein expression sequence with a Gussia luciferase expression sequence through the enzyme cutting site of 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 obtained in the step 2) and an expression system of the virus 3C protease in cells, and characterizing the activity of the virus 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.
10. The method according to claim 9, wherein the viral 3C protease is a 3C protease of a single positive strand RNA virus, preferably a SARS-CoV-2 coronavirus 3C protease.
11. A kit for determining the activity of a SARS-CoV-2 coronavirus 3C protease, comprising the expression vector of claim 7 expressing a substrate for a SARS-CoV-2 coronavirus 3C protease, optionally, the nucleotide sequence of the expression vector expressing a substrate for a SARS-CoV-2 coronavirus 3C protease is set forth in SEQ ID No. 3.
Technical Field
The invention belongs to the field of molecular and cell biology, and particularly relates to a screening method and a screening kit for a virus 3C protease activity inhibitor, in particular to a screening method and a screening kit for a SARS-CoV-2 coronavirus 3C protease activity inhibitor.
Background
The novel coronavirus SARS-CoV-2 is the 7 th coronavirus which is discovered in 2019 and can infect human, is named by the world health organization in 2020, 1, 12, and can cause the novel coronavirus pneumonia COVID-19 to cause serious threat to the health of human.
SARS-COV-2 coronavirus is a single plus-strand RNA virus, like SARS coronavirus (SARS-CoV) and MERS coronavirus (MERS-CoV) which was SARS in 2003. By gene sequence alignment, SARS-COV-2 has about 80% similarity to SARS-CoV and 40% similarity to MERS-CoV. SARS-COV-2 coronavirus is highly contagious and is a great potential threat to human health, so that the search for anti-SARS-COV-2 coronavirus drugs 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 viral polyprotein, cleaving it into multiple active proteins, and finally assembling into new viral particles. Therefore, the catalytic function of inhibiting the 3C protease can effectively inhibit the cutting of virus precursor protein, block the replication of virus and play a role in resisting single plus strand RNA virus. Screening inhibitors capable of inhibiting the activity of 3C protease has become an important approach for developing drugs against single positive strand RNA viruses, so that a method for in vitro establishment of inhibitors for screening the activity of 3C protease against a newly discovered single positive strand RNA virus, SARS-COV-2 coronavirus, has important clinical significance for developing drugs against SARS-COV-2 coronavirus.
Disclosure of Invention
In view of one or more of the problems presented in the prior art, one aspect of the present invention provides a method for screening for inhibitors of viral 3C protease activity, comprising:
incubating an expression vector for expressing active virus 3C protease, an expression vector for expressing a substrate of the virus 3C protease and a candidate reagent in cells to serve as an experimental group, simultaneously setting a negative control without adding the candidate reagent, and screening the virus 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 a fusion protein formed by connecting IL1 beta protein and Gussia luciferase through the enzyme cutting site of the virus 3C protease.
The above virus 3C protease is SARS-CoV-2 coronavirus 3C protease.
The method for constructing the expression vector for expressing the active viral 3C protease comprises the following steps:
A) respectively connecting the NSP7 and NSP8 sequences of SARS-COV-2 coronavirus 3C protease at the 5 'end and the 3' end of the nucleotide sequence of SARS-COV-2 coronavirus 3C protease, and inserting the restriction enzyme cutting sites of SARS-COV-2 coronavirus 3C protease at each connection position to obtain the SARS-COV-2 coronavirus 3C protease recombinant expression sequence;
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 method for constructing the expression vector for expressing the substrate of the viral 3C protease comprises the following steps:
a) connecting the IL1 beta protein expression sequence with the Gussia luciferase expression sequence through the restriction enzyme cutting 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 the fluorescence signal intensity is a decrease in the fluorescence signal intensity.
Expression vectors for expressing the 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 the SARS-COV-2 coronavirus 3C protease with activity is shown as SEQ ID NO. 7.
Expression vectors for expressing a substrate for 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 the substrate of SARS-COV-2 coronavirus 3C protease is shown as SEQ ID NO. 3.
Another aspect of the present invention provides a kit for screening an inhibitor of SARS-CoV-2 coronavirus 3C protease activity, which comprises the above-mentioned expression vector for expressing an SARS-CoV-2 coronavirus 3C protease having activity, and the above-mentioned expression vector for expressing a substrate for SARS-CoV-2 coronavirus 3C protease.
In another aspect, the present invention provides a method for measuring the activity of viral 3C protease, comprising the steps of:
1) connecting an IL1 beta protein expression sequence with a Gussia luciferase expression sequence through the enzyme cutting site of 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 obtained in the step 2) and an expression system of the virus 3C protease in cells, and characterizing the activity of the virus 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 above virus 3C protease is 3C protease of single positive strand RNA virus, preferably SARS-CoV-2 coronavirus 3C protease.
In still another aspect, the present invention provides a kit for measuring the activity of SARS-CoV-2 coronavirus 3C protease, which comprises the above-mentioned expression vector for expressing the SARS-CoV-2 coronavirus 3C protease substrate; 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 the SARS-COV-2 coronavirus 3C protease activity inhibitor based on the technical scheme can evaluate the SARS-COV-2 coronavirus 3C protease activity at the cellular level by establishing a system for expressing SARS-COV-2 coronavirus 3C protease with activity and constructing a report system for activating the luciferase activity based on the 3C protease enzyme digestion, and can further screen the inhibitor of the SARS-COV-2 coronavirus 3C protease activity by utilizing the evaluation system. Based on the provided screening method, a kit for screening the SARS-COV-2 coronavirus 3C protease activity inhibitor is also provided, the inhibitor capable of inhibiting the activity of SARS-COV-2 coronavirus 3C protease can be quickly screened, and the inhibitor can be effectively used for developing the anti-SARS-COV-2 coronavirus medicines.
Drawings
FIG. 1 is a schematic diagram of the construction of a SARS-CoV-2 coronavirus 3C protease activity assay system;
FIG. 2 is a plasmid map of expression vector S1;
FIG. 3 is a plasmid map of expression vector C1;
FIG. 4 is a bar graph comparing the activity 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 the activity of 3C protease;
FIG. 6 is a graph showing inhibition curves of the activity of SARS-CoV-2 coronavirus 3C protease 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 a kit for rapidly screening the SARS-COV-2 coronavirus 3C protease activity inhibitor based on the screening method.
In order to achieve the above object, the present inventors refer to the following principles: an IL1 beta protein precursor is aggregated and precipitated in cells, and must be sheared by a protease CASPASE1 to release a C-terminal soluble IL1, a fusion vector expressed by connecting the IL1 beta protein precursor and Gussia luciferase (Gluc) in series is constructed, as shown in figure 1, the two expressed proteins are connected by a digestion site of 3C protease, when no 3C protease exists in the cells, the fusion protein is aggregated and precipitated, and when the 3C protease shears the digestion site between the IL1 beta and Gussia luciferase, the fusion vector releases an activated soluble Gussia luciferase, so that the activity of the 3C protease can be characterized through the high and low fluorescence (chemiluminescence) signal intensity, and the system which is constructed by the invention and can characterize the activity of the 3C protease also belongs to the content of the invention. In order to screen the inhibitor of the activity of the SARS-COV-2 coronavirus 3C protease, the key is to obtain an expression vector for expressing the 3C protease with higher activity, the inventor utilizes the characteristics that the protein coded by two sequences NSP7 and NSP8 of the SARS-COV-2 coronavirus is shorter in length, and the enzyme cutting site at the boundary has high homology with the enzyme cutting site of the 3C protease to construct and obtain the expression vector capable of expressing the activity of the 3C protease, and the expression vector is used for screening the inhibitor of the activity of the 3C protease.
The present invention will be further described below by taking a method for screening an inhibitor of the activity of SARS-COV-2 coronavirus 3C protease as an example. It should be understood that the specific examples are intended to be illustrative of the invention and are not intended to limit the scope of the invention.
The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: a Molecular Cloning Laboratory Manual (Molecular Cloning: A Laboratory Manual, Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).
The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.
The sequences used were synthesized by Shanghai Producer, Inc.
Example 1: construction of SARS-CoV-2 coronavirus 3C protease activity determination system
In the embodiment, a fusion protein containing a SARS-CoV-2 coronavirus 3C protease cleavage site is constructed as a substrate of SARS-CoV-2 coronavirus 3C protease, as shown in FIG. 1, the substrate comprises an IL1 beta protein-3C cleavage site (the amino acid sequence of the 3C cleavage site is shown in SEQ ID NO:1 in the sequence table) -Gussia luciferase (Gluc), namely IL1 beta protein and Gussia luciferase are connected through the 3C cleavage site, the amino acid sequences are shown in SEQ ID NO:2 in the sequence table, and the specific construction method is as follows:
synthesizing a gene sequence containing an enzyme cutting site of 3C of the coronavirus 3C for expressing IL1 beta protein-SARS-CoV-2-Gussia luciferase, cloning the gene sequence to a vector pCDNA3.1(Thermo Fisher Scientific), obtaining an expression vector for expressing a substrate of the 3C protease, and naming the expression vector as an expression vector S1, wherein the nucleotide sequence of the expression vector S1 is shown as SEQ ID NO:3, and the plasmid map is shown as FIG. 2. The expression vector S1 was transfected into HEK293T cells, and the fusion protein obtained by expression was used as a substrate for SARS-CoV-2 coronavirus 3C protease and used in the following examples to determine the activity of 3C protease.
If the expressed fusion protein is not cut by 3C protease in the system when the expression vector S1 is expressed, the fusion protein is subjected to aggregation precipitation and is inactivated; when the expressed fusion protein is cleaved in the presence of 3C protease, luciferase is activated, so that the activity of 3C protease can be characterized by reading the change in intensity of the fluorescent signal, and an inhibitor that inhibits the activity of 3C protease is screened for according to the activity of 3C protease. Thus, the expression vector S1 obtained based on this example can provide a kit for measuring the activity of SARS-CoV-2 coronavirus 3C protease, which comprises the expression vector S1, when the kit is used, the expression vector S1 can be co-transfected with an expression vector of SARS-CoV-2 coronavirus 3C protease into HEK293T cells for co-expression, and the activity of the 3C protease can be characterized by reading the intensity of a fluorescent signal in a co-expression system.
Example 2: expression of SARS-CoV-2 coronavirus 3C protease active in vitro
After SARS-CoV-2 coronavirus invades human body, its RNA is used as template to make translation synthesis to form polyprotein containing several proteins, and only after the active 3C proteinase enzyme digestion can the polyprotein be cut into independent active protein to make subsequent replication and assembly. However, SARS-CoV-2 coronavirus 3C protease expressed in the prior art usually has very low or even no protease activity, so that the expression structure thereof needs to be designed specifically, so that the expressed 3C protease can undergo self-cleavage and dimerization to obtain the biological function thereof and improve the activity of the protease.
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 as SEQ ID NO: 4) are respectively connected with the NSP7 (the nucleotide sequence is shown as SEQ ID NO: 5) and NSP8 (the nucleotide sequence is shown as SEQ ID NO: 6) sequences of SARS-COV-2 coronavirus, and the expression sequence of the enzyme cutting site of the 3C protease is inserted into the connection position, so as to construct and obtain a recombinant expression sequence, wherein the recombinant expression sequence can promote the 3C protease to complete self-shearing and dimerization after expression, and further become an activated protein with biological function. 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 bioactivity is obtained and named as expression vector C1, the nucleotide sequence of the expression vector is shown as SEQ ID NO. 7 in the sequence table, and as shown in figure 3, 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, which is named as an expression vector C2, and the nucleotide sequence of the expression vector is shown as SEQ ID NO. 8 in a sequence table.
To verify that the expression vector C1 obtained in this example indeed enables the production of 3C protease with higher activity, HEK293T cells were resuspended at 1X 105Cells/ml, 100. mu.l per well plated in 96-well plates, 8 hours later for DNA transfection; 150ng of DNA per well containing 50ng of DNA from expression vector S1Plasmid (S1 plasmid) and gradient diluted plasmids from expression vectors C1 and C2 (0ng, 25ng, 50ng, 100ng, C1/C2 plasmid), the deficit of DNA was filled up with pCDNA3.1 empty vector. The specific transfection procedure was: mu.l of Opti-MEM (Gibco) medium was added to the corresponding DNA, mixed well, and 3-fold DNA mass of lipofectin (Hanbio, Shanghai) was added thereto, and left to stand at room temperature for 10 minutes, and then added dropwise to a 96-well plate in 5% CO2After 24 hours in the incubator, 15. mu.l of Renilla-glo reagent (Promega) was added to each well, and after 5 minutes of reaction, the fluorescence was read.
As shown in fig. 4, it can be seen that the 3C protease expressed after the expression vector C1 constructed in this example is transfected into HEK293T cells can cleave the substrate expressed by the expression vector S1 in a mass-dependent manner, i.e., the more the expression vector C1 is added, the more the cleaved substrate, the higher the fluorescence signal intensity (the higher the fluorescence value), which is significantly higher than the fluorescence value after the substrate is cleaved by the same transfection amount of C2 plasmid, and it is proved that the activity of the 3C protease expressed by the expression vector C1 is much higher than that of the 3C protease expressed by the expression vector C2, and the calculated enzyme cleavage activity of the 3C protease expressed by C1 is about 1000 times that of the 3C protease expressed by C2.
Example 3: screening of SARS-COV-2 coronavirus 3C protease activity inhibitor
In this example, the expression vector S1 constructed in example 1 and the expression vector C1 constructed in example 2 were used to screen the inhibitor of the activity of SARS-COV-2 coronavirus 3C protease, which specifically includes the following steps:
3.1, HEK293T cells were plated in 10 cm cell culture dishes and transfected at a density of 75%.
3.2, 3 μ g of the C1 vector expressing the 3C protease and 6 μ g of the S1 vector expressing the substrate of the 3C protease are mixed and added into 1ml of Opti-MEM culture medium, the mixture is mixed evenly, then 27 μ l of the transfection reagent lipoiter is added, the mixture is well sucked and blown up, the mixture is kept still for 10 minutes at room temperature, and HEK293T cells are dripped into the mixture for protein expression. 8 hours after transfection, cells were digested with 0.25% pancreatin and resuspended by centrifugation and diluted to 1X 105Individual cells/ml.
3.3 separately diluting the gradients(0.0, 3.0, 10.0, 30.0. mu.M) of DMSO-soluble compounds (Arbidol (Arbidol), Lopinavir (Lopinavir), nelfinavir (nelfinavir) and Hydroxychloroquine (Hydroxychloroquine) (all available from MedChemexpress) with Arbidol and Lopinavir as positive drugs (Zhen Zhu et al, Arbidol monoothyripy is to Lopinavir/Ritonavir in treeting COVID-19, Journal of Infectin, i:10.1016/j. jinf.2020.2020.03.060; Bodee Nuo et al, y Are Lopinavir and Ritonavir Effective g the New Ne empty emerald coral electron transfer and invitro biological samples 20160. mu.32. mu.8. mu.M) of the DMSO suspension was added as a buffer suspension of the candidate for culturing of the compound in a volume of the respective mBioquindox, and the mixture was continued with the mixture of the mixture2Culturing in an incubator at 37 ℃ for 16 hours; the 96-well plate was removed, 15. mu.l of Renilla Glo reagent (Promega) was added, incubated at room temperature for 5 minutes, and the fluorescence was read. Simultaneously setting a blank control of a blank medium without adding cells and a negative control without adding the candidate compound or the positive drug, and calculating the relative activity of the 3C protease of each candidate compound or the positive drug under the gradient concentration through respective fluorescence values after deducting the reading value of a blank control hole. The results are shown in fig. 5, which shows the relative activities of the two candidate compounds and the two positive drugs at different concentrations of 3C protease, and it can be seen that both positive drugs exhibit inhibitory effect on the activity of SARS-COV-2 coronavirus 3C protease, and exhibit dose dependence, i.e., the higher the concentration of the positive drug is, the stronger the inhibitory effect on the activity of 3C protease is within a certain concentration range. In addition, the nelfinavir in the two candidate compounds does not show the inhibition of the activity of the SARS-COV-2 coronavirus 3C protease, while the hydroxychloroquine shows the activity capable of inhibiting the SARS-COV-2 coronavirus 3C protease and shows the dose dependence as the two positive medicaments, so that the hydroxychloroquine can be selected as the candidate compound for preparing the medicaments for resisting the SARS-COV-2 coronavirus infection.
3.4 according to the above procedures, the C1 carrier is loaded with hydroxychloroquine with gradient concentration of 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100. mu.MAnd (3) treating a coexpression system of the body and the S1 vector in HEK293T cells, setting a blank control of a blank culture medium without adding cells and a negative control without adding hydroxychloroquine, and calculating the inhibition rate of the 3C protease at each concentration by the respective fluorescence value after deducting the reading value of a blank control hole. The results are shown in FIG. 6, which is a curve showing the inhibition of SARS-COV-2 coronavirus 3C protease activity by hydroxychloroquine at a gradient dilution concentration, and the half inhibition concentration IC of the activity of SARS-COV-2 coronavirus 3C protease by hydroxychloroquine is calculated50Approximately 30 nM.
In summary, the present invention constructs a system capable of measuring 3C protease activity, and constructs a vector expressing 3C protease with higher activity, and the two can be combined as a kit for screening a SARS-COV-2 coronavirus 3C protease activity inhibitor, i.e. the kit comprises an S1 vector expressing a substrate of 3C protease and a C1 vector expressing 3C protease with activity, and the two can be used for rapidly screening the inhibitor of SARS-COV-2 coronavirus 3C protease activity, and provide important data support for the development of drugs for resisting SARS-COV-2 coronavirus infection.
The above examples only describe the screening method of SARS-COV-2 coronavirus 3C protease activity inhibitor, and since all the 3C proteases are present in the single positive strand RNA viruses and each can recognize specific enzyme cutting sites, the respective system for measuring 3C protease activity of the single positive strand RNA viruses and the expression vectors for expressing the respective active 3C proteases can be constructed according to the description of the above examples, and can be used for screening the inhibitors of the 3C protease activity of the respective viruses for the development of antiviral drugs.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement 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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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