CN117106851A - Screening method for EV71 protease inhibitor and inhibition effect evaluation method - Google Patents
Screening method for EV71 protease inhibitor and inhibition effect evaluation method Download PDFInfo
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Abstract
The invention discloses a screening method and an inhibition effect evaluation method of EV71 protease inhibitor, wherein a sequence of a 3C protease related cleavage site of EV71 is inserted into fluorescent protein to form a modified fluorescent protein reporter protein, and the EV71 3C protease is cloned and expressed on a fluorescent protein reporter protein plasmid, so that the screening method and the inhibition effect evaluation method can be used for screening protease inhibitor and evaluating the activity of protease inhibitor, and finally an EV71 protease inhibitor screening and drug effect evaluation platform is formed. The invention can avoid the safety concern caused by using EV71 live virus, has low requirement on biological safety level, can meet the experimental requirement in BSL-1 level laboratory, and is high-efficiency, accurate, stable, high-flux and repeatable for EV71 protease inhibitor screening and drug effect evaluation.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a screening method and an inhibition effect evaluation method of EV71 protease inhibitors.
Background
Hand-foot-and-mouth disease (HFMD) is a common infectious disease of children, and can be caused by various enteroviruses, and is commonly and easily felt by preschool children and infants below three years old. Among them, enterovirus 71 (ev 71) is one of the main pathogens causing hand-foot-and-mouth disease. Of the hand-foot-and-mouth patients, 70% of the severe cases are caused by EV71 infection, with up to 90% of cases fatal to HFMD being caused by EV71 infection. Therefore, development of effective antiviral drugs is urgently required.
EV71 is a non-enveloped virus belonging to the genus Picornavirus (Enterovirus) of the family Picornavirus. The genome of EV71 is a single-stranded positive strand RNA of about 7.4. 7.4 kb, the intermediate ORF of which encodes a polypeptide of about 2194 amino acids. During EV71 replication, the polypeptide is first hydrolyzed into 3 precursor proteins (P1 to P3), and then further cleaved into four structural proteins and seven non-structural proteins by the action of virally encoded 2A protease and 3C protease. After translation of the polyprotein, the 2A protease is mainly responsible for cleavage of the junction sequence between VP1 and 2A, while the 3C protease is responsible for cleavage of the other eight junction sites in the remaining polyprotein, the primary protease. The EV71 3C protease participates in a plurality of pathological processes of EV71 and plays a very important role in various aspects of EV71 virus replication, host cell apoptosis induction, host antiviral immune response inhibition and the like, so the 3C protease can be used as one of the most main targets for the research and development of anti-EV 71 virus medicaments.
The traditional broad-spectrum antiviral medicament is mainly used for treating hand-foot-mouth disease in clinic at present. However, various antiviral drugs have been developed for EV71, including Placonaril and BPR0Z-194, which interfere with viral entry into the host, rupintrivir, which inhibits 3C protease activity, DTriP-22, which targets viral replication, and Kaempferol, which targets viral translation. Among them, 3C protease plays an important role in EV71 replication and host cell apoptosis, and EV71 3C protease has no homology with mammalian protease, which makes 3C protease the most prominent target in antiviral drug development.
Most cellular antiviral experiments of highly infectious viruses need to be performed in a laboratory of BSL-2+ or higher, however the resources of such a laboratory are rather scarce. At present, the activity evaluation method of the EV71 3C protease inhibitor is mainly a fluorescence resonance energy transfer method, and enzymatic and inhibition experiments are carried out in vitro at the in vitro or non-cellular level. Although the fluorescence resonance energy transfer method has the advantages of high flux, rapidness, sensitivity, quantification, good repeatability and the like, many inhibitors with good in vitro activity cannot fully reflect the effect of protease inhibitors in cells because of poor cell membrane permeability or specificity. Therefore, it is necessary to develop a simple, safe, high-throughput and reproducible protease activity and protease inhibitor screening and activity detection method at the cellular level.
Disclosure of Invention
The invention aims to provide a screening method and an inhibition effect evaluation method for EV71 protease inhibitors, which are free from using live viruses, have good safety, and can efficiently, accurately, stably and high-throughput screen and inhibition effect evaluation for EV71 protease inhibitors.
The technical scheme adopted for solving the technical problems is as follows:
a method of screening for an EV71 protease inhibitor, comprising the steps of:
(1) Constructing plasmids for coexpression of modified GFP and EV71 3C protease, wherein the modified GFP is formed by inserting an EV 71C protease cleavage sequence into wild GFP;
(2) Screening: spreading cells to be transfected in a porous plate, then adding plasmids of GFP and EV 71C protease which are subjected to co-expression modification for transfection, culturing the transfected cells, simultaneously adding different groups of inhibitors to be screened into the porous plate, taking the non-added inhibitors to be screened as a control, observing the state of the cells and taking a fluorescent photograph, and detecting the fluorescence intensity; if the fluorescence intensity is increased compared with the control, the inhibitor to be screened has an inhibition effect, and if the fluorescence intensity is increased, the inhibition effect of the inhibitor to be screened is stronger.
According to the invention, the sequence of the cleavage site related to the EV71 3C protease is inserted into Green Fluorescent Protein (GFP) to form a modified fluorescent protein reporter protein system, and the EV71 3C protease is cloned and expressed on the fluorescent protein reporter protein plasmid, so that the modified fluorescent protein reporter protein system can be used for screening protease inhibitors and evaluating the activity of the protease inhibitors, and finally an EV71 protease inhibitor screening and efficacy evaluating platform is formed. The invention can avoid the safety concern caused by using EV71 live virus, has low requirement on biological safety level, can meet the experimental requirement in BSL-1 level laboratory, and is a high-efficiency, accurate, stable, high-throughput and repeatable platform for EV71 protease inhibitor screening and drug effect evaluation.
Insertion sites of the EV71 3C protease cleavage sequence on the wild-type GFP amino acid sequence include site a, site B and site C;
the site A is 158 th to 159 th amino acid of the amino acid sequence of the wild GFP, or an amino acid region corresponding to the amino acid sequence with more than 99% sequence identity compared with the amino acid sequence of the wild GFP;
the site B is 117 th to 118 th amino acid of the amino acid sequence of the wild GFP, or an amino acid region corresponding to the amino acid sequence with more than 99% sequence identity compared with the amino acid sequence of the wild GFP;
the locus C is 159 th to 160 th amino acid of the amino acid sequence of the wild GFP, or an amino acid region corresponding to the amino acid sequence with more than 99% sequence identity compared with the amino acid sequence of the wild GFP;
the amino acid sequence of the wild GFP is shown in SEQ ID No. 1.
In the invention, the site A is 158 th to 159 th amino acid of the amino acid sequence of the wild GFP, or an amino acid region corresponding to the amino acid sequence with more than 99% sequence identity compared with the amino acid sequence of the wild GFP; the corresponding amino acid region refers to the position on the amino acid sequence corresponding to amino acids 158-159 of the wild-type GFP amino acid sequence that has more than 99% sequence identity as compared to the wild-type GFP amino acid sequence. Other similar expressions are referred to herein for explanation.
The inventor researches and discovers that after the three specific sites (site A, site B and site C) are inserted into the EV71 3C protease cleavage sequence, the modified GFP fluorescent protein can still detect the excited fluorescence.
The EV71 3C protease cleavage sequences comprise cut1 and cut2, the amino acid sequence of the cut1 is shown in SEQ ID No.2, and the amino acid sequence of the cut2 is shown in SEQ ID No. 3. The EV71 3C protease cleavage sequence can be accurately recognized and cleaved by EV71 3C protease, and the cleavage can destroy the luminous function of fluorescent protein, so that fluorescence quenching is caused.
The fluorescence excitation was still detectable after expression of the engineered GFP.
The preparation method of the modified GFP plasmid comprises the following steps:
cloning a gene sequence of the wild GFP into a plurality of cloning sites of an expression plasmid vector in a homologous recombination mode to construct a plasmid for expressing the wild GFP;
by homologous recombination, a nucleotide sequence corresponding to the cleavage sequence of EV 71C protease is inserted into the GFP gene sequence on the plasmid expressing wild-type GFP, so as to construct the GFP plasmid with improved expression.
A plasmid co-expressing a engineered GFP and an EV71 3C protease, said plasmid comprising a coding gene sequence that expresses an EV71 3C protease and a coding gene sequence that expresses the engineered GFP; the modified GFP is obtained by inserting an EV71 3C protease cleavage sequence into the wild GFP. After co-expression of the engineered GFP and the EV71 3C protease, the EV 71C protease can cleave the engineered GFP, resulting in fluorescence quenching. If an effective protease inhibitor exists, the protease function is inhibited, and the cleavage sequence of the EV 71C protease cannot be cut, so that the luminous function of fluorescent protein is reserved, which is the principle of screening the protease inhibitor.
A method for evaluating an inhibitory effect of an EV71 protease inhibitor, comprising the steps of:
(1) Constructing plasmids for coexpression of modified GFP and EV71 3C protease, wherein the modified GFP is formed by inserting an EV 71C protease cleavage sequence into wild GFP;
(2) Evaluation: spreading the cells to be transfected in a cell culture dish, then adding plasmids of GFP and EV 71C protease which are co-expressed and modified for transfection, culturing the transfected cells, simultaneously adding inhibitors into the cell culture dish, taking the cells without the inhibitors as a control, observing the state of the cells and taking a fluorescent photograph, and detecting the fluorescence intensity; the fluorescence intensity positively reflects the inhibition effect of the inhibitor, and a higher fluorescence intensity represents a stronger inhibition effect of the inhibitor.
The beneficial effects of the invention are as follows: the method does not need to use live viruses, has good safety, and can efficiently, accurately, stably and high-flux screen EV71 protease inhibitors and evaluate inhibition effects.
Drawings
FIG. 1 is a fluorescent image of EV71 3C protease cleavage sequence inserted into three sites of wild-type GFP and of co-expressed engineered GFP and EV 71C protease, GFP15-3Ccut1 representing engineered GFP formed by insertion of EV71 3C protease cleavage sequence cut1 at site15 of GFP, AVTQGF, and so on; pGFP15-3Ccut1 is a plasmid expressing GFP15-3Ccut1, and so on; pGFP15-3Ccut1-P2A-3C is a plasmid co-expressing GFP15-3Ccut1 and EV71 3C protease, and so on.
FIG. 2 is a schematic diagram of plasmids co-expressing engineered GFP and EV71 3C protease according to the invention.
FIG. 3 is a photograph of the fluorescence of cells 48h after inhibitor addition.
FIG. 4 is a high throughput detection fluorescence expression profile; 01 represents the average fluorescence value of three replicates after transfection of cells with plasmid expressing the engineered GFP, 02 represents the average fluorescence value of three replicates after transfection of cells with plasmid co-expressing the engineered GFP and EV71 3C protease, and 03 represents the average fluorescence value of three replicates after transfection of cells with plasmid disrupting expression of the EV71 3C protease.
Description of the embodiments
The technical scheme of the invention is further specifically described by the following specific examples.
In the present invention, the materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Virus name
Hand-foot-mouth disease virus (Human Enterovirus 71); protease name: protease 3C; protease size 183 aa; cleavage sequence size: 6 aa.
Example 1:
a method of screening for an EV71 protease inhibitor, comprising the steps of:
(1) Constructing a modified GFP plasmid for expression, wherein the modified GFP is formed by inserting a wild GFP amino acid sequence into an EV71 3C protease cleavage sequence, and the modified GFP fluorescent protein still has the function of excitation and luminescence;
first, the gene encoding the wild-type GFP and pcDNA3.1 (+) vector (commercially available) were PCR amplified according to the Primer Star enzyme (Takara) protocol. The wild-type GFP gene sequence (SEQ ID No. 4) was cloned between the Multiple Cloning Sites (MCS) of the pcDNA3.1 (+) vector by homologous recombination (Uniclone One Step Seamless Cloning Kit kit) to construct pGFP plasmids as a subsequent template and control.
The EV 71C protease cleavage sequences cut1: AVTQ ∈GF (SEQ ID No. 2) and cut2: ALFQ ∈GP (SEQ ID No. 3) are inserted into different positions (site A: site 15-1598 aa, site B: site 20-118 aa, site C: site 24-160 aa) of the wild-type GFP amino acid sequence by homologous recombination technology (Uniclone One Step Seamless Cloning Kit kit), and the corresponding nucleotide sequences of cut1 and cut2 are gctcttttccaaggtcca (SEQ ID No. 5) and gcaacagtgcaaggcccg (SEQ ID No. 6), so that plasmids pGFP15/20/24-3Ccut1/2 (remark: GFP15/20/24 and "/" in cut1/2 represent "or" meaning, and the same applies below) are constructed. The constructed pGFP15/20/24-3Ccut1/2 plasmid was transfected into 293T cells. Cell status and fluorescence photographing were observed every 24 th h after transfection (fig. 1), and fluorescence intensity was detected.
(2) Constructing a plasmid co-expressing the engineered GFP and EV71 3C protease (FIG. 2);
based on plasmid pGFP15/20/24-3Ccut1/2, the coding gene sequence (SEQ ID No. 7) of EV71 3C protease and the coding gene sequence (modified GFP) of GFP15/20/24-3Ccut1/2 are connected through a 2A peptide coding gene by a homologous recombination mode to construct pGFP15/20/24-3Ccut1/cut2-P2A-3C plasmid and transfect 293T cells. Cell status and fluorescence photographing were observed every 24 th h after transfection (fig. 1), and fluorescence intensity was detected.
In fluorescence intensity detection, 293T cells were plated in 96-well plates and incubated overnight in a 5% CO2 incubator at 37 ℃; transfecting 0.5ug plasmid per hole, making three repeated holes, and changing liquid after 4-6 h; after culturing in a 5% CO2 incubator at 37℃for 48 hours, the fluorescence value was read on a multifunctional microplate reader (Diken trade Co., ltd.).
(3) Screening: spreading cells to be transfected in a porous plate (96-well plate), then adding pGFP15/20/24-3Ccut1/cut2-P2A-3C plasmid to transfect the cells, culturing the transfected cells, adding different groups of inhibitors to be screened into the porous plate, taking the non-added inhibitors to be screened as a control, observing the state of the cells and performing fluorescence photographing, and detecting the fluorescence intensity; if the fluorescence intensity is increased compared with the control, the inhibitor to be screened has an inhibition effect, and if the fluorescence intensity is increased, the inhibition effect of the inhibitor to be screened is stronger.
As shown in FIG. 1, pGFP15/20/24-3Ccut1/2 showed fluorescence after expression, and pGFP20-3Ccut1 had the weakest fluorescence intensity. This suggests that the modified GFP fluorescent protein formed after insertion of the EV 71C protease cleavage sequence at the specific site of the wild-type GFP, may still be observed with varying degrees of fluorescence intensity. While pGFP15/20/24-3Ccut1/cut2-P2A-3C showed a decrease in fluorescence intensity with co-expression of 3C protease, it was observed that pGFP15-3Ccut2-P2A-3C group cells were still able to observe weak fluorescence, which was determined by both the fluorescence properties of the engineered GFP and the cleavage ability of the 3C protease. Although the background of pGFP15-3Ccut2-P2A-3C group was higher than that of the other experimental groups, it can be seen from FIG. 4b that after GFP15-3Ccut2 and 3C were co-expressed (02 treatment group), the fluorescence intensity was still significantly reduced compared to 01 treatment group, and in other cases, the fluorescence intensities of pGFP15-3Ccut1-P2A-3C vs pGFP15-3Ccut1, pGFP20-3Ccut1-P2A-3Cvs pGFP20-3Ccut1, pGFP20-3Ccut2-P2A-3C vs pGFP20-3Ccut2, GFP24-3Ccut1-P2A-3Cvs pGFP24-3Ccut1 and GFP24-3Ccut2-P2A-3C vs pGFP24-3Ccut2 were all significantly reduced. This suggests that the 3C protease recognizes and cleaves the EV71 3C protease cleavage sequence to affect the fluorescent protein luminescence function.
Coding gene sequence of EV71 3C protease (SEQ ID No. 7):
ggcccgagccttgactttgctctctccctactgagaaggaacatcaggcaagtccaaacagaccaagggcatttcaccatgctgggtgttagggatcgcttagcagtcctcccacgccactcacaacctggcaaaaccatttggattgagcacaaactcgtgaacgtccttgatgcagttgaactggtggatgagcaaggagtcaacctggaattaaccctcatcactcttgataccaacgaaaagtttagggatatcaccaaattcatcccagaaaatatcagcactgctagcgatgccaccctagtgatcaacacggagcacatgccatcaatgtttgtcccggtgggtgacgttgtgcagtatggctttttgaatctcagtggtaagcctacccatcgcaccatgatgtacaattttcctactaaagcaggacagtgtggaggagtggtgacgtctgttgggaaggttgtcggtattcacattggtggcaatggcagacaaggtttttgcgcaggcctcaaaaggagttactttgctagtgaacaa（SEQ ID No.7）。
amino acid sequence of GFP:
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*（SEQ ID No.1）。
GFP gene sequence:
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA（SEQ ID NO.4）。
example 2:
a method for evaluating the inhibitory effect of an EV71 protease inhibitor, which differs from example 1 in that step (1) and step (2) are evaluated in step (3): paving 293T cells to be transfected in a cell culture dish, then adding plasmids pGFP20-3Ccut2-P2A-3C and pGFP24-3Ccut2-P2A-3C for co-expression of modified GFP and EV71 3C protease to respectively transfect the cells, culturing the transfected cells, simultaneously adding 3C protease inhibitors Rupintrivir (commercially available) with different concentrations into the cell culture dish, taking the cells without inhibitors as a control, observing cell states and performing fluorescence photographing every 24 hours, and detecting fluorescence intensity; the fluorescence intensity positively reflects the inhibition effect of the inhibitor, and a higher fluorescence intensity represents a stronger inhibition effect of the inhibitor.
As can be seen from fig. 3, after the inhibitor 1 μm Rupintrivir is added, no fluorescence is observed at 48 hours, and when the inhibitor concentration reaches 10 μm or more, the fluorescence intensity is higher as the inhibitor concentration is increased. Therefore, the plasmid for coexpression of the modified GFP and the EV 71C protease is a very simple and convenient product for evaluating the inhibition effect of the EV71 protease inhibitor at the cellular level.
Example 3: the EV71 3C protease inactivation is simulated, and the modified GFP fluorescence recovery (quantification) is verified to destroy 3C on the basis of pGFP15/20/24-3Ccut1/cut2-P2A-3C plasmid to construct a plasmid:
the specific operation is that 5 continuous stop codons TGA are added after P2A by utilizing PCR amplification (Primer Star) and homologous recombination technology (Uniclone One Step Seamless Cloning Kit kit); the constructed pGFP15/20/24-3Ccut1/cut 2-P2A-5 XSTOP-3C plasmid was transfected. Cell status and fluorescence photographing were observed every 24 th h after transfection, and fluorescence intensity was detected (fig. 4).
As can be seen from FIG. 4, after the cells were transfected with the 3C protease and GFP15/20/24-3Ccut1/cut2 co-expression plasmids (treatment group 02), the fluorescence intensity was significantly reduced compared with that of GFP15/20/24-3Ccut1/cut2 expressed alone (treatment group 03), and the fluorescence intensity was significantly or extremely significantly recovered when the expression of 3C protease was disrupted (treatment group 03), wherein pGFP15-3Ccut2-P2A-3C plasmids of FIG. 4b and pGFP24-3Ccut1-P2A-3C plasmids of FIG. 4e were significantly recovered after the protease was disrupted.
The above-described embodiment is only a preferred embodiment of the present invention, and is not limited in any way, and other variations and modifications may be made without departing from the technical aspects set forth in the claims.
Claims (7)
1. A method for screening an EV71 protease inhibitor, comprising the steps of:
(1) Constructing plasmids for coexpression of modified GFP and EV71 3C protease, wherein the modified GFP is formed by inserting an EV 71C protease cleavage sequence into wild GFP;
(2) Screening: spreading cells to be transfected in a porous plate, then adding plasmids of GFP and EV 71C protease which are subjected to co-expression modification for transfection, culturing the transfected cells, simultaneously adding different groups of inhibitors to be screened into the porous plate, taking the non-added inhibitors to be screened as a control, observing the state of the cells and taking a fluorescent photograph, and detecting the fluorescence intensity; if the fluorescence intensity is increased compared with the control, the inhibitor to be screened has an inhibition effect, and if the fluorescence intensity is increased, the inhibition effect of the inhibitor to be screened is stronger.
2. The method of claim 1, wherein the insertion sites of the EV71 3C protease cleavage sequence on the wild-type GFP amino acid sequence include site a, site B and site C;
the site A is 158 th to 159 th amino acid of the amino acid sequence of the wild GFP, or an amino acid region corresponding to the amino acid sequence with more than 99% sequence identity compared with the amino acid sequence of the wild GFP;
the site B is 117 th to 118 th amino acid of the amino acid sequence of the wild GFP, or an amino acid region corresponding to the amino acid sequence with more than 99% sequence identity compared with the amino acid sequence of the wild GFP;
the locus C is 159 th to 160 th amino acid of the amino acid sequence of the wild GFP, or an amino acid region corresponding to the amino acid sequence with more than 99% sequence identity compared with the amino acid sequence of the wild GFP;
the amino acid sequence of the wild GFP is shown in SEQ ID No. 1.
3. The screening method according to claim 1 or 2, wherein the EV71 3C protease cleavage sequence comprises cut1 and cut2, the cut1 amino acid sequence is shown in SEQ ID No.2, and the cut2 amino acid sequence is shown in SEQ ID No. 3.
4. A plasmid for coexpression of engineered GFP and EV71 3C protease, said plasmid comprising a coding gene sequence for expression of EV71 3C protease and a coding gene sequence for expression of engineered GFP; the modified GFP is obtained by inserting an EV71 3C protease cleavage sequence into the wild GFP.
5. The plasmid of claim 4, wherein the insertion sites of the EV71 3C protease cleavage sequence on the wild-type GFP amino acid sequence comprise site a, site B and site C;
the site A is 158 th to 159 th amino acid of the amino acid sequence of the wild GFP, or an amino acid region corresponding to the amino acid sequence with more than 99% sequence identity compared with the amino acid sequence of the wild GFP;
the site B is 117 th to 118 th amino acid of the amino acid sequence of the wild GFP, or an amino acid region corresponding to the amino acid sequence with more than 99% sequence identity compared with the amino acid sequence of the wild GFP;
the locus C is 159 th to 160 th amino acid of the amino acid sequence of the wild GFP, or an amino acid region corresponding to the amino acid sequence with more than 99% sequence identity compared with the amino acid sequence of the wild GFP;
the amino acid sequence of the wild GFP is shown in SEQ ID No. 1.
6. The plasmid of claim 4, wherein the EV71 3C protease cleavage sequence comprises cut1 and cut2, the cut1 amino acid sequence is shown in SEQ ID No.2, and the cut2 amino acid sequence is shown in SEQ ID No. 3.
7. A method for evaluating an inhibitory effect of an EV71 protease inhibitor, comprising the steps of:
(1) Constructing plasmids for coexpression of modified GFP and EV71 3C protease, wherein the modified GFP is formed by inserting an EV 71C protease cleavage sequence into wild GFP;
(2) Evaluation: spreading the cells to be transfected in a cell culture dish, then adding plasmids of GFP and EV 71C protease which are co-expressed and modified for transfection, culturing the transfected cells, simultaneously adding inhibitors into the cell culture dish, taking the cells without the inhibitors as a control, observing the state of the cells and taking a fluorescent photograph, and detecting the fluorescence intensity; the fluorescence intensity positively reflects the inhibition effect of the inhibitor, and a higher fluorescence intensity represents a stronger inhibition effect of the inhibitor.
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