CN117088987A - Screening method of FMDV protease inhibitor and inhibition effect evaluation method - Google Patents
Screening method of FMDV 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 FMDV protease inhibitor, wherein a sequence of a 3C protease related cleavage site of FMDV is inserted into GFP to form a modified fluorescent protein reporter protein, and the FMDV 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 the protease inhibitor, and finally a FMDV protease inhibitor screening and drug effect evaluation platform is formed. The invention can avoid the safety concern caused by using FMDV living 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 screening FMDV protease inhibitor and evaluating drug effect.
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 an FMDV protease inhibitor.
Background
Foot-and-mouth-disease (FMD) is an infectious disease of artiodactyl animals caused by Foot-and-mouth-disease virus (FMDV), and can infect various even-hooed livestock such as pigs, cattle and sheep and wild animals to cause serious diseases.
FMDV belongs to the genus foot-and-mouth disease virus of the picornaviridae family. The virus particles are round and have a diameter of about 27-30 nm. The genome length of FMDV is about 8.5 kb, with ORF length 7 kb, consisting of four coding segments L, P, P2 and P3. Wherein the P1 encoded protein is cleaved by the 3C protein into 1A (VP 4), 1B (VP 2), 1C (VP 3), 1D (VP 1) during later translation and modification, and thereby constitutes the viral capsid. The main function of the 3C protein is to cleave the viral multimeric precursor protein to form a mature protein, and it can exert the function of cleaving the host protein in the infected host cell, which can cleave off the N-terminal 20 amino acids of histone H3, promoting viral replication. In addition, the 3C protein plays an important role in viral infection and immunosuppression. Therefore, the 3C protein is an important target for developing FMDV protease inhibitor antiviral drugs.
Development of antiviral drugs involves screening of a large number of candidate small/large molecule drugs, whereas cellular antiviral experiments of most highly infectious viruses need to be performed in BSL-2+ or higher-class laboratories, which are however quite resource-starved. At present, the activity evaluation method of the FMDV-3C protease inhibitor is mainly a fluorescence resonance energy transfer method, is a relatively wide high-throughput screening method applied to the activity of the protease inhibitor at present, has the advantages of rapidness, sensitivity, quantification, good repeatability and the like, but a fluorescent agent is easily influenced by a small molecular compound to cause the problem of false positive, and meanwhile, a plurality of inhibitors with good in vitro activity have no activity or weak activity 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 assay platform.
Disclosure of Invention
The invention aims to provide a screening method and an inhibition effect evaluation method of an FMDV protease inhibitor, which do not need to use live viruses, have good safety, and can efficiently, accurately, stably and high-flux screen and evaluate the inhibition effect of the FMDV protease inhibitor.
The technical scheme adopted for solving the technical problems is as follows:
a method of screening for FMDV protease inhibitors comprising the steps of:
(1) Constructing plasmids for coexpression of modified GFP and FMDV 3C protease, wherein the modified GFP is formed by inserting an FMDV 3C protease cleavage sequence into wild GFP;
(2) Screening: spreading cells to be transfected in a porous plate, then adding plasmids of GFP and FMDV 3C 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 3C protease related cleavage site of FMDV is inserted into Green Fluorescent Protein (GFP) to form a modified fluorescent protein report protein, and the FMDV 3C protease is cloned and expressed on the fluorescent protein report protein plasmid, so that the modified fluorescent protein report protein can be used for screening protease inhibitors and evaluating the activity of the protease inhibitors, and finally, a FMDV protease inhibitor screening and efficacy evaluating platform is formed. The invention can avoid the safety concern caused by using FMDV living 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 screening FMDV protease inhibitors and evaluating drug effect.
Insertion sites of FMDV 3C protease cleavage sequence on wild-type GFP amino acid sequence include site a, site C and site D;
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 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 site D is 170 th-171 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 corresponding to amino acids 158-159 of the wild-type GFP amino acid sequence on the amino acid sequence having more than 99% sequence identity 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 C and site D) are inserted into the FMDV 3C protease cleavage sequence, the modified GFP fluorescent protein can still detect the excited fluorescence.
The FMDV 3C protease cleavage sequences include cut1, cut2, cut3, and cut4, wherein the cut1 amino acid sequence is AEKQLKAR, the cut2 amino acid sequence is PHHEGLVV, the cut3 amino acid sequence is KIIAPAKQLLNFDLLK, and the cut4 amino acid sequence is DLERAEKQLKARDIND. The FMDV 3C protease cleavage sequence can be accurately identified and cleaved by FMDV 3C protease, and the cleavage can destroy the luminous function of fluorescent protein, thereby leading to fluorescence quenching.
The FMDV 3C protease cutting sequence inserted in the site A is cut4; the FMDV 3C protease cleavage sequence inserted at site C is cut1; the FMDV 3C protease cleavage sequence inserted at position D is cut3.
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;
inserting nucleotide sequences corresponding to FMDV 3C protease cleavage sequences into GFP gene sequences on plasmids expressing wild GFP by homologous recombination, and constructing the GFP plasmids with improved expression.
A plasmid co-expressing a engineered GFP and FMDV 3C protease, said plasmid comprising a coding gene sequence that expresses FMDV 3C protease and a coding gene sequence that expresses engineered GFP; the modified GFP is formed by inserting an FMDV 3C protease cleavage sequence into wild GFP. After co-expression of the engineered GFP fluorescent protein and the FMDV 3C protease, the FMDV 3C protease can cleave the engineered GFP fluorescent protein, resulting in fluorescence quenching. If an effective protease inhibitor exists, the protease function is inhibited, and the cleavage of the FMDV 3C protease cleavage sequence cannot be performed, 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 FMDV protease inhibitor, comprising the steps of:
(1) Constructing plasmids for coexpression of modified GFP and FMDV 3C protease, wherein the modified GFP is formed by inserting an FMDV 3C 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 FMDV 3C 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 FMDV protease inhibitors and evaluate inhibition effects.
Drawings
FIG. 1 is a fluorescent plot of FMDV 3C protease cleavage sequence inserted into three sites of wild-type GFP and of co-expressed engineered GFP and FMDV 3C protease, GFP15-3Ccut4 representing the engineered GFP formed by insertion of FMDV 3C protease cleavage sequence cut4: DLERAEKQLKARDIND at site15 of GFP, and so on; pGFP15-3Ccut4 is a plasmid expressing GFP15-3Ccut4, and so on; pGFP15-3Ccut4-P2A-3C is a plasmid co-expressing GFP15-3Ccut4 and FMDV 3C protease, and so on.
FIG. 2 is a schematic plasmid diagram of the present invention co-expressing engineered GFP and FMDV 3C protease.
FIG. 3 is a high throughput detection fluorescence expression profile; 01 represents the average fluorescence value of three replicates after pGFP15/24-3Ccut 1/cut 4 transfected cells, 02 represents the average fluorescence value of three replicates after pGFP15/24-3Ccut 1/cut 4-P2A-3C transfected cells, and 03 represents the average fluorescence value of three replicates after pGFP15/24-3Ccut 1/cut 4-P2A-5Xstop-3C transfected cells.
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
Foot-and-mouth disease virus (Foot-and-mouth Disease Virus); protease name: protease 3C; protease size 214 aa; cleavage sequence size: 8aa,16aa. The amino acid sequence of cut1 is AEKQ ∈LKAR, the amino acid sequence of cut2 is PHHE ∈GLVV, the amino acid sequence of cut3 is KIIAPAKQ ∈LLNFDLLK, and the amino acid sequence of cut4 is DLERAEKQ ∈LKARDIND.
Example 1:
construction of plasmids
(1) Constructing a modified GFP plasmid, wherein the modified GFP is formed by inserting an FMDV 3C protease cleavage sequence into a wild GFP amino acid sequence, and the modified GFP fluorescent protein still has the function of exciting 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 GFP gene sequence (SEQ ID No. 10) 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 plasmid as a subsequent template.
The FMDV 3C protease cleavage sequence cut1: AEKQLKAR (SEQ ID No. 2), cut2: PHHEGLVV (SEQ ID No. 3), cut3: KIIAPAKQLLNFDLLK (SEQ ID No. 4), cut4: DLERAEKQLKARDIND (SEQ ID No. 5), nucleotide sequences corresponding to cut1-cut4 being gcagagaaacagctcaaagcacgt (SEQ ID No. 6), ccacaccacgagggtttggttgtc (SEQ ID No. 7), aagattattgcgcccgcaaaacagctgttgaactttgacctacttaag (SEQ ID No. 8), gaccttgagagagcagagaaacagctcaaagcacgtgacattaacgac (SEQ ID No. 9) were inserted into different positions of the wild-type GFP amino acid sequence (site A: site 15-1590 aa, site D: site 170-171aa) by homologous recombination techniques (Uniclone One Step Seamless Cloning Kit kit), and plasmids pGFP15/24/45-3Ccut1/2/3/4 were constructed (remarked: GFP15/24/45 and "/" stands for "or" in cut 1/2/3/4).
Wherein GFP15-3Ccut4 is a modified fluorescent protein formed by inserting FMDV protease cleavage sequence 3Ccut4 between wild-type GFP amino acid sequences 158-1597a (namely between amino acids ADKQ and KNG), pGFP15-3Ccut4 is a plasmid for expressing modified fluorescent protein GFP15-3Ccut4, and the like.
Well-grown 293T cells were prepared, 10% FBS DMEM medium was added to the 293T cells, and the mixture was left at 37℃with 5% CO 2 Culturing in an incubator. Transfection experiments were performed when 293T cells grew to a density of 70% -80% and the 6-well plate plasmid transfection amount was 2. Mu.g. The cell status was observed every 24. 24 h after transfection, fluorescent photographing was performed after 48 hours (fig. 1), and the 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.).
(2) Constructing plasmids co-expressing the engineered GFP and FMDV 3C proteases (fig. 2);
based on plasmid pGFP15/24/45-3Ccut1/2/3/4, the coding gene sequence (SEQ ID No. 11) of FMDV 3C protease and the GFP15/24/45-3Ccut1/2/3/4 coding gene sequence (modified GFP) were linked by a 2A peptide coding gene to construct pGFP15/24/45-3Ccut1/2/3/4-P2A-3C plasmid and transfected into 293T cells by homologous recombination. Cell status and fluorescence photographing were observed every 24 th h after transfection (fig. 1), and fluorescence intensity was detected.
The modified fluorescent proteins formed by pGFP15-3C cut1/2/3, pGFP24-3C cut2/3/4 and pGFP45-3C cut1/2/4 were observed with little or no excitation light (not shown). As can be seen from FIG. 1, the plasmids pGFP15-3C-cut4, pGFP24-3C-cut1 and pGFP45-3Ccut3, all of which, after transfection of cells, were observed to excite green fluorescence of different intensities. The modified GFP formed by inserting the specific FMDV 3C protease cleavage sequence at a specific position above the wild-type GFP is still provided with an excitation light function. Meanwhile, in addition to the decrease in fluorescence intensity of cells transfected with pGFP24-3Ccut1-P2A-3C plasmids, cells co-expressing the modified GFP and 3C protease showed a decrease in fluorescence following expression of the 3C protease after transfection of pGFP15-3Ccut4-P2A-3C and pGF45-3Ccut3-P2A-3C plasmids. This suggests that expression of 3C protease can cleave engineered GFP, resulting in fluorescence quenching. Subsequently, a fluorescence quantification assay was performed based on pGFP15-3C-cut4, pGFP24-3C-cut 1.
Example 2:
a method of screening for FMDV protease inhibitors comprising the steps of:
(1) Plasmids were constructed which co-expressed the engineered GFP and 3C proteases, i.e., pGFP15-3Ccut4-P2A-3C, pGFP24-3C cut1-P2A-3C (for specific construction methods reference example 1).
(2) Screening: spreading cells to be transfected in a porous plate (96-well plate), then adding pGFP15-3Ccut4-P2A-3C, pGFP-3C cut1-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 photography, 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.
Coding gene sequence of FMDV 3C protease:
agtggtgccccaccgaccgacttgcagaagatggtcatgggcaacacaaagcctgttgagcttaacctcgacgggaagacagtagccatctgctgtgctactggagtgttcggcactgcttacctcgtgcctcgtcaccttttcgcagagaagtatgacaagattatgttggacggcagagccatgacagacagtgattacagagtgtttgagttcgagattaaagttaaaaggacaggacatgctctcagacgcggcactcattggttgcttcaccgtgggaactgcgtgagagacatcacgaaacactttcgtgatacagcaagaatgaagaaaggcacccccgtcgttggtgttgtcaacaacgccgatgttgggagactgattttctctggtgaggcccttacctacaaggacattgtagtgtgcatggatggagacaccatgcccggcctctttgcctacaaagccgccaccagggctggctactgtggaggagccgttcttgccaaggacggggctgacacattcatcgtcggcactcactctgcaggtggcaatggagttggatactgctcatgcgtttccaggtccatgcttcaaaagatgaaggctcacgtcgaccctgaaccgcaccacgag（SEQ ID No.11）。
amino acid sequence of GFP:
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*（SEQ ID No.1）。
GFP gene sequence:
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA（SEQ ID NO.10）。
example 3:
simulating the inactivation of FMDV 3C protease, verifying that the modified GFP fluorescence is recovered (quantified) and constructing a plasmid by disrupting 3C on the basis of pGFP15-3Ccut4-P2A-3C, pGFP24-3Ccut1-P2A-3C 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 pGFP15-3Ccut4-P2A-5 XSTOP-3C, pGFP-3 Ccut 1-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. 3). In fluorescence intensity detection, 293T cells were plated in 96-well plates at 37℃with 5% CO 2 Culturing overnight in an incubator; transfecting 0.5 mug plasmid in each hole, taking average value of three repeated holes in each group of treatment, and changing liquid after 4-6 hours; 37 ℃ 5% CO 2 After 48h incubation in incubator, fluorescence values were read on a multifunctional microplate reader (Diken trade Co., ltd.) and subjected to significant difference analysis using T testp<0.05,**p<0.01,***p<0.001）。
As can be seen from fig. 3, after co-expression of 3C protease and GFP15-3Ccut4 or GFP24-3Ccut1 plasmids were transfected in cells (treatment group 02), fluorescence intensity was significantly reduced compared to that of the single expression engineered GFP (treatment group 01), and fluorescence was significantly restored after disruption of functional expression of 3C protease (treatment group 03).
Therefore, the method has high activity sensitivity for evaluating the antiviral drugs of the protease inhibitor, can be used for screening FMDV protease inhibitors in a high-throughput manner and detecting the antiviral activity of the FMDV protease inhibitors in a high-throughput manner, and has wide prospects for developing excellent antiviral drug screening platforms and corresponding protease inhibitor activity detection products.
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 (8)
1. A method of screening for an FMDV protease inhibitor comprising the steps of:
(1) Constructing plasmids for coexpression of modified GFP and FMDV 3C protease, wherein the modified GFP is formed by inserting an FMDV 3C protease cleavage sequence into wild GFP;
(2) Screening: spreading cells to be transfected in a porous plate, then adding plasmids of GFP and FMDV 3C 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 FMDV 3C protease cleavage sequence at the wild-type GFP amino acid sequence include site a, site C and site D;
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 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 site D is 170 th-171 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 FMDV 3C protease cleavage sequence comprises cut1, cut2, cut3, cut4, the cut1 amino acid sequence is AEKQLKAR, the cut2 amino acid sequence is pheglvv, the cut3 amino acid sequence is KIIAPAKQLLNFDLLK, and the cut4 amino acid sequence is DLERAEKQLKARDIND.
4. A screening method according to claim 3, wherein the FMDV 3C protease cleavage sequence inserted at position a is cut4; the FMDV 3C protease cleavage sequence inserted at site C is cut1; the FMDV 3C protease cleavage sequence inserted at position D is cut3.
5. A plasmid for coexpression of engineered GFP and FMDV 3C protease, said plasmid comprising a coding gene sequence for expression of FMDV 3C protease and a coding gene sequence for expression of engineered GFP; the modified GFP is formed by inserting an FMDV 3C protease cleavage sequence into wild GFP.
6. The plasmid of claim 5, wherein the insertion sites of FMDV 3C protease cleavage sequence at the wild-type GFP amino acid sequence comprise site a, site C and site D;
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 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 site D is 170 th-171 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.
7. The plasmid of claim 5, wherein the FMDV 3C protease cleavage sequence comprises cut1, cut2, cut3, cut4, the cut1 amino acid sequence is AEKQLKAR, the cut2 amino acid sequence is PHHEGLVV, the cut3 amino acid sequence is KIIAPAKQLLNFDLLK, and the cut4 amino acid sequence is DLERAEKQLKARDIND.
8. A method for evaluating an inhibitory effect of an FMDV protease inhibitor, comprising the steps of:
(1) Constructing plasmids for coexpression of modified GFP and FMDV 3C protease, wherein the modified GFP is formed by inserting an FMDV 3C 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 FMDV 3C 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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| CN116840201A (en) * | 2023-06-06 | 2023-10-03 | 昆明理工大学 | Screening of human coronavirus major protease 3CL pro Methods of inhibiting |
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| WO2007036056A1 (en) * | 2005-09-27 | 2007-04-05 | Oncalis Ag | Genetic selection system to identify proteases, protease substrates and protease inhibitors |
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