CN117092084B - Screening method of WNV protease inhibitor and inhibition effect evaluation method - Google Patents
Screening method of WNV 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 a WNV protease inhibitor, wherein a sequence of a cleavage site related to the NS2B/NS3 protease of WNV is inserted into GFP to form a modified fluorescent protein report protein, and WNV NS2B/NS3 protease is cloned and expressed on a fluorescent protein report protein plasmid, so that the screening method and the inhibition effect evaluation method can be used for screening the protease inhibitor and evaluating the activity of the protease inhibitor, and finally a WNV protease inhibitor screening and drug effect evaluation platform is formed. The invention can avoid the safety concern caused by using WNV 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 screening WNV 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 a WNV protease inhibitor.
Background
West Nile Virus (WNV) is a recently widely spread yellow virus worldwide, transmitted mainly by culex, and can cause West Nile fever after human infection. The first discovery in 1937 in a female febrile patient in the Undaria was named West Nile virus and later spread on a small scale in Asia, europe and Australia, and after an outbreak in 1999 and rapid spread to other areas of North America, was of great concern. Influenza-like symptoms appear when people are infected by WNV, and few patients progress to severe neuroinvasive diseases such as encephalitis, meningitis and the like, and severe death can result.
WNV belongs to the genus Flaviviridae of the family Flaviviridae, is a single-stranded positive strand RNA virus, has a gene full length of about 11 kb, and has a virus diameter of 40-60 nm. The WNV genome comprises an open reading frame encoding a multimeric protein that is cleaved into 3 structural proteins: capsid protein (C), precursor membrane (prM), envelope protein (E) and 7 nonstructural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5. Wherein NS2B is a cofactor for NS3, forms a heterodimer with the N-terminal serine protease domain of the NS3 protein on the endoplasmic reticulum membrane, constitutes a NS2B/NS3 protease, and is responsible for cleaving the multimeric proteins of NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5. The NS2B/NS3 protease complex plays a critical role in viral replication and assembly, and therefore the NS2B/NS3 protease of WNV is a major target for antiviral drug development. To date, there is no marketed drug specifically for the treatment of west nile virus infection. Therefore, the development of low-toxicity and high-efficiency anti-West Nile virus medicaments has important significance.
There are a number of NS2B/NS3 protease inhibitors of WNV currently being investigated. Two main categories are peptides and non-peptides. The peptide inhibitor has larger relative molecular mass and low stability and selectivity. Whereas non-peptide inhibitors have a relatively smaller molecular mass and higher selectivity and stability, activity is generally lower than peptide inhibitors. The existing west nile virus inhibitor has the defects of activity problem, low safety, poor pharmacokinetic property and the like, and can not meet the requirement of curing west nile virus infection. There is therefore still a need for extensive drug screening efforts to ameliorate such problems. 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. Currently, the activity evaluation method of WNV-NS2B/NS3 protease inhibitor is mainly a fluorescence resonance energy transfer method. The fluorescence resonance energy transfer method is a high-throughput screening method which is widely applied 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 a false positive problem, 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 for WNV protease inhibitor, which are free from using live viruses, have good safety, and can efficiently, accurately, stably and high-flux screen and evaluate the inhibition effect of the WNV protease inhibitor.
The technical scheme adopted for solving the technical problems is as follows:
a method of screening for a WNV protease inhibitor comprising the steps of:
(1) Constructing plasmids for coexpression of modified GFP and WNV NS2B/NS3 protease, wherein the modified GFP is formed by inserting a WNV NS2B/NS3 protease cleavage sequence into wild GFP;
(2) Screening: spreading cells to be transfected in a porous plate, then adding plasmids of GFP and WNV NS2B/NS3 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 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.
The invention inserts the sequence of the related cleavage site of the NS2B/NS3 protease of WNV into Green Fluorescent Protein (GFP) to form a modified fluorescent protein reporter protein system, and clones and expresses the WNV NS2B/NS3 protease on the fluorescent protein reporter protein plasmid, thereby being applicable to screening protease inhibitors and evaluating the activity of the protease inhibitors and finally forming a platform for screening the WNV protease inhibitors and evaluating the drug effect. The invention can avoid the safety concern caused by using WNV 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 screening WNV protease inhibitor and evaluating drug effect.
The insertion site of WNV NS2B/NS3 protease cleavage sequence on the wild-type GFP amino acid sequence comprises site B and site C;
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 B is the 117 th to 118 th amino acid of the wild GFP amino acid sequence, or the corresponding amino acid region on the amino acid sequence with more than 99% sequence consistency compared with the wild GFP amino acid sequence; the corresponding amino acid region refers to the position corresponding to amino acids 117 to 118 of the wild-type GFP amino acid sequence on the amino acid sequence having a sequence identity of 99% or more as compared with the wild-type GFP amino acid sequence. Other similar expressions are referred to herein for explanation.
The inventors have found that after insertion of WNV NS2B/NS3 protease cleavage sequences at the two specific sites (site B and site C), the engineered GFP fluorescent protein can still detect the excitation fluorescence.
WNV NS2B/NS3 protease cleavage sequences include cut1 and cut2, wherein the cut1 amino acid sequence is SGKRSQIG, and the cut2 amino acid sequence is GLKRGGAK. The WNV NS2B/NS3 protease cleavage sequence can be accurately recognized and cleaved by the WNV NS2B/NS3 protease, and the cleavage can destroy the luminous function of fluorescent protein, thereby leading to fluorescence quenching.
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 WNV NS2B/NS3 protease cleavage sequence is inserted into the GFP gene sequence on the plasmid expressing the wild-type GFP, thereby constructing a plasmid expressing the engineered GFP.
A plasmid for coexpression of engineered GFP and WNV NS2B/NS3 protease, said plasmid comprising a coding gene sequence for expression of WNV NS2B/NS3 protease and a coding gene sequence for expression of engineered GFP; the modified GFP is obtained by inserting a WNV NS2B/NS3 protease cleavage sequence into wild GFP. After co-expression of the engineered GFP fluorescent protein and the WNV NS2B/NS3 protease, the WNV NS2B/NS3 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 sequence of WNV NS2B/NS3 protease cannot be used for cleavage, so that the luminous function of fluorescent protein is reserved, which is the principle of the invention for screening protease inhibitors.
A method for evaluating an inhibitory effect of a WNV protease inhibitor, comprising the steps of:
(1) Constructing plasmids for coexpression of modified GFP and WNV NS2B/NS3 protease, wherein the modified GFP is formed by inserting a WNV NS2B/NS3 protease cleavage sequence into wild GFP;
(2) Evaluation: spreading the cells to be transfected in a cell culture dish, then adding plasmids for coexpression modification of GFP and WNV NS2B/NS3 protease for transfection, culturing the transfected cells, simultaneously adding an inhibitor into the cell culture dish, taking the cell state and fluorescence photographing without the inhibitor as a control, 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 WNV protease inhibitors and evaluate inhibition effects.
Drawings
FIG. 1 is a fluorescent plot of WNV NS2B/NS3 protease cleavage sequence after insertion into three sites of wild-type GFP and of co-expression of engineered GFP and WNV NS2B/NS3 protease, GFP15-NS2B/NS3cut1 representing engineered GFP formed by insertion of WNV NS2B/NS3 protease cleavage sequence cut1 at site15 of GFP SGKRQIG, and so on; pGFP15-NS2B/NS3cut1 is a plasmid expressing GFP15-NS2B/NS3cut1, and so on; pGFP15-NS2B/NS3cut1-P2A-NS2B/NS3 is a plasmid that co-expresses GFP15-NS2B/NS3cut1 and WNV NS2B/NS3 protease, and so on.
FIG. 2 is a schematic diagram of plasmids co-expressing engineered GFP and WNV NS2B/NS3 proteases of the invention.
FIG. 3 is a high throughput detection fluorescence expression profile; 01 represents the average fluorescence of three replicates following transfection of cells with plasmid expressing the engineered GFP, 02 represents the average fluorescence of three replicates following transfection of cells with plasmid co-expressing the engineered GFP and WNV NS2B/NS3 protease, and 03 represents the average fluorescence of three replicates following transfection of cells with plasmid disrupting WNV NS2B/NS3 protease expression.
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
West Nile Virus (West Nile Virus); protease name: protease NS2B/NS3; protease size 750 aa; cleavage sequence size: 8aa.
Example 1
Construction of plasmids
(1) Constructing a modified GFP plasmid, wherein the modified GFP is formed by inserting a wild GFP amino acid sequence into a WNV NS2B/NS3 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 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 plasmid as a subsequent template.
The plasmid pGFP15/20/24-NS2B/NS3cut1/2 (remark: GFP15/20/24 and "/" in cut1/2 means "or" as follows) was constructed by inserting the WNV NS2B/NS3 protease cleavage sequences cut1: SGKRGQIG (SEQ ID No. 2) and cut2: GLKRGGGAK (SEQ ID No. 3) into different positions (site A: site 15.158-1590 aa, site B: site 20.117 aa, site C: site 24.159-160 aa) of the amino acid sequence of the wild-type GFP by homologous recombination technique (Uniclone One Step Seamless Cloning Kit kit), and the corresponding nucleotide sequences of cut1 and cut2 are tcggggaaacgatcacaaatcggg (SEQ ID No. 5) and ggcctcaagaggggtggagccaaa (SEQ ID No. 6). Wherein GFP15-NS2B/NS3cut1 is a modified fluorescent protein formed by inserting a west nile virus protease cleavage sequence NS2B/NS3cut1 between wild-type GFP amino acid sequences 158-1597a (namely between amino acids ADKQ and KNG), pGFP15-NS2B/NS3cut1 is a plasmid expressing the modified fluorescent protein GFP15-NS2B/NS3cut1, and so on.
293T cells were transfected with the constructed pGFP15/20/24-NS2B/NS3cut1/2 plasmid. 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.).
(2) Constructing a plasmid co-expressing the engineered GFP and WNV NS2B/NS3 protease (FIG. 2);
based on the plasmid pGFP15/20/24-NS2B/NS3cut1/2, the coding gene sequence (SEQ ID No. 7) of WNV NS2B/NS3 protease and the coding gene sequence (modified GFP) of GFP15/20/24-NS2B/NS3cut1/2 were linked by a 2A peptide coding gene to construct pGFP15/20/24-NS2B/NS3cut1/cut2-P2A-NS2B/NS3 plasmid and transfected 293T cells. After transfection, the cell status was observed every 24. 24 h, fluorescent photographing was performed for 48 hours (fig. 1), and the fluorescence intensity was detected.
As can be seen from FIG. 1, other GFP plasmids after expression modification were excited to observe green fluorescence 48h after cell transfection, except that pGFP24-NS2B/NS3cut1 experimental group was weak in fluorescence intensity. It was shown that the modified GFP formed after insertion of the WNV NS2B/NS3 protease cleavage sequence at the specific site of the wild-type GFP still has an excitation function. After 48h of transfection of pGFP15/20/24-NS2B/NS3cut1/cut2-P2A-NS2B/NS3 plasmid, pGFP20/24-NS2B/NS3cut1/cut2-P2A-NS2B/NS3 transfected groups all showed a decrease in fluorescence intensity with expression of the NS2B/NS3 protease, and only pGFP15-NS2B/NS3cut1/2-P2A-NS2B/NS3 transfected groups cells were still able to observe green fluorescence, it was seen that the NS2B/NS3 protease could not cleave all of the modified GFP, probably due to the fact that the protease cleavage sequence was located at a site where the tertiary structure of the modified GFP was not easily exposed. Thus, the plasmid pGFP20/24-NS2B/NS3cut1/cut2-P2A-NS2B/NS3 is more suitable for subsequent studies.
Example 2:
a method of screening for a WNV protease inhibitor comprising the steps of:
(1) Plasmids co-expressing the engineered GFP and NS2B/NS3 proteases, pGFP20/24-NS2B/NS3cut1/cut2-P2A-NS2B/NS3, were constructed (for specific construction reference example 1).
(2) Screening: spreading cells to be transfected in a porous plate (96-well plate), then adding pGFP20/24-NS2B/NS3cut1/cut2-P2A-NS2B/NS3 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 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.
Coding gene sequence of WNV NS2B/NS3 protease:
ggctggcctgctacagaagtgatgactgcagttggactcatgtttgccatcgttgggggtctggcagaacttgacatagattctatggctatccccatgaccatcgccggacttatgttcgcggcatttgtcatctctggaaagtcaacagacatgtggattgagaggacggctgacattacttgggagagtgatgctgaaatcacaggctctagcgaaagagtagatgtgaggctggatgatgatggaaattttcaactgatgaatgaccccggggcaccatggaaaatttggatgcttaggatggcctgcctggcgataagtgcctacacaccttgggcaattctcccctcggtcatcggattctggataacccttcagtacacaaagagaggaggtgttctttgggacacaccatcacccaaggagtacaagaagggtgataccaccactggcgtttacagaatcatgactcgaggtctgcttggcagttaccaagctggagccggagtgatggtagagggggtgttccacacactatggcacaccactaagggagctgctctcatgagtggtgagggacgtctggatccctactgggggagcgtgaaagaggaccgactttgctatggggggccatggaaactccaacataaatggaatggacatgatgaggtccaaatgattgtcgtggagccagggaaaaatgtgaaaaacgtccagaccaagcccggagtgtttaagacaccagaaggagaaattggggcagttacgctagactatcctaccggaacgtcaggttcccccattgtagacaaaaatggagatgtgattggattgtatgggaacggcgtcatcatgcctaatggttcatacataagcgccattgtgcaaggagagagaatggaagaaccggcaccagctggcttcgaacctgaaatgttgaggaagaaacagatcactgtccttgatctgcaccccggagcaggaaagacacgcaagatacttccccaaatcatcaaggaggccatcaacaaaagattgaggacggctgtgctggcacccaccagggtcgttgctgctgagatgtctgaggccctgagaggacttcccattcggtaccaaacctcagcagtgcacagagagcacagtggaaatgagatcgttgatgtcatgtgccatgccaccctcacacacaggctgatgtctccacacagagtccccaactacaacctgttcataatggatgaagcccatttcacggatccagcgagcatcgcagccagaggatacatagcaaccaaggttgaattgggcgaagccgccgcgattttcatgacggcaacgccacccgggacttctgacccctttccagagtctaatgctcctatctcggacatgcaaacagagatcccagacagagcctggaacactggatatgaatggataactgagtatgttggaaagaccgtttggtttgttccaagtgtgaaaatgggaaatgagattgccctctgtctgcaacgggcggggaagaaggttatccagctgaacagaaagtcctatgagacagagtaccccaagtgtaagaacgatgattgggattttgtcatcaccacagacatatcagaaatgggagccaacttcaaggcgagcagagtgatcgacagccgcaaaagcgtgaaacccaccatcattgaggaaggtgatggaagagtcatcctgggggaaccctcagccatcacggctgccagcgctgctcagcggagaggacgcataggaagaaacccatcacaagttggtgatgagtattgctatggagggcacacaaatgaggatgattccaactttgctcactggacagaggctcgcatcatgctagacaacatcaacatgccgaatggtctggtggctcaactatatcagcctgagcgcgagaaggtgtacaccatggacggggaatacaggctcagaggggaagaacggaagaacttccttgaattcctgagaacagctgatttaccagtctggctcgcttacaaagtggcagcagcaggaatatcataccatgaccggaaatggtgctttgatggacctcgaaccaacacgattcttgaagacaacaatgaagttgaagtcatcacgaagttgggtgagagaaagatcctaagacccaggtgggcagatgctagagtgtactcagaccatcaagctctaaagtccttcaaagattttgcatcggggaaacga（SEQ ID No.7）。
amino acid sequence of GFP:
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*（SEQ ID No.1）。
GFP gene sequence:
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA（SEQ ID NO.4）。
example 3:
a method for evaluating the inhibitory effect of WNV protease inhibitor, step (1) is the same as in example 2, except that step (2) is evaluated: paving 293T cells to be transfected in a cell culture dish, then adding plasmid transfected cells of GFP and WNV NS2B/NS3 protease which are co-expressed and modified, culturing the transfected cells, adding an inhibitor into the cell culture dish, taking the cell state and fluorescence photographing without the inhibitor as a control, 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.
Test example: the WNV NS2B/NS3 protease is simulated to be inactivated, and the modified GFP fluorescence recovery (quantification) is verified to destroy and construct the plasmid NS2B/NS3 on the basis of pGFP20/24-NS2B/NS3cut1/cut2-P2A-NS2B/NS3 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-NS2B/NS3cut1/cut 2-P2A-5 XSTOP-NS 2B/NS3 plasmid was transfected. Cell status and fluorescence photographing were observed every 24 th h after transfection, and fluorescence intensity was detected (fig. 3).
As can be seen from FIG. 3, after transfection of cells with both the NS2B/NS3 protease and GFP24-NS2B/NS3cut2 co-expression plasmids (treatment group 02), the fluorescence intensity was significantly reduced compared to plasmids expressing GFP24-NS2B/NS3cut2 alone, and when the expression of the NS2B/NS3 protease was disrupted (treatment group 03), the fluorescence intensity was significantly restored, and the pGFP24-NS2B/NS3cut2-P2A-NS2B/NS3 plasmid was found to have significantly restored fluorescence levels after disruption of the protease, which was the most potential candidate for developing a screening and evaluation platform for WNV viral protease inhibitors.
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 (2)
1. A method of screening for a WNV protease inhibitor comprising the steps of:
(1) Constructing plasmids for coexpression of modified GFP and WNV NS2B/NS3 protease, wherein the modified GFP is formed by inserting a WNV NS2B/NS3 protease cleavage sequence into wild GFP;
(2) Screening: spreading cells to be transfected in a porous plate, then adding plasmids of GFP and WNV NS2B/NS3 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 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;
the insertion site of WNV NS2B/NS3 protease cleavage sequence on the wild-type GFP amino acid sequence comprises site B and site C;
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;
WNV NS2B/NS3 protease cleavage sequences include cut1 and cut2, wherein the cut1 amino acid sequence is SGKRSQIG, and the cut2 amino acid sequence is GLKRGGAK.
2. A plasmid for coexpression of engineered GFP and WNV NS2B/NS3 protease, said plasmid comprising a coding gene sequence for expression of WNV NS2B/NS3 protease and a coding gene sequence for expression of engineered GFP; the modified GFP is formed by inserting a WNV NS2B/NS3 protease cutting sequence into wild GFP;
the insertion site of WNV NS2B/NS3 protease cleavage sequence on the wild-type GFP amino acid sequence comprises site B and site C;
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;
WNV NS2B/NS3 protease cleavage sequences include cut1 and cut2, wherein the cut1 amino acid sequence is SGKRSQIG, and the cut2 amino acid sequence is GLKRGGAK.
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