CN113621680B - 3C-like protease inhibitor of porcine epidemic diarrhea virus, screening method and application thereof - Google Patents
3C-like protease inhibitor of porcine epidemic diarrhea virus, screening method and application thereof Download PDFInfo
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- CN113621680B CN113621680B CN202110855357.8A CN202110855357A CN113621680B CN 113621680 B CN113621680 B CN 113621680B CN 202110855357 A CN202110855357 A CN 202110855357A CN 113621680 B CN113621680 B CN 113621680B
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Abstract
The invention discloses a 3C-like protease inhibitor of porcine epidemic diarrhea virus, a screening method thereof and application thereof, wherein the 3C-like protease inhibitor comprises at least one of myrobalan acid and isomers thereof, quercetin-3-O-p-coumaroyl rhamnoside, troxerutin, citrate B, apigenin-7-O- (2G-rhamnose) gentian glucoside, brutieridin, heraclenol '-O-beta-D-apiofuranosyl- (1- & gt 6) -beta-D-glucopyranoside, procyanidin B23,3' -di-O-galtate, citrate A and benzyl benzoate glucoside. Because the 3C-like protease plays an important role in transcription and replication of porcine epidemic diarrhea virus and in the host cell defense mechanism resisting process, the 3C-like protease inhibitor provided by the invention can inhibit porcine PEDV 3C-like protease from infecting host cells by inhibiting the activity of porcine epidemic diarrhea virus, and has a good inhibition effect. Furthermore, the 3C-like protease inhibitor provided by the invention can be used for preparing medicines for resisting porcine epidemic diarrhea virus, and a new application way is developed for the molecules.
Description
Technical Field
The invention relates to the technical field of antivirus, in particular to a 3C-like protease inhibitor of porcine epidemic diarrhea virus, a screening method and application thereof.
Background
Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is a highly contagious porcine intestinal infectious disease caused by porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV) and is mainly characterized by vomiting, watery diarrhea and dehydration of the onset pigs. PED affects pigs at all ages, and has high mortality, up to 100% of the death rate, especially for suckling piglets within 7 days of age, and is an important factor currently affecting survival rate of piglets and pig raising production. Vaccines are an effective means of preventing this disease, but vaccines do not provide protection for infected piglets and no therapeutic drug is currently available against this disease.
Disclosure of Invention
The invention mainly aims to provide a 3C-like protease inhibitor of porcine epidemic diarrhea virus, a screening method and application thereof, and aims to provide the 3C-like protease inhibitor capable of treating porcine epidemic diarrhea.
In order to achieve the aim, the invention provides a 3C-like protease inhibitor of porcine epidemic diarrhea virus, wherein the 3C-like protease inhibitor comprises at least one of myrobalan acid and isomers thereof, quercetin-3-O-p-coumaroyl rhamnoside, troxerutin, citrate B, apigenin-7-O- (2G-rhamnose) gentian glucoside, heraclenol3 '-O-beta-D-apiofuranosyl- (1- & gt 6) -beta-D-glucopyranoside, brutieridin, procyanidin B23,3' -di-O-galtate, citrate A and benzyl benzoate glucosyl glucoside.
Further, the invention also provides a screening method of the 3C-like protease inhibitor of the porcine epidemic diarrhea virus, which comprises the following steps:
s10, taking 3C-like protease of porcine epidemic diarrhea virus as a target point, taking a small molecular database as a screening object, carrying out molecular docking on all molecules in the small molecular database and an active region of the 3C-like protease, and screening to obtain natural molecules with affinity ranking of 50-150;
s20, carrying out biological activity screening on the natural molecules obtained by screening outside cells and/or inside cells to obtain a 3C-like protease inhibitor;
wherein the small molecule database comprises a ZINC15 database.
Optionally, the small molecule database is a natural molecule library in the ZINC15 database.
Optionally, step S10 includes:
s11, butting all molecules in the small molecule database with the active region of the 3C-like protease by using Autodock Vina, and primarily screening to obtain primary screening molecules with affinity ranking of 9000-14000;
s12, flexibly butting all the primary screening molecules with the 3C-like protease by using a Discovery studio, and finely screening to obtain the natural molecules with the affinity ranking of 50-150.
Optionally, step S20 includes:
s21, detecting the activity of the 3C-like protease inhibitor outside cells by using a fluorescence resonance energy transfer system; and/or the number of the groups of groups,
s22, detecting the activity of the 3C-like protease inhibitor in the cell by using a dual-fluorescence enzyme report system.
Optionally, step S21 includes:
s210, synthesizing a modified polypeptide Dabcyl-YNSTLQ ∈AGLRKM-E-Edans according to the sites of replicase polyproteins ppra and pplb of the porcine epidemic diarrhea virus cut by 3C-like protease, wherein the modified polypeptide can be cut by the 3C-like protease so that a fluorescent signal of the E-Edans is released;
s211, adding the 3C-like protease, the 3C-like protease inhibitor and a buffer solution to the modified polypeptide, and detecting the emitted light intensity at 480nm under the condition of 340nm excitation light so as to detect the activity of the 3C-like protease inhibitor.
Optionally, step S22 includes:
s220, fusing and expressing the 3C-like protease cleavage polypeptide and firefly luciferase, and inserting intein DnaE to obtain a cyclization report plasmid;
s221, co-transfecting the cyclization report plasmid, the Renilla luciferase plasmid and the plasmid expressing the 3C-like protease into 293T cells, replacing a maintenance solution containing the 3C-like protease inhibitor 5-7 hours after transfection, and detecting the cleavage activity of the 3C-like protease by using a double-luciferase detection kit 20-28 hours after transfection.
In addition, the invention also provides a medicine for resisting the porcine epidemic diarrhea virus, the active ingredient of the medicine comprises a 3C-like protease inhibitor, and the 3C-like protease inhibitor is the 3C-like protease inhibitor of the porcine epidemic diarrhea virus according to claim 1.
According to the technical scheme provided by the invention, the 3C-like protease inhibitor of PEDV comprises at least one of myrobalan acid and isomers thereof, quercetin-3-O-p-coumaroyl rhamnoside, troxerutin, citrate B, apigenin-7-O- (2G-rhamnose) gentian glycoside, brutieridin, heraclenol '-O-beta-D-apiofuranosyl- (1- & gt 6) -beta-D-glucurouranoside, procyanidin B23,3' -di-O-gate, citrate A and phenylmethanolic glucophenolic glycoside, and the replicase polyprotein encoded by PEDV can form mature nonstructural proteins after being cut by the 3C-like protease, and the mature nonstructural proteins play a vital role in the processes of viral transcription, replication and host cell defense mechanism resistance, so that the molecules belonging to the 3C-like protease inhibitor can inhibit the activity of the 3C-like protease in PEDV and inhibit the infection of the PEDV by inhibiting the activity of the 3C-like protease in the PEDV, and have better host cell defense effect. Furthermore, the 3C-like protease inhibitor provided by the invention can be used for preparing anti-PEDV drugs, and a new application way is developed for the molecules.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of expression and purification of PEDV 3C-like protease provided by the present invention;
FIG. 2 is a graph showing the cleavage activity of the purified PEDV 3C-like protease in a FRET system;
FIG. 3 is a graph showing the results of a FRET system for detecting extracellular activity of a 3C-like protease inhibitor;
FIG. 4 is a schematic diagram of a reporter plasmid constructed in step S220 of the present invention;
FIG. 5 is a graph showing Western blot verification results of PEDV 3C-like protease cleavage of the circularized firefly luciferase;
FIG. 6 is a graph showing the cleavage activity of PEDV 3C-like protease detected by a dual-luciferase assay kit;
FIG. 7 is a graph showing the results of a dual-luciferase reporter system for detecting activity of a 3C-like protease inhibitor in a cell;
FIG. 8 is a diagram of the use of TCID 50 Determining the effect result graph of myrobalan acid on PEDV replication;
FIG. 9 is a graph showing the effect of using western blot to determine chebular acid on PEDV replication;
FIG. 10 is a graph showing the effect of myrobalan acid on PEDV replication using an indirect immunofluorescence assay.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a 3C-like protease inhibitor of PEDV, and aims to provide a 3C-like protease inhibitor capable of treating PED. The 3C-like protease inhibitor comprises at least one of myrobalan acid and isomers thereof, quercetin-3-O-p-coumaroyl rhamnoside, troxerutin, citrate B, apigenin-7-O- (2G-rhamnose) gentian glycoside, brutieridin, heraclenol ' -O-beta-D-apiofuranosyl- (1- & gt 6) -beta-D-glucouranosyl, procyanidin B23,3' -di-O-gallate, citrate A, phenylcarbinol glucide benzoate and Heraclenol3' -O-beta-D-apiofuranosyl- (1- & gt 6) -beta-D-apiofuranosyl.
PEDV-encoded replicase polyproteins (Replicase polyprotein 1a, ppra) and (Replicase polyprotein 1ab, pplab) must undergo cleavage by viral proteases to form mature nonstructural proteins, i.e., NSP1 to NSP16 nonstructural proteins produced by cleavage by NSP 3-encoded papain-like protease and NSP 5-encoded 3C-like protease. Wherein papain is responsible for cleaving proteins between NSP 1-NSP 4, and 3C-like protease is responsible for cleaving proteins between NSP 5-NSP 16. In addition, 3C-like proteases can also inhibit the production of interferon by host cells by cleaving NEMO, closely related to immune escape of PEDV. Since mature nonstructural proteins play a critical role in viral transcription, replication and anti-host cell defense mechanisms, 3C-like proteases are critical for PEDV infection process.
Specifically, the 3C-like protease inhibitor is selected from a natural molecular library in a ZINC15 database. Wherein, the ID in the natural molecular library (for convenience of description, hereinafter, the ID will be referred to as ID in the ZINC15 database) of chebulic acid (chebulic acid), namely chebula Lin Rousuan, is: ZINC000299817893; the chebula acid isomer comprises small molecules with ID of ZINC000390835486, ZINC000257667213 and ZINC 000390835480; quercetin-3-O-p-coumaroyl rhamnose glucoside is a small molecule with an ID of ZINC 000253390566; troxerutin (troxertin), a small molecule with an ID of ZINC 000299817570; citrate B (Parishin B), a small molecule with ID ZINC 000085643640; apigenin-7-O- (2G-rhamnose) gentian glycoside is a small molecule with an ID of ZINC 000299817632; brukieridin is a small molecule with the ID of ZINC 000253389678; heraclenol3' -O-beta-D-apiofuranosyl- (1- & gt 6) -beta-D-glucouranoside is a small molecule with ID of ZINC000107428706 and ZINC 000107428702; procyanidin B23,3' -di-O-gate, a small molecule with ID ZINC 000150528319; citrate a (parishin a), a small molecule with ID ZINC 000257616571; benzyl alcohol glucophenolic glycoside benzoate is a small molecule with the ID of ZINC 000253389687.
Preferably, the 3C-like protease inhibitor is chebulic acid and isomer thereof, more preferably, the 3C-like protease inhibitor is chebulic acid, and the 3C-like protease inhibitor is obtained through experimental verification: the chebular acid has better inhibiting effect on 3C-like protease. Further, chebular acid is effective in inhibiting infection of host cells by PEDV.
The PEDV 3C-like protease inhibitor provided by the invention can inhibit PEDV from infecting host cells by inhibiting the activity of PEDV 3C-like protease, and has a good inhibition effect. Furthermore, the 3C-like protease inhibitor provided by the invention can be used for preparing anti-PEDV drugs, and a new application way is developed for the molecules.
Furthermore, the invention also provides a screening method of the 3C-like protease inhibitor of the porcine epidemic diarrhea virus, and aims to provide a high-throughput screening method. The screening method comprises the following steps:
and S10, taking 3C-like protease of porcine epidemic diarrhea virus as a target point, taking a small molecular database as a screening object, carrying out molecular docking on all molecules in the small molecular database and an active region of the 3C-like protease, and screening to obtain natural molecules with affinity ranking of 50-150.
Namely, natural molecules with the affinity ranking of 50-150 with the 3C-like protease active region in the small molecule database are obtained by screening through a virtual screening technology. Virtual screening, also known as computer screening, is to analyze the affinity between a target and a drug molecule using molecular docking software to reduce the number of compounds actually screened and to increase the efficiency of the discovery of lead compounds.
The premise of virtual screening is to obtain the crystal structure and active site of target protein and the database of small molecular compound crystal structure. At present, a plurality of free small molecule databases exist, the invention does not limit the specific sources of the small molecule databases, and the small molecule databases can be used as conventional small molecule databases for drug screening. In this embodiment, the small molecule database is a ZINC15 database, and the ZINC15 database can provide 1300 ten thousand of small molecule structure data for free.
In order to make the small molecule database more targeted to save workload, further, the small molecule database is a natural molecule library in the ZINC15 database. The natural molecule library contains more than 8 ten thousand natural molecule structures, and molecules in the natural molecule library are plant extracts and are mostly harmless to organisms, so that the natural molecule library can be rapidly applied to clinic after being screened and validated to be effective.
Specifically, step S10 includes:
and S11, butting all molecules in the small molecule database with the active region of the 3C-like protease by using Autodock Vina, and primarily screening to obtain the primary screening molecules with the affinity ranking of 9000-14000.
In specific implementation, step S11 includes the following steps:
preparation of receptor molecules: the 3C-like protease (www.rcsb.org, protein number 6U 7K) crystal structure was dehydrated and hydrotreated using autodock1.5.6 software and the molecules stored as PDBQT files. Using Discover Studio 3.5 to open the receptor molecule, select protein active site amino acids, determine the molecular docking region, wherein determining the molecular docking region includes determining the central site coordinates and docking region size;
preparation of ligand molecules: the molecular structure (about 8 ten thousand molecules) in the library of natural molecules was downloaded in ZINC15 and all molecules were individually converted to PDBQT files using Raccon software. And then according to the prepared bat file, performing molecular docking by using an Autodock Vina operation bat file, sequencing according to the molecular docking result, and primarily screening to obtain the primary screening molecules with the affinity ranking of 9000-14000.
It will be appreciated that the specific primary screening of how many of the prescreened molecules with affinity rank may be based on the circumstances, not only the molecules with affinity of 9000-14000. If it is desired to avoid as much as possible that the primary screening eliminates molecules with a possibly better affinity, the number of primary screened molecules obtained by the primary screening may be increased, for example: screening to obtain a primary screening molecule with affinity ranking of 20000; if it is desired to save the amount of work, the number of primary screening molecules for preliminary screening can be appropriately reduced, for example: screening to obtain a prescreened molecule with the affinity ranking of 5000.
Molecular docking using Autodock Vina is a rigid docking method. In the rigid butt joint method, the molecular conformation participating in butt joint is not changed in the calculation process, only the spatial position and the gesture of the molecule are changed, the simplification degree of the rigid butt joint method is highest, and the calculated amount is relatively small.
And step S12, flexibly butting all the primary screening molecules with the 3C-like protease by using a Discovery studio, and finely screening to obtain the natural molecules with the affinity ranking of 50-150.
Because the primary screening in the step S11 is a rigid butt joint method with lower accuracy, according to the primary screening result, all the primary screening molecules are flexibly butt-jointed with the 3C-like protease by using a Discovery studio, and the natural molecules with the affinity ranking of 50-150 are obtained by fine screening.
Step S20, performing biological activity screening on the natural molecules obtained by screening outside cells and/or inside cells to obtain a 3C-like protease inhibitor;
since the natural molecules with affinity ranking of 50-150 are obtained by virtual screening, wherein the possible activity of the 3C-like protease inhibitor is only 20-30%, the natural molecules obtained by screening are also required to be subjected to biological activity screening so as to obtain the active 3C-like protease inhibitor.
The natural molecules can be directly subjected to biological activity screening outside the cells, the operation is simple, the cost is saved, the natural molecules can be subjected to biological activity screening in the cells, and the result is more accurate and convincing. Of course, the selection of biological activity may also be performed both extracellular and intracellular.
The invention is not limited to a specific method for screening for biological activity of the native molecule outside and/or inside the cell, and in one embodiment, step S20 includes:
s21, detecting the activity of the 3C-like protease inhibitor outside cells by using a fluorescence resonance energy transfer system; and/or the number of the groups of groups,
s22, detecting the activity of the 3C-like protease inhibitor in the cell by using a dual-fluorescence enzyme report system.
Wherein, the fluorescence resonance energy transfer system (fluorescence resonance energy transfer, FRET) and the dual-luciferase reporting system are matched with 384 micro-plates and an enzyme label instrument to perform high-throughput screening of the 3C-like protease inhibitor.
It should be noted that, because the 100 natural molecules virtually screened are not easily obtained, the embodiment of the invention uses myrobalan acid as an example only to verify the feasibility of the FRET system and the dual-luciferase reporting system for detecting the biological activity of the natural molecules obtained by screening. It can be understood that natural molecules with different concentrations can be added to obtain natural molecules with the best inhibitory effect on the cleavage activity of the PEDV 3C-like protease and the concentration thereof, and then the natural molecules with the best inhibitory effect on the cleavage activity of the PEDV 3C-like protease and the concentration thereof are selected by combining the factors of cost, dosage, safety and the like.
In one embodiment, step S21 includes:
step S210, synthesizing a modified polypeptide Dabcyl-YNSTLQ ∈AGLRKM-E-Edans according to sites of replicase polyprotein ppra and pprb of PEDV cleaved by 3C-like protease, wherein the modified polypeptide can be cleaved by the 3C-like protease so that a fluorescent signal of the E-Edans is released;
in the complete situation, the fluorescence of the C-terminal modification group Edans of the modified polypeptide is quenched by the N-terminal Dabcyl group due to the FRET effect, and in the presence of the 3C-like protease, the modified polypeptide is cut, the FRET system is destroyed, and the fluorescence signal of the E-Edans is released.
Step S211, adding the 3C-like protease, the 3C-like protease inhibitor and a buffer solution to the modified polypeptide, and detecting the emitted light intensity at 480nm under the condition of 340nm excitation light so as to detect the activity of the 3C-like protease inhibitor.
In another embodiment, step S22 includes the steps of:
s220, fusing and expressing the 3C-like protease cleavage polypeptide and firefly luciferase, and inserting intein DnaE to obtain a cyclization report plasmid;
because of the introduction of intein DnaE, the luciferase protein expressed by the reporter plasmid transfected cells is expressed in a natural state in the form of cyclic proteins, and the cyclic proteins have great steric hindrance and are difficult to approach each other so as to avoid self-activation. Only when the 3C-like protease is expressed in the cell, the cleavage sequence can be recognized and cleaved, the steric hindrance after cleavage is reduced, and the firefly luciferase activity is recovered, so that the cleavage activity of the 3C-like protease can be detected.
S221, co-transfecting the cyclization report plasmid, the Renilla luciferase plasmid and the plasmid expressing the 3C-like protease into 293T cells, replacing a maintenance solution containing the 3C-like protease inhibitor 5-7 hours after transfection, and detecting the cleavage activity of the 3C-like protease by using a double-luciferase detection kit 20-28 hours after transfection.
In the screening method of the 3C-like protease inhibitor of PEDV, which is provided by the invention, a PEDV 3C-like protease is taken as a target point, a natural molecular library in a ZINC15 database is taken as a screening object, molecular docking is carried out through autodock vina and Discovery studio (virtual screening technology), so that natural molecules with the affinity ranking of 50-150 are obtained, and then biological activity detection is carried out through a FRET system and/or a luciferase reporting system, so that the 3C-like protease inhibitor with biological activity is obtained, and the 3C-like protease inhibitor can inhibit the activity of the 3C-like protease, thereby inhibiting replication of PEDV; in addition, the virtual screening technology has the advantages of high throughput, high efficiency and low cost compared with the common screening technology, and the FRET system and the luciferase reporting system can also perform high throughput screening of the 3C-like protease inhibitor.
In addition, the invention also provides an anti-PEDV drug, the active ingredient of the drug comprises a 3C-like protease inhibitor, the 3C-like protease inhibitor comprises at least one of myrobalan acid and isomers thereof, quercetin-3-O-p-coumaroyl rhamnoside, troxerutin, citrate B, apigenin-7-O- (2G-rhamnose) gentian glucoside, brutieridin, heraclenol3 '-O-beta-D-apiofuranosyl- (1- & gt 6) -beta-D-glucopyranoside, procyanidin B23,3' -di-O-galtate, citrate A and phenylmethanolic glucose phenolic glucoside benzoate, and the 3C-like protease inhibitor is preferably myrobalan acid.
The 3C-like protease inhibitor can inhibit the replication of PEDV by inhibiting the activity of 3C-like protease, thereby inhibiting the infection of host cells by porcine epidemic diarrhea virus and treating porcine epidemic diarrhea. In the specific implementation, the 3C-like protease inhibitor can be used as one of main components, and medicinal common auxiliary materials and carriers can be added to prepare the medicine for resisting porcine epidemic diarrhea. Meanwhile, the medicament can be selected into proper dosage forms such as tablets, injection, suppositories, aerosols, sustained release preparations, microcapsules, controlled release preparations, liposomes, nano preparations and the like according to actual needs.
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.
Example 1 screening for PEDV 3C-like protease inhibitors
1. Virtual screening
(1) Preparation of receptor molecules: the 3C-like protease (www.rcsb.org, protein number 6U 7K) crystal structure was dehydrated and hydrotreated using autodock1.5.6 software and the molecules stored as PDBQT files. The receptor molecule was turned on using Discover Studio 3.5, the protein active site amino acids were selected, and the molecular docking region, including the central site coordinates and docking region size, was determined. Preparation of ligand molecules: the molecular structure (about 8 ten thousand molecules) in the library of natural molecules was downloaded in ZINC15 and all molecules were individually converted to PDBQT files using Raccon software. And then according to the prepared bat file, performing molecular docking by using an Autodock Vina operation bat file, extracting results after screening, sequencing the results of all molecular docking, and selecting 10000 molecules with the front affinity, namely the primary screening molecules.
(2) All of the nascent molecules were then flexibly docked with 3C-like protease using Discovery studio, and fine-screened for the native molecule with affinity ranking of top 100, the results of which are shown in table 1 (in table 1/referring to the inability to look into or determine their names).
TABLE 1 virtual screening results
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As can be seen from Table 1, the above-mentioned natural molecules (e.g., chebulic acid) are capable of efficiently binding to the active region of PEDV 3C-like protease.
2. Biological Activity screening
2.1 detection of extracellular Activity of Natural molecules obtained by virtual screening
(1) Construction of fluorescence resonance energy transfer System (FRET)
Materials and methods:
according to the sites of PEDV 3C-like protease cleavage PEDV replicase polyprotein ppra and pprab, modified polypeptide Dabcyl-YNSTLQ ∈AGLRKM-E-Edans is synthesized by Kirschner biological company, and the fluorescence of C-terminal modified group Edans of the modified polypeptide under the complete condition can be quenched by N-terminal Dabcyl group due to FRET effect. In the presence of the 3C-like protease, the polypeptide is cleaved, the FRET system is destroyed, and the fluorescent signal of E-Edans is released.
In order to optimize the 3C-like protease activity detection system, the enzyme digestion reaction system comprises 10 mu M of modified polypeptide and 1-10 mu g of 3C-like protease (purified PEDV 3C-like protease) with different concentrations, the total volume of the reaction system is 200 mu L, and the buffer solution is 20mM Tris/HCl solution (pH 7.5). And continuously detecting 480nm emitted light intensity by using a multifunctional enzyme-labeled instrument under the excitation light condition of 37 ℃ and 340nm, and determining the optimal protein addition amount.
Experimental results: FIG. 1 is a graph showing the results of expression and purification of PEDV 3C-like protease, wherein (a) a protein marker; (b) inclusion bodies; (c) supernatant; (d) a GST-tagged 3C-like protease; (e) The GST-tagged 3C-like protease was removed and as can be seen from FIG. 1, the prokaryotic expression system expressed purified PEDV 3C-like protease. FIG. 2 is a graph showing the detection of cleavage activity of purified PEDV 3C-like protease in a FRET system, and it can be seen from FIG. 2 that purified PEDV 3C-like protease can effectively cleave a FRET substrate, and the cleavage activity is dose-dependent. Taken together, it is shown that the FRET system constructed by the invention can effectively detect the activity of PEDV 3C-like protease outside cells.
(2) The material and the method for detecting the inhibition effect of the natural molecules obtained by virtual screening on the 3C-like protease activity comprise the following steps:
according to the detection method optimized in the step (1) in the step 2.1, chebulinic acid (chebulinic acid, purchased from Chengdu Pu Rui method technology Co., ltd., purity > 95%) with different concentrations is added into the FRET reaction system. The intensity of 480nm emitted light is continuously detected by using a multifunctional enzyme-labeled instrument under the excitation light condition of 340nm at 37 ℃. According to the fluorescence intensity detection result, the inhibition effect of the chemulinic acid on the cleavage activity of the PEDV 3C-like protease under the condition of each concentration is calculated. As can be seen from fig. 3, chebulinic acid (chebulinic acid) can inhibit the activity of PEDV 3C-like protease extracellularly and exhibits a remarkable dose dependency.
2.2 detection of the biological Activity of the Natural molecules in the cells obtained by virtual screening
(1) Construction of a Dual luciferase reporter System
Materials and methods:
the cleavage sequence is recognized only when the 3C-like protease is expressed in the cell, and the cleavage sequence is reduced, and the cleavage activity of the 3C-like protease can be detected by recovering the firefly luciferase activity, as shown in FIG. 4.
The constructed reporter plasmid, renilla luciferase plasmid (pRL-TK) and 3C-like protease-expressing plasmid (pCAGGS-NSP 5) were co-transfected into 293T cells, and 24 hours after transfection, the firefly luciferase and Renilla luciferase activities were detected using the Promega double luciferase assay kit.
Experimental results:
FIG. 5 is a graph showing Western blot verification of cleavage of the circularized firefly luciferase by PEDV 3C-like protease, and it can be seen from FIG. 5 that the circularized firefly luciferase can be efficiently cleaved after transfection of a plasmid (pCAGGS-NSP 5) expressing 3C-like protease. FIG. 6 is a graph showing the results of the double luciferase assay kit for detecting the cleavage activity of PEDV 3C-like protease, and as can be seen from FIG. 6, the firefly luciferase activity after transfection of pCAGGS-NSP5 is significantly higher than that of the control group. Taken together, it was shown that the dual luciferase reporter system can effectively detect the activity of PEDV 3C-like protease in cells.
(2) Detection of inhibition effect of natural molecules obtained by virtual screening on 3C-like protease activity
The reporter plasmid 358DnaE-PEDV constructed in step (1) of pCAGGS-NSP5, 2.2 and pRL-TK were co-transfected into 293T cells, and 6h after transfection was replaced with a culture solution containing Chebulinic acid (Chebulinic acid, purchased from Chebulinib technology Co., ltd., purity > 95%) at various concentrations, and the cleavage activity of 3C-like protease was detected 24h after transfection using a promega double luciferase kit, as shown in FIG. 7, and as a result, chebulinic acid was found to be effective in inhibiting the cleavage activity of PEDV 3C-like protease.
Example 2 verification of anti-PEDV effect of PEDV3 c-like protease inhibitor
The testing method comprises the following steps: vero cells (moi=0.001) were infected with PEDV YN144 strain, with different concentrations of chebulinic acid (chebulinic acid) added simultaneously. Samples were collected 24h after infection and the effect of chebulinic acid (chebulinic acid, purchased from Chenopodial Prime technology Co., ltd., purity > 95%) on PEDV replication was evaluated using TCID50 assay, western blot and indirect immunofluorescence assay, and the results are shown in FIGS. 8 to 10.
As can be seen from fig. 7 to 9, chebulinic acid can effectively inhibit infection of PEDV in vitro.
In summary, in the screening method of the PEDV 3C-like protease inhibitor provided by the invention, the PEDV 3C-like protease inhibitor can be efficiently screened out from a large number of small molecules through virtual screening; by providing a FRET system and a luciferase reporter system, the activity of PEDV 3C-like protease can be effectively detected outside and inside cells. In addition, the PEDV 3C-like protease inhibitor (such as myrobalan acid) obtained by screening can effectively inhibit the infection of PEDV in vitro, and the PEDV can be used as an anti-PEDV drug with good therapeutic effect.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (1)
1. Use of chebular acid for the preparation of a medicament against porcine epidemic diarrhea virus.
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| Inhibition of porcine epidemic diarrhea virus replication and viral 3C-like protease by quercetin;Li,Z.H.等;International journal of Molecular sciences;第21卷;8095 * |
| Molecular docking studies on the anti-viral effects of compounds from Kabasura Kudineer on SARS-CoV-2 3CLpro;Vincent,S等;frontiers in Molecular Biosciences;第7卷;613401 * |
| X-Ray structure and inhibition of 3C-like protease from Porcine Epidemic Diarrhea Virus;John,S.E.S等;Scientific Reports;第6卷;25961 * |
| 猪流行性腹泻病毒3C样蛋白酶(3CLpro)的真核表达及生物信息学分析;梁荣等;畜牧与兽医;第52卷(第9期);80-87页 * |
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