CN113621680A - 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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- CN113621680A CN113621680A CN202110855357.8A CN202110855357A CN113621680A CN 113621680 A CN113621680 A CN 113621680A CN 202110855357 A CN202110855357 A CN 202110855357A CN 113621680 A CN113621680 A CN 113621680A
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Abstract
The invention discloses a 3C-like protease inhibitor of porcine epidemic diarrhea virus, a screening method and an application thereof, wherein the 3C-like protease inhibitor comprises at least one of myrobalic acid and isomers thereof, quercetin-3-O-p-coumaroyl rhamnose glucoside, troxerutin, citrate B, apigenin-7-O- (2G-rhamnose) gentiopicroside, brutinidin, Heraclenol3 '-O-beta-D-apofuranosyl- (1 → 6) -beta-D-glucopyranoside, Procyanidin B23,3' -di-O-gallolate, citrate A and benzyl benzoate glucopyranoside. Because the 3C-like protease plays an important role in the processes of transcription and replication of the porcine epidemic diarrhea virus and host cell defense mechanism resistance, the 3C-like protease inhibitor provided by the invention can inhibit the porcine epidemic diarrhea virus from infecting host cells by inhibiting the activity of the porcine 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 a medicament for resisting porcine epidemic diarrhea virus, and a new application approach 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 (PED) is a highly-contact porcine intestinal infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV), and is mainly characterized by vomiting, watery diarrhea and dehydration of diseased pigs. PED has influence on pigs at all ages, has high lethality particularly to suckling piglets within 7 days of age, has a fatality rate of up to 100 percent, and is an important factor influencing the survival rate of piglets and the pig production at present. The vaccine is an effective means for preventing the disease, but the vaccine cannot provide protection for infected piglets, and no therapeutic medicine is available for the disease at present.
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 a 3C-like protease inhibitor capable of treating porcine epidemic diarrhea.
In order to achieve the above objects, the present invention provides a 3C-like protease inhibitor of porcine epidemic diarrhea virus, wherein the 3C-like protease inhibitor comprises at least one of myrobalic acid and isomers thereof, quercetin-3-O-p-coumaroyl rhamnoside, troxerutin, citrate B, apigenin-7-O- (2G-rhamnose) gentioside, Heraclenol3'-O- β -D-apofuranosyl- (1 → 6) - β -D-glucopyranoside, briuridin, Procyanidin B23,3' -di-O-gallate, citrate a, and benzyl benzoate glucopyranoside.
Further, the present invention also provides a screening method for the 3C-like protease inhibitor of porcine epidemic diarrhea virus as described above, comprising the steps of:
s10, taking 3C-like protease of the porcine epidemic diarrhea virus as a target spot, taking a small molecule database as a screening object, carrying out molecular docking on all molecules in the small molecule database and an active region of the 3C-like protease, and screening to obtain natural molecules with the 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, docking 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 primarily screened molecules with the affinity ranking of 9000-14000;
s12, flexibly docking all the primary-screened molecules with the 3C-like protease by using Discovery studio, and finely screening to obtain natural molecules with the affinity ranking 50-150.
Optionally, step S20 includes:
s21, detecting the activity of the 3C-like protease inhibitor outside the cell by using a fluorescence resonance energy transfer system; and/or the presence of a gas in the gas,
s22, detecting the activity of the 3C-like protease inhibitor in the cell by using a double-fluorescence enzyme report system.
Optionally, step S21 includes:
s210, according to the 3C-like protease to cut the sites of the replicase polyprotein ppla and pplb of the porcine epidemic diarrhea virus, synthesizing a modified polypeptide Dabcyl-YNSTLQ ↓ AGLRKM-E-Edans, wherein the modified polypeptide can be cut by the 3C-like protease so as to release the fluorescence signal of the E-Edans;
s211, adding the 3C-like protease, the 3C-like protease inhibitor and a buffer solution into the modified polypeptide, and detecting the intensity of emitted light at 480nm under the condition of excitation light at 340nm to detect the activity of the 3C-like protease inhibitor.
Optionally, step S22 includes:
s220, performing fusion expression on the 3C-like protease cleavage polypeptide and the firefly luciferase, and inserting an 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 after transfection for 5-7 h, and detecting the cleavage activity of the 3C-like protease by using a dual-luciferase detection kit after transfection for 20-28 h.
In addition, the invention also provides a medicament for resisting the porcine epidemic diarrhea virus, the active ingredient of the medicament comprises a 3C-like protease inhibitor, and the 3C-like protease inhibitor is the 3C-like protease inhibitor of the porcine epidemic diarrhea virus as defined in claim 1.
In the technical scheme provided by the invention, the PEDV 3C-like protease inhibitor comprises myrobalic acid and isomers thereof, quercetin-3-O-p-coumaroyl rhamnose glucoside, troxerutin, citrate B, apigenin-7-O- (2G-rhamnose) gentiopicroside, brutiedin, at least one of Heraclenol3 '-O-beta-D-apofuranosyl- (1 → 6) -beta-D-glucopyranoside, Procyanidin B23,3' -di-O-gallate, citrate A and benzyl benzoate glucoside, PEDV-encoded replicase protein must be cut by 3C-like protease to form mature non-structural protein which plays a vital role in the processes of virus transcription, replication and host cell defense mechanism resistance, therefore, the above-mentioned molecule belonging to the 3C-like protease inhibitor can inhibit infection of host cells by PEDV by inhibiting the activity of 3C-like protease in PEDV, and has a good inhibitory effect. Furthermore, the 3C-like protease inhibitor provided by the invention can be used for preparing anti-PEDV medicines, and a new application approach 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 used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the results of the expression and purification of PEDV 3C-like protease provided by the present invention;
FIG. 2 is a graph showing the cleavage activity of purified PEDV 3C-like protease in FRET system;
FIG. 3 is a graph showing the results of the FRET system detecting the 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 Western blot analysis result of PEDV 3C-like protease cleaved cyclized firefly luciferase;
FIG. 6 is a graph showing the results of detecting the cleavage activity of PEDV 3C-like protease by the dual-luciferase assay kit;
FIG. 7 is a graph showing the results of a dual-luciferase reporter system for detecting the intracellular activity of 3C-like protease inhibitors;
FIG. 8 illustrates the use of TCID50Results plot of the effect of chebulac acid on PEDV replication;
FIG. 9 is a graph showing the results of using a western blot to determine the effect of chebulanic acid on PEDV replication;
figure 10 is a graph of the results of measuring the effect of chebulate acid on PEDV replication using an indirect immunofluorescence assay.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In addition, the meaning of "and/or" appearing throughout includes three juxtapositions, exemplified by "A and/or B" including either A or B or both A and B. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present 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 chebulanic acid and isomers thereof, quercetin-3-O-p-coumaroyl rhamnose glucoside, troxerutin, citrate B, apigenin-7-O- (2G-rhamnose) gentioside, brutieridin, Heraclenol3' -O-beta-D-apofuranosyl- (1 → 6) -beta-D-glucopyranoside, Procyanidin B23,3' -di-O-gallate, citrate A, benzyl benzoate glucopyranoside and Heraclenol3' -O-beta-D-apofuranosyl- (1 → 6) -beta-D-glucopyranoside.
PEDV-encoded Replicase polyproteins (Replicase polyprotein 1a, ppla) and (Replicase polyprotein 1ab, pplab) must be cleaved by viral proteases to form mature non-structural proteins, i.e., sixteen non-structural proteins NSP 1-NSP 16 are generated under the cleavage of papain encoded by NSP3 and 3C-like protease encoded by NSP 5. Wherein the papain is responsible for cutting proteins between NSP1 and NSP4, and the 3C-like protease is responsible for cutting proteins between NSP5 and NSP 16. In addition, 3C-like proteases may also inhibit interferon production by host cells by cleaving NEMO, and are closely associated with the immune escape of PEDV. Since the mature nonstructural proteins play crucial key roles in viral transcription, replication, and defense mechanisms against host cells, 3C-like proteases are essential for the PEDV infection process.
Specifically, the 3C-like protease inhibitor is obtained by screening a natural molecule library in the ZINC15 database. Wherein chebulanic acid (Chebulinic acid) is chebularianic tannic acid, and the ID in the natural molecule library (for convenience of description, the ID in the following refers to the ID in the ZINC15 database) is: ZINC 000299817893; the myrobalic acid isomer comprises micromolecules with IDs of ZINC000390835486, ZINC000257667213 and ZINC 000390835480; quercetin-3-O-p-coumaroyl rhamnose glucoside, namely a small molecule with the ID of ZINC 000253390566; troxerutin (troxerutin) is a small molecule with the ID of ZINC 000299817570; citrate b (parishin b), a small molecule with ID of ZINC 000085643640; apigenin-7-O- (2G-rhamnose) gentiopicroside, namely a micromolecule with the ID of ZINC 000299817632; brutieridin, a small molecule with ID ZINC 000253389678; heraclenol3' -O-beta-D-iodofuranosyl- (1 → 6) -beta-D-glucopyranoside, a small molecule with the 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 of ZINC 000257616571; benzyl benzoate and glucosylchenolide is a small molecule with the ID of ZINC 000253389687.
Preferably, the 3C-like protease inhibitor is myrobalic acid and an isomer thereof, and more preferably, the 3C-like protease inhibitor is myrobalic acid, and experimental verification shows that: the myrobalam acid has good inhibition effect on 3C-like protease. Furthermore, the myrobalamin acid can effectively inhibit the infection of the host cells by PEDV.
The PEDV 3C-like protease inhibitor provided by the invention can inhibit the PEDV from infecting host cells by inhibiting the activity of the 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 medicines, and a new application approach 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 the porcine epidemic diarrhea virus as a target spot, taking a small molecule database as a screening object, carrying out molecular docking on all molecules in the small molecule database and an active region of the 3C-like protease, and screening to obtain natural molecules with the affinity of 50-150 th rank.
Namely, natural molecules which are 50-150 th of the order of the affinity with the 3C-like protease activity region in the small molecule database are obtained by screening through a virtual screening technology. Virtual screening is also called computer screening, namely molecular docking software is used for analyzing the affinity between a target point and a drug molecule so as to reduce the number of actually screened compounds and improve the discovery efficiency of lead compounds.
Wherein, the virtual screening is carried out on the premise of obtaining the crystal structure and the active site of the target protein and a database of the crystal structure of the small molecular compound. At present, a plurality of free small molecule databases exist, and the invention does not limit the specific sources of the small molecule databases and only needs the conventional small molecule databases which can be used for drug screening. In this embodiment, the small molecule database is a zip 15 database, and the zip 15 database can provide 1300 thousands of purchased small molecule structure data for free.
In order to make the small molecule database more targeted and save workload, further, the small molecule database is a natural molecule database in the ZINC15 database. The natural molecule library comprises more than 8 million natural molecule structures, and as the molecules in the natural molecule library are plant extracts and are mostly harmless to organisms, the natural molecule library can be rapidly applied to clinic after being screened and verified to be effective.
Specifically, step S10 includes:
and S11, docking 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-screened 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 No. 6U7K) crystal structure was subjected to dehydration hydrotreating using autodock1.5.6 software and the molecules were stored as PDBQT files. Opening a receptor molecule by using Discover Studio 3.5, selecting protein active site amino acids, and determining a molecule docking region, wherein the determination of the molecule docking region comprises the determination of the coordinates of a central site and the size of the docking region;
preparation of ligand molecules: the molecular structure (about 8 ten thousand molecules) in the natural molecular library was downloaded at zip 15 and all molecules were converted separately into PDBQT files using Raccon software. And then, preparing a bat file according to the bat file, performing molecular docking by using the bat file operated by Autodock Vina, sequencing according to the result of the molecular docking, and preliminarily screening to obtain a primary-screened molecule with the affinity ranking of 9000-14000.
It can be understood that how many primary-screened molecules before the affinity ranking are primarily screened can be determined according to actual conditions, and not only molecules 9000-14000 before the affinity can be screened. If the primary screening is avoided as much as possible to eliminate molecules with possibly better affinity, the number of the primary screened molecules obtained by the primary screening can be increased, such as: screening to obtain a primary sieve molecule with the affinity rank 20000; if the workload is to be saved, the number of preliminarily screened molecules can be appropriately reduced, such as: and screening to obtain the primary screening molecules with the affinity ranking of 5000.
The molecular docking by Autodock Vina is a rigid docking method. In the rigid docking method, in the calculation process, the molecular conformation participating in docking does not change, only the spatial position and the posture of the molecules are changed, the simplification degree of the rigid docking method is highest, and the calculation amount is relatively small.
And S12, flexibly docking all the primary sieve molecules with the 3C-like protease by using Discovery studio, and finely sieving to obtain natural molecules with the affinity being 50-150 th from the top.
Because the primary screening in the step S11 is a rigid docking method and has low accuracy, according to a primary screening result, flexibly docking all the primary screening molecules with the 3C-like protease by using Discovery studio, and finely screening to obtain natural molecules with the affinity ranking 50-150.
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;
because the natural molecules with the affinity ranking of 50-150 finally obtained by adopting virtual screening are only 20-30% of the natural molecules with the 3C-like protease inhibitor activity, the natural molecules obtained by screening also need to be subjected to biological activity screening so as to obtain the 3C-like protease inhibitor with the activity.
The natural molecule can be directly screened for biological activity outside the cell, the operation is simple, the cost is saved, the natural molecule can be screened for biological activity inside the cell, and the result is more accurate and convincing. Of course, biological activity screening can also be performed both extracellularly and intracellularly.
The present invention is not limited to the specific method for screening the natural molecule for biological activity in the extracellular and/or intracellular environment, and in one embodiment, step S20 includes:
s21, detecting the activity of the 3C-like protease inhibitor outside the cell by using a fluorescence resonance energy transfer system; and/or the presence of a gas in the gas,
s22, detecting the activity of the 3C-like protease inhibitor in the cell by using a double-fluorescence enzyme report system.
Among them, the Fluorescence Resonance Energy Transfer (FRET) system and the dual-luciferase reporter system can be used in 384 microwell plates and microplate readers to perform high-throughput screening of 3C-like protease inhibitors.
In addition, because 100 natural molecules virtually screened are not easily obtained, the embodiment of the invention only takes myrobalic acid as an example to verify the feasibility of a FRET system and a dual-luciferase reporter system for detecting the biological activity of the screened natural molecules. It will be appreciated that it is also possible to select the most suitable native molecule for anti-PEDV and its concentration by adding the native molecule at different concentrations to obtain the native molecule with the best inhibitory effect on the cleavage activity of the PEDV 3C-like protease and its concentration, and then combining the cost, dosage, safety and other factors.
In one embodiment, step S21 includes:
step S210, according to the cleavage of the sites of PPpla and pplb of PEDV replicase polyprotein by 3C-like protease, synthesizing a modified polypeptide Dabcyl-YNSTLQ ↓ AGLRKM-E-Edans, wherein the modified polypeptide can be cleaved by the 3C-like protease, so that the fluorescence signal of E-Edans is released;
in the intact case, the fluorescence of the C-terminal modification group Edans is quenched by the N-terminal Dabcyl group due to FRET effect, while in the presence of 3C-like protease, the modified polypeptide is cleaved, the FRET system is destroyed, and the fluorescence signal of 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 intensity of emitted light at 480nm under the excitation light condition of 340nm to detect the activity of the 3C-like protease inhibitor.
In another embodiment, step S22 includes the steps of:
s220, performing fusion expression on the 3C-like protease cleavage polypeptide and the firefly luciferase, and inserting an intein DnaE to obtain a cyclization report plasmid;
due to the introduction of the intein DnaE, the luciferase protein expressed after the reporter plasmid transfects cells is expressed in a form of cyclic protein in a natural state, and the cyclic proteins have great steric hindrance and are difficult to approach each other to avoid self-activation. Only when the 3C-like protease is expressed in cells, the cleavage sequence can be recognized and cleaved, the steric hindrance is reduced after the cleavage, 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 after transfection for 5-7 h, and detecting the cleavage activity of the 3C-like protease by using a dual-luciferase detection kit after transfection for 20-28 h.
According to the screening method of the PEDV 3C-like protease inhibitor, PEDV 3C-like protease is taken as a target spot, 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), natural molecules with the affinity being 50-150 th above the rank are obtained, then biological activity detection is carried out through a FRET system and/or a luciferase report 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, so that the copying of PEDV is inhibited; 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 carry out high throughput screening on the 3C-like protease inhibitor.
In addition, the invention also provides an anti-PEDV medicament, the active component of the medicament comprises a 3C-like protease inhibitor, the 3C-like protease inhibitor comprises myrobalic acid and isomers thereof, quercetin-3-O-p-coumaroyl rhamnose glucoside, troxerutin, citrate B, apigenin-7-O- (2G-rhamnose) gentiopicroside, brutinidine, Heraclenol3 '-O-beta-D-apofuranosyl- (1 → 6) -beta-D-glucopyranoside, Procyanidin B23,3' -di-O-gallate, citrate A and benzyl benzoate glucopyranoside, and preferably, the 3C-like protease inhibitor is myrobalic acid.
The 3C-like protease inhibitor can inhibit the replication of PEDV by inhibiting the activity of the 3C-like protease, thereby inhibiting the infection of the porcine epidemic diarrhea virus on host cells and playing a role in treating the porcine epidemic diarrhea. In specific implementation, the 3C-like protease inhibitor can be used as one of the main components, and common pharmaceutical adjuvants and carriers can be added to prepare the medicine for resisting porcine epidemic diarrhea. Meanwhile, the medicine can be selected into proper dosage forms according to actual requirements, such as tablets, injections, suppositories, aerosols, sustained-release preparations, microcapsules, controlled-release preparations, liposomes, nano-preparations and the like.
The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, it should be understood that the following examples are merely illustrative of 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 No. 6U7K) crystal structure was subjected to dehydration hydrotreating using autodock1.5.6 software and the molecules were stored as PDBQT files. The Discover Studio 3.5 was used to open the receptor molecule, select the protein active site amino acids, and determine the docking region of the molecule, including the central site coordinates and the docking region size. Preparation of ligand molecules: the molecular structure (about 8 ten thousand molecules) in the natural molecular library was downloaded at zip 15 and all molecules were converted separately into PDBQT files using Raccon software. And then, according to the prepared bat file, performing molecular docking by using the Autodock Vina operating bat file, extracting results after screening is finished, sequencing the results of all molecular docking, and selecting 10000 molecules with the highest affinity, namely the primary-screened molecules.
(2) All primary screened molecules were then subjected to flexible docking with 3C-like protease using Discovery studio and fine-screened to give top 100 affinity native molecules, the results of which are shown in table 1 (table 1/indicates that their names cannot be found or determined).
TABLE 1 virtual screening results
As can be seen from Table 1, the above-mentioned native molecules (e.g., chebulac acid) are effective in binding to the active region of the PEDV 3C-like protease.
2. Biological Activity screening
2.1 detection of extracellular biological Activity of Natural molecules obtained by virtual screening
(1) Construction of fluorescence resonance energy transfer System (FRET)
The material and the method are as follows:
according to the cleavage site of the PEDV replicase polyproteins ppla and pplab by the PEDV 3C-like protease, a modified polypeptide Dabcyl-YNSTLQ ↓agLRKM-E-Edans was synthesized by Kirgiz bio-Inc., and the fluorescence of the C-terminal modification group Edans in the complete case was quenched by the N-terminal Dabcyl group due to FRET effect. In the presence of a 3C-like protease, the polypeptide is cleaved, the FRET system is disrupted, and the fluorescent signal of E-Edans is released.
In order to optimize a 3C-like protease activity detection system, an enzyme digestion reaction system comprises 10 mu M 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 a buffer solution is 20mM Tris/HCl solution (pH 7.5). And continuously detecting 480nm emitted light intensity by using a multifunctional microplate reader at 37 ℃ under 340nm exciting light conditions, and determining the optimal protein addition amount.
The experimental results are as follows: FIG. 1 is a graph showing the results of the expression and purification of PEDV 3C-like protease, wherein (a) protein marker; (b) inclusion bodies; (c) supernatant fluid; (d) a GST-tagged 3C-like protease; (e) as can be seen from FIG. 1, the prokaryotic expression system expresses and purifies PEDV 3C-like protease. FIG. 2 is a graph showing the cleavage activity of the purified PEDV 3C-like protease in the FRET system, and it can be seen from FIG. 2 that the purified PEDV 3C-like protease can efficiently cleave a FRET substrate, and the cleavage activity is dose-dependent. In conclusion, the FRET system constructed by the invention can effectively detect the activity of the PEDV 3C-like protease in the cells.
(2) The material and the method for detecting the inhibition effect of the natural molecules obtained by virtual screening on the activity of the 3C-like protease comprise the following steps:
according to the detection method optimized in step (1) of 2.1, chebulinic acid (myrobalic acid, purchased from Kyoto-Dougui-Co., Ltd., purity > 95%) was added at different concentrations to the FRET reaction system. And continuously detecting the 480nm emission light intensity by using a multifunctional microplate reader at 37 ℃ under the condition of 340nm exciting light. And calculating the inhibition effect of chebulinic acid on the PEDV 3C-like protease cleavage activity under various concentration conditions according to the fluorescence intensity detection result. As can be seen from fig. 3, chebulinic acid (myrobalaminic acid) can inhibit the activity of PEDV 3C-like protease extracellularly and exhibit a significant dose dependence.
2.2 detection of the biological Activity of the Natural molecules obtained by virtual screening in cells
(1) Construction of Dual-luciferase reporter System
The material and the method are as follows:
PEDV 3C-like protease cleavage substrate polypeptides (polypeptide sequence: YNSTLQAGLRKM, clean peptides) and firefly luciferase (firefly luciferase) are subjected to fusion expression to construct a reporter plasmid (because intein DnaE is introduced, luciferase protein expressed after cells are transfected by the reporter plasmid is naturally expressed and exists in the form of cyclic protein, great steric hindrance exists between the cyclic protein and is difficult to approach each other so as to avoid self-activation of the cyclic protein).
The constructed reporter plasmid, the Renilla luciferase plasmid (pRL-TK) and a plasmid expressing 3C-like protease (pCAGGS-NSP5) are co-transfected into 293T cells, and are detected by western blot 24h after transfection, and the activities of firefly luciferase and Renilla luciferase are detected by using a Promega dual-luciferase detection kit.
The experimental results are as follows:
FIG. 5 is a graph showing the results of Western blot analysis of cyclized firefly luciferase cleaved by PEDV 3C-like protease, and it can be seen from FIG. 5 that the cyclized firefly luciferase can be efficiently cleaved after transfection of a plasmid expressing 3C-like protease (pCAGGS-NSP 5). FIG. 6 is a graph showing the result of detecting the cleavage activity of PEDV 3C-like protease by using the dual-luciferase assay kit, and it can be seen from FIG. 6 that the luciferase activity of firefly transfected with pCAGGS-NSP5 is significantly higher than that of the control group. Taken together, it was shown that the dual-luciferase reporter system allows efficient intracellular detection of the activity of PEDV 3C-like protease.
(2) Detection of inhibition effect of natural molecules obtained by virtual screening on 3C-like protease activity
pCAGGS-NSP5, the reporter plasmid 358DnaE-PEDV constructed in step (1) of 2.2 and pRL-TK were co-transfected into 293T cells, and 6h after transfection, the culture solution containing different concentrations of Chebulinic acid (myrobalic acid, purchased from Doppel technologies, Inc., purity > 95%) was replaced, and 24h after transfection, the cleavage activity of 3C-like protease was detected using a promega dual-luciferase kit, and the results are shown in FIG. 7, and it can be seen from FIG. 7 that Chebulinic acid can effectively inhibit the cleavage activity of PEDV 3C-like protease.
Example 2 verification of anti-PEDV Effect of PEDV 3C-like protease inhibitors
The test method comprises the following steps: vero cells were infected with PEDV YN144 strain (MOI ═ 0.001) and varying concentrations of chebulaic acid (chebulinic acid) were added at the same time. Samples were collected 24h post infection and evaluated for the effect of chebulinic acid (myrobalic acid, purchased from dodemory technologies ltd., purity > 95%) on PEDV replication using the TCID50 assay, western blot and indirect immunofluorescence assay, with results shown in figures 8 to 10.
As can be seen from figures 7 to 9, Chebulinic acid is effective in inhibiting infection by PEDV in vitro.
In conclusion, 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 from a large number of small molecules through virtual screening; the provided FRET system and luciferase reporter system can effectively detect the activity of the PEDV 3C-like protease in the cells and outside the cells. In addition, the screened PEDV 3C-like protease inhibitor (such as myrobalam acid) can effectively inhibit the infection of PEDV in vitro, and has good treatment effect when being used as an anti-PEDV medicament.
The above is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the scope of the present invention.
Claims (8)
1. A 3C-like protease inhibitor of porcine epidemic diarrhea virus, wherein the 3C-like protease inhibitor comprises at least one of chebulac acid and its isomers, quercetin-3-O-p-coumaroyl rhamnose glucoside, troxerutin, citrate B, apigenin-7-O- (2G-rhamnose) gentioside, brutinidin, Heraclenol3'-O- β -D-apofuranosyl- (1 → 6) - β -D-glucopyranoside, Procyanidin B23,3' -di-O-gallolate, citrate a and benzyl benzoate glucophenol glycoside.
2. A method of screening for a 3C-like protease inhibitor of porcine epidemic diarrhea virus as claimed in claim 1, comprising the steps of:
s10, taking 3C-like protease of the porcine epidemic diarrhea virus as a target spot, taking a small molecule database as a screening object, carrying out molecular docking on all molecules in the small molecule database and an active region of the 3C-like protease, and screening to obtain natural molecules with the 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.
3. The method of screening for a 3C-like protease inhibitor of porcine epidemic diarrhea virus of claim 2, wherein said small molecule database is a natural molecule database of said ZINC15 database.
4. The method for screening a 3C-like protease inhibitor of porcine epidemic diarrhea virus of claim 2, wherein step S10 comprises:
s11, docking 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 primarily screened molecules with the affinity ranking of 9000-14000;
s12, flexibly docking all the primary-screened molecules with the 3C-like protease by using Discovery studio, and finely screening to obtain natural molecules with the affinity ranking 50-150.
5. The method for screening a 3C-like protease inhibitor of porcine epidemic diarrhea virus of claim 2, wherein step S20 comprises:
s21, detecting the activity of the 3C-like protease inhibitor outside the cell by using a fluorescence resonance energy transfer system; and/or the presence of a gas in the gas,
s22, detecting the activity of the 3C-like protease inhibitor in the cell by using a double-fluorescence enzyme report system.
6. The method of screening for a 3C-like protease inhibitor of porcine epidemic diarrhea virus of claim 5, wherein step S21 comprises:
s210, according to the 3C-like protease to cut the sites of the replicase polyprotein ppla and pplb of the porcine epidemic diarrhea virus, synthesizing a modified polypeptide Dabcyl-YNSTLQ ↓ AGLRKM-E-Edans, wherein the modified polypeptide can be cut by the 3C-like protease so as to release the fluorescence signal of the E-Edans;
s211, adding the 3C-like protease, the 3C-like protease inhibitor and a buffer solution into the modified polypeptide, and detecting the intensity of emitted light at 480nm under the condition of excitation light at 340nm to detect the activity of the 3C-like protease inhibitor.
7. The method of screening for a 3C-like protease inhibitor of porcine epidemic diarrhea virus of claim 5, wherein step S22 comprises:
s220, performing fusion expression on the 3C-like protease cleavage polypeptide and the firefly luciferase, and inserting an 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 after transfection for 5-7 h, and detecting the cleavage activity of the 3C-like protease by using a dual-luciferase detection kit after transfection for 20-28 h.
8. A medicament against porcine epidemic diarrhea virus, wherein the active ingredient of the medicament comprises a 3C-like protease inhibitor, and the 3C-like protease inhibitor is the 3C-like protease inhibitor of porcine epidemic diarrhea virus of claim 1.
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