Drug screening model for targeting 3CL protease of porcine torovirus and application of drug screening model

Abstract

The invention relates to the technical field of biomedical engineering, and discloses a drug screening model for targeting 3CL protease of porcine torovirus and application of the drug screening model. The model comprises PToV-3CL protease, a fluorescent polypeptide substrate and an enzymolysis reaction buffer solution, wherein the amino acid sequence of the PToV-3CL protease is SEQ ID NO. 1; the fluorescent polypeptide substrate is MCA-EFEQQSSLDK-Lys (Dnp)-Lys-NH2, and the amino acid sequence of the fluorescent polypeptide substrate is SEQ ID NO. 2. The drug screening model can be used for effectively detecting whether the anti-PToV capability of a compound to be detected with 3CL protein as a target spot or not, and can also be used for quantitatively judging the inhibition capability of a target compound. The model is convenient and rapid in medicine screening and simple to operate, has good specificity and sensitivity, has good application prospects in screening of anti-PToV virus infection medicines, and can also be used for screening of 3CLP inhibitors of EToV, BToV or AToV.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering technology, and particularly relates to a drug screening model targeting porcine tetanus virus 3CL protease and its application. Background Art

[0002] Porcine tetanus virus (PToV) is a nonsegmented, single-stranded, positive-sense RNA virus encapsulated in an envelope. It causes symptoms such as diarrhea and weight loss. PToV has a confirmed global distribution, with a high infection rate in pigs. However, a lack of systems for culturing PToV and infection models makes it difficult to obtain large quantities of viral particles, hindering the development of PToV diagnostics and epidemiology.

[0003] The main approaches to finding anti-PToV drugs are: (1) targeted design based on known viral protein functional domains; (2) extraction and screening from natural sources such as marine organisms, Chinese herbal medicines, and microorganisms. The latter are important sources of antiviral drugs with new structures and mechanisms, but it is difficult to identify the antiviral targets of compounds obtained from these natural resources. Therefore, finding targets and constructing a simple, convenient, and rapid drug screening model are of great significance for screening drugs for treating PToV-related diseases. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a drug screening model targeting porcine tetanus virus 3CL protease and its application. The goal is to screen whether a drug candidate can inhibit PToV-3CL protease, thereby verifying its ability to inhibit PToV virus reproduction and its potential application as an anti-PToV drug.

[0005] 3C-like protease, also known as main protease (Mpro), cleaves the polyproteins pp1a and pp1ab to produce other nonstructural proteins. These NSPs play an important role in regulating viral replication and pathogenesis, making 3CLP a potential target for antiviral drug development. This study used a prokaryotic expression system to express and purify a His-Tga-PToV-3CLP fusion protein with a free amino terminus. The biological activity of the PToV-3CLP protease was determined using fluorescence resonance energy transfer (FRET). The established FRET model was then used to screen for a broad spectrum of 3CLP inhibitors, resulting in the identification of small molecule PToV-3CLP protease inhibitors.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a drug screening model targeting porcine tetanus virus 3CL protease, comprising PToV-3CLP protease, a fluorescent polypeptide substrate, and an enzymatic reaction buffer, wherein the amino acid sequence of the PToV-3CLP protein is shown in SEQ ID NO.1; the polypeptide substrate is MCA-substrate (MCA-EFEQQ↓SSLDK-Lys(Dnp)-Lys-NH2 "↓" indicates the recognition cleavage site), and its amino acid sequence is shown in SEQ ID NO.2.

[0008] Preferably, the enzymatic reaction buffer is a reaction buffer solution comprising the following components: 20 mM Tris, 100 mM NaCl, 1 mM EDTA, 4 mM DTT, and 20% glycerol.

[0009] Preferably, the pH of the enzymatic reaction buffer is 7.0-10.0, most preferably pH 9.

[0010] Preferably, the reaction concentration of the PToV-3CLP protein is 0.5-4 μmol / L, most preferably 2.5 μmol / L.

[0011] Preferably, the reaction temperature of the PToV-3CLP protein and the polypeptide substrate is 4-37°C, most preferably 25°C.

[0012] Preferably, the DMSO content of the PToV-3CLP hydrolysis reaction system in the model is less than 2%.

[0013] Preferably, the reaction time between the PToV-3CLP protein and the polypeptide substrate is 80 min.

[0014] The second object of the present invention is to provide the application of the above-mentioned drug screening model targeting porcine tetanus virus 3CL protease in screening anti-PToV virus drugs.

[0015] The present invention also provides the use of the above-mentioned drug screening model targeting porcine tetanus virus 3CL protease in screening 3CLP small molecule inhibitors of PToV (porcine tetanus virus), EToV (equine tetanus virus), BToV (bovine tetanus virus) or AToV (ovine tetanus virus).

[0016] The third object of the present invention is to provide a method for screening anti-PToV virus drugs using the above-mentioned model, which is as follows: adding the compound to be tested and PToV-3CLP protease to the enzymatic reaction buffer, then adding the fluorescent polypeptide substrate, mixing and reacting, and detecting its fluorescence intensity at 405 nm under excitation at a wavelength of 320 nm; compared with the control group to which only the fluorescent polypeptide substrate and PToV-3CLP protease are added, if a decrease in the fluorescence value is detected, it indicates that the compound to be tested can inhibit 3CLP protease and can be further studied and applied as an anti-PToV virus drug; if there is no obvious change in the fluorescence value, it indicates that the compound to be tested cannot inhibit 3CL protease and cannot be used as an anti-PToV virus drug.

[0017] Preferably, the fluorescent polypeptide substrate is excited at a wavelength of 320 nm to obtain a fluorescence intensity value A at 405 nm, indicating the fluorescence intensity when not degraded; the PToV-3CLP protease is added to the enzymatic reaction buffer, and then the fluorescent polypeptide substrate is added, mixed and reacted, and the fluorescence intensity value B at 405 nm is obtained under excitation at a wavelength of 320 nm, indicating the fluorescence intensity after degradation; a certain concentration of the test compound is added to the above reaction system, and the above steps are repeated to obtain the fluorescence intensity value C in the presence of the compound; by comparing the sizes of the three values A, B, and C, the effect of the test compound on the enzymatic activity of the PToV-3CLP protease is determined.

[0018] The principle behind the model construction of the present invention is as follows: This drug screening model primarily consists of the PToV-3CLP protein and a designed fluorescent peptide substrate. The PToV-3CLP protease can specifically cleave the fluorescent substrate, separating the fluorescent group and quenching group at either end of the substrate. The fluorescent group then resumes fluorescence, generating a larger relative fluorescence value. By comparing the fluorescence emission curves of the negative group and the control group, it is determined whether the test compound affects the degradation activity of the PToV-3CLP protease on the substrate, thereby screening for target compounds that are antagonistic to the PToV-3CLP protease. Subsequently, the inhibitory ability of the target compound can be quantitatively determined by calculating the Michaelis constant (Km) and catalytic efficiency (Kcat) of PToV-3CLP.

[0019] The fourth object of the present invention is to provide a method for quantitatively determining the inhibition rate of a drug targeting porcine convulsion virus 3CL protease, which specifically comprises the following steps: diluting the drug to be tested to 100 μmol / L with a buffer containing 3 μmol / L PToV-3CLP protease, adding 1% DMSO to the PToV-3CLP protease as a negative control, and the drug diluted in Tris buffer as a blank control. 25 μL / well is added to a full black half-bottom 96-well plate and incubated at room temperature for 80 min. Then, a FRET substrate with a working concentration of 40 μmol / L is added, 25 μL / well, and after shaking for 1 min, the gain value is set to 58, the detection temperature is 25 ° C, the excitation light is 320 nm, and the emission light is 405 nm. The fluorescence value is measured in a multifunctional microplate reader, and the measured value is the fluorescence value (F0) at the reaction time 0. The fluorescence values of the negative control group at 0 min and 100 min after the reaction are respectively recorded as F0. C 、F1 C After the 96-well plate was placed at room temperature for 100 min, the fluorescence value was measured again and recorded as F1. The inhibition rate of the drug to be tested was calculated according to the formula: 1-(F1-F0) / (F1 C -F0 C ).

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a drug screening model targeting porcine tetanus virus 3CL protease and its application, which can effectively detect whether the anti-PToV ability of the test compound is targeted by 3CL protease and quantitatively determine the inhibitory ability of the target compound. The model is convenient and fast, simple to operate, and has good specificity and sensitivity. It has good application prospects in screening drugs for anti-PToV viral infection and is also suitable for screening 3CLP small molecule inhibitors of EToV, BToV and AToV. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the result of colony PCR identification of the pET28a(+)-PToV-opti3CLP-His plasmid constructed in the present invention.

[0023] Figure 2 This is the SDS-PAGE result of the natural N-terminal PToV-opti3CLP fusion protein prepared in the present invention.

[0024] Figure 3 The relative fluorescence intensity values of the specific cleavage of substrate by PToV-3CLP are determined.

[0025] Figure 4 The results are for the determination of the specific activity of PToV-3CLP.

[0026] Figure 5 The results of the determination of the Michaelis constant and catalytic constant of PToV-3CLP are shown in Figure 2.

[0027] Figure 6 These are the optimization results for the buffer system, 3CLP working concentration, pH, reaction temperature, DMSO content in the system, and reaction time in the FRET model.

[0028] Figure 7 To screen small molecule inhibitors of PToV-3CLP using the FRET model.

[0029] Figure 8 IC of positive inhibitors for 3CLP enzymatic reaction 50 The measurement results of the value.

[0030] Figure 9 The results of sequence alignment (B) and multiple sequence alignment (A) of the recognition cleavage sites of 3CLP in polyproteins of PToV, EToV, BToV, and AToV. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the examples of the present invention are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0033] Drug screening model targeting porcine tetanus virus 3CL protease and its application

[0034] 1. Methods and steps

[0035] 1.1 Construction of pET28a(+)-PToV-opti3CLP-His plasmid

[0036] Based on the reported amino-terminal self-cleavage sequence of mature PToV-3CLP (Xu et al., 2020), its nucleotide sequence was inserted into the N-terminus of the PToV-3CLP gene sequence. The fragment was then ligated into the pET28a(+) vector to construct the pET28a(+)-PToV-3CLP-His prokaryotic expression plasmid. During induced expression of the pET28a(+)-PToV-3CLP-His plasmid, 3CLP protease recognizes the N-terminal self-cleavage site and cleaves it, producing a 3CLP-His fusion protein with a free N-terminus.

[0037] By PCR, the mature PToV-3CLP amino-terminal self-cleavage recognition site sequence was inserted into the N-terminus of the PToV-3CLP gene, as shown in Figure 1 As shown in lanes 1 to 2 of A, the target band of about 955 bp was amplified, which is consistent with the theoretical value. Lanes 3 to 4 are the linearized PCR products of vector pET28a(+), with a band size of about 5293 bp, corresponding to the theoretical band. The constructed plasmid was identified by colony PCR ( Figure 1 B), the target band was visible, and sequencing results showed that the pET28a(+)-PToV-opti3CLP-His expression vector was successfully constructed.

[0038] 1.2 Expression, purification and concentration of PToV-opti3CLP-His protein

[0039] The recombinant plasmid pET28a(+)-PToV-opti3CLP-His was transformed into E. coli-BL21(DE3) competent cells to construct prokaryotic expression engineering bacteria, and induced expression was performed. The bacteria were collected to extract PToV-3CLP protease, which was then purified and concentrated.

[0040] At 25°C and 180 rpm, the cells were induced with IPTG at a final concentration of 0.4 mM for 18 h, and then purified by nickel affinity chromatography and concentrated by 10 kDa ultrafiltration tube. The results of SDS-PAGE (see Figure 2 ) showed that the native N-terminal PToV-opti3CLP fusion protein was successfully obtained, with a size of approximately 34.4 kDa, but with significant contaminants. The concentration of the concentrated protein was measured using a BCA Protein Quantification Kit to be 3 mg / mL.

[0041] 1.3 Determination of biological activity of PToV-3CLP protease

[0042] Based on the natural substrate sequence of mature PToV-3CLP that cleaves nsp12 and nsp13 in the PToV life cycle, MCA-substrate (MCA-EFEQQ↓SSLDK-Lys(Dnp)-Lys-NH2, "↓" indicates the recognition cleavage site) was used as its simulated substrate, and the biological activity of PToV-3CLP protease was identified based on the principle of fluorescence resonance energy transfer (FRET).

[0043] 1.3.1 Specific cleavage of substrates by PToV-3CLP

[0044] Using prokaryotic expression, the supernatant of induced pET28a-PToV-3CLP-His and the supernatant of pET28a bacterial cell disruption were used as negative controls, and Tris buffer was used as blank controls. 25 μL / well of the above samples were added to a black half-bottom 96-well plate. Then, 25 μL / well of substrate with a working concentration of 40 μmol / L was added for FRET reaction. The gain was set to 58, and the relative fluorescence intensity (RFU) was measured using a multifunctional microplate reader (BioTek).

[0045] like Figure 3 As shown, the protein in the negative control group was unable to cleave the substrate, resulting in a very low relative fluorescence value, close to that of the blank control group. In contrast, the relative fluorescence value of the experimental group was much higher than that of both the negative and blank controls. During the first 180 seconds, the fluorescence value increased over time before reaching a plateau. This indicates that the PToV-3CLP protease can specifically cleave the fluorescent substrate, separating the fluorophore and quencher groups at either end of the substrate, allowing the fluorophore to recover its fluorescence and resulting in a higher relative fluorescence value.

[0046] 1.3.2 Establishment of MCA standard curve

[0047] 40 μmol / L MCA was diluted in Tris buffer in 2-fold dilutions to 7 concentration gradients, with the wells containing only Tris solution as negative control wells. The above MCA dilutions were added to a 96-well plate at 50 μL / well. The total amount of MCA in each well was 0, 31.25, 62.5, 125, 250, 500, 1000, and 2000 pmol, respectively. The gain value was set to 58, and RFU was detected. According to the total amount of MCA in each well and the ΔRFU value (ΔRFU = RFU MCA -RFU0) to fit the regression equation and draw the MCA standard curve.

[0048] 1.3.3 Determination of PToV-3CLP specific activity

[0049] A 10 mmol / L MCA-substrate was diluted to 40 μmol / L with Tris solution, and 3CLP was added to final concentrations of 0, 2.5, 5, and 10 μmol / L, respectively. 50 μL / well of the reaction solution was added to a 96-well plate. The RFU values were measured using a multi-functional microplate reader with a gain of 58, a detection temperature of 25°C, an excitation wavelength of 320 nm, an emission wavelength of 405 nm, a detection interval of 1 s, and a total detection time of 16 min. During the first 30 s of the hydrolysis reaction, the total amount of MCA-EFEQQ produced by 3CLP hydrolysis during the specified time was calculated using an established MCA standard curve according to the literature. The hydrolytic activity of PToV-3CLP was then determined using the formula for calculating the specific protease activity (U / mg).

[0050] like Figure 4 As shown, the specific activity of PToV-3CLP was calculated to be U≈22,814.2 U / mg.

[0051] 1.3.4 Determination of Michaelis-Menten Constant and Catalytic Constant of PToV-3CLP

[0052] PToV-3CLP was diluted to 0.5 μmol L-1 in Tris buffer and added to a 96-well plate at 25 μL / well. FRET substrate was then added to the same 96-well plate at 25 μL / well, followed by 180, 160, 140, 120, 100, 80, 60, 40, 20, 10, and 5 μmol / L, respectively. Each concentration was replicated in triplicate. The reaction time was 3 min, and the RFU values within the first 30 s of the enzymatic reaction were measured. The initial reaction velocity (ΔRFU / s) of each group was calculated based on the slope of the hydrolysis curve. The Michaelis-Menten equation was fitted using GraphPad Prism 9.0 software. The Michaelis constant (Km) and catalytic number (kcat) of PToV-3CLP were calculated.

[0053] According to the catalytic constant (K cat ) is calculated using the formula: Kcat = Vmax / [E]total, and the Kcat of PToV-3CLP is calculated.

[0054] The specificity constant value (Kcat / Km) was calculated according to the calculation formula: Kcat / Km=Vmax / ([E]×Km).

[0055] In the formula, [E] is the total enzyme concentration in the system.

[0056] like Figure 5As shown, Km = 72.07 μmol / L, Vmax = 8.847ΔRFU / s, K cat =0.07 / s.

[0057] 1.4 Optimization of the FRET model

[0058] 1.4.1 Optimal buffer system

[0059] To identify the optimal buffer system, 2.5 μmol of 3CLP was added to the buffer conditions listed in the table and 40 μM of the FRET substrate was added. Enzymatic reactions were performed in a black 96-well plate at room temperature, with triplicate wells for each buffer condition. The gain was set to 58, the detection temperature to 25°C, the excitation wavelength to 320 nm, and the emission wavelength to 405 nm. RFU values were measured using a multifunctional microplate reader for a total assay time of 3 minutes. The slope of the hydrolysis reaction curve during the first 30 seconds was used to calculate the initial velocity (ΔRFU / s) of each enzymatic reaction to determine the optimal buffer system.

[0060]

[0061] 1.4.2 Optimal reaction concentration of PToV-3CLP

[0062] 3CLP was diluted with Tris buffer to 4, 3.5, 3, 2.5, 2, 1.5, 1, and 0.5 μmol / L. FRET substrate was added to each dilution to a final concentration of 40 μmol / L. 50 μL was then added to a black, half-bottom 96-well plate. RFU values were measured using a multi-function microplate reader. 3CLP hydrolysis curves were fitted using GraphPad Prism 9.0, and the half-maximal effective concentration (EC50) of 3CLP was calculated.

[0063] 1.4.3 Optimal pH

[0064] The PToV-3CLP hydrolysis reaction was carried out in buffer solutions with pH values of 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0, with three replicates per group. The working concentration of PToV-3CLP was 2.5 μmol / L, and the working concentration of the FRET substrate was 40 μmol / L. The RFU values were detected using a multifunctional microplate reader, and the initial velocities of the 3CLP enzymatic reaction were compared under different pH conditions.

[0065] 1.4.4 Optimal reaction temperature

[0066] The PToV-3CLP hydrolysis reaction was placed in an environment with temperature (T) = 4°C, 25°C, and 37°C, respectively. The specific operation was to dilute the FRET substrate to 40 μmol / L with 2.5 μmol / L PToV-3CLP, set the reaction temperature to 4°C, 25°C, and 37°C, with 3 replicates in each group. The optimal reaction temperature was determined by calculating the initial velocity of the enzymatic reaction at each reaction temperature.

[0067] 1.4.5 Determination of the maximum tolerated concentration of DMSO

[0068] DMSO was added to the PToV-3CLP hydrolysis reaction system at final concentrations of 0, 1%, 2%, 5%, and 10%, respectively. Three replicates were used in each group. The RFU value was detected by a multifunctional microplate reader, and the initial velocity of the enzymatic reaction was calculated to determine the maximum tolerable DMSO concentration during the 3CLP enzymatic hydrolysis reaction.

[0069] 1.4.6 Optimal Response Time

[0070] A PToV-3CLP hydrolysis reaction system was prepared with a working concentration of 2.5 μmol / L PToV-3CLP and a working concentration of 40 μmol / L FRET substrate. 50 μL was added to a black, half-bottomed 96-well plate in triplicate. RFU values were measured using a multi-functional microplate reader at 0, 20, 40, 80, and 100 minutes of the enzymatic reaction. The optimal reaction time for the 3CLP hydrolysis reaction was determined by calculating the ΔRFU value.

[0071] Result Analysis

[0072] like Figure 6 As shown, in Buffer 4 (20mM Tris, 100mM Nacl, 1mM EDTA, 4mM DTT, 20% glycerol), the initial enzymatic reaction rate of PToV-3CLP is the fastest, so Buffer 4 is selected as the optimal buffer system for 3CLP. 2.5μmol / L is used as the optimal working concentration of 3CLP. When pH = 9.0, the enzymatic reaction activity of PToV-3CLP is the highest. 25°C is the optimal reaction temperature. When the DMSO content in the system is less than 2%, it has no significant effect on the activity of 3CLP protease. To ensure the reliability of inhibitor screening, the DMSO content should be less than 2%. To ensure the accuracy and reliability of 3CLP inhibitor screening, the present invention determines that the optimal reaction time between FRET substrate and 3CLP is 80min. 1.5 Screening of small molecule inhibitors of PToV-3CLP protease using the FRET model

[0073] 1.5.1 Preliminary Screening of Marketed Drugs

[0074] The established FERT model was used to rescreen known drug molecules. The specific operation was as follows: GC376, AG7088, and GST-HG171 were diluted to 100 μmol / L with a buffer containing 3 μmol / L PToV-3CLP protease, PToV-3CLP protease with 1% DMSO added was used as a negative control, and drugs diluted with Tris buffer were used as blank controls. 25 μL / well were added to a fully black half-bottom 96-well plate and incubated at room temperature for 80 minutes. Then, a FRET substrate with a working concentration of 40 μmol / L was added, 25 μL / well, and after shaking for 1 minute, the gain value was set to 58, the detection temperature to 25°C, the excitation light to 320 nm, and the emission light to 405 nm. The fluorescence value was measured in a multifunctional microplate reader, and the measured value was the fluorescence value at reaction time 0 (F0). The fluorescence values of the negative control group at 0 minutes and 100 minutes after reaction were recorded as F0, respectively. C 、F1 C After the 96-well plate was placed at room temperature for 100 minutes, the fluorescence value was measured again and recorded as F1. The inhibition rate was calculated according to the formula = 1-(F1-F0) / (F1 C -F0 C ).

[0075] like Figure 7 As shown, compared with the negative control group, these five compounds all showed a certain inhibitory effect on 3CLP protease, thereby reducing the RFU value, among which GST-HG171 had the most obvious inhibitory effect.

[0076] 1.5.2 IC of positive inhibitors on PToV-3CLP enzymatic reaction 50 Determination of value

[0077] GC376 was diluted in Tris buffer containing 3 μmol / L PToV-3CLP protease by a 2-fold serial dilution method to 6 concentrations: 320 μM, 160 μM, 80 μM, 40 μM, 20 μM, and 10 μM. Each concentration was replicated three times, 25 μL / well. Protease without drug was used as a negative control. After incubation at room temperature for 80 minutes, 25 μL of FRET substrate with a working concentration of 40 μM / L was added and the mixture was shaken. The inhibition rate of GC376 was calculated according to the operating instructions. The inhibition curve of GC376 was fitted with GraphPad Prism9.5 to calculate the half-maximal inhibitory concentration (IC). 50 ) value. The same experimental method was used to determine the IC values of AG7088 and GST-drug. 50 value.

[0078] like Figure 8 As shown, GC376 IC50 The value is 129 μM / L, and the IC of AG7088 50 The IC value of GST-HG171 was 146 μM / L. 50 The value is 124.5 μM / L. The method of the present invention can be used to screen inhibitors of PToV virus PToV-3CLP enzyme activity.

[0079] 1.6 Screening of 3CLP small molecule inhibitors using the FRET model, also applicable to EToV, BToV, and AToV

[0080] The 3CLP genes of PToV, EToV, BToV, and AToV were aligned in MEGA, and the recognition cleavage site sequences of the 3CLP of PToV, EToV, BToV, and AToV in the polyprotein were collected and multiple sequence alignment was also performed.

[0081] Result Analysis

[0082] According to the sequence alignment results (such as Figure 9 As shown in A), the 3CLP gene sequences of PToV, EToV, BToV, and AToV in the Tolonoviridae family are highly conserved, and the enzyme active site residues Ser 160-His 53 of PToV 3CLP are also highly conserved in these four viruses. The sequence alignment results of the recognition cleavage sites of the 3CLP of these four viruses in the polyprotein show (as shown in Figure 9 B), Gln is at the cleavage site P1, and Ser is at the cleavage site P1, both of which are highly conserved. The FRET model established in the present invention is also applicable to the screening of 3CLP small molecule inhibitors of EToV, BToV and AToV.

[0083] Obviously, the specific implementation scheme described above is only a further detailed description of the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above is only a specific example of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A drug screening model targeting porcine tetanus virus 3CL protease, characterized in that: It includes PToV-3CL protease, a fluorescent polypeptide substrate, and an enzymatic reaction buffer. The amino acid sequence of the PToV-3CLP protein is shown in SEQ ID NO.1; the polypeptide substrate is MCA-EFEQQSSLDK-Lys(Dnp)-Lys-NH2, and its amino acid sequence is shown in SEQ ID NO.

2.

2. The drug screening model targeting porcine tetanus virus 3CL protease according to claim 1, characterized in that: The enzymatic reaction buffer is a reaction buffer solution, which includes the following components: 20mM Tris, 100mM NaCl, 1mM EDTA, 4mM DTT, and 20% glycerol.

3. The drug screening model targeting porcine tetanus virus 3CL protease according to claim 1, characterized in that: The pH of the enzymatic reaction buffer is 7.0-10.

0.

4. The drug screening model targeting porcine tetanus virus 3CL protease according to claim 1, characterized in that The reaction concentration of the PToV-3CLP protein is 0.5-4 μmol / L.

5. The drug screening model targeting porcine tetanus virus 3CL protease according to claim 1, characterized in that The reaction temperature of the PToV-3CLP protein and the polypeptide substrate is 4-37°C.

6. The drug screening model targeting porcine tetanus virus 3CL protease according to claim 1, characterized in that: The DMSO content of the PToV-3CLP hydrolysis reaction system in the model is less than 2%.

7. The drug screening model targeting porcine tetanus virus 3CL protease according to claim 1, characterized in that: The reaction time of the PToV-3CLP protein and the polypeptide substrate is 80 minutes.

8. Use of the drug screening model targeting porcine tetanus virus 3CL protease according to any one of claims 1 to 7 in screening small molecule inhibitors of 3CLP of PToV, EToV, BToV or AToV.

9. A method for screening anti-PToV virus drugs, characterized in that: Screening is performed using the model described in any one of claims 1 to 7, and the specific steps are as follows: adding the compound to be tested and PToV-3CLP protease to the enzymatic reaction buffer, then adding the fluorescent polypeptide substrate, mixing and reacting, and detecting its fluorescence intensity at 405 nm under excitation at a wavelength of 320 nm; Compared with the control group to which only fluorescent polypeptide substrate and PToV-3CLP protease are added, if a decrease in fluorescence value is detected, it indicates that the compound to be tested can inhibit 3CLP protease and can be further studied and applied as an anti-PToV virus drug; if there is no obvious change in the fluorescence value, it indicates that the compound to be tested cannot inhibit 3CL protease and cannot be used as an anti-PToV virus drug.

10. The method according to claim 9, characterized in that When the fluorescent polypeptide substrate is excited at a wavelength of 320 nm, its fluorescence intensity value A at 405 nm is obtained, which indicates the fluorescence intensity when it is not degraded; PToV-3CLP protease is added to the enzymatic hydrolysis reaction buffer, and then the fluorescent polypeptide substrate is added. After mixing and reaction, its fluorescence intensity value B at 405 nm is obtained under excitation at a wavelength of 320 nm, which indicates the fluorescence intensity after degradation; a certain concentration of the test compound is added to the above reaction system, and the above steps are repeated to obtain the fluorescence intensity value C in the presence of the compound; by comparing the sizes of the three values of A, B, and C, the effect of the test compound on the enzymatic activity of the PToV-3CLP protease is determined.

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