Use of nucleic acid molecules and related biological materials in the preparation of coronavirus mpro inhibitor screening products

Abstract

The application discloses a nucleic acid molecule and application thereof in preparation of a coronavirus Mpro inhibitor screening product. The core element of the coronavirus Mpro inhibitor screening system in the application comprises a nucleic acid molecule coding a fusion protein, the nucleic acid molecule comprises, in sequence, a coding gene of split EX, a coding gene of a cleavage site of Mpro enzyme, a coding gene of Mpro enzyme and a coding gene of split AP, wherein the split AP and the split EX are connected to form a complete peroxidase to realize the catalytic function of the enzyme. The application can be used for preparing a coronavirus inhibitor screening product with high safety, high sensitivity and specificity, and can be used for screening a drug for inhibiting the coronavirus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of nucleic acid molecules and related biomaterials in the preparation of coronavirus Mpro inhibitor screening products. Background Technology

[0002] APEX2 is an engineered horseradish peroxidase that has become an indispensable and powerful tool in modern cell biology and proteomics research. At its core, APEX2's ability is not luminescence, but catalysis. When hydrogen peroxide (H2O2) and a biotin-phenol substrate are added to cells expressing the APEX2 target protein fusion, a bright red fluorescent signal is produced.

[0003] The pcDNA3.1(+) vector is one of the most commonly used mammalian cell expression vectors. The vector uses a cytomegalovirus (CMV) enhancer promoter for high-level expression.

[0004] Human coronaviruses (CoVs) are enveloped viruses with a positive-sense single-stranded RNA genome. Currently, six human coronaviruses have been reported, causing moderate to severe respiratory and intestinal infections in humans, posing a significant challenge to human health and causing substantial economic losses. Although effective vaccines and therapeutic monoclonal antibodies exist, a large portion of the global population remains unvaccinated and without access to monoclonal antibody therapies due to cost and limited supply. Small molecule drugs are urgently needed to curb the progression of the COVID-19 pandemic. Therefore, there is an urgent need in this field to develop a reporter system capable of real-time, efficient, and visually screening and evaluating coronavirus main protease (Mpro) inhibitors in living cells.

[0005] Mpro plays a crucial role in viral replication, responsible for cleaving viral polyproteins, and is a highly attractive key target for developing broad-spectrum anti-coronavirus drugs. Currently, methods for screening Mpro inhibitors mainly include in vitro enzyme activity assays and cytotoxicity assays. However, these methods may have limitations such as low throughput, high cost, or inability to accurately reflect inhibitory effects in a living cell environment. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to screen for coronavirus Mpro inhibitors and / or how to efficiently, sensitively and specifically screen for coronavirus Mpro inhibitors at the cellular level.

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a reporter system based on protein fragment complementation technology, which can efficiently, sensitively and specifically screen for coronavirus Mpro inhibitors at the cellular level.

[0008] This invention relates to the fields of molecular biology and drug screening technology, specifically to a reporter system for screening inhibitors of coronavirus main protease (Mpro, also known as 3CLpro), as well as a method for constructing and applying the reporter system. To address the aforementioned technical problems, this invention first provides a nucleic acid molecule that encodes a fusion protein, the fusion protein comprising a coronavirus Mpro enzyme, a cleavage EX, a cleavage site of the Mpro enzyme, and a cleavage AP. The cleavage AP may be a polypeptide fragment at the N-terminus of a peroxidase; the cleavage EX may be the remaining polypeptide fragment after cleavage of the cleavage AP from the peroxidase.

[0009] In the aforementioned nucleic acid molecule, the splitting AP and the splitting EX can be linked to form the complete peroxidase to achieve the catalytic function of the peroxidase. In a specific embodiment of the present invention, the length of the splitting EX is 50 amino acid residues, the length of the splitting AP is 199 amino acid residues, and the total length of the two is 249 amino acid residues. During construction, the splitting EX fragment (50 amino acid residues) is placed at the N-terminus (5' end), and the splitting AP fragment (199 amino acid residues) is placed at the C-terminus (3' end).

[0010] The aforementioned nucleic acid molecule may contain the coding gene for the split EX, the coding gene for the cleavage site of the Mpro enzyme, and the coding gene for the split AP. The coding gene for the cleavage site of the Mpro enzyme may be located between the coding gene for the split EX and the coding gene for the split AP, that is, the coding gene for the split EX and the coding gene for the split AP are separated by the coding gene for the cleavage site of the Mpro enzyme.

[0011] The aforementioned nucleic acid molecule may also contain a coronavirus Mpro enzyme encoding gene. The coronavirus Mpro enzyme encoding gene may be located between the EX cleavage encoding gene and the AP cleavage encoding gene, i.e., the EX cleavage encoding gene and the AP cleavage encoding gene are separated by the Mpro enzyme cleavage site encoding gene and the coronavirus Mpro enzyme encoding gene.

[0012] In the nucleic acid molecule described above, the coding gene for the split EX may be located upstream of the coding gene for the cleavage site of the Mpro enzyme. The coding gene for the cleavage site of the Mpro enzyme may be located upstream of the coding gene for the split AP.

[0013] In the nucleic acid molecule described above, the peroxidase can be any of the following proteins: A1) The amino acid sequence is that of the protein in sequence 3 of the sequence listing; A2) A protein derived from A1) or having more than 80% identity with and the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) Proteins obtained by attaching protein tags to the N-terminus and / or C-terminus of A1) or A2); The Mpro enzyme can be any of the following proteins: B1) The amino acid sequence is the protein consisting of amino acid residues 62-367 of sequence 2 in the sequence listing; B2) A protein derived from B1) or having more than 80% identity with and the same function as the protein shown in B1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in B1). B3) is a protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2).

[0014] The above-mentioned nucleic acid molecule is characterized in that: the nucleic acid molecule may be any of the following: 1) The coronavirus Mpro enzyme encoding gene is nucleotides 196-1113 of sequence 1 in the sequence listing, and / or, 2) The Mpro cleavage site encodes nucleotides 181-195 of sequence 1 in the sequence listing, and / or, 3) The coding gene for the split EX is nucleotides 16-165 of sequence 1 in the sequence listing, and / or, 4) The coding gene for the split AP is nucleotides 1129-1725 of sequence 1 in the sequence listing.

[0015] The nucleic acid molecules mentioned above can be the DNA molecules shown in Sequence 1 of the sequence listing.

[0016] To address the aforementioned technical problems, the present invention also provides a biomaterial, which may be any of the following: B1) Expression cassettes containing the nucleic acid molecules described above; B2) A recombinant vector containing the nucleic acid molecules described above, or a recombinant vector containing the expression cassette described in B1); B3) Recombinant microorganisms containing the nucleic acid molecules described above, or recombinant microorganisms containing the expression cassette described in B1), or recombinant microorganisms containing the recombinant vector described in B2); B4) Recombinant cell lines containing the nucleic acid molecules described above, or recombinant cell lines containing the expression cassette described in B1), or recombinant cell lines containing the recombinant vector described in B2), or recombinant cell lines containing the recombinant microorganisms described in B3).

[0017] To address the aforementioned technical problems, the present invention also provides a fusion protein, which may be any of the fusion proteins described above.

[0018] To address the aforementioned technical problems, the present invention also provides applications, which may be the applications of the nucleic acid molecules described above, the applications of the biological materials described above, or the applications of the fusion proteins described above, and the applications may be any of the following: P1. Application in the preparation of screening products for coronavirus Mpro inhibitors; P2. Application in the preparation of screening products for anti-coronavirus drugs; P3. Application in screening inhibitors for coronavirus Mpro; P4. Application in screening anti-coronavirus drugs.

[0019] This invention provides a reporter system for screening coronavirus Mpro inhibitors. The reporter system uses pcDNA3.1 (or other suitable eukaryotic expression vectors) as its vector, and its core elements are sequentially linked from the N-terminus to the C-terminus as follows: The split AP fragment: namely, the N-terminal fragment of Ascorbate Peroxidase (APEX2).

[0020] Mpro cleavage sequence: an amino acid sequence that can be specifically recognized and cleaved by coronavirus Mpro (e.g., a conserved cleavage site sequence derived from coronavirus polyprotein).

[0021] Mpro gene: The gene encoding the main protease of coronaviruses.

[0022] The split EX segment: the other half of the APEX2 segment that is complementary to the AP segment.

[0023] Preferably, the linkage sequence of the reporter system is: split EX-Mpro cleavage sequence-Mpro-split AP. This design ensures that the complete APEX enzyme can only be correctly assembled when Mpro is effectively inhibited.

[0024] Secondly, the present invention provides the working principle of the above-mentioned reporting system: Inhibitor-free case (negative control): After transfecting the reporter system into host cells (e.g., HEK293T cells), the system expresses a fusion protein. The Mpro contained within autonomously recognizes and cleaves the preceding Mpro cleavage sequence, resulting in the separation of the AP and EX fragments. Since the function of the APEX enzyme depends on the physical binding of the two fragments, the separated fragments cannot reconstruct the catalytically active APEX enzyme. Therefore, even with the addition of an APEX substrate (e.g., a fluorescent substrate), no fluorescent signal can be detected.

[0025] In the presence of inhibitors (test sample): When an effective Mpro inhibitor is added to the cell culture medium, the inhibitor molecules enter the cells and inhibit the protease activity of Mpro. Inactivated Mpro cannot cleave its preceding cleavage sequence, allowing the entire fusion protein to be preserved intact. At this point, the AP and EX fragments located on the same polypeptide chain can spontaneously fold and assemble into an APEX enzyme with complete catalytic activity. Subsequently, the addition of an APEX substrate allows the active APEX enzyme to catalyze a reaction that produces a strong fluorescent signal.

[0026] The intensity of the fluorescence signal is positively correlated with the inhibitory efficacy of Mpro inhibitors, thus enabling efficient screening and efficacy evaluation of inhibitor compounds.

[0027] Thirdly, the present invention provides the application of the reporting system in screening anti-coronavirus drugs.

[0028] The beneficial effects of this invention are as follows: High sensitivity and specificity: Based on the catalytic amplification effect of APEX, it can generate extremely strong fluorescence signals and has high detection sensitivity; and the reaction depends on the specific cleavage of Mpro, resulting in a low false positive rate.

[0029] Visualization and high throughput: Fluorescent signals are easy to detect, and their distribution within cells can be observed using a fluorescence microscope, or high-throughput screening can be performed using an ELISA reader, making it ideal for screening large-scale drug libraries.

[0030] High safety: The system contains only reporter genes and viral proteases, and does not require handling live viruses. It can be operated in a biosafety level 1 (BSL-1) laboratory, ensuring high safety.

[0031] High versatility: By replacing the Mpro cleavage sequence and Mpro gene, it can be adapted to the screening of inhibitors for other coronaviruses, and has broad applicability. Attached Figure Description

[0032] Figure 1 The figures above and below are schematic diagrams illustrating the working principle and plasmid vector structure of the reporting system of the present invention.

[0033] Figure 2 This document presents the fluorescence detection results and quantification of fluorescence results for inhibitor validation using the reporting system of this invention. A shows the fluorescence detection results for inhibitor validation using the reporting system of this invention. B is a quantification graph of fluorescence results, with the vertical axis representing mean fluorescence intensity (MFI) and the horizontal axis representing experimental negative and positive controls.

[0034] Figure 3 The image shows the fluorescence detection results used to verify the specificity of the reporting system of this invention.

[0035] Figure 4 The reporting system of this invention is used to screen drug ICs based on the fluorescence detection results of different drugs. 50 Evaluation. A shows the fluorescence detection results of different drugs by the reporting system; B shows the IC50 of three different Mpro inhibitors screened by the reporting system. 50 The evaluation is as follows: the left vertical axis represents the drug inhibition rate, the right vertical axis represents the cell viability rate, and the horizontal axis represents the drug concentration (μM).

[0036] Figure 5 This is a Z-factor assessment plot for the reporting system. The vertical axis represents the mean fluorescence intensity (MFI), and the horizontal axis represents the number of positive inhibitors and the number of inhibitors-free (number 50). Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] Virus: The public may obtain the biological material from the applicant in accordance with the relevant national biosafety regulations. The biological material is only for repeating the relevant experiments of this invention and may not be used for other purposes.

[0040] The coronavirus Mpro inhibitor (GC376) in this embodiment of the invention is from the manufacturer: sellect, catalog number: S0475.

[0041] The nucleotide sequence of the coronavirus Mpro inhibitor screening report system in this embodiment of the invention was provided by the laboratory of Sangon Biotech Co., Ltd. (China).

[0042] Example 1. Construction of a report system for screening coronavirus Mpro inhibitors The functional target fragment of the reporter gene expression plasmid in the coronavirus Mpro inhibitor detection and reporting system of the present invention was synthesized by Shanghai Sangon Biotech. The functional target fragment (Sequence 1 in the sequence listing) consists of the coding gene M1 for split EX, the coding gene M2 for the coronavirus (SARS-CoV-2 in this embodiment) Mpro cleavage site (VAKLQ in this embodiment) connected to M1, the coding gene M3 for Mpro connected to M2, and the coding gene M4 for split AP connected to M3.

[0043] In this context, the split AP is a C-terminal or N-terminal polypeptide fragment of Ascorbate Peroxidase (APEX2, amino acid sequence of sequence 3 in the sequence listing); the split EX is the remaining APEX2 polypeptide fragment after the split AP polypeptide fragment is extracted from APEX2; the split AP and the split EX can be linked to form the complete APEX2; the Mpro cleavage site is an amino acid sequence that can be specifically recognized and cleaved by coronavirus Mpro (in this embodiment, it is a conserved cleavage site sequence derived from coronavirus polyprotein); Mpro is the coronavirus main protease.

[0044] This functional fragment expresses a fusion protein (amino acid sequence of sequence 2 in the sequence listing) consisting of the cleavage EX, the coronavirus Mpro cleavage site, the Mpro enzyme, and the cleavage AP. Amino acid residues 62-367 of sequence 2 are the Mpro sequence, which is the coronavirus main protease sequence.

[0045] The synthesized functional target fragment was inserted into the multiple cloning site after the CMV promoter of the pcDNA3.1 vector between the recognition sites of restriction endonucleases NheI and XbaI, resulting in the recombinant reporter gene expression plasmid (pcDNA3.1(+)-EX-Mpro cs-Mpro-AP), which was verified to be correct by sequencing.

[0046] Sequence 1 (5'-3'):

[0047] Sequence 1 includes the NheI recognition site sequence (nucleotides 1-6 of sequence 1), the Kozak sequence (nucleotides 7-15 of sequence 1), the gene encoding the split EX (nucleotides 16-165 of sequence 1), the linker (nucleotides 166-180 of sequence 1), the gene encoding the Mpro cleavage site (nucleotides 181-195 of sequence 1), the gene encoding the Mpro (nucleotides 196-1113 of sequence 1), the linker (nucleotides 1114-1128 of sequence 1), the gene encoding the split AP (nucleotides 1129-1725 of sequence 1), the stop codon site sequence (nucleotides 1726-1728), and the XbaI recognition site sequence (nucleotides 1728-1734 of sequence 1).

[0048] Sequence 2 (NC): .

[0049] Amino acid residues 2-51 of sequence 2 correspond to the EX sequence, amino acid residues 52-56 of sequence 2 correspond to the linker sequence, amino acid residues 57-61 of sequence 2 correspond to the Mpro cleavage site, amino acid residues 62-367 of sequence 2 correspond to the Mpro protein, amino acid residues 368-372 of sequence 2 correspond to the linker sequence, and amino acid residues 373-571 of sequence 2 correspond to the cleavage AP sequence.

[0050] Sequence 3 (NC): GKSYPTVSADYQDAVEKAKKRLGGFIAEKRCAPLMLRLAFHSAGTFDKRTKTGGPFGTIRRPAELAHSANSGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPP PEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTLGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKASELGFADA.

[0051] The AP sequence (amino acids 1-199 of sequence 3) and the EX sequence (amino acids 200-249 of sequence 3) are in sequence 3.

[0052] Example 2. Functional verification of the reporting system of the present invention Prepare 293T cells one day in advance. Dilute the prepared 293T cells with complete culture medium and seed them at 100 μL / well in a 96-well plate, with a cell count of approximately 2.5 × 10⁶ cells per well. 4 They were then incubated overnight in a 37°C, 5% CO2 (volume fraction, the meaning of CO2 is the same below) cell culture incubator.

[0053] After culturing 293T cells for 18 hours, when the cell density reached 70%-80%, cell transfection was performed. The pcDNA3.1(+)-EX-Mpro cs-Mpro-AP plasmid was pre-diluted to 50 ng / μL and transfected into 293T cells using a Lipofectamine 3000 transfection kit. The transfection system is as follows: Liposome system:

[0054] DNA system:

[0055] After allowing the above systems to stand for 5 minutes, the liposome system and the DNA system were mixed to obtain the DNA-liposome complex, and then allowed to stand for another 15 minutes. 10 μL of the DNA-liposome complex was added to each well of 293T cells and cultured in a 5% CO2, 37°C incubator.

[0056] Six hours after transfection, two groups were set up: a negative control group and an experimental group. The cells in the negative control group were replaced with fresh medium without coronavirus inhibitors, and the cells in the experimental group were replaced with fresh medium containing coronavirus inhibitors. The cells were cultured in a 5% CO2 incubator at 37°C.

[0057] After 24 hours, the cells were washed three times with PBS, and 50 μM Amplex Ultrared and PBS containing 6.7 mM H2O2 were added to the cells and the cells were ice-cold for 25 minutes. The reaction is quenched by removing the substrate solution through suction. The cells were then washed three times with PBS, fixed on ice with 4.0% formaldehyde for 30 minutes, washed twice with PBS, and the fluorescence signal was observed.

[0058] Figure 2 In Figure A, the fluorescence signal was measured 24 hours after transfection. A negative result indicates that only the reporter gene expression plasmid was transfected. The experimental group received the coronavirus inhibitor GC376 (10 μM). The negative control group showed no fluorescence signal, while the experimental group showed a fluorescence signal. This indicates that the reporter system constructed in this invention can recognize coronavirus Mpro inhibitors and can be effectively used for screening coronavirus Mpro inhibitors. Quantitative analysis results are shown below. Figure 2 As shown in Figure B, the fluorescence signal of the reporting system of the present invention under the experimental conditions was 3274 ± 131, which was significantly different from the fluorescence signal of the control group. p <0.0001).

[0059] Example 3. Specificity verification of the reporting system of the present invention 1. Site-directed mutagenesis to obtain non-restriction cleavage site mutations and Mpro inactivation cleavage sites. 1.1 pcDNA3.1(+)-EX-Mpro cs-Mpro-AP without restriction site mutation A reporter vector containing the VAKLQ cleavage site was obtained using site-directed mutagenesis. Following the manufacturer's recommendations, the VAKLQ cleavage site mutation was removed using the Mut Express II Fast Mutagenesis Kit V2. Specifically, the coding sequence of the VAKLQ cleavage site was mutated to delete (removing the cleavage site) nucleotides (i.e., nucleotides 181-195 of sequence 1 in the sequence listing were deleted). Primers used are shown below: EX-Mpro-AP-F: 5'-AAGCTGCAGAGCGGCTTCGGAAGATGGCTTTC-3'; EX-Mpro-AP-R: 5'-AAAGCCGCTCTGCAGCTTGGCCACAGAGCCGCC-3'; The specific steps are as follows: Prepare the PCR system: 2× Max Buffer 25μL; dNTP Mix (10μM) 1μL; pcDNA3.1(+)-EX-Mpro cs-Mpro-AP 1μL; forward primer EX-Mpro-AP-F, 2μL; reverse primer EX-Mpro-AP-R, 2μL; Phanta Max Super-Fidelity DNA Polymerase 1μL; ddH2O 17μL. Add to a final volume of 50μL.

[0060] PCR reaction program: pre-denaturation 95℃ for 30s, 1 cycle; denaturation 95℃ for 15s, annealing 62℃ for 15s, extension 72℃ for 2min, 30 cycles; final extension 72℃ for 5min, 1 cycle; final reaction at 12℃.

[0061] After PCR is complete, remove the PCR tubes from the PCR instrument, and then add 1 μL of PCR product to each tube. Dpn I was digested with enzymes, gently mixed, briefly centrifuged, and then placed at 37°C for 1.5 h. After the reaction, agarose gel electrophoresis was performed. The agarose was dissolved in 1×TAE buffer and melted in a microwave oven to prepare a 1% agarose gel, which yielded the PCR product after enzyme digestion.

[0062] The linearized reporter plasmid vector pcDNA3.1(+) was recovered using a gel recovery kit.

[0063] According to the instructions for the site-directed mutagenesis kit, the formula for calculating the amount of template added to the subsequent recombinant PCR system is: Template amount = 0.02 × number of bases. The recombinant reaction system is prepared by placing it on ice. The PCR reaction system is as follows: 2 μL of the product from the enzyme digestion of plasmid (pcDNA3.1(+)-EX-Mprocs-Mpro-AP digested with restriction endonucleases NheI and XbaI) was recovered from the gel; 3.5 μL of the PCR product after enzyme digestion was also recovered; 4 μL of 5×CE II Buffer, 2 μL of Exnase II, and 10.5 μL of ddH2O were added to bring the total volume to 20 μL; the mixture was gently shaken to mix, briefly centrifuged, and incubated at 37°C for 30 min. The mixture was immediately placed on ice after the reaction was completed to obtain recombinant mutant plasmid 1.

[0064] Transformation with recombinant mutant plasmid 1: Thaw one tube of competent cells (DH5α) on ice. Prepare one EP tube, aliquot 50 μL of competent cells, add 5 μL of recombinant mutant plasmid 1 to the labeled competent cells, gently tap the bottom of the tube to mix, incubate on ice for 30 min, heat shock at 42℃ for 60 s, immediately place on ice for 2 min, add 500 μL of antibiotic-free LB medium, shake on a shaker for 1 h (37℃, 200 rpm), after shaking, spread on plates, and incubate for 13 h. After colony formation, pick single colonies and transfer to LB medium containing 1 ml of ampicillin antibiotic, shake and incubate for 6-8 h. After shaking, send for sequencing, select the correctly sequenced bacterial cultures for further expansion and plasmid extraction. This yields the final recombinant mutant plasmid 1 with the Mpro cleavage site deleted (pcDNA3.1(+)-EX-Mpro-AP).

[0065] 1.2 pcDNA3.1(+)-EX-Mpro cs-Mpro-AP reporter plasmid inactivates Mpro (C145A) site Reporter vectors containing a mutant Mpro-encoding gene lacking Mpro enzyme activity were obtained using site-directed mutagenesis. Following the manufacturer's recommendations, the codon sequence corresponding to cysteine ​​(C) at position 145 of the coronavirus Mpro protease on the pcDNA3.1(+)-EX-Mpro cs-Mpro-AP reporter plasmid was mutated to the codon sequence corresponding to alanine (A) using the Mut Express II Fast Mutagenesis Kit V2. This mutation inactivates the Mpro enzyme, rendering it inactive. Primers used are shown below: EX-Mpro cs-Mpro-C145A-AP-F: 5'-TTCCTGAACGGCAGCgcTGGCAGCGTGGGCTTCAA-3'; EX-Mpro cs-Mpro-C145A-AP-R: 5'-AgcGCTGCCGTTCAGGAAGGAGCCCTTGATGGT-3'.

[0066] The specific steps are as follows: Prepare the PCR system: 2× Max Buffer 25μL; dNTP Mix (10μM) 1μL; pcDNA3.1(+)-EX-Mpro cs-Mpro-AP 1μL; forward primer EX-Mpro cs-Mpro-C145A-AP-F, 2μL; reverse primer EX-Mpro cs-Mpro-C145A-AP-R, 2μL; Phanta Max Super-Fidelity DNA Polymerase 1μL; ddH2O 17μL. Add to a final volume of 50μL.

[0067] PCR reaction program: pre-denaturation 95℃, 30s, 1 cycle; denaturation 95℃, 15s, annealing 62℃, 15s, extension 72℃, 2min, 30 cycles; final extension 72℃, 5min, 1 cycle; final reaction at 12℃.

[0068] After PCR is complete, remove the PCR tubes from the PCR instrument, and then add 1 μL of PCR product to each tube. Dpn I was digested with enzymes, gently mixed, briefly centrifuged, and then placed at 37°C for 1.5 h. After the reaction, agarose gel electrophoresis was performed. The agarose was dissolved in 1×TAE buffer and melted in a microwave oven to prepare a 1% agarose gel, which yielded the PCR product after enzyme digestion.

[0069] The linearized reporter plasmid vector pcDNA3.1(+) was recovered using a gel recovery kit.

[0070] According to the instructions for the site-directed mutagenesis kit, the formula for calculating the amount of template added to the subsequent recombinant PCR system is: Template amount = 0.02 × number of bases. The recombinant reaction system is prepared by placing it on ice. The PCR reaction system is as follows: 2 μL of the gel recovery product after digestion of the plasmid (pcDNA3.1(+)-EX-Mpro cs-Mpro(C145A)-AP using restriction endonucleases NheI and XbaI); 3.5 μL of the PCR product after digestion; 4 μL of 5×CE II Buffer, 2 μL of Exnase II, and 10.5 μL of ddH2O were added to bring the total volume to 20 μL; the mixture was gently shaken, briefly centrifuged, and incubated at 37°C for 30 min. The mixture was immediately placed on ice after the reaction to obtain recombinant mutant plasmid 2.

[0071] 1.3 Transformation of recombinant mutant plasmids: Thaw one tube of competent cells (DH5α) on ice. Prepare one EP tube, aliquot 50 μL of competent cells, add 5 μL of recombinant mutant plasmid 2 to the labeled competent cells, gently tap the bottom of the tube to mix, incubate on ice for 30 min, heat shock at 42℃ for 60 s, immediately place on ice for 2 min, add 500 μL of antibiotic-free LB medium, shake on a shaker for 1 h (37℃, 200 rpm), after shaking, spread on a plate and incubate for 13 h. After colony formation, pick single colonies and transfer to LB medium containing 1 ml of ampicillin, shake and incubate for 6-8 h. After shaking, send for sequencing, and select the correctly sequenced bacterial cultures for further expansion. This yields the final Mpro mutant inactivated recombinant mutant plasmid 2 (pcDNA3.1(+)-EX-Mpro cs-Mpro(C145A)-AP).

[0072] 2. Specificity verification Prepare 293T cells one day in advance. Dilute the prepared 293T cells with complete culture medium and add 100 μL / well to a 96-well plate. The cell count per well is approximately 2.5 × 10⁻⁶. 4 They were then incubated overnight in a 37°C, 5% CO2 (volume fraction, the meaning of CO2 is the same below) cell culture incubator.

[0073] The reporter system (including the pcDNA3.1(+)-EX-Mpro cs-Mpro-AP and recombinant mutant plasmid 1 and recombinant mutant plasmid 2 of the present invention) was transfected and seeded into 96-well plates.

[0074] Liposome system:

[0075] DNA system:

[0076] After allowing the above systems to stand for 5 min each, the liposome system and the DNA system were mixed to obtain a DNA-liposome complex. This was then allowed to stand for another 15 min, and 10 μL of the DNA-liposome complex per well was added to 293T cells. The cells were then incubated in a 5% CO2, 37°C incubator. A negative control cell line (the non-mutated reporter system pcDNA3.1(+)-EX-Mpro cs-Mpro-AP constructed in this invention) was set up. The experimental groups consisted of pcDNA3.1(+)-EX-Mpro cs-Mpro-AP + GC376, the Mpro mutant inactivated cell (C145A) pcDNA3.1(+)-EX-Mpro cs-Mpro(C145A)-AP (recombinant mutant plasmid 2 obtained in step 1), and the restriction site deletion mutant pcDNA3.1(+)-EX-Mpro-AP (recombinant mutant plasmid 1 obtained in step 1). After 24 hours, the cells were washed three times with PBS, and 50 μM Amplex Ultrared and PBS containing 6.7 mM H2O2 were added to the cells and the cells were ice-cold for 25 minutes. The reaction is quenched by removing the substrate solution through suction. The cells were then washed three times with PBS, fixed on ice with 4.0% formaldehyde for 30 minutes, washed twice more with PBS, and the fluorescence signal was observed. The results are as follows: Figure 3 As shown.

[0077] Figure 3 It can be observed that the Mpro (report system C145A mutation inactivates) Figure 3 Fluorescence can be observed in the CM-MS-Mpro (C145A)-AP (represented by CM-MS-Mpro cs-Mpro(C145A)-AP), indicating that the reporter system depends on Mpro-specific cleavage. After Mpro is inactivated, it cannot cleave the restriction site. APEX is located on the same strand and is relatively close together, thus it can exert APEX enzyme activity. The mutated Mpro restriction site (… Figure 3 The EX-Mpro-AP (represented by the Chinese character EX-Mpro-AP) exhibited fluorescence even without the addition of an inhibitor. The uncleaved Mpro restriction site is key to the separation of APEX. The uncleavable site means that even if Mpro is present, it cannot be cleaved, indicating that the reporter system depends on the specific cleavage of Mpro.

[0078] Example 4. Validation of Drug Inhibitors Prepare 293T cells one day in advance. Dilute the prepared 293T cells with complete culture medium and add 100 μL / well to a 96-well plate. The cell count per well is approximately 2.5 × 10⁻⁶. 4 Cells were cultured overnight at 37°C in a 5% CO2 (volume fraction, the meaning of CO2 is the same below) cell culture incubator.

[0079] The reporter system (including the pcDNA3.1(+)-EX-Mpro cs-Mpro-AP plasmid constructed according to this invention) was transfected into 96-well plates. Liposome system:

[0080] DNA system:

[0081] After allowing the above systems to stand for 5 minutes each, mix them and let them stand for another 15 minutes. Add 10 μL of the DNA-liposome complex / well to 293T cells and incubate in a 5% CO2, 37°C incubator. A positive control group (cells obtained by transfecting 293T cells with the recombinant mutant plasmid 1 obtained in step 1 of Example 3) and an experimental group (cells obtained by transfecting 293T cells with the pcDNA3.1(+)-EX-Mpro cs-Mpro-AP plasmid of this invention) were established.

[0082] Six hours later, the negative control cells were replaced with fresh culture medium, while the experimental groups were replaced with 10-fold diluted coronavirus inhibitors GC376, Lufotrelvir (TargetMoI, T9574), and Ebselen (MCE, HY-13750) (100, 10, 1, 0.1, 0.01), and cultured in a 5% CO2, 37°C incubator.

[0083] After 24 hours, the cells were washed three times with PBS, and 50 μM Amplex Ultrared and PBS containing 6.7 mM H2O2 were added to the cells and the cells were ice-cold for 25 minutes. The reaction is quenched by removing the substrate solution through suction. The cells were then washed three times with PBS, fixed on ice with 4.0% formaldehyde for 30 minutes, washed twice more with PBS, and the fluorescence signal was observed. The results are as follows: Figure 3 As shown.

[0084] Calculations yield the following results: Figure 4 As shown, the reporting system screened drug inhibitors IC 50 Consistent with literature reports, the reporting system can be used for screening coronavirus Mpro inhibitors.

[0085] In the C3SIR reporting system, the inhibitory activity of the same compound was verified in this invention. The results are as follows: Figure 4 As shown in Figure A, the red fluorescence signal in the experimental group showed a significant dose-dependent increase with increasing concentrations of Lufotrelvir and GC376. When the inhibitor concentration was increased to 1 μM, obvious fluorescence signals were observed for both Lufotrelvir and GC376, indicating that the two inhibitors effectively inhibited Mpro activity. In contrast, the fluorescence signal of the Ebselen treatment group at each concentration did not show significant changes compared to the untreated control group, indicating that no specific inhibitory activity was detected in this cell reporter system.

[0086] Further quantitative analysis of the fluorescence images and fitting of the drug effect curves yielded the following results: Figure 5As shown in Figure B, the relative fluorescence units of the Lufotrelvir and GC376 treatment groups exhibited a typical dose-dependent decrease with increasing drug concentration. The half-maximal inhibitory concentrations (IC50) were calculated to be 2.852 μM and 2.609 μM, respectively, using nonlinear regression fitting. The IC50 of GC376 in similar reporter systems is reported in the literature. 50 The result was 0.99 μM (related literature: Kim, Y., Lovell, S., Tiew, KC, Mandadapu, SR, Alliston, KR, Battaile, KP, Groutas, WC, Chang, KO, 2012. Broad-spectrum antivirals against 3C or 3C-like proteases of picoviruses, noroviruses, and coronaviruses. J Virol, 86, 11754-11762), which is on the same order of magnitude as the result obtained in this invention. This further supports the stability and reliability of the report system of this invention for inhibitor screening. It is worth noting that Ebselen did not show specific inhibitory activity in this system, suggesting that it may not be an effective inhibitor of coronavirus Mpro. This result also verifies that this system can accurately identify the activity of known inhibitors. Notably, even at concentrations as high as 100 μmol / L with Ebselen, no significant change in fluorescence signal was observed, indicating that this system possesses good specificity for targeted inhibitor detection and is not easily affected by changes in protein spatial conformation. It more closely resembles the actual enzymatic reaction process in vivo, rather than relying solely on signal output generated by protein-protein interactions. In contrast, FRET-based detection methods have previously validated Ebselen as an inhibitor of coronavirus Mpro.

[0087] Example 5. Determination of the Z-factor 1) Prepare 293T cells one day in advance. Dilute the prepared 293T cells with complete culture medium and seed them into 96-well plates at 100 μL / well, with a cell count of approximately 2.5 × 10⁻⁶ cells per well. 4 They were then incubated overnight in a 37°C, 5% CO2 (volume fraction, the meaning of CO2 is the same below) cell culture incubator.

[0088] 2) The recombinant plasmid of the reporter system of the present invention was transfected and seeded into a 96-well plate. Liposome system:

[0089] DNA system:

[0090] After allowing the above systems to stand for 5 min each, mix them and let them stand for another 15 min. Add 10 μL of DNA-liposome complex / well to 293T cells and incubate in a 5% CO2, 37°C incubator. After 6 h, half of the cells were set up as negative control cells (i.e., replaced with fresh medium without coronavirus inhibitors), and the other half were set up as experimental groups (i.e., replaced with fresh medium containing the coronavirus Mpro inhibitor GC376 (20 μM)).

[0091] After 24 hours, the cells were washed three times with PBS, and 50 μM Amplex Ultrared and PBS containing 6.7 mM H2O2 were added to the cells and the cells were ice-cold for 25 minutes. The reaction is quenched by removing the substrate solution through suction. The cells were then washed three times with PBS, fixed on ice with 4.0% formaldehyde for 30 minutes, washed twice more with PBS, and the fluorescence signal was observed. The results are as follows: Figure 5 As shown.

[0092]

[0093] Figure 5 The Z-factor of the reporting system is approximately 0.80, which is greater than 0.5. This indicates that the reporting system constructed in this invention has a certain degree of stability in screening coronavirus Mpro inhibitors and can perform high-throughput drug screening.

[0094] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A nucleic acid molecule, characterized by: The nucleic acid molecule encodes a fusion protein, which includes a coronavirus Mpro enzyme, a split EX, a cleavage site of the Mpro enzyme, and a split AP; the split AP is a polypeptide fragment at the N-terminus of the peroxidase; the split EX is the remaining polypeptide fragment after the split AP is cleaved from the peroxidase.

2. The nucleic acid molecule according to claim 1, characterized in that: The nucleic acid molecule contains the coding gene for the split EX, the coding gene for the cleavage site of the Mpro enzyme, and the coding gene for the split AP; the coding gene for the cleavage site of the Mpro enzyme is located between the coding gene for the split EX and the coding gene for the split AP, that is, the coding gene for the split EX and the coding gene for the split AP are separated by the coding gene for the cleavage site of the Mpro enzyme.

3. The nucleic acid molecule according to claim 1 or 2, characterized in that: The nucleic acid molecule also contains a coronavirus Mpro enzyme encoding gene, which is located between the EX splitting gene and the AP splitting gene, i.e., the EX splitting gene and the AP splitting gene are separated by the Mpro enzyme cleavage site encoding gene and the coronavirus Mpro enzyme encoding gene.

4. The nucleic acid molecule according to any one of claims 1-3, characterized in that: In the nucleic acid molecule, the coding gene for the split EX is located upstream of the coding gene for the cleavage site of the Mpro enzyme, and the coding gene for the cleavage site of the Mpro enzyme is located upstream of the coding gene for the split AP.

5. The nucleic acid molecule according to any one of claims 1-4, characterized in that: The peroxidase is any of the following proteins: A1) The amino acid sequence is that of the protein in sequence 3 of the sequence listing; A2) A protein derived from A1) or having more than 80% identity with and the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) Proteins obtained by attaching protein tags to the N-terminus and / or C-terminus of A1) or A2); The Mpro enzyme is any of the following proteins: B1) The amino acid sequence is the protein consisting of amino acid residues 62-367 of sequence 2 in the sequence listing; B2) A protein derived from B1) or having more than 80% identity with and the same function as the protein shown in B1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in B1). B3) is a protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2).

6. The nucleic acid molecule according to any one of claims 1-5, characterized in that: The nucleic acid molecule is any one of the following: 1) The coronavirus Mpro enzyme encoding gene is nucleotides 196-1113 of sequence 1 in the sequence listing, and / or, 2) The Mpro cleavage site encodes nucleotides 181-195 of sequence 1 in the sequence listing, and / or, 3) The coding gene for the split EX is nucleotides 16-165 of sequence 1 in the sequence listing, and / or, 4) The coding gene for the split AP is nucleotides 1129-1725 of sequence 1 in the sequence listing.

7. The nucleic acid molecule according to any one of claims 1-6, characterized in that: The nucleic acid molecule is the DNA molecule shown in Sequence 1 of the sequence listing.

8. A biomaterial, characterized in that: The biomaterial is any one of the following: B1) An expression cassette containing the nucleic acid molecule described in any one of claims 1-7; B2) A recombinant vector containing the nucleic acid molecule of any one of claims 1-7, or a recombinant vector containing the expression cassette of claim B1; B3) A recombinant microorganism containing the nucleic acid molecule of any one of claims 1-7, or a recombinant microorganism containing the expression cassette of B1), or a recombinant microorganism containing the recombinant vector of B2); B4) A recombinant cell line containing the nucleic acid molecule of any one of claims 1-7, or a recombinant cell line containing the expression cassette of B1), or a recombinant cell line containing the recombinant vector of B2), or a recombinant cell line containing the recombinant microorganism of B3).

9. A fusion protein, characterized by: The fusion protein is the fusion protein described in any one of claims 1-7.

10. Application, characterized in that: The application is the application of the nucleic acid molecule described in any one of claims 1-7, or the application of the biomaterial described in claim 8, or the application of the fusion protein described in claim 9, wherein the application is any one of the following: P1. Application in the preparation of screening products for coronavirus Mpro inhibitors; P2. Application in the preparation of screening products for anti-coronavirus drugs; P3. Application in screening inhibitors for coronavirus Mpro; P4. Application in screening anti-coronavirus drugs.

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