Active reporter plasmid of RNA dependent RNA polymerase of human coronavirus and application of active reporter plasmid

Abstract

The invention discloses an activity reporter plasmid pCMV-5 'UTR-Luc-3' UTR of human coronavirus RNA-dependent RNA polymerase, the nucleotide sequence of the activity reporter plasmid is shown as SEQ ID NO: 1, the activity reporter plasmid can indicate the activity of the human coronavirus RNA-dependent RNA polymerase RdRp, the RdRp activity reporter plasmid is applied to screening of anti-human coronavirus drugs, and the activity of the human coronavirus RNA-dependent RNA polymerase can be obviously improved. Experimental results show that the inhibition effect of the to-be-screened medicine can be visually observed through fluorescence intensity, the method can achieve high-throughput screening of the anti-human coronavirus medicine, time and cost consumed for screening the anti-human coronavirus medicine are greatly saved, operation is easy and convenient, and the method is suitable for application and popularization.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug screening, and in particular relates to a reporter plasmid for screening human coronavirus RNA-dependent RNA polymerase (RdRp) activity and an application of the reporter plasmid in screening anti-human coronavirus drugs. Background Art

[0002] Coronavirus is a single-stranded positive-sense RNA virus, and its infection has caused many outbreaks in human history, greatly affecting human survival and development. Although a variety of coronavirus vaccines have been developed to prevent the spread of coronavirus, due to the specificity of its genome, the vaccine protection is easily ineffective, so antiviral therapy is very important to reduce the morbidity and mortality of infected people. Currently, the FDA has approved remdesivir as an anti-COVID-19 drug, but studies have shown that its efficacy is not ideal, so it is very important to screen new and effective anti-coronavirus drugs to treat and respond to emerging and re-emerging coronaviruses.

[0003] HCoV-OC43 infection mainly causes mild upper respiratory tract symptoms, which can be cured by the patient's own immunity without special treatment. It is a beta coronavirus like SARS-CoV-2. Comparison of nucleotide and amino acid sequences shows that the two viruses have similarities in important motifs involved in viral replication and pathogenesis (such as RdRp, helicase motif and 3CL). pro ) and does not have the disadvantages of Class III biological laboratory restrictions, so it is a good high-throughput drug screening model. Coronavirus nonstructural protein 12 (nsp12) has RNA-dependent RNA polymerase activity (RNA-dependent RNA polymerase, RdRp), which is a core component of the coronavirus replication and transcription mechanism and can catalyze the synthesis of viral RNA; RdRp has a highly conserved core structure in coronaviruses and is therefore an important antiviral drug target. Inhibitors developed against RdRp are expected to become broad-spectrum anti-coronavirus drugs.

[0004] Firefly luciferase is a protein with a molecular weight of about 61kD. In the presence of ATP, magnesium ions and oxygen, it can catalyze the oxidation of luciferin to oxyluciferin. During the oxidation of luciferin, bioluminescence is emitted, and it is widely used as a fluorescent marker in biomedical research. The bioluminescence system of luciferin and luciferase can detect gene expression extremely sensitively and efficiently. Summary of the invention

[0005] The present invention provides an activity reporter plasmid of human coronavirus RNA-dependent RNA polymerase RdRp. By inserting a firefly luciferin reporter gene into the 5' non-coding region and the 3' non-coding region of the human coronavirus, an activity reporter plasmid pCMV-5'UTR-Luc-3'UTR of human coronavirus RNA-dependent RNA polymerase is constructed. The nucleotide sequence of the plasmid pCMV-5'UTR-Luc-3'UTR is shown in SEQ ID NO: 1.

[0006] Another object of the present invention is to apply the above-mentioned reporter plasmid of human coronavirus RNA-dependent RNA polymerase to the screening of anti-human coronavirus drugs, and the activity reporter plasmid pCMV-5'UTR-Luc-3'UTR and the expression plasmid pCMV-nsp12 are used for drug screening at the same time, and the nucleotide sequence of the expression plasmid pCMV-nsp12 is shown in SEQ ID NO: 2; the object of the present invention is achieved by the following technical solutions: 1. Construction of human coronavirus RNA-dependent RNA polymerase activity reporter plasmid pCMV-5'UTR-Luc-3'UTR: The 5' non-coding DNA sequence (5'UTR), 3' non-coding DNA sequence (3'UTR) (GenBank: ON376724.1) and coding DNA sequence of firefly luciferin reporter gene of HCoV-OC43 were downloaded from NCBI (National Center for Biotechnology Information (nih.gov)), sent to Sangon Biotech for full gene synthesis, and ligated to pCMV-c-flag to obtain plasmid pCMV-5'UTR-Luc-3'UTR; 2. Construction of human coronavirus RNA-dependent RNA polymerase expression plasmid (1) Download the coding DNA sequence of HCoV-OC43 nsp12 from NCBI, analyze the restriction sites and design specific primers using Primer Primer 5.0; (2) Extracting HCoV-OC43 RNA and reversely transcribing it to obtain cDNA, using the cDNA as a template and using primers nsp12-F and nsp12-R to amplify the nsp12 gene fragment; (3) Use an agarose gel DNA recovery kit to recover the target fragment and use XOt Ⅰ and Not Ⅰ Double enzyme digestion of the target fragment and the vector pCMV-c-flag, gel recovery and purification of the fragment; (4) Use T4 DNA ligase to connect, transform the ligation product into competent cells, and pick a single clone; extract the plasmid and perform sequencing to verify the successful construction of the plasmid, then extract the endotoxin-free plasmid to obtain the plasmid pCMV-nsp12; 3. Screening of anti-human coronavirus RdRp drugs using human coronavirus RNA-dependent RNA polymerase activity reporter plasmid (1) Screening of anti-human coronavirus drugs using the activity reporter plasmid pCMV-5'UTR-Luc-3'UTR and expression plasmid pCMV-nsp12 ① One day before transfection, the density is 2×10 5 cells / mL HEK-293T cells (human embryonic kidney epithelial cells) were plated into a cell well plate, 1 mL of cell suspension was added to each well, and an experimental group with the drug to be screened and a control group without the drug to be screened were set up. After the cell adhesion and fusion reached 60-70%, the RdRp activity reporter plasmid pCMV-5'UTR-Luc-3'UTR and the expression plasmid pCMV-nsp12 were co-transfected into the experimental and control group cells; ② 6 hours after transfection, the experimental group replaced the culture medium containing the drug to be screened, and the control group replaced the normal culture medium. After 48 hours of continuous culture, the firefly luciferase activity was detected using an ELISA instrument. When the firefly luciferase activity of the drug-added group cells was lower than that of the control group cells, the drug to be screened had RdRp inhibitory activity; In the present invention, HEK-293T cells are cultured in a complete DMEM medium containing 10% serum, 1% penicillin-streptomycin, and 1% Hepes at a constant temperature of 37° C. and 5% CO 2 .

[0007] Advantages and technical effects of the present invention: 1. The present invention constructs an activity reporter plasmid of RdRp by inserting a firefly luciferase reporter gene into the 5' non-coding region and the 3' non-coding region of human coronavirus HCoV-OC43. After the activity reporter plasmid and the sea renilla plasmid as an internal reference are co-transfected into HEK-293T cells and HEK-293T cells stably expressing RdRp, the fluorescence intensity of the co-transfected HEK-293T cells stably expressing RdRp is enhanced compared with the co-transfected HEK-293T cells, indicating that the firefly luciferase activity of the activity reporter plasmid can indicate the RNA-dependent RNA polymerase RdRp activity of human coronavirus; 2. The RdRp activity reporter plasmid prepared by the present invention is co-transfected with the expression plasmid pCMV-nsp12. When the firefly luciferase activity of the drug-added group cells is lower than that of the control group cells without drug addition, the drug to be screened has RdRp inhibitory activity. The method of the present invention can intuitively observe the inhibitory effect through the fluorescent firefly luciferase activity, and can achieve high-throughput screening of anti-human coronavirus drugs, greatly saving the time and cost of screening anti-human coronavirus drugs. 3. The HCoV-OC43 used in the method of the present invention is a low-pathogenic human coronavirus, which is not subject to the restrictions of the Class III biosafety laboratory, and is simple and convenient to operate, which can more effectively save labor costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is the electrophoresis diagram of the amplification results of the nsp12 gene fragment, lane 1 is the DNA marker, and lanes 2-4 are the nsp12 gene fragments; Figure 2 This is the electrophoresis result of bacterial solution PCR of pCMV-nsp12 plasmid, where lane 1 is DL 5000 DNA marker, and the remaining lanes are nsp12 bands obtained by bacterial solution amplification; Figure 3 The expression of RdRp gene mRNA was detected by PCR in cells stably expressing RdRp, where lanes AD are cell lines stably expressing RdRp, lane E is the original HEK-293T cell line, lane F is the negative control group, lane G is the positive control, and lane M is the DL 2000 DNA Marker; Figure 4 The results of Western-blot detection of cells stably expressing RdRp (left) and internal reference gapdh (gapdh size is 30-40kDa, right), lanes A and E are protein markers (Thermo Fisher 26616), lanes B and C are constructed stably expressing RdRp cell lines (RdRp-Flag size is 107.2 kDa), and lane D is HEK-293T cell line; Figure 5 The activity reporter plasmid and the Renilla plasmid as an internal reference were co-transfected into HEK-293T cells stably expressing RdRp or original HEK-293T cells, and the firefly luciferase activity was detected using a microplate reader; Figure 6 The results of detecting firefly luciferase activity after HEK-293T cells were co-transfected with plasmids pCMV-5'UTR-Luc-3'UTR + pCMV-nsp12 at a mass ratio of 1:2 and HEK-293T cells were transfected with pCMV-5'UTR-Luc-3'UTR alone.

[0009] Figure 7 This is the result of using the activity reporter plasmid pCMV-5'UTR-Luc-3'UTR to screen anti-human coronavirus drugs. DETAILED DESCRIPTION

[0010] The present invention is further described below by means of the accompanying drawings and examples, but the protection scope of the present invention is not limited to the above contents. The methods in the present embodiments are operated according to conventional methods unless otherwise specified, and the reagents used are conventional reagents or reagents prepared according to conventional methods unless otherwise specified; Example 1: Construction of expression plasmid pCMV-nsp12 for human coronavirus RNA-dependent RNA polymerase RdRp The HCoV-OC43 nsp12 encoding DNA sequence (GenBank: ON376724.1) was downloaded from NCBI, and specific primers were designed using PrimerPrimer 5.0. The primer sequences are shown in the following table (the underlined sequences in the figure indicate the enzyme sites);

[0011] 2. The RNA of the virus HCoV-OC43 was extracted using the OMEGA EZNA®Total RNA Kit I. The extraction steps were carried out according to the instructions of the kit; 3. Using RNA as a template, reverse transcribe the cDNA using TAKARA One Step PrimeScript™ RT-PCR Ki. Follow the instructions of the kit. 4. Using cDNA as template, primers nsp12-F and nsp12-R were used, and Novazon high-fidelity enzyme was used to amplify the nsp12 DNA fragment with restriction sites. The amplification systems were: nsp12-F 2 mL, nsp12-R 2 mL, cDNA 1 mL, 2 × Phanta Flash Master Mix 25 mL, ddH2O 20 mL; amplification conditions: 98℃ 30s; 98℃ 10s, 62℃ 5s, 72℃ 15s, 30 cycles; 72℃ 1min; the amplification results were as follows: Figure 1 As shown, the target fragment was recovered using Tiangen Agarose Gel DNA Recovery Kit; 5. Use XOt Ⅰ and Not Ⅰ Double-digest the target fragment and vector pCMV-c-flag in step 4, and then purify the fragment using Tiangen agarose gel DNA recovery kit, where the enzyme digestion system is: XOt Ⅰ 2mL 、 NotⅠ 2mL, DNA 2mg, 10×M buffer 4mL, ddH2O add to 40mL, treat at 37℃ for 10h; 6. Use T4 DNA ligase to connect the target fragment and the vector. After connecting at 16°C overnight, take 20 μL of the ligation product and add it to 100 μL of E. coli DH5α. After ice bathing for 30 min, transfer it to a 42°C water bath for heat shock for 90 s, then place it on ice for 5 min, and then make up to 1 mL with liquid LB medium. Then, place it on a shaker at 37°C and 200 rpm for 1 hour. Finally, take out the culture medium, centrifuge it at 4000 rpm for 5 min, discard 900 mL of culture medium, resuspend the cells with the remaining culture medium, apply the cell suspension to solid LB medium containing ampicillin resistance, and culture it at 37°C overnight. 7. Pick a single clone and culture it at 37℃ and 200 rpm for 3h. Then use nsp12 specific primers to perform bacterial solution PCR. The electrophoresis results preliminarily confirm that the nsp12 gene fragment is successfully connected to the vector. Then expand the bacterial solution, use Tiangen plasmid extraction kit to extract the plasmid and perform sequencing verification. The bacterial solution PCR electrophoresis results are shown in the figure below. Figure 2 ; The sequencing results confirmed that the plasmid pCMV-nsp12 was successfully constructed, and then the endotoxin-free plasmid was extracted using the Tiangen Endotoxin-Free Plasmid Midi Extraction Kit.

[0012] Example 2: Construction of human coronavirus RNA-dependent RNA polymerase activity reporter plasmid pCMV-5'UTR-Luc-3'UTR The 5' noncoding DNA sequence, 3' noncoding DNA sequence (GenBank: ON376724.1) and firefly luciferase gene sequence of HCoV-OC43 were downloaded from NCBI and sent to Sangon Biotech for full gene synthesis, and then ligated to pCMV-c-flag to obtain the plasmid pCMV-5'UTR-Luc-3'UTR.

[0013] Example 3: Activity verification experiment of the RNA-dependent RNA polymerase activity reporter plasmid pCMV-5'UTR-Luc-3'UTR constructed in Example 2 1. Construction of HEK-293T cells stably expressing RdRp (1) Using the cDNA of HCoV-OC43 as a template (prepared as in Example 1), the RdRp gene was amplified using primers RdRp-F: ccctcgagatgTCAAAAGATACTAATTTTTTAAACcGGGTTC; RdRp-R: CGGGATCCttacttgtcatcgtcgtccttgtagtcCTGCATAACTGCACTTCTTAAATACATG (introduced at the 5' and 3' ends of the RdRp gene, respectively); XOt Ⅰ and Bam HI), through XOt Ⅰ and Bam HⅠ double enzyme digestion, enzyme digestion system is XOt Ⅰ 1 mL, Bam 1 mL of HⅠ, 2 mL of 1×KBuffer, DNA ≤ 1 mg, make up to 20 mL with ddH2O, and digest at 37℃ for 8 h; perform agarose gel electrophoresis, gel purification, and concentration measurement of the digestion product; The RdRp gene was connected to the lentiviral shuttle plasmid PLVX-IRES-Puro (purchased from Beyotime Biotechnology Co., Ltd.); Takara T4 DNA ligase was used for ligation, the reaction system was T4 Buffer 2 mL, T4 enzyme 2 mL, target fragment: vector molar ratio = 1:1, ddH2O was added to 20 mL; 16℃ ligation for 12 h, the ligation product was transformed into competent cell culture, plated, single clones were picked, PCR amplified, and sent for sequencing verification. The RdRp gene was successfully connected to the lentiviral shuttle plasmid PLVX-IRES-Puro.

[0014] Lentiviral packaging A. HEK-293T cells were seeded in 6-well plates at a seeding density of 2×10 5 cells / mL, and when the cells reached a confluence of about 70-80%, they were transfected using Lipofectamine 3000; ① Preparation of transfection mixture for 6 wells Solution A: Add 20 µg DNA plasmids (10 µg of shuttle plasmid PLVX-IRES-RdRp-Puro, 2.5 µg of packaging plasmid pSPAX2 (purchased from Beyotime Biotech) and 7.5 µg of envelope plasmid pMD2G (purchased from Beyotime Biotech)) to 1.5 mL of serum-free DMEM medium, and add 36 µL of P3000 reagent; Solution B: Add 42 µL of Lipofectamine 3000 reagent to 1.5 mL of serum-free DMEM medium; ② After incubating solutions A and B at room temperature for 5 min each, mix solution A and B in a 1:1 volume ratio and incubate at room temperature for 20 min; ③ Remove 1 mL of culture medium from each well of the 6-well plate, add 500 µL / well of the solution obtained in step ②, and gently shake the 6-well plate in a cross-shaped pattern; ④Incubate at 37 ℃ for 6 h; B. After 6 hours, remove the culture medium containing the transfection reagent, wash three times with PBS, add 2 mL of complete culture medium to each well, incubate for 24 hours, and collect all cell supernatants, which is the first harvest (stored at -80°C).

[0015] C. After the first harvest, continue to add 2 mL of complete medium to each well of the cells, incubate for 24 hours, and collect the cell supernatant, which is the second harvest; After the second harvest, 2 mL of complete medium was added to each well of the cells, and the cell supernatant was collected after 24 h of incubation, which was the third harvest. The supernatants harvested three times were mixed and centrifuged at 4000 rpm for 10 min, then filtered through a 0.45 μm filter membrane and centrifuged through a 100 kD ultrafiltration tube at 4000 rpm for 20 min to concentrate the virus; (3) Screening of cells that stably express RdRp A. One day before infection, HEK-293T cells were plated at 2×10 5 cells / well density was plated in 12-well plates; B. Infection ① Thaw the virus, dilute it with serum-free DMEM medium, add polybrene (the working concentration of polybrene is 8 μg / mL), and gently pipette to mix; ② Add 500 μL of virus solution directly to the well plate. In addition, keep one well of cells without adding virus as a blank control, that is, add 500 μL of serum-free DMEM medium and culture overnight in a 37°C, 5% CO2 incubator; ③Replace complete culture medium and incubate in a 37°C cell culture incubator for 24 h; C. Replacement of screening medium ① Prepare DMEM medium containing 2 μg / mL puromycin, 10% serum, 1% penicillin-streptomycin, and 1% Hepes as the screening medium; ② Remove the culture medium containing the virus from the well plate, gently rinse the cells 2-3 times with PBS, and add the screening medium to continue culturing; ③Replace the screening medium every two days; ④ After several days of screening, you can see that there are no living cells in the control cell group, while one or more wells with added virus will have puromycin-resistant clones. (Note: The number of days required for screening should be based on the death of all cells in the control cell group) D. Isolate clones to obtain the maximum number of cells Remove the old culture medium from the well plate, gently rinse the cells in the wells where clones appear with PBS 2-3 times, and transfer them to a 6-well plate. Incubate them in a 37°C, 5% CO2 incubator. This can effectively reduce the chance of monoclonal cells being contaminated by other cells, thereby increasing the possibility of clone survival and successfully isolating surviving clones.

[0016] (4) Expand the culture of surviving clones and conduct a second round of stable strain screening The isolated HEK-293T cells stably expressing RdRp were expanded and frozen, and the cells were collected and RdRp was detected at the molecular level and protein level using PCR and Western-blot, respectively.

[0017] 2. Detection of HEK-293T cells stably expressing RdRp A. PCR detection of RdRp gene mRNA expression (1) Extraction of total cellular RNA using the Trizol method a. Discard the cell culture medium and wash the cells twice with PBS.

[0018] b. Aspirate the PBS and then 8 -2×10 7 Add 1 mL of Trizol to the cultured cells and gently shake the culture dish to ensure that the solution is evenly distributed on the cell surface; c. Use a pipette to repeatedly blow and swirl to remove the cells, then transfer the lysate containing the cells to a centrifuge tube, and then use a pipette to blow and swirl until there is no obvious precipitation in the lysate; d. Leave at room temperature for 5-10 min; e. Add 1 / 5 volume of chloroform of Trizol to the above lysate, mix thoroughly (vortex or mix with a pipette), let stand at room temperature for 15 min, centrifuge at 12000 rpm and 4℃ for 15 min, carefully remove the centrifuge tube, at this time the homogenate is divided into three layers, namely: supernatant (containing RNA), middle protein layer and lower organic phase. Transfer the supernatant to another new centrifuge tube; f. Precipitate RNA: add the same volume of isopropanol, mix by inversion, place at -20°C for 10 min, centrifuge at 12,000 rpm and 4°C for 10 min, and discard the supernatant; g. Wash RNA: Add 500 μL of pre-cooled anhydrous ethanol to the precipitate, blow off the precipitate to wash the RNA, wash twice, centrifuge at 12000 rpm at 4°C and discard the supernatant; h. Open the centrifuge tube and dry the precipitate at room temperature for about 5 min; i. Add 20 μL DPEC water to the centrifuge tube to dissolve RNA, measure the RNA concentration and then reverse it; j. Prepare 10 μL system in an RNasc-free centrifuge tube: 2 μL of 5×gDNA Eraser Buffer, 1 μL of Buller 2.0 μl gDNA Eraser, 1 μg of Total RNA, and 10 μL of RNase Free ddH2O; mix well and react at 42°C for 2 min.

[0019] k. Prepare 20 μL system in an RNase-free centrifuge tube: 10 μL of the reaction solution in step j, 1 μL of Prime ScriptRT Enzyme Mix I, 1 μL of RT Primer Mix, 4 μL of 5× PrimeScripl Buffer 2 (for RealTime), 4 μL of RNase Free ddH2O; PCR program: 37℃ 15 min; 85℃ 5 sec; store at 4℃.

[0020] The product obtained by the above reversal was subjected to ordinary PCR detection, and the results were as follows Figure 3 As shown in the figure, it can be seen that the amplification R The gene fragment size is 197 bp, which is consistent with the expected fragment size, indicating that it is stably expressed at the molecular level. R The HEK-293T cells were successfully constructed.

[0021] B. Westen-blot detection ① Collect some HEK-293T cells that stably express RdRp, place the cells on ice, add appropriate RIPA lysis buffer and PMSF protease inhibitor (RIPA: PMSF ratio is 100:1) according to the cell amount, and then place them on ice for 2 h, then centrifuge at 4°C and 12000 rpm for 20 min, transfer the supernatant to a new centrifuge tube to prepare the protein sample; ② Use the Bio-Tech BCA Protein Concentration Assay Kit to determine the concentration of the protein sample. The specific steps are as follows: a. First, prepare the working solution according to the ratio of BCA:BCB=1:50, where each sample requires 100 μL of working solution. Then add 100 μL of working solution to each well of the 96-well plate, and then take 10 μL of each protein sample to be tested and add it to the prepared working solution, incubate at 37°C for 30 min, and then use an enzyme reader to detect the absorbance at 562 nm. According to the test results, calculate the protein concentration.

[0022] b. Denaturation of protein samples: Based on the calculated concentration, the protein loading amount was unified to 20 μg. Total protein, PBS and loading buffer were added to the PCR tube in proportion, mixed well, and denatured at 98°C for 5 min. c. For SDS-PAGE electrophoresis, place the prepared gel in the electrophoresis solution, remove the comb, and use a 10μL pipette to spot the sample into the gel wells. First, run at 90 V for 30 min, then at 120 V for 1 h. Run until the protein pre-staining buffer reaches the bottom of the separation gel.

[0023] d. Transfer: Soak the PVDF membrane in methanol for 1-2 min, start with the transparent transfer gel plate, and place the sponge, filter paper, PVDF membrane, gel, filter paper, and sponge in the order, and then place it in the electrophoresis tank with a constant current of 130 mA. The transfer time is adjusted according to the size of the protein. e. Immunohistochemical hybridization reaction: prepare 5% skim milk blocking solution, block for 2 h, wash with PBST 3 times, 10 min each time, dilute the primary antibody according to the proportion, incubate at 4°C overnight, recover the primary antibody the next day, wash with PBST 3 times, 10 min each time, dilute the secondary antibody according to the proportion, and incubate at room temperature for 1 h; f. Development: Prepare the developer according to the instructions (A solution: B solution = 1:1). The result is as follows Figure 4 As shown in the figure, it can be seen that the protein size of the constructed cell line stably expressing RdRp is 107.2 kDa, indicating that HEK-293T cells stably expressing RdRp were successfully constructed.

[0024] 3. Activity verification of activity reporter plasmid pCMV-5'UTR-Luc-3'UTR (1) Set up an experimental group (transfect the activity reporter plasmid pCMV-5'UTR-Luc-3'UTR and the sea renilla plasmid PGMLR-TK (purchased from Biotech) at a mass ratio of 10:1 into HEK-293T cells stably expressing RdRp HEK-293T-RdRp), a negative control group (transfect the pCMV-5'UTR-Luc-3'UTR activity reporter plasmid and the sea renilla plasmid PGMLR-TK at a mass ratio of 10:1 into HEK-293T cells), and a blank control group, namely HEK-293T-RdRp-NC cells (cells not transfected); (2) One day before transfection, the density of 5 cells / mL cells HEK-293T-RdRp or HEK-293T cells were plated in 12-well plates; (3) After the cells were cultured at 37°C and 5% CO2 until they adhered to the wall, transfection was performed according to different treatment groups. The transfection was carried out using the LipofectamineTM3000 transfection kit. The transfection system was as follows: Reagent A: 50 mL serum-free DMEM + 1 mg plasmid DNA + 2 mL p3000; Reagent B: 50 mL serum-free DMEM + 1.5 mL lip3000; After reagents A and B are prepared, incubate at room temperature for 5 minutes, transfer solution A to solution B, mix well, and incubate at room temperature for 15 minutes; add the mixed solution to the cell culture medium, 100 mL per well; continue to culture for 6 hours after transfection, then replace the DMEM medium containing 10% serum, and then continue to culture for 48 hours before collecting the cells;

[0025] (4) Use the dual luciferase detection kit of Biyuntian to detect the firefly luciferase activity according to its instructions. The specific steps are as follows: A. Cell lysis: After thoroughly mixing the reporter gene cell lysis buffer, add 300 mL of reporter gene cell lysis buffer to each well, fully lyse the cells, centrifuge at 12000g for 5 minutes, and take the supernatant for determination; B. Dissolve the firefly luciferase detection reagent and Renilla luciferase detection buffer and bring them to room temperature. Place the Renilla luciferase detection substrate (100X) on an ice bath or ice box for later use. C. Take an appropriate amount of Renilla luciferase assay buffer according to the amount of 100 mL required for each sample, and add Renilla luciferase assay substrate (100X) at a ratio of 1:100 to prepare Renilla luciferase assay working solution; D. Add the supernatant solution to be tested in step A to a 96-well ELISA plate, 50 mL per well, and set three replicate wells for each sample, then add 100 mL of firefly luciferase detection reagent, mix well with a gun, and measure RLU (Relative Light Unit); after completing the above firefly luciferase determination step, add 100 mL of Renilla luciferase detection working solution, mix well with a gun, and measure RLU (Relative Light Unit); E, using Renilla luciferase as an internal reference, the RLU value obtained by the firefly luciferase assay was divided by the RLU value obtained by the Renilla luciferase assay to obtain the relative fluorescence intensity; F. One-way ANOVA, multiple comparisons and graph drawing were performed using Graphpad Prism 8. The results are shown in Figure 5 ,It can be seen from the figure that the firefly luciferase activity of the experimental group was significantly higher than that of the negative control group. The results showed that the firefly luciferase activity of the active reporter plasmid can indicate the RNA-dependent RNA polymerase RdRp activity of human coronavirus.

[0026] Example 4: Screening of inhibitors using RNA-dependent RNA polymerase activity reporter plasmid pCMV-5'UTR-Luc-3'UTR 1. Set up the control group NC: no treatment was given to HEK-293T cells; Co-transfection group (pCMV-nsp12+pCMV-5'UTR-Luc-3'UTR): activity reporter plasmid pCMV-5'UTR-Luc-3'UTR and expression plasmid pCMV-nsp12 were co-transfected into HEK-293T cells at a mass ratio of 1:2; Single transfection group (pCMV-5'UTR-Luc-3'UTR): single transfection of activity reporter plasmid pCMV-5'UTR-Luc-3'UTR; 2. One day before transfection, the density was 2×10 5 cells / mL HEK-293T cells were plated in 12-well plates; 3. After the cells were cultured at 37°C and 5% CO2 until they adhered to the wall, transfection was performed according to different treatment groups. The transfection was carried out using the LipofectamineTM3000 transfection kit. The transfection system was as follows: Reagent A: 50 mL serum-free DMEM + 1 mg plasmid DNA + 2 mL p3000; Reagent B: 50 mL serum-free DMEM + 1.5 mL lip3000; After reagents A and B are prepared, incubate at room temperature for 5 minutes, transfer solution A to solution B, mix well, and incubate at room temperature for 15 minutes; add the mixed solution to the cell culture medium, 100 mL per well; continue to culture for 6 hours after transfection, then replace the DMEM medium containing 10% serum, and then continue to culture for 48 hours before collecting the cells; 4. Use the dual luciferase detection kit of Biyuntian to detect the firefly luciferase activity according to its instructions. The specific steps are as follows: (1) Cell lysis: After thoroughly mixing the reporter gene cell lysis buffer, add 300 mL of reporter gene cell lysis buffer to each well to fully lyse the cells. Centrifuge at 12,000 g for 5 minutes and take the supernatant for determination. (2) Add the supernatant solution to be tested in step (1) to a 96-well ELISA plate, 50 mL per well, with two replicate wells for each sample, and then add 100 mL of firefly luciferase detection reagent, mix well with a gun, and measure the RLU (Relative Light Unit); Results Figure 6 The experimental results showed that the firefly luciferase activity of the co-transfection group was higher than that of the single transfection group, proving that the expression level of firefly luciferase expressed by the pCMV-5'UTR-Luc-3'UTR activity reporter plasmid can indicate the activity of RdRp.

[0027] 5. Drug screening Set up the experimental group (co-transfect the activity reporter plasmid pCMV-5'UTR-Luc-3'UTR and the expression plasmid pCMV-nsp12 at a ratio of 1:2, and add remdesivir); Control group (co-transfection of activity reporter plasmid pCMV-5'UTR-Luc-3'UTR and expression plasmid pCMV-nsp12 at a ratio of 1:2, without adding remdesivir) The results are as follows Figure 7 As shown, the firefly luciferase activity of the control group was significantly higher than that of the experimental group. The experimental results showed that the firefly luciferase activity expressed by the activity reporter plasmid pCMV-5'UTR-Luc-3'UTR can indicate the activity of RdRp and can screen out anti-human coronavirus drugs.

[0028] The above experimental results show that the activity reporter plasmid of the human coronavirus RNA-dependent RNA polymerase RdRp of the present invention has the potential to screen anti-human coronavirus drugs.

Claims

1. A human coronavirus RNA-dependent RNA polymerase activity reporter plasmid pCMV-5'UTR-Luc-3'UTR, whose nucleotide sequence is shown in SEQ ID NO:

1.

2. Application of the activity reporter plasmid of human coronavirus RNA-dependent RNA polymerase according to claim 1 in screening anti-human coronavirus drugs.

3. The use according to claim 2, characterized in that: When in use, the activity reporter plasmid pCMV-5'UTR-Luc-3'UTR and the expression plasmid pCMV-nsp12 are used together to screen anti-human coronavirus drugs. The two plasmids are transfected into HEK-293T cells at the same time, and the drugs to be screened are added. At the same time, a control group without drug addition is set up. After transfection, the firefly luciferase activity is detected using an ELISA instrument. When the firefly luciferase activity of the drug-added group cells is lower than that of the control group cells, the drug to be screened has inhibitory activity.

4. The use according to claim 3, characterized in that: The nucleotide sequence of the expression plasmid pCMV-nsp12 is shown in SEQ ID NO:2.

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