CN117363654A - Human coronavirus main protease 3CL pro Active reporter plasmid of (2) and use thereof - Google Patents

Abstract

The invention discloses a reporter plasmid of human coronavirus main protease 3CL ^pro , which is plasmid pCMV-GFP-β8-LQSG-9-LQSG-10 or plasmid pCMV-GFP-β9-RLQSGF-10, plasmid pCMV-GFP-β8- The nucleotide sequence of LQSG‑9‑LQSG‑10 is shown in SEQ ID NO:1, and the nucleotide sequence of plasmid pCMV‑GFP‑β9‑RLQSGF‑10 is shown in SEQ ID NO:2; the reporter plasmid is used to screen antibodies. Among human coronavirus drugs, the inhibitory effect of the reagent can be visually observed through the fluorescence intensity. The present invention can achieve the purpose of high-throughput screening of anti-human coronavirus drugs, and can significantly save the time and cost of screening anti-human coronavirus drugs. .

Description

Activity reporter plasmid of human coronavirus main protease 3CLpro and its application

Technical field

The invention belongs to the technical field of drug screening, and in particular relates to a method for screening inhibitors of 3CL ^pro , the main protease of human coronavirus.

Background technique

Coronavirus is a single-stranded positive-strand RNA virus. Although vaccines for a variety of coronaviruses have been developed, vaccine protection is prone to failure due to the specificity of their genomes. Therefore, antiviral therapy is very important to reduce morbidity and mortality in infected individuals.

Current methods for coronavirus drug screening mainly include computer-aided drug design targeting single viral proteins or small-scale drug screening of some approved antiviral drugs using wild-type coronaviruses. Although these two methods have achieved positive results, they also have obvious limitations. Computer-aided drug design can only conduct drug screening for single or multiple viral targets, but cannot simulate the entire viral life cycle, which may result in the loss of a large number of potential antiviral drugs, and pharmacological experiments are also required to evaluate the actual antiviral properties of candidate drugs. Viral activity. Considering the biosafety requirements of wild-type coronavirus, live virus drug screening methods are usually used to identify effective therapeutic drugs from several existing antiviral drugs based on clinical experience. Therefore, this method is not suitable for high-throughput drugs. filter.

The main protease of coronavirus, 3CL ^pro , is one of the key proteolytic enzymes of coronavirus. It can specifically recognize and cleave LQ↓S (↓ indicates the cleavage site) in non-structural proteins, releasing it and is mainly responsible for the replication, transcription and participation of the viral genome. Viral protein translation, modification and nucleic acid synthesis proteins, therefore inhibiting the activity of 3CL ^pro protease can effectively inhibit viral replication and transcription. In addition, 3CL ^pro has no homologues found in human cells and is highly conserved in the human coronavirus genome sequence, making it an ideal anti-coronavirus target.

Green fluorescent protein (GFP) has a molecular weight of approximately 27kDa and is a single-chain polypeptide composed of 238 amino acids. The maximum excitation wavelength is 395nm, the emission peak is 509nm, and there is a shoulder peak at 470nm. It has stable chemical properties and is widely used in biomedical research as a fluorescent label. Its secondary structure contains 11 β-strands and a central α-helix. Only when the GFP structure is complete can it fluoresce normally.

Contents of the invention

The invention provides an activity reporter plasmid for human coronavirus major protease 3CL ^pro , by inserting the cleavage site LQ↓SG or RLQ↓SGF of human coronavirus major protease 3CL ^pro into the β chain of GFP (↓ represents the cleavage site ), two mutants of GFP were constructed, namely plasmid pCMV-GFP-β8-LQSG-9-LQSG-10 or plasmid pCMV-GFP-β9-RLQSGF-10, plasmid pCMV-GFP-β8-LQSG-9-LQSG The nucleotide sequence of -10 is shown in SEQ ID NO:1, and the nucleotide sequence of plasmid pCMV-GFP-β9-RLQSGF-10 is shown in SEQ ID NO:2;

Plasmid pCMV-GFP-β8-LQSG-9-LQSG-10 is to insert LQ↓SG between β chains 8, 9, and 10 of GFP. After transfection of this mutant into 293T cells, the fluorescence intensity is weakened compared with the wild type. , when cleaved by 3CL ^pro , the main coronavirus protease, the fluorescence intensity becomes significantly weaker. Plasmid pCMV-GFP-β9-RLQSGF-10 inserts RLQ↓SGF between β chains 9 and 10 of GFP. This mutant is transfected into 293T cells. Compared with the wild type, the difference in fluorescence intensity is not obvious. When it is mainly infected by coronavirus After cleavage by protease 3CL ^pro , its fluorescence intensity is greatly reduced. Therefore, the green fluorescence intensity of the two GFP mutants can indicate the activity of the main coronavirus protease 3CL ^pro , that is, the green fluorescence intensity is inversely proportional to the activity of 3CL ^pro .

Another object of the present invention is to apply the above-mentioned reporter plasmid of human coronavirus main protease 3CL ^pro in screening anti-human coronavirus drugs.

The present invention achieves the purpose of the present invention through the following technical solutions:

1. Construction of reporter plasmid for human coronavirus main protease 3CL ^pro

The construction method of plasmid pCMV-GFP-β8-LQSG-9-LQSG-10 is as follows:

(1) Use pEGFP-N3 plasmid (commercially available) as a template, use primers GFP-F: cgggaattcatggtgagcaagggcgaggagc; GFP-R: ccgctcgagcttgtacagctcgtccatgccgagagtg to amplify the GFP gene (introduce EcoRⅠ and XhoⅠ at the 5' end and 3' end of the GFP gene respectively) ), through double enzyme digestion with EcoRⅠ and XhoⅠ, connect the GFP gene to the pCMV-c-flag plasmid to obtain the pCMV-GFP-c-Flag plasmid;

(2) Use pCMV-GFP-c-Flag plasmid as template and use plasmid mutation primers GFPβ8-9-LQS-F: cacaacatcgaggacctgcagagcggcagcgtgcagctcgccgaccactac, GFPβ8-9-LQS-R: cgctgccgctctgcaggtcctcgatgttgtggcggatcttgaagttcaccttgatgccgtt c;

GFPβ9-10-F: CATCGGCGACCTGCAGCGCGCCCCCCCTGCCCCCCCCCCACCCTAC, GFPβ9-10-R: CACGGGGGCCGCGCGGGGGGGGGGTGGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT DPN Ⅰ enzyme treatment, remove the plasmids that have not produced unsatisfactory mutations, use agarose gel DNA to recover the DPN Ⅰ enzyme treatment DNA;

The plasmid mutation primer used in this step is a point mutation primer containing the 3CL ^pro cleavage site LQ↓SG (↓ indicates the cleavage site). After amplification, LQ↓SG is inserted between the 8, 9, and 10 chains of the GFP gene β chain. the goal of;

(3) Use the plasmid treated with DpnⅠ enzyme to transform into competent cells, pick single clones, extract the plasmid and perform sequencing to verify that the plasmid is constructed successfully. Use an endotoxin-free plasmid extraction kit to extract the plasmid pCMV-GFP-β8-LQSG. -9-LQSG-10.

The construction method of plasmid pCMV-GFP-β9-RLQSGF-10 is the same as the construction of plasmid pCMV-GFP-β8-LQSG-9-LQSG-10. The difference is that the plasmid mutation primer used in step (2) is GFP9-10-RLQSGF-F: catcggcgacaggctgcagagcggcttccccgtgctgctgcccgacaaccactac, GFP9-10-RLQSGF-R: gccgctctgcagcctgtcgccgatgggggtgttctg; the plasmid mutation primer used is a point mutation primer containing the 3CL ^pro cleavage site RLQ↓SGF (↓ indicates the cleavage site). After amplification, RLQ↓SGF is inserted into GFP The gene beta chain targets between 9 and 10.

2. Use the pCMV-GFP-β8-LQSG-9-LQSG-10 reporter plasmid or pCMV-GFP-β9-RLQSGF-10 reporter plasmid constructed above that has reduced green fluorescence intensity after being cleaved by 3CL ^pro , the main human coronavirus protease, to screen for antibodies. Human coronavirus drugs, here’s how:

(1) Use human coronavirus (HCoV-OC43) culture medium with an adsorption concentration of 100TCID ₅₀ to infect 293T cells (human kidney epithelial cells). The virus-infected cells are cultured in 2 groups. One group is added with the drug to be screened and is the experimental group. Adding screening drugs is the control group;

(2) Use pCMV-GFP-β8-LQSG-9-LQSG-10 reporter plasmid or pCMV-GFP-β9-RLQSGF-10 reporter plasmid to transfect 293T cells in the experimental group and control group in step (1);

(3) After transfection, use a laser confocal detection microscope to measure the intensity of green fluorescence. If the green fluorescence intensity of the experimental group is higher than that of the control group, then the drug to be screened is determined to be an inhibitor of 3CL ^pro .

The screening method can also replace human coronavirus with the expression plasmid of human coronavirus main protease 3CL ^pro (pCMV-3CL ^pro (OC43)-c-Flag), and use it to transfect 293T cells, and then transfect pCMV-GFP-β8- LQSG-9-LQSG-10 reporter plasmid or pCMV-GFP-β9-RLQSGF-10 reporter plasmid, in which the expression plasmid of human coronavirus main protease 3CL ^pro is constructed according to the conventional method, and the required human coronavirus is downloaded from NCBI ( HCoV-OC43) Main protease 3CL ^pro coding DNA sequence and primer design, extract human coronavirus RNA, reverse the obtained cDNA, use primers to amplify the main protease 3CL ^pro gene fragment, connect it to the initial vector after enzyme digestion, and obtain after transformation verification Successfully constructed expression plasmid.

Advantages and technical effects of the present invention:

1. The present invention constructs two mutants (reporter plasmids) of GFP by inserting the cleavage site LQ↓SG or RLQ↓SGF (↓ represents the cleavage site) of the human coronavirus main protease 3CL ^pro into the β chain of GFP. , after the mutant is transfected into cells, the fluorescence intensity is weakened or does not change significantly compared with the wild type. When cleaved by 3CL ^pro , the main protease of human coronavirus, the fluorescence intensity becomes significantly weaker. The green fluorescence intensity of the two reporter plasmids can indicate human The activity of 3CL ^pro , the main coronavirus protease, is used to screen anti-human coronavirus drugs. If the reagent has antiviral activity, compared with the control group (no drug added, but 3CL ^pro is cleaved), the experimental group (drug added, In the presence of 3CL ^pro at the same time, the green fluorescence intensity is higher;

2. Compared with the method of screening antiviral drugs using live viruses, the method of the present invention can intuitively observe the inhibitory effect through fluorescence intensity, can achieve high-throughput screening of anti-human coronavirus drugs, and greatly save the cost of screening anti-human coronavirus drugs. time and cost;

3. The HCoV-OC43 used in the method of the present invention is a human coronavirus with low pathogenicity and is not restricted by the third-level biosafety laboratory. The operation is simple and convenient, and can more effectively save labor costs.

Description of the drawings

Figure 1 is an electrophoresis diagram of the amplification result of 3CL ^pro gene fragment, 1 in the figure is the 3CL ^pro band;

Figure 2 is a picture of the PCR electrophoresis results of pCMV-3CL ^pro -c-Flag bacterial liquid, in which 1 is the DL2000 DNA marker, and 2-13 are the 3CL ^pro bands obtained by amplification of the bacterial liquid;

Figure 3 is the comparison result of the upstream and downstream primer sequencing peak charts of the pCMV-GFP-β8-LQSG-9-LQSG-10 plasmid, in which 1 is the upstream primer, 2 is the GFP reference sequence, 3 is the downstream primer, and A is β chain 9. , the LQSG coding sequence inserted between 10 and 10, B is the LQSG coding sequence inserted between 8 and 9 of the β chain;

Figure 4 is the comparison result of sequencing peak charts of the upstream and downstream primers of pCMV-GFP-β9-RLQSGF-10 plasmid, in which 1 is the upstream primer, 2 is the GFP reference sequence, 3 is the downstream primer, and A is between β chain 9 and 10. Inserting the RLQSGF coding sequence;

Figure 5 is a picture of the results of laser confocal microscopy of transfected 3CL ^pro activity reporter plasmid pCMV-GFP-β8-LQSG-9-LQSG-10, in which A, B, and C are negative groups (not infected with viruses and no drugs are added). ) GFP, DAPI and Merge pictures of pCMV-GFP-β8-LQSG-9-LQSG-10 plasmid transfected; D, E and F are the transfection reports of the control group (24h infection with OC43 virus, no GC-376 added) respectively. GFP, DAPI and Merge diagrams of the plasmid pCMV-GFP-β8-LQSG-9-LQSG-10 group; Figures G, H, and I respectively show the experimental group (infected with OC43 virus, added GC-376) transfected with the reporter plasmid pCMV- GFP, DAPI and Merge images of GFP-β8-LQSG-9-LQSG-10;

Figure 6 is a picture of the results of laser confocal microscopy of the transfected plasmid pCMV-GFP-β9-RLQSGF-10, in which A, B, and C are the negative groups (not infected with viruses and without adding drugs) transfected with pCMV-GFP- GFP, DAPI and Merge diagrams of β9-RLQSGF-10; D, E and F are respectively the GFP, DAPI and Merge diagrams of the control group (infected with OC43 virus for 24 hours, without adding GC-376) transfected with the reporter plasmid pCMV-GFP-β9-RLQSGF-10. DAPI and Merge diagrams; Figures G, H, and I are respectively the GFP, DAPI, and Merge diagrams of the experimental group (infected with OC43 virus, added with GC-376) transfected with the reporter plasmid pCMV-GFP-β9-RLQSGF-10;

Figure 7 is a picture of the results of laser confocal microscopy of the transfected plasmid pCMV-GFP-β8-LQSG-9-LQSG-10 in Example 4, in which A, B, and C are the GFP, DAPI, and Merge pictures of the negative group respectively; D, E, and F are the GFP, DAPI, and Merge images of the control group respectively; Figures G, H, and I are the GFP, DAPI, and Merge images of the experimental group, respectively.

Figure 8 is a picture of the results of laser confocal microscopy of the 3CL ^pro activity reporter plasmid group pCMV-GFP-β9-RLQSGF-10 transfected in Example 4, where A, B, and C are the GFP, DAPI and Merge of the negative group respectively. Pictures; D, E, and F are the GFP, DAPI, and Merge pictures of the control group respectively; Pictures G, H, and I are the GFP, DAPI, and Merge pictures of the experimental group, respectively.

Detailed ways

The present invention will be further described below through the drawings and examples, but the protection scope of the present invention is not limited to the content described. The methods in this example are operated according to conventional methods unless otherwise specified. The reagents used are conventional reagents or reagents unless otherwise specified. Reagents configured according to conventional methods.

Example 1: Construction of expression plasmid pCMV-3CL ^pro (OC43)-c-Flag of human coronavirus main protease 3CLpro

1. Download the HCoV-OC43 main protease 3CL ^pro coding DNA sequence (GenBank: ON376724.1) from NCBI (National Center for Biotechnology Information (nih.gov)), use Primer Primer 5.0 to analyze the enzyme cleavage site and design specific primers. And add EcoRⅠ and KpnⅠ to the 5' ends of the upstream and downstream primers respectively. The primer sequences are as shown in the table below;

2. Use kit OMEGA Total RNA Kit I extracts the RNA of viral HCoV-OC43, and the extraction steps are carried out according to the kit instructions;

3. Use RNA as a template and use TAKARA One Step PrimeScript ^TM RT-PCR Ki to reverse-transcribe the cDNA obtained. The operation is carried out according to the kit instructions;

4. Use cDNA as a template and use Novozantn high-fidelity enzyme to amplify the 3CL ^pro fragment with enzyme cutting sites. The amplification system is: 3CL ^pro -F 2μL, 3CL ^pro -R 2μL, cDNA 1μL, 2× Phanta Flash Master Mix 25μL, ddH ₂ O20μL; amplification conditions: 98℃ 30s; 30 cycles of 98℃ 10s, 65℃ 5s, 72℃ 5s; 72℃ 1min; the amplification electrophoresis diagram is shown in Figure 1, using daily Root agarose gel DNA recovery kit recovers the target fragment;

5. Use EcoRⅠ and KpnⅠ to double-digest the target fragment and vector pCMV-c-flag, and then use Tiangen agarose gel DNA recovery kit to purify the fragment. The enzyme cutting system is: EcoRⅠ2μL, KpnⅠ2μL, DNA 2μg, 10×M Add buffer4μL and ddH ₂ O to 40μL, and treat at 37°C for 5h;

6. Use T4 DNA ligase to ligate the target fragment and the vector at a molar ratio of 1:1. After ligation overnight at 16°C, add 20 μL of the ligation product to 100 μL E. coli DH5α, incubate on ice for 30 minutes, and then transfer to a 42°C water bath. Heat shock for 90 seconds, then place on ice for 5 minutes, then fill it up to 1 mL with liquid LB medium, and then place it on a shaker at 37°C and 200 rpm for 1 hour. Finally, take out the culture medium, centrifuge at 4000 rpm for 5 minutes, discard 900 μL of medium, and use the remaining Resuspend the cells in the medium, spread the cell suspension in solid LB medium containing ampicillin resistance, and culture at 37°C overnight;

7. Pick a single clone and culture it at 37°C and 200rpm for 3 hours. Then use 3CL ^pro- specific primers to perform bacterial liquid PCR. The electrophoresis results preliminarily confirm that 3CL ^pro is successfully connected to the vector. Then the bacterial liquid is expanded and Tiangen plasmid miniprep is used. The plasmid was extracted with the kit and verified by sequencing. The result of PCR electrophoresis of the bacterial liquid is shown in Figure 2; the sequencing result confirmed that the 3CL ^pro expression plasmid was successfully constructed, and then the endotoxin-free plasmid was extracted using the Tiangen endotoxin-free plasmid medium extraction kit. .

Example 2: Construction of reporter plasmid for human coronavirus main protease 3CL ^pro

1. Construction of plasmid pCMV-GFP-β8-LQSG-9-LQSG-10

(1) Use pEGFP-N3 plasmid (commercially available) as a template, use primers GFP-F: cgggaattcatggtgagcaagggcgaggagc; GFP-R: ccgctcgagcttgtacagctcgtccatgccgagagtg to amplify the GFP gene (introduce EcoRⅠ and XhoⅠ at the 5' end and 3' end of the GFP gene respectively) ), the amplification system is: 2 μL of GFP-F, 2 μL of GFP-R, 20 ng of pEGFP-N3 plasmid, 25 μL of 2×Phanta Flash Master Mix, and ddH ₂ O. Make up to 50 μL; amplification conditions: 98°C for 30s; 98°C for 10s. , 65℃ for 5s, 72℃ for 5s, 30 cycles; 72℃ for 1min; use Tiangen agarose gel DNA recovery kit to recover GFP gene fragments;

Use EcoRⅠ and XhoⅠ to double-digest the GFP gene fragment and pCMV-c-flag plasmid, and connect the GFP gene to the pCMV-c-flag plasmid to obtain the pCMV-GFP-c-Flag plasmid;

(2) First, use the pCMV-GFP-c-Flag plasmid as a template, and use the plasmid mutation primers GFPβ8-9-LQS-F: cacaacatcgaggacctgcagagcggcagcgtgcagctcgccgaccactac, GFPβ8-9-LQS-R: cgctgccgctctgcaggtcctCgatgttgtggcggatcttgaagttcaccttgatgccgt tc, Novozymes high-fidelity enzyme amplification, amplification The amplification system is: GFPβ8-9-LQS-F2μL, GFPβ8-9-LQS-R 2μL, pCMV-GFP-c-Flag plasmid 20ng, 2×Phanta FlashMaster Mix 25μL, ddH ₂ O, make up to 50μL; amplification conditions: 98℃ for 30s; 98℃ for 10s, 72℃ for 25s, 72℃ for 5s, 30 cycles; 72℃ for 1min, in order to first insert LQSG into the β chain 8-9 of GFP;

(3) The amplification product is treated with TAKARA DpnⅠ enzyme. The system is: 19 μL PCR product and 1 μL DpnⅠ, react at 37°C to remove non-mutated plasmid;

(4) Transform the processed PCR product into E. coli DH5α, incubate on ice for 30 minutes, transfer to a 42°C water bath and heat shock for 90 seconds, then place on ice for 5 minutes, then fill up to mL with liquid LB medium, and then Place the culture medium on a shaker at 37°C and 200 rpm for 1 hour. Finally, take out the culture medium and centrifuge it at 4000 rpm for 5 minutes. Discard the upper 900 μL medium, resuspend the cells with the remaining medium, and spread the cell suspension on solid LB containing ampicillin resistance. culture medium at 37°C overnight;

(5) Pick a single clone, culture it at 37°C and 200rpm for 3 hours, and use 3CL ^pro -specific primers to perform bacterial liquid PCR. The electrophoresis results preliminarily confirm that 3CL ^pro is successfully connected to the vector, and then expand the bacterial liquid, using Tiangen plasmid mini The plasmid was extracted with the kit and verified by sequencing. The sequencing results confirmed that LQSG was successfully inserted between β chains 8-9 of GFP, and the pCMV-GFP-β8-LQSG-9 plasmid was obtained;

(6) Use pCMV-GFP-β8-LQSG-9 plasmid as the amplification template, use primers GFPβ9-10-F: catcggcgacctgcagagcggccccgtgctgctgcccgacaaccactac, GFPβ9-10-R: cacggggccgctctgcaggtcgccgatgggggtgttctgctggtag for amplification, the amplification system is: GFPβ9-10-LQS -F 2μL, GFPβ9-10-LQS-R 2μL, pCMV-GFP-β8-LQSG-9 plasmid 20ng, 2×Phanta Flash Master Mix 25μL, ddH ₂ O. Make up to 50μL; amplification conditions: 98°C for 30s; 98 ℃10s, 72℃25s, 72℃5s, 30 cycles; 72℃1min;

(7) The amplification product is treated with DpnⅠ enzyme according to step (3) to remove the non-mutated plasmid, and then operates according to steps (4) and (5) to obtain pCMV-GFP-β8-LQSG-9-LQSG-10, The sequencing results are shown in Figure 3, and the nucleotide sequence is shown in SEQ ID NO: 1.

2. Construction of plasmid pCMV-GFP-β9-RLQSGF-10

(1) Use pCMV-GFP-c-Flag plasmid as template and use plasmid mutation primers GFP9-10-RLQSGF-F: catcggcgacaggctgcagagcggcttccccgtgctgctgcccgacaaccactac, GFP9-10-RLQSGF-R: gccgctctgcagcctgtcgccgatgggggtgttctg; according to the plasmid pCMV-GFP-β8- LQSG-9 -The construction method of LQSG-10 was carried out, and pCMV-GFP-β9-RLQSGF-10 was obtained. The sequencing result is shown in Figure 4, and its nucleotide sequence is shown in SEQ ID NO: 2.

Example 3: Feasibility verification experiment of screening human coronavirus major protease 3CL ^pro inhibitors using the reporter plasmid of human coronavirus major protease 3CL ^pro

1. One day before virus infection, spread the well-growing 293T cells at 2×10 ⁵ cells/mL into a 12-well plate where the cell sheets have been placed in advance;

2. Take out the HCoV-OC43 virus stock solution from the -80°C refrigerator and place it on ice to thaw. During infection, use serum-free DMEM medium to dilute the virus stock solution to a suspension with an adsorption degree of 100TCID ₅₀ , and then remove 12 wells. For the cell supernatant in the plate, wash the cells twice with PBS buffer, then add the virus suspension to the 12-well plate at an amount of 1 mL per well, and incubate it in a constant temperature incubator at 34°C for 2 hours, then remove the supernatant from the 12-well plate. Clear, replace with DMEM medium containing 2% serum and continue culturing;

3. Set up an experimental group (infected with OC43 virus, while adding inhibitor GC-376), a control group (infected with OC43 virus, but without adding inhibitor GC-376), and a negative group (not infected with virus and without adding GC-376);

4. Use sterile water to dissolve the coronavirus 3CL ^pro protease inhibitor GC-376 to 1mmol/L. After 24 hours of virus infection, add GC376 solution to the cells in the experimental group, add 50 μL to each well, and continue to culture for 30 minutes;

5. Use Lipofectamine ^TM 3000 transfection kit to transfect the reporter plasmid pCMV-GFP-β8-LQSG-9-LQSG-10 or pCMV-GFP-β9-RLQSGF-10 into the cells in step 4. The transfection system is as follows:

①Configure transfection reagent (loading amount of each well):

Reagent A: 50μL serum-free DMEM+1μg DNA+2μL p3000

Reagent B: 50μL serum-free DMEM+1.5μL lip3000;

② 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;

③Add the mixed solution to the cell culture medium, 50 μL per well;

6. After transfection, continue to culture for 6 hours, then replace the DMEM medium containing 2% serum (the experimental group needs to continue to add 50 μL GC-376), then continue to culture for 48 hours, remove the culture supernatant, and wash with PBS buffer. Cells were added twice, and then 400 μL of 4% paraformaldehyde was added to each well and left at room temperature for 10 min to fix the cells;

7. Use PBS buffer to dilute DAPI to 0.5μg/mL according to the instructions;

8. Remove the fixative, wash the cells twice with PBS buffer, then add 400 μL of DAPI solution to each well, and leave at room temperature for 10 minutes;

9. Remove the DAPI solution, wash twice with PBS buffer, then take out the cell slide, place it upside down on a glass slide (cell side down), then fix the slide, mark it, and use an inverted fluorescence microscope to observe the green fluorescence. the amount of expression;

The experimental results are shown in Figures 5 and 6. The figures show that the green fluorescence intensity of the control group (infected with OC43 virus but without the addition of drugs) is significantly lower than that of the negative group (not infected with the virus and without the addition of GC-376), indicating that 3CL ^pro protease The cleavage weakened the green fluorescence intensity expressed by the activity reporter plasmid. Compared with the control group, the green fluorescence intensity of the experimental group (infected with OC43 virus and added with GC-376) was significantly stronger. The results showed that the plasmid pCMV-GFP-β8-LQSG -9-LQSG-10 or pCMV-GFP-β9-RLQSGF-10 can indicate the activity of 3CL ^pro , the main protease of OC43 virus, that is, the green fluorescence intensity is inversely proportional to the 3CL ^pro protease activity.

Example 4: The 3CLpro expression plasmid pCMV-3CL ^pro (OC43)-c-Flag constructed in Example 1 was used to transfect 293T cells to verify the feasibility of the human coronavirus main protease 3CL ^pro reporter plasmid of the present invention for screening inhibitors. sexual experimentation

The method of this example is the same as that of Example 3, except that step 2 is replaced by using 1 μg of pCMV-3CL ^pro (OC43)-c-Flag plasmid to transfect 293T cells and pCMV-3CL ^pro (OC43)-c-Flag plasmid. 24 hours later, 1 μg of reporter plasmid pCMV-GFP-β8-LQSG-9-LQSG-10 or pCMV-GFP-β9-RLQSGF-10 was used to transfect 293T cells;

Set up an experimental group (transfected with 3CL ^pro expression vector, adding inhibitor GC-376), a control group (transfected with 3CL ^pro expression vector, without adding GC-376), and a negative group (not transfected with 3CL ^pro expression vector and without adding drugs );

The results are shown in Figures 7 and 8. The results are consistent with Example 3, that is, the green fluorescence intensity of the control group (co-transfected with 3CL ^pro expression plasmid and reporter plasmid, without adding GC-376) is significantly lower than that of the negative group ( without transfection of 3CL ^pro expression vector and without adding drugs), indicating that the cleavage of 3CL ^pro protease weakens the green fluorescence intensity expressed by the activity reporter plasmid. Compared with the control group, the experimental group (co-transfected with 3CL ^pro protease and activity reporter plasmid (adding GC-376), the green fluorescence intensity is significantly stronger. The experimental results show that the green fluorescent protein expressed by the two reporter plasmids pCMV-GFP-β8-LQSG-9-LQSG-10 or pCMV-GFP-β9-RLQSGF-10 can Indicates the activity of 3CL ^pro protease, that is, the green fluorescence intensity is inversely proportional to the activity of 3CL ^pro protease.

The above experimental results show that the reporter plasmid of the human coronavirus main protease 3CL ^pro of the present invention has the potential to screen anti-human coronavirus drugs.

Claims (2)

1. An activity reporter plasmid for the main human coronavirus protease 3CL ^pro , which is characterized in that: it is plasmid pCMV-GFP-β8-LQSG-9-LQSG-10 or plasmid pCMV-GFP-β9-RLQSGF-10, plasmid pCMV- The nucleotide sequence of GFP-β8-LQSG-9-LQSG-10 is shown in SEQ ID NO:1, and the nucleotide sequence of plasmid pCMV-GFP-β9-RLQSGF-10 is shown in SEQ ID NO:2. 2. Application of the activity reporter plasmid of human coronavirus main protease 3CL ^pro according to claim 1 in screening anti-human coronavirus drugs.