Patent Application
20240192196
This application includes a sequence listing in computer readable form (a “txt” file) that is submitted herewith on ASCII text file named P23GZINW00010US_ST25.txt, created on Aug. 21, 2023 and 69,974 bytes in size. This sequence listing is incorporated by reference herein.
The present disclosure belongs to the field of biotechnology, and more particularly relates to a novel coronavirus SARS-COV-2 safe replicon system and use thereof.
As of Jul. 23, 2020, novel coronavirus SARS-COV-2 had infected more than 15 million people worldwide and killed more than 140,000 people. However, there are currently very limited clinical therapeutic drugs applicable to SARS-COV-2 infection. For biosafety reasons, drug development and screening against wild-type SARS-COV-2 can only be carried out in biosafety level 3 laboratories (P3 laboratories), which greatly limits the development of antiviral drugs against SARS-COV-2.
Previous studies have shown that a safe replicon system constructed by inserting E protein-deleted coronavirus genome into a Bacterial Artificial Chromosome (BAC) can simulate the replication of coronaviruses. This system has been applied to drug verification and drug screening against SARS-COV. However, the system is based on BAC plasmid. BAC plasmid, which has a relatively large molecular weight and is unstable, cannot reach an ideal expression level after transduction into cells and is also time-consuming and laborious to operate.
Therefore, it is urgent to develop a tool that can simulate SARS-COV-2 virus replication and can be simply operated in low-level biosafety laboratories.
The object of a first aspect of the present disclosure is to provide a novel SARS-COV-2 safe replicon structure which can make up for the blank of novel coronavirus SARS-COV-2 safe replicons while overcoming the deficiency of BAC replicon systems.
The object of a second aspect of the present disclosure is to provide a novel coronavirus SARS-COV-2 safe replicon system comprising the above-mentioned replicon structure.
The object of a third aspect of the present disclosure is to provide a packaging cell comprising the above-mentioned replicon structure or replicon system.
The object of a fourth aspect of the present disclosure is to provide use of the above-mentioned novel coronavirus SARS-COV-2 safe replicon structure, replicon system or packaging cell in drug detection or drug screening against novel coronavirus SARS-COV-2.
The object of a fifth aspect of the present disclosure is to provide a method for screening an anti-novel coronavirus SARS-COV-2 drug.
The object of a sixth aspect of the present disclosure is to provide a kit for screening an anti-novel coronavirus SARS-COV-2 drug.
The object of a seventh aspect of the present disclosure is to provide a system for screening an anti-novel coronavirus SARS-COV-2 drug.
The object of an eighth aspect of the present disclosure is to provide a novel coronavirus SARS-COV-2 molecular epidemiological monitoring system.
The following technical solutions are used in the present disclosure:
In the first aspect of the present disclosure, provided is a novel coronavirus SARS-CoV-2 replicon structure, comprising the following nucleic acid sequences:
Preferably, according to the replicon structure of the first aspect of the present disclosure, the non-structural protein is at least one of novel coronavirus SARS-COV-2 protein nsps 1-16.
Preferably, according to the replicon structure of the first aspect of the present disclosure, the transcription regulatory region is at least one of transcription regulatory regions (TRSs) of S, ORF3a, M, ORF7a, ORF8 or N genes of novel coronavirus SARS-COV-2.
Furthermore, a core sequence (AAACGAAC) of the transcription regulatory region (TRS) alone, or a combination of the core sequence (AAACGAAC) of the transcription regulatory region and another sequence, also falls within the scope of protection.
Furthermore, according to the replicon structure of the first aspect of the present disclosure, the transcription regulatory region is connected upstream of the reporter gene.
Furthermore, the replicon structure of the first aspect of the present disclosure further comprises a nucleic acid sequence of an additional reporter gene as a reference.
Moreover, according to the replicon structure of the first aspect of the present disclosure, the additional reporter gene as the reference is connected to a stop codon and located upstream of the transcription regulatory region.
Preferably, according to the replicon structure of the first aspect of the present disclosure, the nucleic acid is DNA or RNA, preferably antisense RNA.
In the second aspect of the present disclosure, provided is a novel coronavirus SARS-CoV-2 replicon system, comprising an expression vector in which the replicon structure according to the first aspect of the present disclosure is inserted.
Preferably, the replicon system according to the second aspect of the present disclosure comprises the following two expression vectors comprising:
More preferably, according to the replicon system of the second aspect of the present disclosure, nucleic acid sequences of the 5′ UTR of novel coronavirus SARS-COV-2, the transcription regulatory region on which the novel coronavirus SARS-COV-2 non-structural protein can act, the reporter gene, and the 3′ UTR of novel coronavirus SARS-COV-2 are inserted in expression vector (ii) in order.
Further preferably, according to the replicon system of the second aspect of the present disclosure, nucleic acid sequences of the 5′ UTR of novel coronavirus SARS-COV-2, a reporter gene A, the transcription regulatory region on which the novel coronavirus SARS-COV-2 non-structural protein can act, a reporter gene B, and the 3′ UTR of novel coronavirus SARS-COV-2 are inserted in expression vector (ii) in order, wherein the reporter gene A is different from the reporter gene B.
More preferably, the reporter gene A is a nucleic acid sequence of fluorescent protein; and the reporter gene B is a nucleic acid sequence encoding luciferase.
Moreover, according to the replicon system of the second aspect of the present disclosure, a nucleic acid sequence of a ribosome entry site (IRES) is further connected between the 5′ UTR of novel coronavirus SARS-COV-2 and the reporter gene A.
Moreover, according to the replicon system of the second aspect of the present disclosure, a translation stop codon, preferably four stop codons, is inserted at an end of reporter gene A.
Specifically, according to the replicon system of the second aspect of the present disclosure, nucleic acid sequences of the 5′ UTR of novel coronavirus SARS-COV-2, the reporter gene A, the transcription regulatory region on which the novel coronavirus SARS-COV-2 non-structural protein can act, the reporter gene B, and the 3′ UTR of novel coronavirus SARS-COV-2 are inserted in expression vector (ii) in order, wherein the reporter gene A is a nucleic acid sequence of fluorescent protein; and the reporter gene B is a nucleic acid sequence encoding luciferase.
Furthermore, the transcription regulatory region is a transcription regulatory region upstream of S, ORF3a, M, ORF7a, ORF8, or N genes of novel coronavirus SARS-COV-2.
Moreover, the nucleotide sequence of the transcription regulatory region for S protein (S-TRS) is shown in SEQ ID NO:20; the nucleic acid sequence of the transcription regulatory region for ORF3a protein (ORF3a-TRS) is shown in SEQ ID NO: 21: the nucleic acid sequence of the transcription regulatory region for protein M (M-TRS) is shown in SEQ ID NO:22: the nucleic acid sequence of the transcription regulatory region for ORF7a protein (ORF7a-TRS) is shown in SEQ ID NO: 23: the nucleic acid sequence of the transcription regulatory region for ORF8 protein (ORF8-TRS) is shown in SEQ ID NO: 24; and the nucleic acid sequence of the transcription regulatory region for N protein (N-TRS) is shown in SEQ ID NO: 25.
The nucleotide sequence of the 5′ UTR of novel coronavirus SARS-COV-2 is shown in SEQ ID NO: 26.
The nucleotide sequence of the 3′ UTR of novel coronavirus SARS-COV-2 is shown in SEQ ID NO: 27.
The nucleotide sequence of the inserted ribosome entry site (IRES) is preferably shown in SEQ ID NO: 28.
The nucleotide sequence of the inserted four stop codons is preferably shown in SEQ ID NO: 29.
More specifically, the nucleotide sequence of ps2V in expression vector (ii) is shown in SEQ ID NO: 30.
Preferably, according to the replicon system of the second aspect of the present disclosure, the encoded novel coronavirus SARS-COV-2 non-structural protein is novel coronavirus SARS-COV-2 protein nsps 1-16.
More preferably, according to the replicon system of the second aspect of the present disclosure, the reporter gene A is a nucleic acid sequence of fluorescent protein; and the reporter gene B is a nucleic acid sequence encoding luciferase. Expression vector (i) comprises three expression vectors, in which nucleic acid sequences encoding one or more of novel coronavirus SARS-COV-2 protein nsps 1-16 are respectively inserted.
Further preferably, the nucleic acid sequences of protein nsps 1-16 are codon-optimized.
More specifically, after codon optimization, the nucleotide sequence of nsp 1 is shown in SEQ ID NO: 1: the nucleotide sequence of nsp 2 is shown in SEQ ID NO: 2: the nucleotide sequence of nsp 3 is shown in SEQ ID NO: 3: the nucleotide sequence of nsp 4 is shown in SEQ ID NO: 4: the nucleotide sequence of nsp 5 is shown in SEQ ID NO: 5: the nucleotide sequence of nsp 6 is shown in SEQ ID NO: 6: the nucleotide sequence of nsp 7 is shown in SEQ ID NO: 7: the nucleotide sequence of nsp 8 is shown in SEQ ID NO: 8; the nucleotide sequence of nsp 9 is shown in SEQ ID NO: 9; the nucleotide sequence of nsp 10 is shown in SEQ ID NO: 10; the nucleotide sequence of nsp 11 is shown in SEQ ID NO: 11: the nucleotide sequence of nsp 12 is shown in SEQ ID NO: 12; the nucleotide sequence of nsp 13 is shown in SEQ ID NO: 13; the nucleotide sequence of nsp 14 is shown in SEQ ID NO: 14; the nucleotide sequence of nsp 15 is shown in SEQ ID NO: 15; and the nucleotide sequence of nsp 16 is shown in SEQ ID NO: 16.
Further preferably, according to the replicon system of the second aspect of the present disclosure, a nucleic acid sequence encoding novel coronavirus SARS-COV-2 protein nsps 1-4, a nucleic acid sequence encoding novel coronavirus SARS-COV-2 protein nsps 5-11, and a nucleic acid sequence encoding novel coronavirus SARS-COV-2 protein nsps 12-16 are respectively inserted in the three expression vectors.
Moreover, according to the replicon system of the second aspect of the present disclosure, the nucleic acid sequences are codon-optimized.
Specifically, according to the replicon system of the second aspect of the present disclosure, expression vector (i) comprises three expression vectors, in which three segments of nucleic acid sequences, i.e., ps2AN, ps2AC, and ps2B, are inserted respectively.
Moreover, the nucleotide sequence of ps2AN is shown in SEQ ID NO: 17; the nucleotide sequence of ps2AC is shown in SEQ ID NO: 18; and the nucleotide sequence of ps2B is shown in SEQ ID NO: 19.
Preferably, according to the replicon system of the second aspect of the present disclosure, the expression vector is preferably but not limited to pcDNA3.1 plasmid.
More preferably, the ratio of the plasmids respectively comprising ps2AN, ps2AC, ps2B and ps2V is (0.01-1 μg): (0.01-1 μg): (0.01-1 μg): (0.01-1 μg).
In the third aspect of the present disclosure, provided is a packaging cell comprising the replicon structure of the first aspect of the present disclosure or the replicon system of the second aspect of the present disclosure.
Preferably, according to the packaging cell of the third aspect of the present disclosure, the cell is a human-derived cell.
More preferably, according to the packaging cell of the third aspect of the present disclosure, the cell is preferably but not limited to an HEK293T cell.
Preferably, according to the packaging cell of the third aspect of the present disclosure, the replicon structure or replicon system is codon-optimized.
Furthermore, the replicon structure or replicon system is transfected into a cell, to form a packaging cell.
Moreover, during transfection, the ratio of the plasmids respectively comprising ps2AN, ps2AC, ps2B and ps2V is (0.01-1 μg): (0.01-1 μg): (0.01-1 μg): (0.01-1 μg).
The ratio of the plasmids is (0.01-1 μg): (0.01-1 μg): (0.01-1 μg): (0.01-1 μg) on a concentration basis.
In the fourth aspect of the present disclosure, provided is use of the replicon structure of the first aspect of the present disclosure, the replicon system of the second aspect of the present disclosure, or the packaging cell of the third aspect of the present disclosure in drug detection or drug screening against novel coronavirus SARS-COV-2.
In the fifth aspect of the present disclosure, provided is a method for screening an anti-novel coronavirus SARS-COV-2 drug, comprising adding a drug to be tested to an expression system comprising the replicon structure of the first aspect of the present disclosure, the replicon system of the second aspect of the present disclosure or the packaging cell of the third aspect of the present disclosure to detect the differential expression of a reporter gene and evaluate an anti-novel coronavirus SARS-COV-2 effect of the drug to be tested.
In the sixth aspect of the present disclosure, provided is a kit for screening an anti-novel coronavirus SARS-COV-2 drug, comprising the replicon structure of the first aspect of the present disclosure, the replicon system of the second aspect of the present disclosure, or the packaging cell of the third aspect of the present disclosure.
In the seventh aspect of the present disclosure, provided is a system for screening an anti-novel coronavirus SARS-COV-2 drug, comprising the replicon structure of the first aspect of the present disclosure, the replicon system of the second aspect of the present disclosure, or the packaging cell of the third aspect of the present disclosure.
Furthermore, the drug screening system according to the seventh aspect of the present disclosure further comprises a luciferase detection device.
Preferably, the drug screening system further comprises a fluorescent protein detection device.
Preferably, the drug screening system further comprises a fully automatic robotic arm drug screening platform.
In the eighth aspect of the present disclosure, provided is a novel coronavirus SARS-CoV-2 molecular epidemiological monitoring system, comprising the replicon structure of the first aspect of the present disclosure, the replicon system of the second aspect of the present disclosure, or the packaging cell of the third aspect of the present disclosure.
The novel coronavirus SARS-COV-2 molecular epidemiological monitoring system according to the eighth aspect of the present disclosure utilizes the replicon system to monitor an effect of a mutation produced in SARS-COV-2 during an epidemic on SARS-COV-2 virus replication.
The present disclosure has the following beneficial effects.
The present disclosure provides a novel coronavirus SARS-COV-2 safe replicon structure, a novel coronavirus SARS-COV-2 safe replicon system, and a packaging cell thereof which can make up for the blank of novel coronavirus SARS-COV-2 safe replicons while overcoming the technical deficiency of BAC replicon systems. The molecule necessary for the synthesis of SARS-COV-2 RNA is artificially split, optimized for nucleotide sequence, so that four plasmids are used for co-expression, which destroys the original SARS-COV-2 sequence and provides safer operation, without the need for operation in a high-level biosafety laboratory.
The novel coronavirus SARS-COV-2 safe replicon system constructed by the present disclosure can highly simulate the response of wild-type SARS-COV-2 to a drug.
The present disclosure further provides a method for screening an anti-novel coronavirus SARS-COV-2 drug, a corresponding kit, and a detection system. The present disclosure provides the possibility of screening an anti-novel coronavirus SARS-COV-2 drug in laboratories below biosafety level 3, greatly promotes the research and screening of anti-novel coronavirus SARS-COV-2 drugs, and has broad application prospects.
The novel coronavirus SARS-COV-2 safe replicon system constructed by the present disclosure can highly simulate the replication characteristics of wild-type SARS-COV-2. Another potential application of the present disclosure is that point mutation can be artificially performed in the replicon system according to the mutation characteristics of epidemic strains, and the effect of the epidemic mutation on virus replication can then be detected and evaluated, which is of positive significance for molecular epidemiological monitoring of novel coronavirus SARS-COV-2.
In order to understand the technical content of the present disclosure more clearly, the following examples are particularly given in conjunction with the attached drawings for detailed description. It should be understood that these examples are only used to illustrate the present disclosure, rather than limiting the scope of the present disclosure. The experimental methods without specific conditions indicated in the following examples usually follow conventional conditions, such as those in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those suggested by the manufacturer. Various conventional chemical reagents used in the examples are all commercially available products.
The genome of novel coronavirus SARS-COV-2 is shown in
After novel coronavirus SARS-COV-2 enters a cell via ACE2 receptor:
The original sequence of novel coronavirus SARS-COV-2 is based on the sequence of SARS-COV-2 Wuhan-Hu-1 (Genbank: NC_045512.2).
Based on the composition of the genome of novel coronavirus and the principle and process of viral RNA synthesis (replication and transcription process), a novel coronavirus SARS-COV-2 safe replicon, including the following two expression structures is creatively constructed:
The expression structure comprising a nucleic acid sequence encoding the novel coronavirus SARS-COV-2 non-structural protein in (I) was an expression vector encoding the sequences of protein nsps 1-16.
In the genome of novel coronavirus, the sequences of rep1a and rep1b, totaling about 20000 bp, accounted for about ⅔ of the viral genome. Considering the efficiency of transfection and expression, as well as the function of each of protein nsps 1-16 in the transcription complex, the nucleotide sequences encoding protein neps 1-16 were codon-optimized and inserted into three expression vectors, respectively, named ps2AN, ps2AC, and ps2B respectively.
In this example, the ps2AN molecule was derived from NSP1-NSP4 sequences on N′ end of ORF1a of SARS-COV-2, and the sequences had been codon-optimized for human; the ps2AN molecule was derived from NSP5-NSP11 sequences on C′ end of ORF1a of SARS-CoV-2, and the sequences had been codon-optimized for human; and the ps2B molecule was derived from NSP12-NSP16 sequences on C′ end of ORF1ab of SARS-COV-2, and the sequences had been codon-optimized for human.
(II) The expression structure comprising nucleotide sequences of 5′ UTR and 3′ UTR of novel coronavirus SARS-COV-2, a transcription regulatory region on which the novel coronavirus SARS-COV-2 non-structural protein could act, and a reporter gene.
Since the expression of S protein, ORF3a, M, ORF7a, ORF8, or N protein of novel coronavirus SARS-COV-2 depended on the participation of the 16 proteins, i.e., proteins nsps 1-16, which matured to form a viral transcriptase/replicase complex, and on the 5′ UTR sequence, 3′ UTR sequence, and transcription regulatory region (TRS) sequence in the viral genome, the transcription regulatory region (TRS) sequence in (II) could be at least one TRS sequences of S, ORF3a, M, ORF7a, ORF8, or N, and the core sequence (AAACGAAC) of the TRS region could be used either alone or in combination with other sequences. Since a transcription regulatory region on which the novel coronavirus SARS-COV-2 non-structural protein could act was connected upstream of reporter gene B, the expression of reporter gene B was dependent on an Nsp1-Nsp16 replicase/transcriptase complex formed by ps2AN, ps2AC, and ps2B transcription, translation and maturing.
The nucleotide sequence of the transcription regulatory region for S protein (S-TRS) was shown in SEQ ID NO: 20; the nucleic acid sequence of the transcription regulatory region for ORF3a protein (ORF3a-TRS) was shown in SEQ ID NO: 21: the nucleic acid sequence of the transcription regulatory region for protein M (M-TRS) was shown in SEQ ID NO: 22; the nucleic acid sequence of the transcription regulatory region for ORF7a protein (ORF7a-TRS) was shown in SEQ ID NO: 23: the nucleic acid sequence of the transcription regulatory region for ORF8 protein (ORF8-TRS) was shown in SEQ ID NO: 24; and the nucleic acid sequence of the transcription regulatory region for N protein (N-TRS) was shown in SEQ ID NO: 25.
In order to make the replicon system comprising the above-mentioned expression structure more accurate, an additional reporter gene was introduced into the expression structure (II) as a control.
Nucleic acid sequences of 5′ UTR of novel coronavirus SARS-COV-2, reporter gene A as a control, a transcription regulatory region on which the novel coronavirus SARS-COV-2 non-structural protein could act, reporter gene B, and 3′ UTR of novel coronavirus SARS-COV-2 were connected in the expression structure in order, wherein the reporter gene A and the reporter gene B were different types of reporter genes. For example, the reporter gene A was fluorescent protein and the reporter gene B was luciferase.
A nucleic acid sequence of a ribosome entry site (IRES) was further connected between the 5′ UTR of novel coronavirus SARS-COV-2 and reporter gene A. A translation stop codon was inserted at an end of reporter gene A.
In this example, reporter gene A was green fluorescent protein (GFP) with four stop codons inserted at an end; reporter gene B was luciferase; and the TRS sequence was a transcription regulatory region (M-TRS) sequence for M protein.
In this example, the 5′ end of the Ps2V molecule was the non-coding region 5′-UTR at the 5′ end of SARS-COV-2; downstream was a ribosome entry site (IRES); further downstream was GFP reporter gene, wherein four translation stop codons were inserted at an end of the GFP reporter gene; further downstream was firefly luciferase gene connected to the transcription regulatory region (TRS) for M protein of SARS-COV-2; and the 3′ end was the non-coding region 3′-UTR at the 3′ end of SARS-COV-2.
The replicon structures in (I) and (II) mentioned above were inserted into expression vectors to construct a replicon system comprising:
The expression vector could be an eukaryotic expression vector or a prokaryotic expression vector depending on the detection purpose.
In this example, pcDNA3.1 plasmids were selected as expression vectors, and ps2V, ps2AN, ps2AC, and ps2B were respectively inserted into the pcDNA3.1 plasmids by means of double digestion with NheI and XbaI (the map of the plasmid was shown in
The purpose of the construction of the replicon system in Example 1 was to screen an anti-novel coronavirus SARS-COV-2 drug, especially a human drug, so HEK 293T cell line was used as a packaging cell for verification. The schematic diagram of the working principles of the four expression vectors, ps2V, ps2AN, ps2AC, and ps2B, in the human body or human cells was shown in
HEK293T cells in a good growth state were evenly plated in a 12-well culture plate treated with polylysine (at a cell density of about 6.5×104/cm2), and the cells were uniformly distributed individually. After about 24 h of culture, the cell confluence was close to 80%. At this point, an Opti-Lipo2000-DNA mixed liquid as shown in Table 1 was prepared and used for transfection.
The concentrations of the four vectors could be between 0.01 μg/μL and 1 μg/μL, and the ratio of the four vectors could be adjusted within the above range.
After transfection, the transfection effect could be evaluated by observing the expression of green fluorescent protein in the cells. As could be seen from
Subsequently, according to the detection time point, 200 μl of Promega cell lysate was added to the cells, the cells were repeatedly pipetted with a pipette, and the lysate was put into a 1.5 mL Ep tube and oscillated on an oscillator for 20 min at room temperature. The intracellular luciferase activities at different time points were detected by luciferase detection system, and the results were shown in
According to the steps in Example 2, transfection was carried out with ps2V, ps2AN, ps2AC, and ps2B plasmids. 6 h after transfection, Remdesivir, Lopinavir, and Ritonavir were added according to the concentration gradient (20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM, 0.078125 μM, and 0.0390625 μM). After 24 h of drug treatment, the cellar luciferase activity was detected, the inhibition rate was calculated with DMSO control as a baseline, and the semi-inhibitory concentration (hereinafter referred to as IC50) of the drug was calculated using Graphpad Prism 7.0 software. The specific results were shown in
The results in
The results of the above data indicated that the replicon system constructed in Example 1 could reproduce the response of wild-type SARS-COV-2 to a drug, with a closer IC50, indicating that the constructed novel coronavirus SARS-COV-2 replicon system could highly simulate the response of wild-type SARS-COV-2 to a drug.
HEK293T cells in a good growth state were evenly plated in a 96-well culture plate treated with polylysine (at a cell density of about 6.5×104/cm2), and the cells were uniformly distributed individually. After about 24 h of culture, the cell confluence was close to 80%. According to the steps in Example 2, transfection was carried out with ps2V, ps2AN, ps2AC, and ps2B plasmids in proportion. 6 h after transfection, each well was charged with a drug from a library of proprietary drugs. 24 h after drug treatment, the cellar luciferase activity was detected, and the inhibition rate was calculated with DMSO control as a baseline. After four rounds of screening, it was preliminarily determined that the drugs M01, A01, and R01 had inhibitory effects on viral RNA replication, and the IC50 of the drug was calculated using Graphpad Prism 7.0 software. The specific results were shown in
Subsequently, the inhibitory effects of the candidate drugs M01, A01, and R01 on wild-type novel coronavirus SARS-COV-2 were further verified. HEK293T cells in a good growth state were evenly plated in a 48-well culture plate treated with polylysine (at a cell density of about 6.5×104/cm2). After 16 h of cell growth (the cell density was about 1.6×105/mL), the cells were transfected with 0.1 g of the plasmid pCMV-ACE2-FLAG expressing SARS-COV-2 binding receptor ACE2 gene. 24 h after transfection, the cells were rinsed with PBS and infected with wild-type novel coronavirus SARS-COV-2 (MOI=0.1, 37° C., 1 h). Subsequently, DMEM (2% FBS) comprising the drugs M01, A01, and R01 with different concentration gradients (20) μM, 5 μM, 1.25 μM, 0.3125 μM, 0.078125 μM, and 0.01953125 μM) was replaced. After 24 h of drug treatment, cell RNA was extracted by TRIZOL, and RNA copies of SARS-COV-2 were detected by means of novel coronavirus 2019-nCOV nucleic acid detection (PCR-fluorescence probe method) from Daan Gene. The Ct value was obtained, the virus copy number was calculated based on a standard curve, the inhibition rate was calculated, and the IC50 of the drug was calculated using Graphpad Prism 7.0 software. The results were shown in
It could be seen that when inhibiting the growth of wild-type SARS-COV-2, the IC50 of M01 was 0.597±0.341 μM, the IC50 of A01 was 0.1396±0.0913 μM, and the IC50 of R01 was 11.25±1.89 μM, showing obvious resistance.
The above experimental results further indicated that the candidate drugs screened by the SARS-COV-2 replicon system constructed in Example 1 could effectively inhibit wild-type SARS-COV-2, and the SARS-COV-2 replicon system could be used as a reliable anti-SARS-CoV-2 drug screening system.
According to the results of the above-mentioned examples, it could also be expected that the replicon system constructed in Example 1 could be used to monitor the effect of a mutation produced in SARS-COV-2 during an epidemic on SARS-COV-2 virus replication.
Study on viral molecule evolution was shown in
In the replicon system constructed in Example 1, 5′ UTR was located on the ps2V molecule. C at position 241 in 5′ UTR of ps2V was mutated into T by using Mut Express II Fast Mutagenesis kit from Vazyme to construct 5′ UTR_241 T_ps2V. Transfection with 5′ UTR_241 T_ps2V was carried out according to the experimental method of Example 2, and 5′ UTR_241 C_ps2V was used as an experimental control. The intracellular luciferase activity was detected by luciferase detection system, and the results were shown in
As could be seen from
The above examples only express several embodiments of the present disclosure, and the description thereof is relatively specific and detailed, but they cannot be understood as a limitation on the scope of protection for the present disclosure. It should be pointed out that for those of ordinary skill in the art, a number of modifications and improvements could also be made without departing from the concept of the present disclosure, and these all fall within the scope of protection of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010818896.X | Aug 2020 | CN | national |
The present application is a national phase entry under 35 USC § 371 of International Application PCT/CN2020/119544, filed Sep. 30, 2020, which claims the benefit of and priority to Chinese Patent Application No. 202010818896.X, filed Aug. 14, 2020, the entire disclosures of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/119544 | 9/30/2020 | WO |
