JP2022177804A - Cell line and method for producing the same - Google Patents

Abstract

To provide cells that stably express coronavirus non-structural proteins and structural proteins or nucleic acids encoding these proteins, and that enable highly sensitive and highly reproducible screening of anti-coronavirus therapeutic agents.SOLUTION: There is provided a cell line with an accession number NITE P-03458. Also there is provided a method for producing a cell line, comprising the steps of: transfecting a cell with a vector having a nucleic acid sequence encoding a nonstructural protein of a coronavirus and a reporter protein including a drug resistance protein and a coronavirus structural protein; and selecting the cells obtained in the transfection step using a drug.SELECTED DRAWING: None

Description

Patent Law Article 30, Paragraph 2 application filed Antiviral Research Volume 199, March 2022, 105268, published on March 7, 2020 https://doi. org/10.1016/j. antiviral. 2022.105268

Patent Law Article 30, Paragraph 2 application filed Antiviral Research Volume 199, March 2022, 105268, published on March 7, 2022 https://doi. org/10.1016/j. antiviral. 2022.105268

The present invention relates to cell lines and methods for producing the same.

Due to the worldwide spread of the new coronavirus, there is a demand for the development of virus inhibitors that can suppress the growth of the virus. In general, the development of virus inhibitors has high hurdles and requires large-scale screening experiments using compound libraries and the like. In the case of the new coronavirus, it is possible to conduct virus infection experiments using cultured cells, so the above-mentioned screening experiments are currently being conducted using infectious viruses.

However, since the infectious new coronavirus can only be handled in BSL3 facilities, it is necessary to bring equipment that may be contaminated with the virus into the BSL3 facility. Against this background, it is thought that current novel coronavirus drug discovery research is often conducted under restrictions.

Therefore, a system for expressing the replicon RNA of the novel coronavirus was developed (Non-Patent Documents 1 to 3).
However, this system is a system for transiently expressing replicon RNA, and a cell line stably expressing replicon RNA cannot be obtained. Therefore, it is necessary to synthesize a replicon expression vector and transfect cells each time. In addition, the size of the replicon expression vector is huge, which causes variations in preparation and transfection efficiency (Non-Patent Document 4).
These limitations make it difficult to implement large-scale screening experiments such as drug discovery research.

On the other hand, as a system for expressing hepatitis C virus (HCV) replicon RNA, a cell line stably expressing HCV replicon RNA, prepared by a method of transfecting HCV replicon RNA, has been reported ( Non-Patent Document 5).
In Non-Patent Document 1, similar to the method described in Non-Patent Document 5, coronavirus replicon RNA is transiently expressed in cultured cells by a method of transfecting coronavirus replicon RNA. Cell lines stably expressing viral replicon RNA have not been generated.

Therefore, there is a strong demand for rapid provision of cells that stably express coronavirus replicon RNA and that enable highly sensitive and highly reproducible screening of anti-coronavirus therapeutic agents.

Scientific Reports 2021 11:2229 Antiviral Research 2021 185:104974 mBio 2021 12:e02754-20 BioTechniques 2003 35:796-807 Science 1999 285:110-3

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned conventional problems and to achieve the following objects. That is, an object of the present invention is to provide cells that stably express coronavirus replicon RNA and that enable highly sensitive and highly reproducible anti-coronavirus therapeutic drug screening.

In order to solve the above-mentioned problems, the present inventors conducted intensive studies and found that a vector having a nucleic acid sequence encoding a coronavirus nonstructural protein, a reporter protein containing a drug resistance protein, and a coronavirus structural protein was developed. , a cell line preparation method comprising a transfection step of transfecting cells, and a selection step of selecting the cells obtained in the transfection step using a drug, stably expressing coronavirus replicon RNA. It was found that cells that enable highly sensitive and highly reproducible screening of anti-coronavirus therapeutic agents can be provided.

The present invention is based on the findings of the present inventors, and means for solving the above problems are as follows. Namely
<1> A cell line characterized by having accession number NITE P-03458.
<2> a transfection step of transfecting a cell with a vector having a nucleic acid sequence encoding a coronavirus nonstructural protein, a reporter protein containing a drug resistance protein, and a coronavirus structural protein; A method for producing a cell line, comprising a selection step of selecting the cells obtained in the step using a drug.
<3> A vector characterized by having a nucleic acid sequence encoding a coronavirus nonstructural protein, a reporter protein containing a drug resistance protein, and a coronavirus structural protein.
<4> An introduced cell characterized by introducing the vector according to <3> above.
<5> A method of screening for an anti-coronavirus therapeutic drug, comprising using the introduced cells according to <4> above.
<6> An anti-coronavirus therapeutic agent screening kit comprising the introduced cells according to <4> above.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to solve the above-mentioned conventional problems and achieve the above-mentioned objects, stably express coronavirus replicon RNA, and perform highly sensitive and highly reproducible anti-coronavirus therapeutic drug screening. It is possible to provide cells that enable

FIG. 1A is a diagram showing an overall schematic diagram of a replicon expression vector. FIG. 1B is a diagram showing details of the portion corresponding to CMV to BGH in FIG. 1A. FIG. 2A is a diagram showing the results of Western blot analysis of replicon protein expression before and after transfection of a replicon expression vector and before and after addition of a drug. FIG. 2B is a diagram showing the results of analysis by Northern blotting of replicon RNA expression before and after transfection of a replicon expression vector and before and after addition of a drug. FIG. 2C is a diagram showing the results of analyzing the replicon replication-suppressing effect of drugs by reporter assay. FIG. 3 is a schematic diagram showing the method for producing the cell line of the present invention. FIG. 4A shows the results of reporter assay analysis of replicon expression in Huh7 cells and clone cells derived from Huh7.5.1 cells. FIG. 4B is a diagram showing the results of RT-PCR analysis of expression of replicon protein mRNA in clone cells derived from Huh7.5.1 cells. FIG. 4C is a diagram showing the results of analysis of replicon expression in clone cells derived from VeroE6 cells by reporter assay. FIG. 4D shows the results of RT-PCR analysis of replicon protein mRNA expression in clone cells derived from VeroE6 cells. FIG. 5A is a diagram showing the results of Western blot analysis of replicon protein expression before and after drug addition to Huh7.5.1/Rep#4 cell line. FIG. 5B is a diagram showing the results of Western blot analysis of replicon protein expression before and after drug addition to the VeroE6/Rep#3 cell line. FIG. 5C shows the results of Northern blot analysis of replicon RNA expression in the VeroE6/Rep#3 cell line. FIG. 6A shows the results of crystal violet staining of the VeroE6/Rep#3 cell line. FIG. 6B is a diagram showing the results of analysis of replicon expression in VeroE6/Rep#3 cell line by reporter assay. FIG. 7A is a diagram showing the results of Northern blot analysis of replicon RNA expression before and after drug addition to VeroE6/Rep#3 cell line. FIG. 7B is a diagram showing the results of analysis of replicon expression by reporter assay before and after drug addition to VeroE6/Rep#3 cell line. 8A shows the results of screening using compound library 1. FIG. FIG. 8B is a magnified view for the 75 compounds of FIG. 8A and EIDD-2801. 8C shows the results of screening using compound library 2. FIG. FIG. 8D is a magnified view for 41 compounds and EIDD-2801 in FIG. 8C.

(cell line)
Said cell line is the cell line with accession number NITE P-03458.
The cell line with the accession number NITE P-03458 is a cell line stably expressing coronavirus replicon RNA.
The cell line having the accession number NITE P-03458 can be prepared by the following cell line preparation method.

<Cell line with accession number NITE P-03458>
1) Receiving organization name: National Institute of Technology and Evaluation Patent Microorganism Depositary Center (〒292-0818) 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan
2) Receipt date: April 8, 2021 3) Receipt number: NITE AP-03458
4) Accession number: NITE P-03458

(Method for producing cell lines)
The method for producing the cell line includes a transfection step and a selection step, and may further include other steps.

<Transfection step>
The transfection step is a step of transfecting a cell with a vector having a nucleic acid sequence encoding a coronavirus nonstructural protein, a reporter protein including a drug resistance protein, and a coronavirus structural protein.

<<Coronavirus>>
The coronavirus is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include viruses belonging to the Coronaviridae family, such as the subfamily Retovirinae and the subfamily Orthocoronavirus.
Among these, viruses belonging to the subfamily Orthocoronavirus are preferred.

The virus belonging to the orthocoronavirus subfamily is not particularly limited and can be appropriately selected according to the purpose. viruses to which it belongs.
Among these, viruses belonging to the genus Betacoronavirus are preferred.

The virus belonging to the genus Betacoronavirus is not particularly limited and can be appropriately selected according to the purpose. Examples include viruses belonging to the subgenus Becovirus.
Among these, viruses belonging to the subgenus Sarbecovirus are preferred.

The virus belonging to the subgenus Sarbecovirus is not particularly limited and can be appropriately selected depending on the purpose. A virus (Severe acute respiratory syndrome coronavirus: SARS-CoV), a novel coronavirus (Severe acute respiratory syndrome coronavirus 2: SARS-CoV-2), and the like.
Among these, the novel coronavirus (SARS-CoV-2) is preferred.

<<Vector>>
The vector has nucleic acid sequences encoding coronavirus nonstructural proteins, reporter proteins, including drug resistance proteins, and coronavirus structural proteins, and can have other sequences.
Said vector can be used as a coronavirus replicon RNA expression vector.

From the viewpoint of ease of handling, the vector preferably does not contain all of the nonstructural proteins, structural proteins, and accessory proteins of the coronavirus, and more preferably does not contain all of the structural proteins of the coronavirus. .

The vector is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include DNA vectors and RNA vectors.
Among these, DNA vectors are preferred, and circular DNA vectors are more preferred.

The type of vector is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include artificial chromosomes, plasmids, cosmids, and lambda phage.
Among these, artificial chromosomes are preferred, and BACs (bacterial artificial chromosomes) are more preferred.

－Nonstructural protein of coronavirus－
The non-structural protein of the coronavirus is not particularly limited and can be appropriately selected according to the purpose. A protein is mentioned.
Among these, polymerase or replicase is preferred.

When the coronavirus is a novel coronavirus (SARS-CoV-2), the nonstructural proteins of the coronavirus are not particularly limited and can be appropriately selected depending on the purpose. Preferred, and more preferred is ORF1ab.

The nucleic acid sequence encoding ORF1ab is not particularly limited and can be appropriately selected depending on the purpose. /19-020 (GenBank ID: LC528232.1) 269th to 21558th nucleic acid sequence, a homologue of the nucleic acid sequence having the sequence of SEQ ID NO: 1, a fragment of the nucleic acid sequence having the sequence of SEQ ID NO: 1, etc. are mentioned.

The homologue of the nucleic acid sequence having the sequence of SEQ ID NO: 1 is not particularly limited and can be appropriately selected depending on the purpose. is preferred, 85% or more of the nucleic acid sequence is more preferred, 90% or more of the nucleic acid sequence is even more preferred, 95% or more of the nucleic acid sequence is particularly preferred, and 99% or more of the nucleic acid sequence is most preferred.

The fragment of the nucleic acid sequence having the sequence of SEQ ID NO: 1 is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include partial sequences of the nucleic acid sequence having the sequence of SEQ ID NO: 1.
The partial sequence of the nucleic acid sequence having the sequence of SEQ ID NO: 1 is not particularly limited and can be appropriately selected depending on the purpose. Sequences are preferred, with nucleic acid sequences of 60% or greater being more preferred, nucleic acid sequences of 65% or greater being even more preferred, nucleic acid sequences of 70% or greater being particularly preferred, and nucleic acid sequences of 75% or greater being most preferred.

- Reporter protein containing drug resistance protein -
The drug resistance protein is not particularly limited and can be appropriately selected depending on the purpose. Zeocin resistant protein and the like.
Among these, the G418 resistant protein is preferred.

The reporter protein containing the drug resistance protein preferably contains a luminescent protein or a fluorescent protein in addition to the drug resistance protein, and more preferably contains a luminescent protein.
A fusion protein of a drug-resistant protein and a luminescent protein or a fluorescent protein may also be used, and a fusion protein of a drug-resistant protein and a luminescent protein is more preferable.

The luminescent protein is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include luminescent enzymes such as Renilla luciferase and firefly luciferase.
The fluorescent protein is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include green fluorescent protein, yellow fluorescent protein, red fluorescent protein, and blue fluorescent protein.

The nucleic acid sequence encoding the fusion protein of the drug-resistant protein and the light-emitting protein is not particularly limited and can be appropriately selected according to the purpose. luciferase-neomycin resistant gene: Reo gene), a homologue of the nucleic acid sequence having the sequence of SEQ ID NO:2, a fragment of the nucleic acid sequence having the sequence of SEQ ID NO:2, and the like.

The homologue of the nucleic acid sequence having the sequence of SEQ ID NO: 2 is not particularly limited and can be appropriately selected depending on the purpose. is preferred, 85% or more of the nucleic acid sequence is more preferred, 90% or more of the nucleic acid sequence is even more preferred, 95% or more of the nucleic acid sequence is particularly preferred, and 99% or more of the nucleic acid sequence is most preferred.

The fragment of the nucleic acid sequence having the sequence of SEQ ID NO:2 is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include partial sequences of the nucleic acid sequence having the sequence of SEQ ID NO:2.
The partial sequence of the nucleic acid sequence having the sequence of SEQ ID NO: 2 is not particularly limited and can be appropriately selected depending on the purpose. Sequences are preferred, with nucleic acid sequences of 60% or greater being more preferred, nucleic acid sequences of 65% or greater being even more preferred, nucleic acid sequences of 70% or greater being particularly preferred, and nucleic acid sequences of 75% or greater being most preferred.

-Structural protein of coronavirus-
The structural protein of the coronavirus is not particularly limited and can be appropriately selected according to the purpose. Examples include proteins produced by viruses.
Among these, capsid proteins are preferred.

The vector preferably does not contain all of the structural proteins of the coronavirus from the viewpoint of ease of handling. That is, one that does not contain any of envelope protein, spike protein, or membrane protein is preferred, and one that does not contain envelope protein, spike protein, and membrane protein is more preferred.

When the coronavirus is a novel coronavirus (SARS-CoV-2), the structural protein of the coronavirus is not particularly limited and can be appropriately selected depending on the purpose. S protein or M protein is preferred, and N protein is more preferred.

The vector preferably does not contain all of the structural proteins of the coronavirus from the viewpoint of ease of handling. That is, one that does not contain any of E protein, S protein, or M protein is preferred, and one that does not contain E protein, S protein, and M protein is more preferred.

The nucleic acid sequence encoding the N protein is not particularly limited and can be appropriately selected depending on the purpose. Homologs of the nucleic acid sequence having the sequence of SEQ ID NO:3, fragments of the nucleic acid sequence having the sequence of SEQ ID NO:3, and the like are included.

The homologue of the nucleic acid sequence having the sequence of SEQ ID NO: 3 is not particularly limited and can be appropriately selected depending on the purpose. is preferred, 85% or more of the nucleic acid sequence is more preferred, 90% or more of the nucleic acid sequence is even more preferred, 95% or more of the nucleic acid sequence is particularly preferred, and 99% or more of the nucleic acid sequence is most preferred.

The fragment of the nucleic acid sequence having the sequence of SEQ ID NO:3 is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include partial sequences of the nucleic acid sequence having the sequence of SEQ ID NO:3.
The partial sequence of the nucleic acid sequence having the sequence of SEQ ID NO: 3 is not particularly limited and can be appropriately selected depending on the purpose. Sequences are preferred, with nucleic acid sequences of 60% or greater being more preferred, nucleic acid sequences of 65% or greater being even more preferred, nucleic acid sequences of 70% or greater being particularly preferred, and nucleic acid sequences of 75% or greater being most preferred.

-Other arrays-
The other sequences are not particularly limited and can be appropriately selected depending on the intended purpose. , regulatory sequences, restriction enzyme sequences, spacer sequences and the like.

The regulatory sequence is not particularly limited and can be appropriately selected according to the purpose, but preferably includes the sequence of ACGAAC (SEQ ID NO: 4), and the latter 129 bp of ACGAAC (SEQ ID NO: 4) and the ORF8 gene. (SEQ ID NO: 5), more preferably ACGAAC (SEQ ID NO: 4), the latter 129 bp of the ORF8 gene (SEQ ID NO: 5), and AAACTAAA (SEQ ID NO: 6).

<<Cells>>
The cells are not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include animal cells, insect cells, plant cells, yeast cells and the like.
Among these, animal cells are preferred.

The animal cells are not particularly limited and can be appropriately selected depending on the purpose. cells, mesenchymal stem cells, and the like.
Among these, VeroE6 cells, Huh7 cells, or Huh7.5.1 cells are preferred, VeroE6 cells or Huh7.5.1 cells are more preferred, and VeroE6 cells are even more preferred.

Commercially available animal cells such as HEK293T (ATCC #CRL-11268) and VeroE6 (ATCC #CRL-1586) can be used.

<<Transfection>>
The transfection method is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include lipofection, calcium phosphate, electroporation, and microinjection.
Among these, the lipofection method is preferred.

<Selection process>
The selection step is a step of selecting the cells obtained in the transfection step using a drug.

The selection method is not particularly limited and can be appropriately selected according to the purpose. For example, a method of culturing cells by adding a drug to the culture medium of the cells obtained in the transfection step. mentioned.
When the nucleic acid sequence encoding a reporter protein containing a drug resistance protein contained in the vector is a nucleic acid sequence encoding a reporter protein containing a G418 resistance protein, in the culture medium of the cells obtained in the transfection step , G418 can be added.

The selection period is not particularly limited and can be appropriately selected according to the purpose, but is preferably 1 week or longer, more preferably 2 weeks or longer, further preferably 3 weeks or longer, and particularly preferably 4 weeks or longer. .

<Other processes>
The other steps are not particularly limited and can be appropriately selected according to the purpose. For example, the culture step after the selection step, the cloning step after the selection step, the cell screening step after the selection step, etc. is mentioned.

<<Culturing process after selection process>>
The culture step after the selection step is not particularly limited and can be appropriately selected according to the purpose. For example, in cells cultured by adding the drug, the drug is removed from the culture medium A step of culturing cells and the like can be mentioned.

The period of the culturing step after the selection step is not particularly limited and can be appropriately selected according to the purpose, but is preferably one week or longer, more preferably two weeks or longer.

<<Cloning step after selection step>>
The cloning step after the selection step is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include a step of cloning cells derived from single cells by limiting dilution method.

<<Cell screening step after selection step>>
The cell screening step after the selection step is not particularly limited and can be appropriately selected according to the purpose. A step of screening the cells using the expression level of a nucleic acid sequence encoding a coronavirus non-structural protein or a nucleic acid sequence encoding a coronavirus structural protein as an indicator.
When the reporter protein contains a luminescent protein or fluorescent protein, the cells can be screened using the luminescence or fluorescence as an index.

(introduced cells)
The transfected cells are cells into which the vector has been introduced.
Said vector is as described above.

The introduced cells are not particularly limited and can be appropriately selected depending on the intended purpose. Cells expressing coronavirus replicon RNA are preferable, and cells expressing coronavirus replicon RNA and structural proteins of coronavirus are preferred. Cells are more preferred.

The introduced cells can be prepared by the method for preparing the cell line.

An example of the transfected cell is the cell line with the accession number NITE P-03458, which expresses the replicon RNA of the coronavirus and the structural proteins of the coronavirus.

(Screening method for anti-coronavirus therapeutic agent)
The screening method for the therapeutic agent for coronavirus is a screening method using the introduced cells.
The transfected cells are as described above.

The screening method for the therapeutic drug for coronavirus is not particularly limited and can be appropriately selected according to the purpose. The expression level of the non-structural protein of the coronavirus or the structural protein of the coronavirus in the cell, or the expression level of the nucleic acid sequence encoding the non-structural protein of the coronavirus or the nucleic acid sequence encoding the structural protein of the coronavirus can be used as an index to screen the therapeutic drug for coronavirus.
When the reporter protein contains a luminescent protein or fluorescent protein, the therapeutic agent for coronavirus can be screened using the luminescence or fluorescence as an indicator.
In screening using the expression level of the protein or nucleic acid sequence, the luminescence, fluorescence, or the like as an index, the efficacy rate of the compound can be calculated by standardizing with the cell viability of the introduced cells.

(Anti-coronavirus drug screening kit)
The anti-coronavirus therapeutic drug screening kit contains the introduced cells and can further contain other components.
The transfected cells are as described above.
The other components are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include protocols, instructions, culture media, media additives and the like.

Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Example 1 Preparation of replicon RNA expression vector pBAC-SCoV2-Rep-Reo)
The full length of the early expression vector is divided into 10 fragments shown below (fragment 1 to fragment 10), each fragment is amplified by the PCR method, and these are subjected to the Circular Polymerase Extension Reaction method (Journal of Virology, 2013 87: 2367 -2372), introduced into Escherichia coli competent cells (BAC-Optimized Replicator v2.0 Electrocompetent Cells: manufactured by Lucigen), and cloned.

The fragment 1 (SEQ ID NO: 7) is part of the ORF1ab gene (389th to 1661st sequence of SEQ ID NO: 1), that is, the 657th to 1929th sequence of SARS-CoV-2 (GenBank ID: LC528232.1) including.
The fragment 2 (SEQ ID NO: 8) is part of the ORF1ab gene (1632 to 4054 sequences of SEQ ID NO: 1), that is, the 1900 to 4322 sequences of SARS-CoV-2 (GenBank ID: LC528232.1) including.
The fragment 3 (SEQ ID NO: 9) is part of the ORF1ab gene (4025 to 6494 sequences of SEQ ID NO: 1), that is, the 4293 to 6762 sequences of SARS-CoV-2 (GenBank ID: LC528232.1) including.
The fragment 4 (SEQ ID NO: 10) is part of the ORF1ab gene (sequences from 6465 to 8595 of SEQ ID NO: 1), that is, the sequence from 6733 to 8863 of SARS-CoV-2 (GenBank ID: LC528232.1) including.
The fragment 5 (SEQ ID NO: 11) is part of the ORF1ab gene (8566 to 10829 sequences of SEQ ID NO: 1), that is, the 8834 to 11097 sequences of SARS-CoV-2 (GenBank ID: LC528232.1) including.
The fragment 6 (SEQ ID NO: 12) is part of the ORF1ab gene (10800 to 13601 sequences of SEQ ID NO: 1), that is, the 11068 to 13869 sequences of SARS-CoV-2 (GenBank ID: LC528232.1) including.
The fragment 7 (SEQ ID NO: 13) is part of the ORF1ab gene (13569 to 17084 sequences of SEQ ID NO: 1), that is, the 13837 to 17352 sequences of SARS-CoV-2 (GenBank ID: LC528232.1) including.
The fragment 8 (SEQ ID NO: 14) is a part of the ORF1ab gene (17053 to 21290 sequences of SEQ ID NO: 1, that is, the 17307 to 21558 sequences of SARS-CoV-2 (GenBank ID: LC528232.1), and artificial spacer sequences.
The fragment 9 (SEQ ID NO: 15) is an artificial spacer sequence, part of the ORF8 gene (SEQ ID NO: 5), that is, the sequence from 28134 to 28262 of SARS-CoV-2 (GenBank ID: LC528232.1), SEQ ID NO: 4, SEQ ID NO: 6, and the Reo gene (SEQ ID NO: 2).
The fragment 10 (SEQ ID NO: 16) is part of the ORF8 gene (SEQ ID NO: 5), namely the sequence from 28134 to 28262 of SARS-CoV-2 (GenBank ID: LC528232.1), the sequence of SEQ ID NO: 4, The sequence of SEQ ID NO: 6, the sequence from the N gene (SEQ ID NO: 3) to the 3'UTR, that is, the sequence from 28277 to 29873 of SARS-CoV-2 (GenBank ID: LC528232.1), from the poly (A) sequence The sequence up to the transcription termination signal (BGH), the pSMART BAC 2.0 vector full-length sequence, the CMV promoter sequence, and a portion of the ORF1ab gene from the 5'UTR (sequences 1 to 418 of SEQ ID NO: 1), that is, SARS- Contains sequences 1 to 686 of CoV-2 (GenBank ID: LC528232.1).

The fragment 1 was amplified by PCR using primer 1 (SEQ ID NO: 17 forward primer) and primer 2 (SEQ ID NO: 18 reverse primer) using SARS-CoV-2 genomic cDNA as a template.

The fragment 2 was amplified by PCR using primer 3 (SEQ ID NO: 19 forward primer) and primer 4 (SEQ ID NO: 20 reverse primer) using SARS-CoV-2 genomic cDNA as a template.

The fragment 3 was amplified by PCR using primer 5 (SEQ ID NO: 21 forward primer) and primer 6 (SEQ ID NO: 22 reverse primer) using SARS-CoV-2 genomic cDNA as a template.

The fragment 4 was amplified by PCR using primer 7 (SEQ ID NO: 23 forward primer) and primer 8 (SEQ ID NO: 24 reverse primer) using SARS-CoV-2 genomic cDNA as a template.

The fragment 5 was amplified by PCR using primer 9 (SEQ ID NO: 25 forward primer) and primer 10 (SEQ ID NO: 26 reverse primer) using SARS-CoV-2 genomic cDNA as a template.

The fragment 6 was amplified by PCR using primer 11 (SEQ ID NO: 27 forward primer) and primer 12 (SEQ ID NO: 28 reverse primer) using SARS-CoV-2 genomic cDNA as a template.

The fragment 7 was amplified by PCR using primer 13 (SEQ ID NO: 29 forward primer) and primer 14 (SEQ ID NO: 30 reverse primer) using SARS-CoV-2 genomic cDNA as a template.

The fragment 8 was amplified by PCR using primer 15 (SEQ ID NO: 31 forward primer) and primer 16 (SEQ ID NO: 32 reverse primer) using SARS-CoV-2 genomic cDNA as a template.

The fragment 9 was amplified by PCR using the Reo gene subcloning vector as a template and primer 17 (SEQ ID NO: 33 forward primer) and primer 18 (SEQ ID NO: 34 reverse primer).

The fragment 10 uses pSMART BAC 2.0, SARS-CoV-2 genomic cDNA, poly (A) sequence (pA), Ribozyme sequence (Rz), and an artificial gene containing a transcription termination signal (BGH) as a template, Amplification was performed by PCR using primer 19 (SEQ ID NO: 35 forward primer) and primer 20 (SEQ ID NO: 36 reverse primer).

The SARS-CoV-2 genomic cDNA was provided by Professor Toru Okamoto, Institute for Advanced Co-Creation Research, Osaka University.
As the Reo gene subcloning vector, pGL4.72 reporter vector (manufactured by Promega Corporation, #E6901) and pcDNA3.1 cloning vector (manufactured by Invitrogen, #V79020) were used and synthesized by PCR.
The pSMART BAC 2.0 was purchased from Lucigen (BAC-Optimized Replicator v2.0).
The poly(A) sequence (pA) was synthesized by Oligo DNA Synthesis Service (Fasmac).
The Ribozyme sequence (Rz) was synthesized by Oligo DNA Synthesis Service (Fasmac).
A pcDNA3.1 cloning vector (manufactured by Invitrogen, #V79020) was used as an artificial gene containing the transcription termination signal (BGH).

The PCR was performed using PrimeSTAR GXL DNA Polymerase (#R050 manufactured by Takara Bio Inc.) at an annealing temperature of 60° C. for 30 cycles.

This produced the construct (pBAC-SCoV2-Rep-Reo) shown in FIG. 1A (SEQ ID NO: 37).

As shown in FIG. 1A, pBAC-SCoV2-Rep-Reo contains a DNA cloning vector (pSMART BAC 2.0; black line) with a promoter (CMV), novel coronavirus replicon (SCoV-Rep-Reo), transcription termination A signal (BGH) is inserted.

FIG. 1B shows details of the portion corresponding to CMV to BGH in FIG. 1A.
5'-UTR (5'), ORF1ab, N protein (N), and 3'-UTR (3') are regions necessary for genome replication derived from novel coronavirus.

“ORF8 (129 bp) ACGAAC AAACTAAA” upstream of the Reo gene in FIG. 1B is a nucleic acid sequence derived from the SARS-CoV2 genome and is a regulatory sequence necessary for expressing the Reo gene.
The ORF8 (129 bp) is the latter 129 bp of the ORF8 gene, and the ACGAAC is a transcription regulation sequence (TRS). "CACTGGCGCGCC" is an artificial spacer sequence containing a confirming restriction enzyme sequence.

Downstream of the Reo gene in FIG. 1B, “ORF8 (129 bp) ACGAAC AAACTAAA” is a nucleic acid sequence derived from the SARS-CoV2 genome, and is a regulatory sequence required to express the N gene.
The ORF8 (129 bp) is the latter 129 bp of the ORF8 gene, and the ACGAAC is a transcription regulation sequence (TRS). "GGATCC" is a restriction enzyme sequence inserted for confirmation.

(Example 2 Introduction of replicon RNA expression vector pBAC-SCoV2-Rep-Reo)
The pBAC-SCoV2-Rep-Reo was transfected into cultured cells (HEK293T cells, Huh7 cells, or VeroE6 cells) by the lipofection method as follows, a cell lysate was prepared, and N protein, nsp8, and actin were transfected. Expression levels were analyzed by Western blotting (Fig. 2A).

The anti-N protein antibody is an antibody provided by Professor Toru Okamoto, Institute for Advanced Co-Creation Research, Osaka University, and the anti-nsp8 antibody is Anti-SARS-CoV/SARS-CoV-2 (COVID-19) NSP8 (#GTX632696) (GeneTex) (manufactured by Sigma-Aldrich), and Monoclonal Anti-β-Actin antibody (#A5441) (manufactured by Sigma-Aldrich) was used as an anti-actin antibody.
Lane 1 of FIG. 2A is a sample of cells with no added drug, lane 2 is a sample of cells with interferon added, and lane 3 is a sample of cells with remdesivir added.

In addition, HEK293T cells were transfected with the pBAC-SCoV2-Rep-Reo by the lipofection method as follows to prepare RNA, subgenomic RNA1 (pp1a, pp1ab), subgenomic RNA2 (reporter gene) and subgenomic RNA3 (N gene) was analyzed by Northern blotting (Fig. 2B).

Subgenomic RNAs 1-3 were detected using a common RNA probe complementary to the N gene.
The progenitor RNA probe was synthesized using the DNA sequence of SEQ ID NO:38 as a template for RNA probe synthesis.
In FIG. 2B, "-" indicates a sample of cells with no drug added, "IFN" indicates a sample of cells with interferon added, and "Rem" indicates a sample of cells with remdesivir.

Furthermore, the pBAC-SCoV2-Rep-Reo was transfected into cultured cells (HEK293T cells, Huh7 cells, or VeroE6 cells) by the lipofection method as follows, and the effect of suppressing replicon replication was analyzed by reporter assay (Fig. 2C).
Statistical significance was evaluated by Student's t-test. "n.s" indicates no significant difference.

As shown in FIG. 2C, it was confirmed that Renilla luciferase activity was reduced by administering interferon or remdesivir, which have been reported to inhibit replication of the novel coronavirus. From this, it was confirmed that pBAC-SCoV2-Rep-Reo can monitor the intracellular replicon RNA replication level by a simple reporter assay (Renilla luciferase assay).

-Transfection-
The HEK293T and Huh7 cells were provided by Professor Yoshiharu Matsuura, Research Institute for Microbial Diseases, Osaka University. The VeroE6 cells were obtained from the American Type Culture Collection (ATCC #CRL-1586).

The cultured cells were seeded in multi-well plates such as 24-well or 6-well and cultured overnight at 37°C in the presence of 5% _CO2 .
After about 16 hours, pBAC-SCoV2-Rep-Reo and TransIT-LT1 (manufactured by Mirus Bio) in an amount three times the amount of DNA were mixed and allowed to stand at room temperature for 30 minutes.
The mixture was added to the cultured cells with medium exchange and cultured at 37° C. in the presence of 5% CO ₂ for 1 or 2 days.

-Preparation of cell lysate-
One or two days after transfection, the cultured cells on the culture plate were washed with PBS, and a cell lysis solution (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet-P40, 0.5% deoxy Sodium cholate, 0.1% SDS, complete protease inhibitor cocktail (manufactured by Roche)) was added, and the cells transiently transfected with the replicon RNA expression vector and the untransfected cells were scraped with a scraper. The cells were detached, treated on ice for 20 minutes, and the supernatant was recovered by centrifugation to obtain a cell lysate.

The medium was changed 6 hours after transfection, and interferon (100 U/mL, PeproTech) or remdesivir (5 μg/mL, Cayman Chemical) was added for 1 or 2 days, and cells were not transfected. Cells and cells to which interferon (100 U/mL, manufactured by PeproTech) or remdesivir (5 μg/mL, manufactured by Cayman Chemical) were added for 1 or 2 days to cells and untransfected cells. got

- Preparation of RNA -
One or two days after transfection, the cultured cells on the culture plate were washed with PBS, 1 mL of TRIzol (manufactured by Invitrogen) was added, and the cells into which the replicon RNA expression vector was transiently introduced were lysed, RNA was purified to obtain RNA.

Six hours after transfection, cells added with interferon (100 U/mL, PeproTech) or remdesivir (5 μg/mL, Cayman Chemical) for 1 or 2 days, non-transfected cells, and transfected cells. RNA was similarly obtained from cells to which interferon (100 U/mL, PeproTech) or remdesivir (5 μg/mL, Cayman Chemical) was added for 2 days to uninfected cells.

-Reporter Assay-
One or two days after transfection, the cultured cells in the culture dish were washed with PBS, diluted Renilla Luciferase Assay Lysis Buffer (manufactured by Promega Corporation) was added, the precultured cells were detached with a scraper, and the precultured cells were scraped off at room temperature for 15 minutes. The cells were treated for minutes, and the supernatant was recovered by centrifugation to obtain a cell lysate.
The luciferase activity of the cell lysate was measured using Renilla Luciferase Assay System (manufactured by Promega Corporation, #E2810).

Six hours after transfection, cells added with interferon (100 U/mL, PeproTech) or remdesivir (5 μg/mL, Cayman Chemical) for 1 or 2 days, non-transfected cells, and transfected cells. A reporter assay was also performed on cells to which interferon (100 U/mL, PeproTech) or remdesivir (5 μg/mL, Cayman Chemical) was added for 2 days to uninfected cells.

(Example 3 Preparation of replicon RNA-expressing cell line)
A replicon RNA-expressing cell line was generated according to the steps shown in FIG.
In the same manner as in Example 2, cultured cells (Huh7 cells, Huh7.5.1 cells, or VeroE6 cells) were transfected with the pBAC-SCoV2-Rep-Reo by the lipofection method, and then the cultured cells were treated with G418 (neomycin (# 09380), Nacalai Tesque Co., Ltd.) containing medium (1 mg/mL).
The Huh7.5.1 cells were a gift from Professor Ralf Bartenschlager, University of Heidelberg.
The G418 kills cultured cells that do not have resistance, but cultured cells expressing a reporter gene (Reo gene) derived from pBAC-SCoV2-Rep-Reo acquire resistance to G418. It is possible to grow inside.

After culturing in the G418-containing medium, G418-resistant cell colonies were picked up to obtain cell lines (clone cells).

Figure 4A shows reporter assays (Renilla luciferase assay) results are shown. The reporter assay was performed in the same manner as in Example 2.
Several Huh7.5.1/Rep cell lines showed high luciferase activity, including clone #4 (Huh7.5.1/Rep#4 cells) derived from Huh7.5.1 cells.

FIG. 4B shows the replicon RNA (N protein) in Huh7.5.1 cell-derived cell lines (clone #1-4, 6-8, 12-14) and, as a control, RT-positive for GAPDH in host cells. Analysis results by PCR method are shown. RNA was prepared using RNeasy mini kit (manufactured by Qiagen).
Sustained expression of replicon RNA was confirmed in Huh7.5.1 cell-derived clone #4 (Huh7.5.1/Rep#4 cells).

FIG. 4C shows the results of the reporter assay (Renilla luciferase assay) in VeroE6 cell-derived cell lines (clone #1-14). The reporter assay was performed in the same manner as in Example 2.
Several VeroE6/Rep cell lines showed high luciferase activity, including clone #3 derived from VeroE6 cells (VeroE6/Rep#3 cells: cell line with accession number NITE P-03458).

FIG. 4D shows the analysis results of replicon RNA (N protein) in VeroE6 cell-derived cell lines (clone #1 to 4, 6, 9, 11) and GAPDH of host cells as a control by RT-PCR method. Indicated. RNA was prepared using RNeasy mini kit (manufactured by Qiagen).
In VeroE6 cell-derived clone #3 (VeroE6/Rep#3 cells: cell line with accession number NITE P-03458), sustained expression of replicon RNA was confirmed.

As a result, clone #4 derived from Huh7.5.1 cells (Huh7.5.1/Rep#4 cells) and clone #3 derived from VeroE6 cells (VeroE6/Rep#3 cells: accession number NITE P-03458 It was confirmed that Renilla luciferase and replicon RNA were expressed in a cell line).

Primer 21 (SEQ ID NO: 39 forward primer) and primer 22 (SEQ ID NO: 40 reverse primer) were used for RT-PCR of mRNA encoding N protein.

Primer 23 (SEQ ID NO: 41 forward primer) and primer 24 (SEQ ID NO: 42 reverse primer) were used for RT-PCR of mRNA encoding GAPDH.

(Example 4 Expression of replicon proteins in cell lines)
A cell lysate of Huh7.5.1/Rep#4 cell line and a cell lysate of Huh7.5.1/Rep#4 cell line supplemented with interferon or remdesivir were prepared in the same manner as in Example 2. , N protein, nsp8, and actin expression levels were analyzed by Western blotting (Fig. 5A).
As a control for N protein expression, Huh7.5.1 cells transfected with pBAC-SCoV2-Rep-Reo were similarly analyzed (Fig. 5A).

Lane P of FIG. 5A is a sample of Huh7.5.1 cells transfected with pBAC-SCoV2-Rep-Reo, lane N is a sample of Huh7.5.1 cells, and lane 1 is Huh7.5.1. Lane 2 is a sample of Huh7.5.1/Rep#4 cell line with interferon added, Lane 3 is a sample of Huh7.5.1/Rep#4 with remdesivir added. Cell line samples.

As shown in FIG. 5A, in Huh7.5.1/Rep#4, expression of N protein could not be confirmed.

Cell lysate of VeroE6/Rep#3 cell line and cell lysate of VeroE6/Rep#3 cell line supplemented with interferon or remdesivir were prepared in the same manner as in Example 2, and N protein, nsp8, and actin was analyzed by Western blotting (Fig. 5B).
As a control for N protein expression, VeroE6 cells transfected with pBAC-SCoV2-Rep-Reo were similarly analyzed (Fig. 5B).

Lane P in FIG. 5B is a sample of VeroE6 cells transfected with pBAC-SCoV2-Rep-Reo, lane N is a sample of VeroE6 cells, lane 1 is a sample of VeroE6/Rep#3 cell lines, lane Lane 2 is a sample of VeroE6/Rep#3 cell line with interferon and Lane 3 is a sample of VeroE6/Rep#3 cell line with remdesivir.

As shown in FIG. 5B, N protein was expressed in the VeroE6/Rep#3 cell line, and the addition of interferon or remdesivir decreased the expression of N protein.

In addition, in the same manner as in Example 2, RNA was prepared from VeroE6 cells and VeroE6/Rep#3 cell lines, and subgenomic RNA1 (pp1a, pp1ab), subgenomic RNA2 (reporter gene) and subgenomic RNA3 (N gene) were expressed. Quantities were analyzed by Northern blotting.
As controls for the amount of RNA, 28S ribosomal RNA and 18S ribosomal RNA were analyzed by ethidium bromide staining (#E8751, Sigma-Aldrich) (Fig. 5C).

These findings suggested that replication of the novel coronavirus replicon occurred in the VeroE6/Rep#3 cell line.

(Example 5 Stability of VeroE6/Rep#3 cell line)
Persistence of replicon expression in the VeroE6/Rep#3 cell line was confirmed.
VeroE6 cells or VeroE6/Rep#3 cell lines were cultured in the absence of G418 for 2 weeks, seeded in a 10 cm dish, and cultured in a G418-containing medium for 2 weeks.
Surviving cells were stained blue with crystal violet staining (031-04852, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). VeroE6 cells died (Fig. 6A left), whereas the VeroE6/Rep#3 cell line was resistant to G418 (Fig. 6A right).

In addition, Renilla luciferase When assayed, VeroE6/Rep#3 cultured for 4 weeks or more in the presence of G418 maintained high luciferase activity (Fig. 6B middle).
VeroE6/Rep#3 cells cultured for two weeks in the absence of G418 (Fig. 6B, right) also maintained high activity compared to VeroE6 cells (Fig. 6B, left).

These results confirmed that VeroE6/Rep#3 replicon expression was stably maintained for at least 2 weeks in the absence of G418 and at least 4 weeks in the presence of G418.

(Example 6 Suitability evaluation of VeroE6/Rep#3 cell line)
The suitability of the VeroE6/Rep#3 cell line for drug screening (monitoring the level of replication of the novel coronavirus replicon) was confirmed.
As in Examples 2 and 4, VeroE6/Rep#3 cell line, interferon (100 U/mL, PeproTech) for 3 days, or remdesivir (5 μg/mL, Cayman Chemical) for 3 days VeroE6/ RNA was prepared from the Rep#3 cell line, and the expression levels of subgenomic RNA1 (pp1a, pp1ab), subgenomic RNA2 (reporter gene) and subgenomic RNA3 (N gene) were analyzed by Northern blotting.
As controls for the amount of RNA, 28S ribosomal RNA and 18S ribosomal RNA were analyzed by ethidium bromide staining (#E8751, Sigma-Aldrich) (Fig. 7A).

As shown in FIG. 7A, treatment of VeroE6/Rep#3 cell lines with interferon or remdesivir, which have been confirmed to inhibit replication of novel coronavirus, decreased intracellular replicon RNA.

In addition, in the same manner as in Example 2, VeroE6/Rep#3 cell line, interferon (100 U/mL, manufactured by PeproTech) for 3 days, or remdesivir (5 μg/mL, manufactured by Cayman Chemical) for 3 days was added to VeroE6/ Reporter assay was performed using Rep#3 cell line to investigate whether it is possible to monitor the replication level, and a decrease in Renilla luciferase activity was observed in VeroE6/Rep#3 cell line administered with interferon or remdesivir ( FIG. 7B center and right).
Statistical significance was evaluated by Student's t-test.

A strong decrease in Renilla luciferase activity was observed with interferon administration, and a mild decrease was observed with remdesivir. 2A, C).
In addition, the VeroE6/Rep#3 cell line was able to evaluate drug sensitivity with high sensitivity compared to VeroE6 cells with respect to the mild decrease in Renilla luciferase activity by remdesivir (FIG. 7B p<0.05).

Based on these facts, the replicon system using the VeroE6/Rep#3 cell line has screening aptitude similar to that of the conventional replicon system (method of transiently transfecting cells with a replicon expression vector). It was suggested that screening results with higher sensitivity and higher reproducibility can be obtained than with the replicon system (a method of transiently transfecting cells with a replicon expression vector).

(Example 7 Screening of anti-coronavirus therapeutic agents)
-Example 7-1: Evaluation of intracellular replicon replication level-
VeroE6/Rep#3 cells were seeded in 96-well plates at 6×10 ³ cells per well and cultured overnight for cell engraftment on the plates. The next day, remove the culture medium of the 96-well plate, compound library 1 (Protein-Protein Interaction Inhibitor Library (manufactured by Selleck): library containing 177 compounds such as inhibitors) and compound library 2 (Diversity Set ( Enamine): a library containing 493 compounds with various structures), a culture solution mixed with each compound (each compound at a final concentration of 10 μM and DMSO at a final concentration of 0.1% containing) and cultured for 72 hours at 37°C in the presence of 5% _CO2 .
After that, in order to evaluate the intracellular replicon replication level, luciferase assay was performed using Renilla Luciferase Assay System (manufactured by Promega Corporation, #E2810). Specifically, the cell culture medium mixed with each compound was removed, 20 μL of diluted Renilla Luciferase Assay Lysis Buffer (manufactured by Promega Corp.) was added per well, and the cells were incubated at room temperature for 30 minutes. Thereafter, 100 μL of diluted Renilla Luciferase Assay Substrate (manufactured by Promega Corporation) was added per well, and luciferase activity was measured using a GloMax (registered trademark) microplate luminometer (manufactured by Promega Corporation). Each compound was evaluated in n=4 wells and the average value was calculated. In addition, the compound whose luciferase activity decreased at the final concentration of 10 μM was evaluated by lowering the final concentration of the compound.

-Example 7-2: Evaluation of cell viability-
In the same manner as in Example 7-1, VeroE6/Rep#3 cells were cultured in a culture solution in which each compound of compound library 1 and compound library 2 was mixed.
After that, in order to evaluate cell viability after treatment with each compound, MTS assay was performed using CellTiter 96 (registered trademark) AQueous One Solution (manufactured by Promega Corporation). Specifically, the cell culture medium mixed with each of the above compounds was removed and replaced with a culture medium mixed with an MTS reagent (CellTiter 96 (registered trademark) AQueous One Solution (manufactured by Promega Corporation)), followed by 5% CO _{2 .} Incubated for 2 hours at 37°C in the presence. Then, using a microplate reader (manufactured by Bio-rad), absorbance at a wavelength of 490 nm was measured. Each compound was evaluated in n=4 wells and the average value was calculated. In addition, the final concentration of the compound was lowered and evaluated about the compound which the reduction of the cell survival rate was recognized in the final concentration of 10 micromol.

Instead of each of the above compounds, the coronavirus replication inhibitor EIDD-2801 (molnuplavir, manufactured by Medchem Express, HY-135853) at a final concentration of 20 μM as a positive control and DMSO at a final concentration of 0.1%, and a final concentration as a negative control A culture solution mixed with 0.1% DMSO was prepared, and luciferase assay and MTS assay were performed in the same manner as in Examples 7-1 and 7-2. Positive and negative controls were evaluated in n=4 wells and the average value was calculated.
Based on the luciferase activity and cell viability of the negative control group, the replication inhibition rate and cell viability of each compound-treated group were calculated.

ＥＩＤＤ－２８０１の効果率は約０．２だった。
ＶｅｒｏＥ６／Ｒｅｐ＃３を用いたスクリーニングにおける効果率のカットオフ値を０．５とした場合、図８Ａに示したとおり、上記の化合物ライブラリー１では、１７７化合物中７５化合物が該当し（効果率が０．５未満）、うち５５化合物がＥＩＤＤ－２８０１より低い効果率を示した。図８Ｂに、図８Ａの７５化合物及びＥＩＤＤ－２８０１についての拡大図を示した。また、図８Ｃに示したとおり、化合物ライブラリー２では、４９３化合物中４１化合物が該当し（効果率が０．５未満）、うち２化合物がＥＩＤＤ－２８０１より低い効果率を示した。図８Ｄに、図８Ｃの４１化合物及びＥＩＤＤ－２８０１についての拡大図を示した。 Compared to the DMSO-treated negative control group, the EIDD-2801-treated positive control group showed a significant decrease in luciferase activity, which is an indicator of the level of replicon replication, but no decrease in cell proliferation. This suggests that EIDD-2801 has an inhibitory effect on replicon replication.
In contrast, groups treated with library compounds included those with significant reductions in luciferase activity and cell proliferation levels. In order to distinguish between compounds that reduce luciferase activity due to non-specific effects due to cytotoxicity and compounds that significantly inhibit replicon replication, as shown in Equation 1 below, the replicon replication inhibition rate calculated from the luciferase activity is It was decided to calculate the "efficacy rate" of the compound, normalized by the survival rate.

The efficacy rate of EIDD-2801 was about 0.2.
When the cutoff value of the efficacy rate in screening using VeroE6/Rep#3 is set to 0.5, as shown in FIG. is less than 0.5), of which 55 compounds showed a lower efficacy rate than EIDD-2801. FIG. 8B shows a magnified view for the 75 compounds in FIG. 8A and EIDD-2801. In addition, as shown in FIG. 8C, in compound library 2, 41 compounds out of 493 compounds corresponded (efficacy rate of less than 0.5), of which 2 compounds showed a lower efficacy rate than EIDD-2801. FIG. 8D shows a magnified view for 41 compounds in FIG. 8C and EIDD-2801.

Embodiments of the present invention include, for example, the following.
<1> A cell line with accession number NITE P-03458.
<2> a transfection step of transfecting a cell with a vector having a nucleic acid sequence encoding a coronavirus nonstructural protein, a reporter protein containing a drug resistance protein, and a coronavirus structural protein; A method for producing a cell line, comprising a selection step of selecting the cells obtained in the step using a drug.
<3> The method for producing a cell line according to <2>, wherein the vector does not contain all structural proteins of the coronavirus.
<4> The method for producing a cell line according to <2> or <3>, wherein the reporter protein is a fusion protein of a drug-resistant protein and a luminescent protein or a fluorescent protein.
<5> The method for producing a cell line according to any one of <2> to <4>, wherein the vector is an artificial chromosome vector.
<6> The method for producing a cell line according to any one of <2> to <5>, wherein the coronavirus is SARS-CoV-2.
<7> The method for producing a cell line according to <6>, wherein the structural protein of the coronavirus comprises the N structural protein of SARS-CoV-2.
<8> The method for producing a cell line according to any one of <2> to <7>, wherein the cells are VeroE6 cells.
<9> A vector characterized by having a nucleic acid sequence encoding a coronavirus nonstructural protein, a reporter protein containing a drug resistance protein, and a coronavirus structural protein.
<10> An introduced cell characterized by introducing the vector according to <9> above.
<11> A screening method for an anti-coronavirus therapeutic agent, comprising using the introduced cells according to <10> above.
<12> An anti-coronavirus therapeutic drug screening kit comprising the introduced cells according to <10> above.

Claims (12)

A cell line with accession number NITE P-03458. a transfection step of transfecting a cell with a vector having a nucleic acid sequence encoding a coronavirus nonstructural protein, a reporter protein including a drug resistance protein, and a coronavirus structural protein;
A method for producing a cell line, comprising a selection step of selecting the cells obtained in the transfection step using a drug. 3. The method of generating a cell line of claim 2, wherein said vector does not contain all of the structural proteins of said coronavirus. 4. The method of producing a cell line according to claim 2 or 3, wherein the reporter protein is a fusion protein of a drug resistance protein and a luminescent protein or fluorescent protein. The method for producing a cell line according to claim 2, wherein the vector is an artificial chromosome vector. 3. The method of generating a cell line according to claim 2, wherein said coronavirus is SARS-CoV-2. 7. The method of generating a cell line of claim 6, wherein the structural protein of the coronavirus comprises the N structural protein of SARS-CoV-2. 3. The method of generating a cell line according to claim 2, wherein said cells are VeroE6 cells. A vector comprising a nucleic acid sequence encoding a coronavirus nonstructural protein, a reporter protein including a drug resistance protein, and a coronavirus structural protein. An introduced cell into which the vector according to claim 9 has been introduced. A screening method for an anti-coronavirus therapeutic drug, which comprises using the introduced cell according to claim 10 . 11. An anti-coronavirus therapeutic drug screening kit, comprising the introduced cells according to claim 10.

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