Replicon DNA, cloning vector, method for manufacturing cloning vector, screening kit and screening method for COVID-19 therapeutic drugs

Abstract

This replicon DNA includes: a CMV promoter sequence that functions in vertebrate cells; a non-structural region that is positioned on the 3' side from the CMV promoter sequence and includes non-structural protein genes 1a and 1b from SARS-CoV-2; and the N gene from SARS-CoV-2, which is positioned on the 3' side from the non-structural region.

Description

Technical Field

[0001] The present invention relates to replicon DNA, a cloning vector, a method for producing a cloning vector, COVID-19 treatment drugs a screening kit, and a screening method.

Background Art

[0002] Coronavirus disease 2019 (COVID-19) caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes SARS (severe acute respiratory syndrome), has become a pandemic worldwide. COVID-19 is not only a medical problem but also has a great impact on the economy and lifestyle, and thus has become an urgent problem to be overcome.

[0003] For assay systems for the development of COVID-19 therapeutic agents, wild-type SARS-CoV-2, infectious clones of SARS-CoV-2, and replicons of SARS-CoV-2 are used. Infectious clones are highly infectious artificial viruses produced by reverse genetics using wild-type SARS-CoV-2 as a template. When using wild-type SARS-CoV-2 and infectious clones, handling in facilities with a biosafety level (BSL) of 3 or higher is required.

[0004] Since the replicon of a virus maintains the non-structural region and can thus self-propagate, while lacking the structural region and losing infectivity, the replicon of SARS-CoV-2 can be obtained by replacing the structural region in the genome of SARS-CoV-2 with a reporter gene or the like. Since a replicon lacking infectivity can be handled even in a facility with a BSL of 2, the use of replicons enables many researchers to participate in the development of COVID-19 therapeutic agents.

[0005] Non-patent documents 1 and 2 disclose replicon RNA having the non-structural region of SARS-CoV-2 and the N gene necessary for replication. In this replicon RNA, genes other than the N gene in the structural region are replaced with reporter genes, etc. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Hongjie Xia, 8 others, “Evasion of Type I Interferon by SARS-CoV-2”, Cell Reports, 2020, 33, 108234 [Non-Patent Document 2] Yang Zhang and 4 others, “A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2”, Antiviral Research, 2021, 185, 104974 [Overview of the project] [Problems that the invention aims to solve]

[0007] The replicon disclosed in Non-Patent Document 1 is constructed by ligating six fragments, including fragments excised from a plasmid, each time. On the other hand, the replicon disclosed in Non-Patent Document 2 is incorporated into an Escherichia coli artificial chromosome (BAC) vector, eliminating the need for repeated ligation and allowing for convenient use in experiments.

[0008] The replicons disclosed in Non-Patent Documents 1 and 2 require obtaining the replicon RNA by in vitro transcription at least before introducing it into cells. Furthermore, the replicon RNA needs to be introduced into cells by electroporation. Introducing RNA into cells is a complicated process and can result in low introduction efficiency.

[0009] Furthermore, regarding the replication level of replicon RNA introduced into cells, in the case of the replicon disclosed in Non-Patent Document 1, mRNA of the N gene was additionally introduced into the cells to increase the replication level, and compared to the background, it was 10 4 It does not reach a multiple. The replication level of replicon RNA disclosed in Non-Patent Document 2 is 10 times the background. 3 Even if the mRNA of the N gene is added to the cells, the difference is less than double, and the difference is 10 4 It is less than double. A higher replication level is preferable for accurately evaluating the efficacy of therapeutic drugs.

[0010] As mentioned above, the replicas disclosed in Non-Patent Documents 1 and 2 are difficult to handle due to the complicated procedures required before introduction into cells, and their replication level is insufficient, making them impractical.

[0011] This invention has been made in view of the above circumstances, and provides a highly practical replicon DNA, a cloning vector, a method for producing a cloning vector, COVID-19 treatment drugs The objective is to provide screening kits and screening methods. [Means for solving the problem]

[0012] Replicon DNA according to the first aspect of the present invention is Cytomegalovirus Promoter sequence and The aforementioned Cytomegalovirus The non-structural region containing SARS-CoV-2 non-structural protein genes 1a and 1b located 3' to the promoter sequence, The N gene of SARS-CoV-2 located 3' to the non-structural region, It has, Derived from HuH-7 cells from which HCV RNA replicating in the HuH-7 cell line has been eliminated. Hepatitis C virus-cured cells This involves cloning the HuH-7.6c cell line via a cloning vector. introduction by , the above HuH-7.6c cell KK Replication level of replicon RNA transcribed in of The decrease occurs at least 96 hours after the introduction of the cloning vector. Let None It is for that purpose. .

[0013] The replicon DNA according to the first aspect of the present invention may further have a reporter gene. This may also be the case.

[0014] [[ID=??]] Also, the replicon DNA according to the first aspect of the present invention may have a recognition site of a type IIS restriction enzyme that has been synonymous substituted. This may also be the case.

[0015] The cloning vector according to the second aspect of the present invention has the replicon DNA according to the first aspect of the present invention.

[0016] The method for producing a cloning vector according to the third aspect of the present invention has each of a plurality of fragments obtained by dividing the replicon DNA according to the first aspect of the present invention into a plurality, and a plurality of nucleic acid constructs having recognition sites of the type IIS restriction enzyme only at both ends of the fragments, and a cloning vector having recognition sites of the type IIS restriction enzyme only at both ends of a cloning site, and a cleavage step of cleaving with the type IIS restriction enzyme, and a ligation step of inserting the replicon DNA into the cloning site by ligating the fragment excised in the cleavage step and the cloning vector with a DNA ligase. including.

[0017] The screening kit for a COVID-19 therapeutic agent according to the fourth aspect of the present invention has the cloning vector according to the second aspect of the present invention, cells into which the cloning vector has been introduced and exposed to a test substance The aforementioned HuH-7.6c cells KK and [[ID=??]] is provided.

[0018] It should be noted that there is an unclear "??" in the original text which might be an error. I've translated it as best as possible based on the context.A screening method according to the fifth aspect of the present invention is: A cloning vector according to a second aspect of the present invention, The aforementioned HuH-7.6c cell KK The implementation steps to be taken, The cloning vector is introduced into the HuH-7.6c cell KK An exposure step in which the subject is exposed to the test substance, The aforementioned HuH-7.6c cell KK A quantitative step of quantifying the replicon RNA replicated from the replicon RNA on which the replicon DNA was transcribed, The quantitative step includes a selection step of selecting a test substance that suppresses the replication of the replicon RNA more than the control, Includes. [Effects of the Invention]

[0019] According to the present invention, the practicality of replicas related to SARS-CoV-2 can be improved. [Brief explanation of the drawing]

[0020] [Figure 1] (A) is a diagram showing the structure of the SARS-CoV-2 genome. (B) is a diagram showing an example of the structure of replicon DNA. (C) is a diagram showing fragments F1 to F10 obtained by dividing the replicon DNA shown in (B) into multiple parts. [Figure 2] This is a conceptual diagram illustrating the first step in introducing a fragment into a cloning vector. [Figure 3] This is a conceptual diagram illustrating the second stage of introducing a fragment into a cloning vector. [Figure 4] This figure shows the replication levels of replicon DNA in different cell lines. (A) to (F) show the results for HuH-7 cell line, Hep3B cell line, HEK293 cell line, Vero cell line, MDCK cell line, and BHK-21 cell line, respectively. [Figure 5] This figure shows the replication levels of replicon RNA in hepatitis C virus (HCV)-cured cell lines. [Figure 6] This figure shows the remdesivir concentration response at the replication level of replicon RNA. [Figure 7] This figure shows the cell survival rate in response to remdesivir. [Modes for carrying out the invention]

[0021] Embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to the embodiments and drawings described below. In the embodiments described below, expressions such as “having,” “including,” or “containing” also include the meaning of “consisting of” or “composed of.”

[0022] (Embodiment 1) Figure 1(A) shows the structure of the SARS-CoV-2 genome. The SARS-CoV-2 genome has a non-structural region containing the non-structural protein genes 1a (ORF1a) and 1b (ORF1b), and a structural region containing the S gene, ORF3a, E gene, M gene, ORF6, ORF7a, ORF7b, ORF8, N gene, and ORF10. The SARS-CoV-2 genome has untranslated regions (5'UTR and 3'UTR) at its 5' and 3' ends, respectively. The nucleotide sequence of the SARS-CoV-2 genome is exemplified by Sequence ID No. 1.

[0023] The replicon DNA according to this embodiment is a DNA construct built based on the nucleotide sequence of the SARS-CoV-2 genome. Generally, viral replicons maintain non-structural regions and lack structural regions, and therefore are not infectious but autonomously replicate. An example of the structure of the replicon DNA according to this embodiment is shown in Figure 1(B). This replicon DNA has the non-structural regions of SARS-CoV-2 and does not have genes included in the structural regions except for the N gene. In detail, this replicon DNA has a promoter sequence, non-structural regions including ORF1a and ORF1b, and the N gene.

[0024] The promoter sequence is a promoter sequence that functions in vertebrate cells. Preferably, the promoter sequence is located at the 5' end of the replicon DNA. Examples of such promoter sequences include the cytomegalovirus (CMV) promoter, SV40 promoter, retrovirus promoter, metallothionein promoter, heat shock protein promoter, CAG promoter, elongation factor 1α promoter, actin promoter, ubiquitin promoter, albumin promoter, and MHC class promoter, as shown as "CMV" in Figure 1(B).

[0025] The non-structural region is located 3' to the promoter sequence. The N gene, located 3' to the non-structural region, encodes a nucleoprotein. This nucleoprotein binds to the SARS-CoV-2 genomic RNA, forming a helical nucleocapsid. The N gene enhances the replication level of the genomic RNA.

[0026] The replicon DNA according to this embodiment is transcribed into RNA (replicon RNA) within the cell, then translated and replicated. The replicon DNA may have a reporter gene for quantifying the replicated replicon RNA. Known reporter genes can be used, such as genes encoding green fluorescent protein, genes encoding β-glucuronidase, and genes encoding luciferase. Preferably, the reporter gene is a secreted luciferase gene (sNLuc).

[0027] Furthermore, the replicon DNA may include marker genes as needed. The marker genes are not particularly limited and include, for example, drug resistance genes. Examples of drug resistance genes include neomycin resistance genes (Neo), hygromycin resistance genes, and puromycin resistance genes.

[0028] Furthermore, the replicon DNA may also contain other sequences used in the gene expression system. These other sequences include a poly(A) region (pA) that contributes to the stability of the transcript, the hepatitis delta virus (HDV) ribozyme gene (Rz) necessary for excision by self-cleavage after replication, and the bovine growth hormone poly(A) signaling molecule (BGH) that terminates transcription. Preferably, the replicon DNA according to this embodiment has sNLuc and Neo between ORF1b and the N gene, as shown in Figure 1(B), and has pA, Rz, and BGH 3' to the N gene. The base sequence of the replicon DNA with the configuration shown in Figure 1(B) is exemplified in Sequence ID No. 2.

[0029] The replicon DNA according to this embodiment is chemically synthesized according to its base sequence by any known nucleic acid synthesis method, such as the solid-phase phosphoramidite method. Various automated nucleotide synthesizers are commercially available, and the replicon DNA can also be synthesized using such automated nucleotide synthesizers. Multinucleotide synthesis methods can also be used as appropriate.

[0030] The following describes a suitable manufacturing method for the replicon DNA according to this embodiment. This manufacturing method uses a type IIS restriction enzyme (Golden Gate method). First, as shown in Figure 1(C), fragments F1 to F10 are defined by dividing the replicon DNA shown in Figure 1(B) into multiple parts. The base sequences at both ends of each fragment are not identical. The size of the fragments is arbitrary, but for example, it is 2 to 3 kbp.

[0031] Preferably, the recognition site of the type IIS restriction enzyme is synonymously substituted in the replicon DNA. That is, the replicon DNA does not have the recognition site of the type IIS restriction enzyme used in the production of the replicon DNA. A type IIS restriction enzyme is an enzyme that cleaves at least one of the two strands downstream of the recognition site. Examples of type IIS restriction enzymes include AlwI, AlwXI, Alw26I, BbsI, BbvI, BbvII, BcefI, BccI, BcgI, BciVI, BinI, BmrI, BpmI, BsaI, BseRI, BsgI, BsmAI, BsmBI, BspMI, BsrDI, BstF5I, EarI, Eco31I, Eco57I, Esp3I, Esp3I, FauI, FokI, GsuI, HgaI, HinGUII, HphI, Ksp632I, MboII, MmeI, Mn1I, NgoVIII, PleI, PsrI, RleAI, SapI, SfaNI, TaqII, Tth111II, BsmI, BsrI, BsmFI, and BseMII.

[0032] In the following, we will use BsaI as the type IIS restriction enzyme. BsaI can cleave downstream of the recognition sequence and design the base sequence of the 4-base overhang. Since the BsaI recognition sequence does not remain in the DNA fragment after cleavage, the 4-base overhang can be used to ligate fragments F1 to F10 and construct replicon DNA.

[0033] Synonymous substitution is a base substitution that does not change the encoded amino acid, and is also called a silent mutation. By synonymizing the recognition site of a type IIS restriction enzyme, it is possible to avoid cleaving fragments at unnecessary locations when the type IIS restriction enzyme is applied during the manufacturing process. Specifically, the BsaI site (gagacc) in ORF1b of the replicon DNA shown in Figure 1(B) is modified to "gGgacc" or similar so that no amino acid substitution occurs.

[0034] Fragments F1 to F10 are introduced into a plasmid vector (nucleic acid construct) by adding the nucleotide sequences of BsaI sites to both ends of each fragment. Any plasmid vector can be used, but preferably one that does not have a BsaI site. If a plasmid vector with a BsaI site is used, the BsaI site should be deleted by synonymous substitution of the nucleotide sequence before use. The plasmid vector contains one of fragments F1 to F10, and has BsaI sites only at both ends of the fragment. The plasmid vectors into which each of F1 to F10 is introduced may all be the same, or some fragments may be introduced into different types of plasmid vectors.

[0035] Next, fragments F1-F10 are introduced into the cloning site of a cloning vector having BsaI sites only at both ends of the cloning site. In detail, the method for producing the cloning vector includes a cleavage step and a ligation step. In the cleavage step, the above-mentioned plasmid vectors and the cloning vector having BsaI sites only at both ends of the cloning site are cleaved at the BsaI sites. In the ligation step, the fragments F1-F10 excised in the cleavage step and the cloning vector are ligated with DNA ligase to insert the replicon DNA into the cloning site.

[0036] Preferably, the introduction of fragments F1-F10 into the cloning vector is carried out in two steps. First, in the first step, 3-4 adjacent fragments are introduced into the cloning site of a first cloning vector that has BsaI sites only at both ends of the cloning site. For example, fragments F1-F3 are introduced into cloning vector A, fragments F4-F6 are introduced into cloning vector B, and fragments F7-F10 are introduced into cloning vector C. To introduce fragments F1-F3 into cloning vector A, as shown in Figure 2, the plasmid vectors containing fragments F1, F2, and F3, respectively, and cloning vector A are cut with BsaI, and DNA ligase is applied to ligate fragments F1, F2, and F3 to the cloning site of cloning vector A. The same procedure is followed for introduction into cloning vectors B and C.

[0037] In the second step, fragments F1-F3, F4-F6, and F7-F10 are introduced into the cloning site of a second cloning vector D, which has BsaI sites only at both ends of its cloning site. In the second step, as in the first step, as shown in Figure 3, cloning vectors A, B, and C and cloning vector D are cleaved with BsaI and treated with DNA ligase. As a result, fragments F1-F10 are ligated and introduced into the cloning site of a single cloning vector D. Preferably, cloning vector D is BAC.

[0038] Cloning vector D is transduced into E. coli or other bacteria using a known method, and the transduced clones are selected using a known colony screening method.

[0039] The replicon DNA according to this embodiment can be introduced into mammalian cells by known methods, for example via cloning vector D, and will autonomously proliferate within the cells. This allows for the evaluation of compounds that suppress SARS-CoV-2 replication.

[0040] The replicon DNA according to this embodiment can be manufactured without using natural viruses as a template, thus avoiding infection and leakage incidents. Furthermore, this replicon DNA can be manufactured and used even in facilities with a BSL level of less than 3. In addition, because this replicon DNA has a promoter sequence that functions in vertebrate cells, it can be introduced into mammalian cells as DNA and replicated. This eliminates the need for in vitro transcription before introduction into cells, and also eliminates the need to introduce RNA into cells, which is a complicated and relatively difficult procedure. As a result, the replicon DNA according to this embodiment achieves higher introduction efficiency than directly introducing replicon RNA into cells, and is highly practical.

[0041] The replicon DNA according to this embodiment may further contain a reporter gene. This allows the replication level to be evaluated via the reporter gene. Furthermore, the replicon DNA may have synonymous substitutions at the recognition site of type IIS restriction enzymes. By not having a recognition site for type IIS restriction enzymes in the replicon DNA manufacturing method described above, the replicon DNA can be efficiently constructed using type IIS restriction enzymes.

[0042] The replicon DNA according to this embodiment is contained in a cloning vector, eliminating the need to ligate fragments each time it is used, making it easy to handle. The replicon DNA according to this embodiment is prepared by 10 times the amount of background, as shown in the example below. 4 A replication level more than double can be obtained, and moreover, the replication level increases or decreases depending on the concentration of the drug. For this reason, the replicon DNA according to this embodiment is highly practical.

[0043] (Embodiment 2) The screening kit according to this embodiment comprises the cloning vector according to Embodiment 1 described above and HCV-healed cells. "HCV-healed cells" are cells obtained by culturing cells that replicate subgenomic HCV replicons or cells that replicate the full-length HCV genome in the presence of an antiviral agent, and are cells from which the subgenomic HCV replicons or full-length HCV genome have been eliminated. Preferably, HCV-healed cells do not express HCV RNA and HCV protein in the cells. Whether or not HCV-derived RNA is contained in the cells can be easily confirmed by reverse transcription polymerase chain reaction (RT-PCR) or Northern blotting. Whether or not HCV protein is expressed in the cells can be easily confirmed by Western blotting.

[0044] HCV-healed cells can be established, for example, by the following method: After introducing full-length HCV RNA into mammalian cells such as HuH-7 cells by electroporation, G418 is added, and cells with high HCV RNA replication levels are selected to form colonies. By adding interferon (IFN) to the cell line of cloned cells with the highest HCV RNA replication level among the colonies, a healed cell line with HCV RNA eliminated can be obtained. Healed cell lines are known to have higher HCV RNA replication levels than the parent cell line.

[0045] The screening kit according to this embodiment may further include a culture medium and culture medium additives that can be used for culturing HCV-cured cells.

[0046] Next, a screening method provided in another embodiment will be described as a method for using the screening kit. This screening method is particularly suitable for screening for COVID-19 therapeutic drugs. This screening method includes an introduction step, an exposure step, a quantification step, and a selection step.

[0047] In the introduction step, a cloning vector containing the replicon DNA according to Embodiment 1 described above is introduced into HCV-healed cells. The cloning vector can be introduced into HCV-healed cells by known methods. For example, the cloning vector can be introduced into HCV-healed cells using commercially available transfection reagents.

[0048] In the exposure step, HCV-cured cells into which the cloning vector has been introduced are exposed to the test substance. In the exposure step, the test substance can be added to the culture medium containing the HCV-cured cells. The test substance is not particularly limited and can be any substance such as compounds, peptides, and nucleic acids.

[0049] In the quantification step, the amount of replicon RNA replicated from replicon RNA transcribed from replicon DNA in HCV-cured cells is quantified. The method for quantifying nucleic acids is not particularly limited and can be quantified using methods such as RT-PCR. In particular, if the replicon DNA contains a secreted luciferase gene, the replicated replicon RNA can be quantified by collecting the culture supernatant and measuring the luciferase activity using a commercially available kit. Note that "quantification" includes not only determining the amount of replicated replicon RNA but also evaluating the relative amount.

[0050] In the selection step, a test substance is selected that suppresses replicon RNA replication more than the control in the quantification step. The control is, for example, HCV-cured cells that have not been exposed to the test substance. Preferably, the control is a COVID-19 therapeutic agent that suppresses SARS-CoV-2 infection or replication. When quantifying replicated nucleic acids by the magnitude of luciferase activity, the selection step can be performed by comparing the luciferase activity between the control and the test substance. In this case, the test substance whose luciferase activity is higher than that of the control is selected as a COVID-19 therapeutic agent or a candidate substance thereof.

[0051] According to the screening kit of this embodiment and the screening method of another embodiment, since HCV-healed cells in which the above-mentioned replicon RNA is particularly strongly replicated are used, it is possible to accurately select test substances that suppress replicon RNA replication. As shown in the example below, since the replication of replicon RNA in HCV-healed cells responds to the concentration of a drug having anti-SARS-CoV-2 activity, the concentration dependence of the test substance can also be evaluated. In the selection step, a reference value set in advance may be compared with the measured value for the test substance.

[0052] Furthermore, the test substances selected by the above screening method can be used as treatments for COVID-19. For this reason, the above screening method constitutes at least a part of a method for manufacturing treatments for COVID-19.

[0053] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. [Examples]

[0054] (Preparation of BsaI(-) vector) The restriction enzyme BsaI site (ggtctc) within the ampicillin resistance gene of pUC19 was modified to "gAtctc" using QuickChange mutagenesis (Stratagene), thereby constructing the BsaI-deficient plasmid pUC19b.

[0055] The restriction enzyme BsaI site (ggtctc) within the sopB gene of pSMART BAC was modified to "gAtctc" using QuickChange mutagenesis, thereby constructing the BsaI-deficient plasmid pSMART BACb.

[0056] (Design of artificial genes) Without using a natural virus as a template, a SARS-CoV-2 replicon (hereinafter referred to as "SC2R") whose base sequence is shown in SEQ ID NO: 2 was artificially synthesized based on the SARS-CoV-2 gene information (NCBI Reference Sequence: NC_045512; SEQ ID NO: 1) in the National Center for Biotechnology Information (NCBI) database, as follows. As shown in Figure 1(B), SC2R contains the CMV promoter sequence, the non-structural regions of SARS-CoV-2 (ORF1a and ORF1b), sNLuc, Neo, N genes, pA, Rz, and BGH from the 5' end.

[0057] In the non-structural region of the SARS-CoV-2 replicon, the nucleotide sequence corresponding to the BsaI site (gagacc) from position 17972 to 17977 from the 5' end of the nucleotide sequence shown in Sequence ID No. 1 was replaced with "gGgacc" for synonymous substitution.

[0058] An artificial gene was synthesized by splitting SC2R (25562 bp) with a deleted BsaI site into 10 fragments F1 to F10, each 2-3 kb in length. In detail, the 2735 bp from position 1 to 2735 from the 5' end of the SC2R base sequence shown in Sequence ID No. 2 were designated as F1, the 2829 bp from position 2732 to 5360 as F2, the 2795 bp from position 5357 to 8151 as F3, the 2446 bp from position 8148 to 10593 as F4, the 2790 bp from position 10590 to 13379 as F5, the 2502 bp from position 13376 to 15877 as F6, the 2835 bp from position 15874 to 18708 as F7, the 2410 bp from position 18705 to 21114 as F8, the 2178 bp from position 21111 to 23288 as F9, and the 2278 bp from position 23285 to 25562 as F10. We designed each fragment to seamlessly integrate SC2R into the BAC vector by adding the BsaI site sequence to both ends.

[0059] (Synthesis of artificial genes) BsaI sites were introduced at the 5' and 3' ends of each of the 10 fragments. Fractions F1-4 and F6-10 were introduced into pUC19b (pUC19b / SC2R-F1,F2,F3,F4,F6,F7,F8,F9,F10). Fraction F5 was introduced into the pCC1-4k vector (pCC1-4k / SC2R-F5).

[0060] (Preparation of the cloning vector pSMART BACbc) The pUC57 NotI-BsaIc-BsaI-EcoRI-HindIII-BamHI-XhoI-AvrII molecule, which had an artificially synthesized multi-cloning site introduced into it, was cleaved with restriction enzymes NotI and AvrII to obtain a 955bp artificial gene shown in SEQ ID NO: 3. The artificial gene was ligated into the NotI-AvrII site of pSMART BACb to create pSMART BACbc.

[0061] (Preparation of cloning vectors pHSG298c and pCC1-4kc) To introduce the BsaI site into the pHSG298 vector, PCR fragments A and B containing BsaI were prepared inside EcoRI and BamHI. For the preparation of PCR fragment A, the HRP gene shown in SEQ ID NO: 4 was used as a template, and PCR was performed using forward primers (SEQ ID NO: 5) and reverse primers (SEQ ID NO: 6). The nucleotide sequence of PCR fragment A is shown in SEQ ID NO: 7. For the preparation of PCR fragment B, the HRP gene shown in SEQ ID NO: 4 was used as a template, and PCR was performed using forward primers (SEQ ID NO: 8) and reverse primers (SEQ ID NO: 9). The nucleotide sequence of PCR fragment B is shown in SEQ ID NO: 10.

[0062] Furthermore, in order to introduce the BsaI site into the pCC1-4k vector, PCR fragment C containing BsaI inside HindIII and BamHI was constructed. For the construction of PCR fragment C, the HRP gene shown in SEQ ID NO: 4 was used as a template, and PCR was performed using forward primers (SEQ ID NO: 11) and reverse primers (SEQ ID NO: 12). The nucleotide sequence of PCR fragment C is shown in SEQ ID NO: 13.

[0063] pHSG298 was cleaved with EcoRI and BamHI, and PCR fragment A was ligated to construct pHSG298c vector A. Similarly, pHSG298 was cleaved with EcoRI and BamHI, and PCR fragment B was ligated to construct pHSG298c vector B. pCC1-4k was cleaved with HindIII and BamHI, and PCR fragment C was ligated to construct pCC1-4kc vector.

[0064] (Preparation of intermediate fragments pHSG298c / F123, pCC1-4kc / F456, and pHSG298c / F7-10) pUC19b / F1, F2, F3, F4, F6, F7, F8, F9, F10 and pCC1-4k / F5 were each cleaved with BsaI. F1, F2, and F3 were introduced into pHSG298c vector A to form pHSG298c / F123. F4, F5, and F6 were introduced into pCC1-4kc vector to form pCC1-4kc / F456. F7, F8, F9, and F10 were introduced into pHSG298c vector B to form pHSG298c / F7-10.

[0065] (Preparation of SC2R vector) pHSG298c / F123, pCC1-4kc / F456, and pHSG298c / F7-10 were cleaved with BsaI, and each fragment was ligated to the BsaI-BsaI site of pSMART BACbc. After ligation, the cells were introduced into BAC-Optimized Replicator v2.0 Electrocompetent Cells (Lucigen) by electroporation and cloned. This process yielded pSMART BACbc / SC2R.

[0066] (Preparation of pSMART BACbc / SC2R SAA vector) The active site of RNA-dependent RNA polymerase (RdRP) present in F7 contains the motif "SDD" (tct gac gat). Using QuickChange mutagenesis, a substitution was made from "SDD" to "SAA" (tct gCc gCt), and pSMART BACbc / SC2R SAA lacking RdRP activity was constructed in the same manner as pSMART BACbc / SC2R.

[0067] (Replication of SC2R in different cell lines) HuH-7, Hep3B, HEK293, Vero, MDCK, and BHK-21 cell lines were seeded in 6-well plates. After 24 hours of culture, the culture supernatant was collected, and each cell was transfected with 1 μg of pSMART BACbc / SC2R or pSMART BACbc / SC2R SAA using Fugene HD reagent (Promega). After 24 hours of culture, the culture supernatant was collected, cells were detached using trypsin, and the cells were seeded in 24-well plates and culture was continued. Subsequently, the culture supernatant was collected every 24 hours. Luciferase activity was measured in the culture supernatant using the Nano-Glo Luciferase Assay System (Promega).

[0068] (result) As shown in Figure 4, the highest replication level was observed in human hepatocellular carcinoma cells (HuH-7) (see Figure 4(A)). Therefore, we decided to investigate SC2R replication in HCV-cured cells obtained by eliminating HCV RNA with IFN from the HuH-7 cell line, which exhibits high levels of HCV RNA replication.

[0069] (Establishment of HuH-7.6c cells) After introducing full-length reporter HCV RNA into HuH-7 cells by electroporation, G418 is added, and cells with high HCV RNA replication levels are selected to form colonies. The OR6 cell line is established by cloning the cells with the highest HCV RNA replication levels among the colonies. Next, HCV RNA is eliminated by adding IFN to OR6 cells to establish a healed cell line (HuH-7.6c cell line). Healed cell lines are known to have higher HCV RNA replication levels than the parental HuH-7 cell line (Masanori Ikeda, et al., "Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system", Biochem Biophys Res Commun., 2005, 329(4):1350-9).

[0070] (SC2R replication in healing cell lines) The replication levels of SC2R in healed cells (HuH-7.RSc and HuH-7.6c) from which HCV RNA replicated in HuH-7 were eliminated were compared with those of the parental HuH-7 strain. HuH-7, HuH-7.RSc, and HuH-7.6c cells were seeded in 6-well plates, cultured for 24 hours, and the culture supernatant was collected. Each cell was then transfected with 1 μg of pSMART BACbc / SC2R or pSMART BACbc / SC2R SAA using Fugene HD reagent. The culture supernatant was collected again in the same manner as above, and luciferase activity was measured using the Nano-Glo Luciferase Assay System.

[0071] (result) As shown in Figure 5, replicon RNA transcribed from SC2R showed a higher replication level in HuH-7.6c cells than in the parental HuH-7 cells. Furthermore, HuH-7.RSc, another healed cell line, also showed a higher replication level of replicon RNA transcribed from SC2R than in the parental HuH-7 cells. HCV healed cells tended to have a more favorable intracellular environment for SC2R-derived replicon RNA than the parental HuH-7 cell line. However, it has been reported that the replication level of SARS-CoV-2 replicon RNA in another healed cell line, HuH-7.5, is lower than in the parental HuH-7 cell line (Non-Patent Literature 2). Therefore, HuH-7.6c cells are considered to be healed cells possessing a unique intracellular environment that enables a high replication level of replicon RNA transcribed from SC2R.

[0072] (Evaluation of anti-SARS-CoV-2 activity) HuH-7.6c cells were placed in a 6-well plate at a rate of 1 × 10⁶ 5 Cells were seeded to a density of cells / well, and the following day, 1 μg of pSMART BACbc / SC2R was transfected into the cells using Fugene HD reagent. After 24 hours, the cells were detached using trypsin and placed in a 24-well plate in a 3 × 10⁶ configuration. 4 Cells were seeded to a specific cell-to-well ratio. After another 24 hours, remdesivir was added at concentrations of 7.81, 15.6, 31.3, 62.5, and 125 nM. After 48 hours of incubation, the culture supernatant was collected, and luciferase activity was measured using the Nano-Glo Luciferase Assay System.

[0073] (result) As shown in Figure 6, SC2R replication was suppressed in a concentration-dependent manner with remdesivir. The EC50 of remdesivir was 16.4 nM.

[0074] (Evaluation of cytotoxicity) HuH-7.6c cells were placed in 5 × 10⁶ well plates in a 96-well plate. 3The cells were seeded to a specific concentration. 24 hours after the start of culture, remdesivir was added to the cells to the predetermined concentration. After 48 hours of culture, 10 μl of Premix WST-1 Cell Proliferation Assay System (Takara Bio Inc.) was added to the culture medium, and after 2 hours of culture at 37°C, the absorbance at 450 nm was measured using a microplate reader.

[0075] (result) Figure 7 shows the cell viability rate as a function of remdesivir concentration. 50 The concentration was 1.13 μM.

[0076] This invention allows for various embodiments and modifications without departing from the broad spirit and scope of the invention. Furthermore, the embodiments described above are for illustrative purposes only and do not limit the scope of the invention. In other words, the scope of the invention is indicated not by the embodiments, but by the claims. Various modifications made within the scope of the claims and the equivalent scope of the meaning of the invention are considered to be within the scope of the invention.

[0077] This application is based on Japanese Patent Application No. 2021-006988, filed on January 20, 2021. The entire specification, claims, and drawings of Japanese Patent Application No. 2021-006988 are incorporated herein by reference. [Industrial applicability]

[0078] The present invention is suitable for screening COVID-19 treatments.

Claims

1. A promoter sequence of cytomegalovirus, A non-structural region including SARS-CoV-2 non-structural protein genes 1a and 1b located 3' to the promoter sequence of the cytomegalovirus, The N gene of SARS-CoV-2 located 3' to the non-structural region, It has, To prevent a decrease in the replication level of replicon RNA transcribed in the HuH-7.6c cell line, which is a hepatitis C virus-cured cell line derived from HuH-7 cells from which HCV RNA replicates in the HuH-7 cell line has been eliminated, by introducing a cloning vector into the HuH-7.6c cell line, for at least 96 hours after introduction of the cloning vector, Replicant DNA.

2. Furthermore, possessing the reporter gene, The replicon DNA according to claim 1.

3. The recognition site of the type IIS restriction enzyme is synonymously substituted. Replicon DNA according to claim 1 or 2.

4. Having the replicon DNA according to any one of claims 1 to 3, Cloning vector.

5. A cleavage step of cleaving a plurality of nucleic acid constructs, each having multiple fragments obtained by dividing the replicon DNA described in claim 3 into a plurality of parts, each having a recognition site for the type IIS restriction enzyme only at both ends of the fragment, and a cloning vector having a recognition site for the type IIS restriction enzyme only at both ends of the cloning site, with the type IIS restriction enzyme, The ligation step involves linking the fragment cut out in the cutting step and the cloning vector with DNA ligase to insert the replicon DNA into the cloning site, A method for producing a cloning vector, including the method described above.

6. The cloning vector according to claim 4, The HuH-7.6c cell line into which the cloning vector is introduced and exposed to the test substance, A screening kit for COVID-19 treatment drugs that includes [features / equipment].

7. An introduction step of introducing the cloning vector according to claim 4 into the HuH-7.6c cell line, An exposure step in which the HuH-7.6c cell line into which the cloning vector has been introduced is exposed to a test substance, A quantitative step of quantifying replicon RNA replicated from replicon RNA transcribed with replicon DNA in the HuH-7.6c cell line, The quantitative step includes a selection step of selecting a test substance that suppresses the replication of the replicon RNA more than the control, A screening method that includes this.

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