Drug screening models and methods for targeting HBV cccDNA

Abstract

The present invention relates to the field of virology, particularly to the field of hepatitis B virus treatment. A model and method for screening HBV cccDNA inhibitors are provided. According to this screening model and method, split luciferase detection is used as a surrogate indicator for HBV cccDNA detection, enabling high-throughput screening of cccDNA-targeting drugs.

Description

[Technical Field]

[0001] The present invention relates to the field of virology, in particular to the field of treatment of Hepatitis B. In particular, the present invention relates to a model and method for screening HBV cccDNA inhibitors. [Background technology]

[0002] Chronic hepatitis B (CHB), caused by hepatitis B virus (HBV), is one of the world's most serious public health problems. Hepatitis B virus-induced liver diseases, including chronic active hepatitis, cirrhosis, and hepatocellular carcinoma, result in over 800,000 deaths annually. Currently, the two main therapeutic agents in clinical practice (nucleoside analogs and interferon) have difficulty achieving a clinical cure. The stable presence of HBV cccDNA is one of the main reasons why chronic hepatitis B is difficult to cure. Currently, there are no clinical drugs that can effectively eliminate cccDNA, and the cccDNA present in cells continues to function as a template for viral replication and transcription.

[0003] Because the mechanism of HBV cccDNA formation and maintenance is complex, designing drugs directly against cccDNA is extremely difficult. A screening model that can be used for high-throughput screening of cccDNA inhibitors provides a new approach to developing drugs that can eliminate cccDNA. Detection methods for cccDNA are complex. Southern blot is the gold standard for cccDNA detection, but it requires large numbers of cells, involves complex procedures, and is time-consuming, making it unsuitable for high-throughput drug screening. Compared to Southern blot, fluorescent quantitative PCR detection is simpler, faster, and has higher throughput, but it is difficult to apply to large-scale drug screening, and detection can be hindered by rcDNA. Using a more easily detectable marker as a surrogate marker for cccDNA detection can reduce detection costs, improve detection efficiency, and increase detection throughput.

[0004] An ideal cccDNA reporter model would not only satisfy the requirement of a stable cccDNA source, but also the requirement of easy detection of surrogate detection markers with a high signal-to-noise ratio. Therefore, it is necessary to develop an HBV cccDNA reporter model suitable for high-throughput screening. Summary of the Invention

[0005] After many experiments and repeated investigations, the inventors of the present application have constructed an HBV cccDNA reporter model that uses split luciferase as a surrogate indicator for detecting HBV cccDNA, which is easy to operate, requires a short time, and can realize high-throughput drug screening. Therefore, this model can be used for preliminary screening of candidate drugs with inhibitory ability against HBV cccDNA, which can be further verified in in vitro and in vivo research models of HBV.

[0006] Reporter Model I In some cases, the first fragment sequence (e.g., HiBiT) in luciferase fragment complementation assay (LFCA) can be integrated into the HBV genome to form an HBV mutant, and the mRNA transcribed from the HBV mutant as a template lacks the initiation codon for the expression of the first fragment (e.g., HiBiT), and therefore cannot translate the protein attached to the first fragment (e.g., HiBiT) tag. Only after cccDNA is formed by reverse transcription of the pgRNA transcribed from the HBV mutant, can the mRNA transcribed from the cccDNA as a template translate the protein attached to the first fragment (e.g., HiBiT) tag. Therefore, the expression level of the first fragment (e.g., HiBiT) can be measured by luciferase fragment complementation assay (LFCA), thereby indicating the formation of HBV cccDNA.

[0007] 1. Isolated Nucleic Acid Molecules Thus, in a first aspect, the present invention provides an isolated nucleic acid molecule comprising a variant of an HBV genome sequence (e.g., a wild-type HBV genome), the variant comprising an HBV genome fragment including a C-ORF, an S-ORF, and a P-ORF, the C-ORF comprising an exogenous insertion sequence between the pre-core gene and the core gene, the exogenous insertion sequence comprising a nucleotide sequence encoding a first fragment of luciferase, the first fragment of luciferase capable of binding to a corresponding second fragment of luciferase in a luciferase fragment complementation assay (LFCA), thereby generating luciferase activity.

[0008] As used herein, the term "luciferase fragment complementation assay (LFCA)" has the meaning commonly understood by those skilled in the art, in which luciferase is divided into a first and a second fragment, each of which is enzymatically inactive, and when the two fragments interact with each other, they complement each other, resulting in luciferase activity and thereby emitting a luminescent signal in the presence of a luciferase substrate. In certain exemplary embodiments, the luciferase fragment complementation assay is based on LgBiT from Promega Corporation and a complementary sub-fragment capable of binding to it (e.g., HiBiT or SmBiT), where structural complementation of LgBiT with HiBiT or SmBiT results in the generation of a functional enzyme.

[0009] In certain embodiments, the first fragment of luciferase is LgBiT and the second fragment of luciferase is a complementary sub-fragment (eg, HiBiT or SmBiT) that is capable of binding to LgBiT.

[0010] In certain embodiments, the first fragment of luciferase is a complementary sub-fragment capable of binding to LgBiT (e.g., HiBiT or SmBiT), and the second fragment of luciferase is LgBiT. In certain embodiments, the first fragment of luciferase is HiBiT and the second fragment of luciferase is LgBiT. In certain embodiments, HiBiT has the sequence set forth in SEQ ID NO:2. In certain embodiments, the nucleotide sequence encoding HiBiT is set forth in SEQ ID NO:3.

[0011] In certain embodiments, the HBV genome fragment further comprises an X-ORF.

[0012] In certain embodiments, the variant comprises an exogenous insertion sequence between the pre-core gene and the core gene of the HBV genome sequence (eg, a wild-type HBV genome).

[0013] In certain embodiments, the exogenous insertion sequence comprises multiple copies of a nucleotide sequence encoding a first fragment of a luciferase (e.g., HiBiT) in tandem repeats, hi certain embodiments, the exogenous insertion sequence comprises three copies of a nucleotide sequence encoding a first fragment of a luciferase (e.g., HiBiT) in tandem repeats.

[0014] In certain embodiments, each copy of the multiple copies of the nucleotide sequence encoding the first fragment of luciferase (e.g., HiBiT) in the tandem repeat comprises a sequence encoding a linker peptide at its 5' end. In certain embodiments, the linker peptide is a flexible peptide linker. In certain embodiments, the linker peptide consists of G (glycine) and / or S (serine). In certain embodiments, the linker peptide is GSG.

[0015] In certain embodiments, the exogenous insert sequence comprises the sequence shown in SEQ ID NO:4.

[0016] In certain embodiments, the HBV genome is a full-length genome, for example, a genome of HBV genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, or genotype H. In certain embodiments, the HBV genome is an over-length genome, for example, a 1.1-fold genome or a 1.3-fold genome. In certain embodiments, the HBV genome is, for example, the 1.1-fold genome set forth in SEQ ID NO: 1.

[0017] In certain embodiments, the exogenous insertion sequence is operably linked to an inducible promoter.

[0018] In certain embodiments, the exogenous inserted sequence has expression regulated by the Tet-On gene expression system. Thus, in certain embodiments, the inducible promoter is a Tet operator (TetO) or promoter in the Tet-On gene expression system, which requires doxycycline to bind to its corresponding transactivator to initiate transcription.

[0019] In certain embodiments, the inducible promoter is the TRE3G promoter (e.g., as set forth in SEQ ID NO: 5) and the corresponding transactivator is the Tet-On 3G transactivator (e.g., as set forth in SEQ ID NO: 9).

[0020] In certain embodiments, the inducible promoter is one or more repeats of the Tet operator (TetO) sequence, and the corresponding transactivator can be a reverse Tet repressor (rTetR) or a reverse Tet transcription activator (rtTA).

[0021] In certain embodiments, the inducible promoter has bidirectional promoter activity.

[0022] In certain embodiments, the inducible promoter is the TRE3G promoter, which has bidirectional promoter activity.

[0023] In certain embodiments, the inducible promoter is operably linked to a reporter gene. In certain embodiments, the reporter gene is oriented opposite to the exogenous insertion sequence. In certain embodiments, the reporter gene is selected from a fluorescent protein gene and / or an antibiotic resistance gene.

[0024] In certain embodiments, the fluorescent protein is selected from the group consisting of green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, orange or red fluorescent protein, near-infrared fluorescent protein, or long Stokes shift fluorescent protein. In certain embodiments, the fluorescent protein is selected from the group consisting of red fluorescent protein, near-infrared fluorescent protein, or long Stokes shift fluorescent protein, such as mRuby3, mApple, FusionRed, mCherry, mScarlet, RFP, iRFP670, mBeRFP, or CyOFP1. In certain embodiments, the fluorescent protein is selected from the group consisting of green fluorescent protein, e.g., mGamillus, mNeonGreen, EGFP, mClover, UnaG, TurboGFP, TagGFP, Venus, EYFP, RFP, iRFP670, mBeRFP, or CyOFP1.

[0025] In certain embodiments, the antibiotic resistance gene is selected from the group consisting of genes capable of conferring resistance to hygromycin, neomycin, G418, blasticidin, puromycin, or ouabain.

[0026] In certain embodiments, the reporter gene comprises a fluorescent protein gene and an antibiotic resistance gene, hi certain embodiments, the reporter gene comprises a gene encoding iRFP (e.g., as set forth in SEQ ID NO: 11) and a blasticidin resistance gene (e.g., as set forth in SEQ ID NO: 13).

[0027] In certain embodiments, the fluorescent protein gene and the antibiotic resistance gene are optionally linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, or T2A). In certain embodiments, the cleavage peptide is P2A, e.g., as set forth in SEQ ID NO:6.

[0028] In certain embodiments, the isolated nucleic acid molecule comprises the sequence set forth in SEQ ID NO:8.

[0029] 2. Recombinant HBV cccDNA The HBV variant contained in the isolated nucleic acid molecule described in the first aspect of the present invention can be used as a template to be transcribed to form pgRNA and reverse transcribed to form cccDNA.

[0030] Thus, in a second aspect, the present invention also provides a recombinant HBV cccDNA comprising the isolated nucleic acid molecule of the first aspect.

[0031] In certain embodiments, the recombinant HBV cccDNA comprises a variant of the HBV genomic sequence described in the first aspect.

[0032] In certain embodiments, the recombinant HBV cccDNA is formed by circularization of the isolated nucleic acid molecule of the first aspect.

[0033] 3. Expression system In a third aspect, the present invention provides an expression system comprising the isolated nucleic acid molecule of the first aspect.

[0034] In certain embodiments, the isolated nucleic acid molecule comprises an inducible promoter operably linked to an exogenous insert sequence, and the expression system comprises the isolated nucleic acid molecule as a first nucleic acid sequence and a second nucleic acid sequence, the second nucleic acid sequence comprising a nucleotide sequence encoding a transactivator corresponding to the inducible promoter.

[0035] In certain embodiments, the transactivator is selected from the group consisting of Tet-On 3G transactivator, rTetR, and rtTA.

[0036] In certain embodiments, the second nucleic acid sequence further comprises an expression control element sequence, such as a promoter (e.g., a constitutive promoter) and / or an enhancer, operably linked to the nucleotide sequence encoding the transactivator.

[0037] In certain embodiments, the first nucleic acid sequence comprises a TRE3G promoter as an inducible promoter, and the second nucleic acid sequence comprises a nucleotide sequence encoding a Tet-On 3G transactivator. In certain embodiments, the TRE3G promoter comprises the sequence set forth in SEQ ID NO: 5. In certain embodiments, the nucleotide sequence encoding the Tet-On 3G transactivator comprises the sequence set forth in SEQ ID NO: 10.

[0038] 4. Vector In a fourth aspect, the present invention also provides a vector comprising the isolated nucleic acid molecule of the first aspect, or the expression system of the third aspect.

[0039] In certain embodiments, the vector comprises the expression system of the third aspect, wherein the first nucleic acid sequence and the second nucleic acid sequence are provided on the same or different vectors, hi certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are provided on the same vector.

[0040] In certain embodiments, the vector is a transposon vector, such as a PiggyBac transposon vector. In certain embodiments, the first nucleic acid sequence and / or the second nucleic acid sequence can be inserted into any commercially available PiggyBac transposon vector, such as PB-CMV-MCS-EF1α-RedPuro (catalog no. PB514B-1). In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are located between two ITR sequences of the transposon vector.

[0041] 5. Co-transfection System In a fifth aspect, the present invention provides a co-transfection system comprising the vector of the fourth aspect, which is a transposon-based vector, and a transposase expression vector.

[0042] In certain embodiments, the transposase expression vector is a PiggyBac transposase expression vector, which is well known in the art and widely available commercially. In certain embodiments, the PiggyBac transposase expression vector is PB210PA-1 (System Biosciences).

[0043] 6.Host cells In a sixth aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the first aspect, or the recombinant HBV cccDNA of the second aspect, or the expression system of the third aspect, or the vector of the fourth aspect, or the co-transfection system of the fifth aspect.

[0044] In certain embodiments, the host cell is a eukaryotic cell. In certain embodiments, the host cell supports the formation and transcription of functional HBV cccDNA.

[0045] In certain embodiments, the host cell is a eukaryotic cell of hepatic origin, such as a hepatoma cell or a hepatocyte, hi certain embodiments, the host cell is selected from HepaRG, HepG2, or Huh7.

[0046] In certain embodiments, the host cell may also be a non-hepatic cell, so long as it supports hepadnavirus cccDNA formation (or, more broadly, hepadnavirus DNA replication). For example, such non-hepatic cells / hosts can be engineered to support hepadnavirus cccDNA formation (or hepadnavirus DNA replication) when viral pregenomic RNA is introduced into the cell or transcribed from a DNA template via an exogenous promoter.

[0047] In certain embodiments, the host cell comprises in its genome an expression system of the third aspect.

[0048] In certain embodiments, the host cell is capable of stably expressing HBV cccDNA formed by a variant of the HBV genomic sequence in the presence of an inducer (e.g., doxycycline) and a transactivator corresponding to the inducible promoter.

[0049] 7. Kit In a seventh aspect, the present invention provides a kit comprising the isolated nucleic acid molecule of the first aspect, or the expression system of the third aspect, or the vector of the fourth aspect, or the co-transfection system of the fifth aspect, or the host cell of the sixth aspect.

[0050] In certain embodiments, the kit comprises a vector of the fourth aspect or a co-transfection system of the fifth aspect.

[0051] In certain embodiments, the kit comprises a host cell of the sixth aspect.

[0052] In certain embodiments, the kit further comprises an LgBiT protein. Optionally, the kit may also comprise a luciferase substrate.

[0053] In certain embodiments, the kit further comprises an inducer (eg, doxycycline) and a transactivator corresponding to the inducible promoter.

[0054] 8. Screening Method In an eighth embodiment, there is provided a method for screening an HBV cccDNA inhibitor, comprising: (1) providing a host cell of the sixth aspect; wherein the host cell comprises in its genome an expression system as described in the third aspect; (2) contacting the host cell with an inducer; The inducer is an inducer (e.g., doxycycline) corresponding to the inducible promoter and a transactivator contained in the host cell; (3) contacting the test agent with the host cell; It should be noted that steps (2) and (3) may be performed simultaneously or in any order; (4) detecting the level of a first fragment of luciferase (e.g., HiBiT) in the cell supernatant of the host cell; A method is provided, comprising:

[0055] In certain embodiments, step (1) comprises: (1a) introducing the first nucleotide sequence and the second nucleotide sequence in the expression system of the third aspect into a host cell; the first nucleotide sequence and the second nucleotide sequence are provided on the same or different expression vectors, and the first nucleic acid sequence is the isolated nucleic acid molecule described in the first aspect, comprising an inducible promoter operably linked to an exogenous insert sequence; (1b) culturing the host cells; Includes.

[0056] In certain embodiments, the host cell is selected from eukaryotic cells of liver cell origin, such as hepatoma cells or hepatocytes, preferably the host cell is selected from HepaRG, HepG2, or Huh7.

[0057] In certain embodiments, in step (1a), the expression vector is a transposon vector (e.g., a PiggyBac transposon vector), and the step further comprises introducing a transposase expression vector (e.g., a PiggyBac transposase expression vector) into the host cell.

[0058] In certain embodiments, step (1) further comprises (1c) identifying and selecting host cells that have integrated into their genome the expression system of the third aspect. In certain embodiments, whether the expression system has been integrated into the genome of the host cell is determined by detecting a reporter gene contained in the first nucleic acid sequence.

[0059] In certain embodiments, in step (2), the inducer activates the inducible promoter, thereby initiating transcription and replication of the downstream HBV genome variant, resulting in recombinant HBV cccDNA, wherein the recombinant HBV cccDNA contains the first fragment of luciferase as a marker.

[0060] In certain embodiments, step (2) comprises (i) synthesis of HBV pregenomic (pg) RNA, (ii) reverse transcription of the synthesized pg RNA into minus-strand DNA, (iii) synthesis of a second plus-strand DNA such that the minus-strand DNA and the plus-strand DNA form a double-stranded imperfect circular DNA, and (iv) culturing the host cell under conditions that allow the formation of cccDNA from the double-stranded imperfect circular DNA.

[0061] In certain embodiments, in step (4), the level of a first fragment of luciferase is detected by a luciferase fragment complementation assay (i.e., by providing a second fragment of luciferase that is structurally complementary to the first fragment and a luciferase substrate).

[0062] In certain embodiments, a second fragment of luciferase that is complementary to the first fragment of luciferase is used for detection.

[0063] In certain embodiments, the first fragment of luciferase is a complementary sub-fragment capable of binding to LgBiT, such as HiBiT or SmBiT, and the second fragment of luciferase is an LgBiT protein. In certain embodiments, the first fragment of luciferase is HiBiT or SmBiT and the second fragment of luciferase is an LgBiT protein.

[0064] In certain embodiments, the method comprises: (5) comparing the detection result of step (4) with the level of the first fragment of luciferase detected in the absence of the test agent; Here, if the detection result in step (4) is lower than the detection result obtained in the absence of the test agent, this indicates that the test agent is an HBV cccDNA inhibitor.

[0065] The present invention also relates to the use of the above-mentioned isolated nucleic acid molecules, expression systems, vectors, co-transfection systems, host cells and kits for screening for HBV cccDNA inhibitors.

[0066] Reporter Model II In other cases, recombinase technology can be used to circularize a linear HBV replicon to form cccDNA. The linear HBV replicon contains a first fragment sequence (e.g., HiBiT sequence) of a luciferase fragment complementation assay (LFCA) incorporated therein. Without expression of the recombinase, the first fragment (e.g., HiBiT) tag cannot be expressed because it lacks a promoter, whereas with expression of the recombinase, it can mediate recombination of double-stranded DNA, causing the linear HBV genomic DNA to form a closed circular DNA. After the circular DNA is formed, the first fragment (e.g., HiBiT) tag can initiate expression of the protein attached to the first fragment (e.g., HiBiT) tag using the HBV endogenous promoter. Therefore, the signal of the first fragment (e.g., HiBiT) tag can be determined by luciferase fragment complementation assay (LFCA) to indicate the formation of recombinant HBV cccDNA, i.e., HBV rcccDNA.

[0067] 9. Isolated Nucleic Acid Molecules Accordingly, in a ninth aspect, the present invention provides an isolated nucleic acid molecule comprising a variant of an HBV genome sequence (e.g., a wild-type HBV genome), wherein the variant comprises, in the 5' to 3' direction: (i) a nucleotide sequence encoding a first fragment of luciferase; Note that a first fragment of luciferase can bind to a corresponding second fragment of luciferase in a luciferase fragment complementation assay (LFCA), thereby generating luciferase activity; (ii) the sequence of the 3'-terminal region of the C-ORF of the HBV genome; (iii) an HBV genome fragment containing the S-ORF and the P-ORF; (iv) a sequence of the 5'-terminal region of the C-ORF of the HBV genome, which can form a complete C-ORF sequence together with the sequence described in (ii); and the variant is located between two site-specific recombinase recognition sequences arranged in the same orientation.

[0068] In certain embodiments, the first luciferase fragment is a complementary sub-fragment capable of binding to LgBiT, such as HiBiT or SmBiT, and the second luciferase fragment is LgBiT. In certain embodiments, the first luciferase fragment is HiBiT and the second luciferase fragment is LgBiT. In certain embodiments, HiBiT has the sequence set forth in SEQ ID NO:2. In certain embodiments, the nucleotide sequence encoding HiBiT is set forth in SEQ ID NO:3.

[0069] In certain embodiments, the HBV genome is a full-length genome, for example, a genome of HBV genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, or genotype H. In certain embodiments, the HBV genome is an over-length genome, for example, a 1.1-fold genome or a 1.3-fold genome. In certain embodiments, the HBV genome is, for example, the 1.1-fold genome set forth in SEQ ID NO: 1.

[0070] In certain embodiments, the sequence of (iii) further comprises an X-ORF.

[0071] In certain embodiments, the sequence of (iii) comprises an HBV genome fragment with the C-ORF deleted.

[0072] In certain embodiments, the sequence of (iii) comprises the sequence set forth in SEQ ID NO:16.

[0073] In certain embodiments, the sequence of (ii) comprises a core gene and the sequence of (iv) comprises a pre-core gene. In certain embodiments, the sequence of (ii) comprises the sequence set forth in SEQ ID NO: 14. In certain embodiments, the sequence of (iv) comprises the sequence set forth in SEQ ID NO: 15.

[0074] In certain embodiments, the site-specific recombinase recognition sequence is selected from a loxP sequence or an FRT sequence.

[0075] In certain embodiments, the isolated nucleic acid molecule comprises the sequence set forth in SEQ ID NO:17.

[0076] 10. Recombinant HBV cccDNA The isolated nucleic acid molecule according to the ninth aspect of the invention may be circularised under the action of a recombinase to form cccDNA.

[0077] Thus, in a tenth aspect, the present invention also provides a recombinant HBV cccDNA formed by circularization of a variant of the HBV genomic sequence contained in the isolated nucleic acid molecule of the ninth aspect.

[0078] In certain embodiments, the recombinant HBV cccDNA is formed by circularizing the isolated nucleic acid molecule of the ninth aspect in the presence of a site-specific recombinase (e.g., Cre recombinase or FLP recombinase) that corresponds to the site-specific recombinase recognition sequence.

[0079] In certain embodiments, the recombinant HBV cccDNA comprises a C-ORF, an S-ORF, and a P-ORF, wherein the C-ORF comprises a nucleotide sequence encoding a first fragment of luciferase (e.g., HiBiT).

[0080] In certain embodiments, the recombinant HBV cccDNA further comprises an X-ORF.

[0081] In certain embodiments, the recombinant HBV cccDNA comprises a C-ORF comprising a nucleotide sequence encoding a first fragment of luciferase (e.g., HiBiT) and an HBV genome fragment from which the C-ORF has been removed (e.g., the sequence set forth in SEQ ID NO: 16).

[0082] In certain embodiments, the recombinant cccDNA comprises the sequence shown in SEQ ID NO:18.

[0083] 11. Vector In an eleventh aspect, the present invention also provides a vector comprising the isolated nucleic acid molecule of the ninth aspect.

[0084] In certain embodiments, the vector is a transposon-based vector, such as a PiggyBac transposon-based vector. In certain embodiments, the isolated nucleic acid molecule of the ninth aspect can be inserted into any commercially available PiggyBac transposon-based vector, such as PB-CMV-MCS-EF1α-RedPuro (catalog no. PB514B-1). In certain embodiments, the isolated nucleic acid molecule is located between two ITR sequences of the transposon-based vector.

[0085] 12. Co-transfection system In a twelfth aspect, the present invention also provides a co-transfection system comprising a vector described in the eleventh aspect and a transposase expression vector.

[0086] In certain embodiments, the transposase expression vector is a PiggyBac transposase expression vector, which is well known in the art and widely available commercially. In certain embodiments, the PiggyBac transposase expression vector is PB210PA-1 (System Biosciences).

[0087] 13.Host cells In a thirteenth aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the ninth aspect, or the recombinant cccDNA of the tenth aspect, or the vector of the eleventh aspect, or the co-transfection system of the twelfth aspect.

[0088] In certain embodiments, the host cell is a eukaryotic cell. In certain embodiments, the host cell supports the formation and transcription of functional HBV cccDNA.

[0089] In certain embodiments, the host cell is a eukaryotic cell derived from a liver cell, such as a hepatoma cell or a hepatocyte, hi certain embodiments, the host cell is selected from HepaRG, HepG2, or Huh7.

[0090] In certain embodiments, the host cell may also be a non-hepatic cell, so long as it supports hepadnavirus cccDNA formation (or, more broadly, hepadnavirus DNA replication). For example, such non-hepatic cells / hosts can be engineered to support hepadnavirus cccDNA formation (or hepadnavirus DNA replication) when viral pregenomic RNA is introduced into the cell or transcribed from a DNA template via an exogenous promoter.

[0091] In certain embodiments, the host cell comprises the isolated nucleic acid molecule of the ninth aspect in its genome.

[0092] In certain embodiments, the host cell can stably express recombinant HBV cccDNA formed by circularization of a variant of the HBV genomic sequence when a site-specific recombinase (e.g., Cre recombinase or FLP recombinase) corresponding to the site-specific recombinase recognition sequence is present.

[0093] 14. Kit In a fourteenth aspect, the present invention provides a kit comprising the isolated nucleic acid molecule of the ninth aspect, or the recombinant cccDNA of the tenth aspect, or the vector of the eleventh aspect, or the co-transfection system of the twelfth aspect, or the host cell of the thirteenth aspect.

[0094] In certain embodiments, the kit comprises a vector of the eleventh aspect or a co-transfection system of the twelfth aspect.

[0095] In certain embodiments, the kit comprises a host cell of the thirteenth aspect.

[0096] In certain embodiments, the kit further comprises an LgBiT protein. Optionally, the kit further comprises a luciferase substrate.

[0097] In certain embodiments, the kit further comprises a recombinase (eg, Cre recombinase or FLP recombinase) or a recombinase (eg, Cre recombinase or FLP recombinase) expression vector.

[0098] 15. Screening Method In a fifteenth aspect, the present invention provides a method for screening an HBV cccDNA inhibitor, comprising: (1) providing a host cell of the thirteenth aspect; wherein the host cell comprises in its genome an isolated nucleic acid molecule of the ninth aspect; (2) introducing the recombinase or a recombinase expression vector into a host cell; The recombinase corresponds to a site-specific recombinase recognition sequence contained in the host cell; (3) contacting the test agent with the host cell; (4) detecting the level of a first fragment of luciferase (e.g., HiBiT) in the cell supernatant of the host cell; The present invention provides a method comprising:

[0099] In certain embodiments, step (1) comprises: (1a) introducing an isolated nucleic acid molecule as described in the ninth aspect or a vector as described in the eleventh aspect into a host cell; (1b) culturing the host cells; Includes.

[0100] In certain embodiments, the host cell is selected from eukaryotic cells of liver cell origin, such as hepatoma cells or hepatocytes, preferably the host cell is selected from HepaRG, HepG2, or Huh7.

[0101] In certain embodiments, in step (1a), the expression vector is a transposon vector (e.g., a PiggyBac transposon vector), and the step further comprises introducing a transposase expression vector (e.g., a PiggyBac transposase expression vector) into the host cell.

[0102] In certain embodiments, in step (2), the mutant HBV genomic sequence contained in the host cell is circularized under the action of a recombinase to form cccDNA.

[0103] In certain embodiments, in step (4), the level of a first fragment of luciferase is detected by a luciferase fragment complementation assay (i.e., by providing a second fragment of luciferase that is structurally complementary to the first fragment and a luciferase substrate).

[0104] In certain embodiments, a second fragment of luciferase that is complementary to the first fragment of luciferase is used for detection.

[0105] In certain embodiments, the first fragment of luciferase is a complementary sub-fragment capable of binding to LgBiT, such as HiBiT or SmBiT, and the second fragment of luciferase is an LgBiT protein. In certain embodiments, the first fragment of luciferase is HiBiT and the second fragment of luciferase is an LgBiT protein.

[0106] In certain embodiments, the method comprises: (5) comparing the detection result of step (4) with the level of the first fragment of luciferase detected in the absence of the test agent; Here, if the detection result in step (4) is lower than the detection result obtained in the absence of the test agent, this indicates that the test agent is an HBV cccDNA inhibitor.

[0107] The present invention also relates to the use of the above-mentioned isolated nucleic acid molecules, vectors, co-transfection systems, host cells and kits for screening for HBV cccDNA inhibitors.

[0108] Definition of Terms In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory methods used herein, such as virology, cell culture, biochemistry, nucleic acid chemistry, immunology, etc., are all routine processes widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0109] As used herein, the term "hepatitis B virus (HBV)" refers to a member of the Hepadnaviridae family, which has a small, double-stranded DNA genome of approximately 3,200 base pairs and a tropism for hepatocellular carcinomas. "HBV" includes any hepatitis B virus that infects any of a variety of mammalian (e.g., humans, non-human primates, etc.) and avian (e.g., ducks) hosts. "HBV" includes any known HBV genotype, such as serotype A, serotype B, serotype C, serotype D, serotype E, serotype F, and serotype G; any HBV serotype or HBV subtype; any HBV isolate; HBV mutants, such as HBeAg-negative mutants; and drug-resistant HBV mutants (e.g., lamivudine-resistant mutants, adefovir-resistant mutants, tenofovir-resistant mutants, entecavir-resistant mutants, etc.).

[0110] As used herein, "HBV genome" includes not only full-length genomes (single-unit genomes) but also over-length HBV genomes (more than one-unit genomes, i.e., genomes longer than one unit). HBV genomes contain all the information required for the establishment and maintenance of HBV replication. These genome sequences are available from all genotypes in the literature and in GenBank. "Over-length HBV genomes" or "over-full-length HBV genomes" refer to sequences containing full-length genomes and portions of genomes, which may vary depending on the desired genome unit and specific HBV strain. Furthermore, methods for obtaining over-full-length HBV genomes and determining genome sequences have been described in the prior art, e.g., in European Patent No. EP 1543168.

[0111] In certain exemplary embodiments, HBV refers to human HBV, whose genome comprises four major overlapping open reading frames (ORFs): S-ORF, C-ORF, P-ORF, and X-ORF. S-ORF is divided into the S gene, the pre-S2 region, and the pre-S1 region, each with its own initiation codon ATG. C-ORF is divided into the C gene and the pre-C region, each with its own initiation codon ATG. P-ORF is the longest reading frame, whose initiation segment overlaps with C-ORF, whose middle segment overlaps with S-ORF, and whose end segment overlaps with X-ORF.

[0112] As used herein, "HBV genome fragment" refers to a portion of the HBV genome. The fragment may have at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, or 3200 consecutive nucleotides of the HBV genome. The fragment may also be a partial genome comprising one or more genes contained in the HBV genome, for example, the fragment may be a nucleic acid encoding the envelope protein, core / prenucleoprotein, x protein, and / or HBV polymerase protein. Additionally, the fragment may be a nucleic acid encoding one or more portions of the envelope protein, core / prenucleoprotein, x protein, and / or polymerase protein of HBV.

[0113] As used herein, the terms "covalently closed circular DNA" and "cccDNA" are well known in the art and are used interchangeably herein. Generally, "covalently closed circular DNA" or "cccDNA" refers to a replication intermediate of the hepadnavirus genome, which is a template for the synthesis of hepadnavirus mRNA and pregenomic RNA.

[0114] As used herein, the term "cccDNA inhibitor" means that it is capable of inhibiting the stability of cccDNA (i.e., reducing the stability of cccDNA), inhibiting the transcriptional activity of cccDNA (i.e., reducing the transcription of hepadnavirus mRNA using cccDNA as a transcription template), and / or inhibiting cccDNA formation (i.e., eliminating or reducing cccDNA formation).

[0115] As used herein, the term "variant" is used to refer to a polypeptide or polynucleotide that has a certain degree of amino acid / nucleotide sequence identity to a parent polypeptide sequence or polynucleotide. A variant is similar to the parent sequence but differs from the parent polypeptide or polynucleotide sequence by having at least one or several or more substitutions, deletions, or insertions in its amino acid or nucleotide sequence. In some cases, a variant has been treated and / or engineered to contain at least one substitution, deletion, or insertion in its amino acid or nucleotide sequence, thereby differing from the parent sequence. Furthermore, a variant may retain the functional characteristics or activity of the parent polypeptide or polynucleotide, for example, it may retain at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the biological activity of the parent polypeptide or polynucleotide.

[0116] As used herein, the term "recombinant" DNA molecule refers to a DNA molecule formed by laboratory methods of genetic recombination (e.g., molecular cloning) that brings together genetic material from multiple sources to produce a sequence not found in an organism. The term "site-specific recombination" refers to recombination between two nucleotide sequences, each containing at least one recognition site. "Site-specific" refers to a specific nucleotide sequence that may be located at a specific location in the genome of a host cell. The nucleotide sequence may be endogenous to the host cell, located in its natural location in the host genome or at another location in the genome, or it may be a heterologous nucleotide sequence that has been previously inserted into the host cell genome by any of a variety of known methods.

[0117] As used herein, the term "recombinase" refers to genetic recombinases, generally derived from bacteria and fungi, that catalyze orientation-dependent DNA exchange reactions between short (30-40 nucleotide) target site sequences specific for each recombinase.

[0118] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, thereby enabling the genetic material element sequence carried by the vector to be expressed in the host cell. Vectors are known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors can contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection element sequences, and reporter genes. Additionally, vectors can also contain an origin of replication site. Methods for introducing vectors and / or the nucleic acid molecules carried thereby into cells are known in the art, such as viral infection / transduction, conjugation, nanoparticle delivery, electroporation, particle gun technology, calcium phosphate precipitation, direct injection, etc. The choice of method generally depends on the type of cell to be transfected and the environment in which the transfection will be performed (i.e., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, Wiley & Sons, 1995.

[0119] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell such as E. coli or Bacillus subtilis, a fungal cell such as a yeast cell or an Aspergillus oryzae cell, an insect cell such as an S2 Drosophila cell or an Sf9 cell, or an animal cell such as a fibroblast, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK293 cell, or a human cell.

[0120] As used herein, the term "identity" refers to the degree of identity between two polypeptides or two nucleic acids. If two compared sequences have the same monomer of a base or amino acid subunit at a particular site (e.g., two DNA molecules each have an adenine at a particular site, or two polypeptides each have a lysine at a particular site), the two molecules are identical at that site. The percent identity between two sequences is a function of the number of identical sites shared by the two sequences times 100 relative to the total number of sites compared. For example, if six out of ten sites in two sequences are identical, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six sites are identical). Generally, two sequences are compared to maximize identity. Such alignments can be performed using computer programs, such as the Align program (DNAstar, Inc.), which is based on the method of Needleman et al. (J. Mol. Biol. 48:443-453, 1970). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which is incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The percentage of identity between two amino acid sequences can also be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which is incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0121] The 20 common amino acids referred to herein are described according to their conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala. [Effects of the Invention]

[0122] The HBV cccDNA inhibitor screening model of the present invention uses split luciferase as a surrogate marker for HBV cccDNA detection. Detection is simple and rapid, allowing for high-throughput drug screening. This detection can be used in many fields, including research, therapy, and diagnosis, and has broad application prospects and clinical value.

[0123]

[0023] The embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are used only to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments. [Brief explanation of the drawings]

[0124] [Figure 1] FIG. 1 shows a schematic diagram of the PiggyBac transposon vector used in the construction of stable integrated cell lines in Example 1. [Figure 2]FIG. 1 shows a schematic diagram of HBV modification of the reporter model, where the cccDNA generated during the HBV replication process is represented by HiBiT in Example 1. [Figure 3] FIG. 1 shows the viral replication and expression of HBV mutants incorporating HiBiT tags of various sequences, as evaluated in Example 1. [Figure 4] FIG. 1 shows the evaluation of RG-Hibit16 cells to screen for inhibitors that inhibit HBV cccDNA formation in Example 1. [Figure 5] FIG. 1 shows a schematic diagram and functional validation of HiBiT as a rcccDNA reporter model in Example 2. [Figure 6] FIG. 1 shows the screening of compounds targeting HBV cccDNA using RG-Hibit16 and G2-Rccc1 in Example 3. [Figure 7] FIG. 1 shows the verification of compounds having inhibitory effects on HBV cccDNA in a HepG2-hNTCP-2B1 infection model in Example 3. [Figure 8] FIG. 1 shows the verification of compounds having a promoting effect on HBV cccDNA in a HepG2-hNTCP-2B1 infection model in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0125] Sequence information Information about partial sequences relevant to the present invention is provided below.

[0126] [Table 1] [Example]

[0127] The invention will now be described with reference to the following examples, which are intended to illustrate but not limit the invention.

[0128] Unless otherwise specified, molecular biology experiments and immunoassays used in the present invention were essentially performed with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Refined Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995, and restriction enzymes were used according to the conditions recommended by the manufacturer. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the claimed invention.

[0129] The main detection methods involved in the following examples are described below:

[0130] Detection of HiBiT: Promega Corporation's Nano Glo HiBiT Extracellular Detection System (Cat. No. N2421) was used for detecting HiBiT, and the detection process was carried out according to the kit's instructions.

[0131] Detection of HBsAg / HBeAg: The detection procedures for hepatitis B surface antigen (chemiluminescence method CLEIA, product specification number: YZB / Guo 0346-2014) and e antigen (enzyme-linked immunosorbent assay ELISA, product specification number: YZB / Guo 0216-2013) were both carried out according to the detection methods of the kits from Beijing Wantai Company.

[0132] Detection of HBV DNA: For HBV DNA extraction, the collected cells were washed with PBS and then subjected to automated extraction using a viral DNA & RNA extraction kit (Beijing GenMagBio) on a nucleic acid extraction workstation. HBV DNA was quantified using a Roche LightCycler™ 96 instrument with Premix Ex Taq™ (Takara Bio Inc.) for the probe method. The primer sequences used are listed in Table 1.

[0133] [Table 2]

[0134] Detection of HBV cccDNA: A modified Hirt method was used for HBV cccDNA extraction using the Tiangen Plasmid Mini Kit. The relevant lysis buffers were Buffer I (50 mM Tris, 10 mM EDTA, pH 7.5), Buffer II (1.2% SDS), and Buffer III (3 M CsCl, 1 M potassium acetate, 0.67 M acetic acid), which were used instead of P1, P2, and P3, respectively, in the Tiangen Plasmid Mini Kit. The extraction steps and methods were performed by referring to the plasmid extraction method in the kit. The primers used for fluorescence quantification are listed in Table 1. The instrument used was a Roche LightCycler™ 96. HBV cccDNA and mitochondrial DNA (mtDNA) were quantified, and the relative values ​​of HBV cccDNA and mtDNA were calculated.

[0135] HBV DNA was detected by DNA immunoblotting as follows: DNA extraction: After treatment, the cells were washed once with PBS and lysed in NET buffer (50 mM Tris (pH 8.0), 1 mM EDTA, 100 mM NaCl, 0.5% NP-40) at 4°C for 1 hour. The cell lysate supernatant was collected and added with 33 μg / mL micrococcal nuclease and 6 mM CaCl2 (final concentrations). The cells were then placed in a 37°C water bath for 30 minutes. 25 mM EDTA was added and the cells were placed in a 65°C water bath for 15 minutes. 200 μg / mL proteinase K and 0.5% SDS (final concentrations) were added and the cells were placed in a 50°C water bath for 12 hours. DNA was extracted with phenol-chloroform. Detection: DNA was separated by electrophoresis in 1.2% agarose for 2 hours. The gel was then sequentially treated with 0.2 N HCl, 0.5 M NaOH / 1.5 M NaCl, and 1 M Tris-HCl to denature the DNA. The nucleic acids were then transferred to a nylon membrane using a vacuum blotter. The nucleic acids were fixed by UV crosslinking, followed by prehybridization and hybridization. Excess probe was washed away, blocking was performed by adding a blocking solution, followed by addition of anti-Dig-Ap antibody, and finally CDP-star was added for color development. The target strips were then detected by sequential exposure to light.

[0136] Example 1: Construction of a reporter model showing cccDNA generated during HBV replication using HiBiT In this example, the PiggyBac transposon system PB-CMV-MCS-EF1α-RedPuro (catalog number PB514B-1) was used as the vector. When this vector was cotransfected with PiggyBac transposase (System Biosciences, PB210PA-1), the sequence between the two "ITR sequences" on the vector plasmid was integrated into the cell genome, achieving integration of the target gene. To achieve controlled expression of HBV, the "CMV promoter" on the vector was replaced with the "TRE3G promoter" (Takara Bio Inc., Tet-On 3G Inducible Expression System). Because this promoter requires the combination of doxycycline and the Tet-On 3G transactivator to initiate transcription, target protein expression could be regulated by doxycycline. To avoid partial loss of the target sequence during integration, we selected the TRE3G promoter, which has bidirectional promoter activity, and introduced an iRFP fluorescent marker and a blasticidin resistance selection marker at the N-terminus. Dual resistance and dual fluorescence were used as screening conditions for integrated cells. Because TRE3G promoter transcription requires the Tet-On 3G protein, an expression cassette was introduced into the vector to express the Tet-On 3G protein. The final vector is shown in Figure 1. The sequence enclosed in the red dashed box is the sequence integrated into the cell genome. "GOI" stands for the ligated target gene (gene of interest). In this example, the ligated sequence is the HBV 1.1x genome sequence into which a reporter gene was inserted.

[0137] Because the open reading frames of the HBV genome are highly overlapping, HBV modification imposes strict limitations on the location of foreign gene insertion. In this example, HiBiT-containing HBV mutants were prepared by inserting various copy numbers of HiBiT sequences connected by various linker peptides between the precore and core regions. A schematic diagram of the insertion is shown in Figure 2. The HiBiT signal, viral protein expression, and viral replication of these insertion mutants were verified. Various HBV mutants were transfected into the hepatoma cell lines HepG2 and Huh7, respectively, and the HiBiT signal, expression of the HBV antigens HBsAg and HBeAg in the cell supernatant (A), and viral replication (B) were detected by Southern blot analysis. The results are shown in Figure 3. Hibit16, an insertion mutant with three copies of the HiBiT tag (SEQ ID NO: 4), was found to be a better choice. A decrease in the HiBiT copy number may weaken the HiBiT signal, while an increase in the HiBiT copy number may affect viral protein expression or replication. When various linker peptides were compared, inserting the peptide "GSG" before and after the HiBiT sequence was superior to inserting the peptide "G" at one end or no linker peptide at all. Therefore, in this example, we constructed HepaRG cells stably incorporating the HBV mutant Hibit16 and screened for inhibitors that could inhibit cccDNA formation.

[0138] HepaRG cells were transfected with the Hibit16 plasmid and PiggyBac transposase to integrate the HBV mutant sequence (Hibit16) and a selectable marker into the cell genome, resulting in HepaRG-Hibit16 cells, which were screened for puromycin resistance and red fluorescent markers. As shown in Figure 4A, doxycycline activated the TRE3G promoter, initiating not only iRFP670 expression but also HBV transcription and replication, thereby generating HiBiT. To evaluate the function of HBV inhibitors in cells, cells were first seeded and doxycycline was added to induce viral transcription and replication, while various HBV inhibitors were added to control the effects. Cell supernatants were collected every two days, and the medium was replaced. The HiBiT signal, HBsAg, and HBeAg in the cell supernatant, as well as HBV DNA and HBV cccDNA in the cell lysates, were detected. The cellular responses to HBV inhibitors are shown in Figures 4B–F. Tenofovir (tenofovir disoproxil fumarate, TDF) is a nucleotide reverse transcriptase inhibitor, and morphotiazine (Mor) is a core particle assembly regulator. Both are currently undergoing clinical Phase II / III evaluation and can inhibit cccDNA formation. In the cell model constructed in this study, TDF and Mor were able to inhibit cccDNA formation, and HiBiT expression levels in the Mor-treated group were significantly lower than in the control group, indicating that this model can be used to screen inhibitors such as Mor that inhibit cccDNA formation.

[0139] Example 2: Construction of a reporter model for recombinant rcccDNA revealed by HiBiT In this example, we used HiBiT to construct a reporter model representing recombinant cccDNA. Recombinant cccDNA, or rcccDNA, was a closed circular DNA formed by circularizing linear HBV DNA using the Cre / loxP recombinase system. The HBV mutant shown in Figure 5A was constructed. Here, the C-terminal partial sequence of the HBV core ORF (SEQ ID NO: 14) was placed at the N-terminus of the entire replicon, while the N-terminal partial sequences of the core ORF and the core promoter (SEQ ID NO: 15) were placed at the C-terminus of the replicon. The reporter gene sequence, HiBiT, was placed at the N-terminus of the replicon, and loxP sequences (SEQ ID NO: 19) were added before and after the replicon. Without Cre recombinase expression, HiBiT lacked a promoter and could not be expressed. However, after rcccDNA formation, HiBiT was able to express HBeAg and HBcAg fused with the HiBiT tag using the core promoter. Therefore, HiBiT could be used as a surrogate marker for rcccDNA detection. The HBV mutant was constructed using the PiggyBac transposon system as a vector, which facilitated integration of the HBV mutant containing the target gene (i.e., a reporter gene) into the cellular genome. This clone was designated Rccc1a. HepG2 cells were transfected with this plasmid, and after 6 hours, the medium was replaced and infected with the adenovirus Adv-Cre to express Cre recombinase (SEQ ID NO: 20). After 48 hours, HiBiT was detected in the cell supernatant, and the cells were lysed. The lysate was used as a template to amplify a partial sequence of the rcccDNA before and after the loxP sequence to verify the formation of the expected rcccDNA. The amplification and sequencing primers were cccDNA-F and cccDNA-R (Table 1). Figure 5B shows the results of HiBiT detection in the cell supernatant of HepG2 cells transfected with Rccc1a with or without Cre recombinase expression, and Figure 5C shows the sequencing results of rcccDNA formed after Cre recombinase expression in HepG2 cells transfected with Rccc1a.The above results demonstrated that Cre recombinase could generate the predicted rcccDNA and initiate the expression of a HiBiT-tagged protein.

[0140] Example 3: Use of reporter models to screen cccDNA inhibitors The reporter model prepared in Example 1 included an HBV mutant incorporating three repeats of the HiBiT sequence. mRNA directly transcribed from the HBV mutant as a template lacked the initiation codon for HiBiT expression and could not translate the protein attached to the HiBiT tag. Only after reverse transcription of pgRNA transcribed from the HBV mutant to form cccDNA could mRNA transcribed from the cccDNA as a template translate the protein attached to the HiBiT tag, so the expression level of HiBiT could be used to indicate the formation of HBV cccDNA. In the reporter model prepared in Example 2, the HiBiT sequence was inserted into the HBV core sequence, and the N- and C-terminal sequences of the core were attached to the C- and N-termini of the HBV replicon, respectively. LoxP sequences were ligated to both ends of the HBV replicon. Therefore, without Cre recombinase expression, the HiBiT tag lacked a promoter and could not be expressed. However, with Cre recombinase expression, it could mediate recombination of double-stranded DNA, causing the linear HBV genomic DNA to form a closed circular DNA. After the circular DNA was formed, the HiBiT tag could initiate expression of the protein attached to the HiBiT tag using the HBV endogenous promoter. Thus, the signal from the HiBiT tag could be used to indicate the formation of recombinant HBV cccDNA, i.e., HBV rcccDNA.

[0141] In this example, we investigated the reporter model of Example 1 (HepaRG-Hibit16, constructed based on HepaRG cells and incorporating Hibit16) and the reporter model of Example 2 (HepG2-Rccc1a, constructed based on HepG2 cells and incorporating Rccc1a) to screen for cccDNA inhibitors. Using these two cell models, we evaluated the ability of 189 drugs, including clinical drugs or drugs under clinical research for various diseases, to inhibit cccDNA. Screening was performed in 96-well cell culture plates. RG-Hibit16 cells were seeded at a cell density of 15,000 cells / well, and G2-Rccc1a cells were seeded at a cell density of 35,000 cells / well. RG-Hibit16 cells were treated with drugs one week after seeding. For RG-Hibit16, various compounds were added for treatment when Dox was added to induce viral expression. The day after seeding, G2-Rccc1a cells were infected with the adenovirus Adv-Cre to express the Cre recombinase protein. Two days later, various compounds were added for treatment. All compounds were diluted to a final concentration of 1 μM (200 μL per well). Fresh medium was replaced every two days. HiBiT expression in the cell supernatant was detected 4 days after compound treatment, and CCK8 detection reagent was added at a 10:1 ratio to detect compound cytotoxicity. The results of compound screening using RG-Hibit16 and G2-Rccc1a cells are shown in Figure 6. Among the 189 compounds, the three compounds that most significantly inhibited HiBiT expression in RG-Hibit16 cells were cetylpyridinium, guanfacine, and palovarotene. The four compounds that most significantly inhibited HiBiT expression in G2-Rccc1a cells were crizotinib, doxifluridine, palovarotene, and dithranol. These six drugs had no obvious cytotoxicity to the two cell types based on CCK8 detection. Palovarotene showed inhibitory effects on HBV cccDNA in both models.

[0142] The functions of these six compounds were further validated in the HepG2-hNTCP-2B1 infection model. HepG2-hNTCP-2B1 was a single clone (2B1) selected for its highest susceptibility to HBV, generated by overexpressing hNTCP in HepG2 cells. In this model, cells were first infected with HBV virus, washed with PBS the next day to remove residual virus, and then added to the cells two days later. The cell supernatant was then collected and replaced with fresh medium every two days. Viral antigens were detected in the cell supernatant. Eight days after infection, cells were lysed and intracellular HBV cccDNA was detected. The results are shown in Figure 7. While the inhibitory effect of palovarotene on HBsAg was relatively weak, the inhibitory rates for both HBsAg and HBV cccDNA were all above 60%, which was better than those of the other five compounds. Furthermore, doxifluridine also had a significant inhibitory effect on cccDNA in this infection model. Based on the evaluation results in RG-Hibit16 and G2-Rccc1a cells, five compounds were also selected that showed up-regulatory effects on HBV cccDNA in both models: vincristine, naftopidil, cinchophen, mebhydroline, and cladribine. The function of these five compounds was verified in the HepG2-hNTCP-2B1 infection model, using the same evaluation strategy as the inhibitors described above. The results are shown in Figure 8. The five compounds had a weak promoting effect on HBeAg, with vincristine and cladribine significantly up-regulating HBsAg expression levels, and vincristine, cinchophen, mebhydroline, and cladribine up-regulating intracellular cccDNA levels. The results of the infection model validation demonstrated that the two cccDNA reporter models constructed in this study can be used to screen for compounds that can promote or inhibit cccDNA. Although some of the screened compounds did not show corresponding effects in the infection model, the range of candidate compounds was significantly narrowed, allowing them to be used for preliminary screening of compounds targeting HBV cccDNA.

[0143] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand, in light of all the teachings published, that various modifications and changes can be made to the details and that all such modifications are within the scope of the present invention, the full extent of which is given by the appended claims and their equivalents. This specification discloses the following inventions. [1] 1. An isolated nucleic acid molecule comprising a variant of an HBV genome sequence, wherein the variant comprises an HBV genome fragment including a C-ORF, an S-ORF, and a P-ORF, wherein the C-ORF comprises an exogenous insertion sequence between a precore gene and a core gene, and wherein the exogenous insertion sequence comprises a nucleotide sequence encoding a first fragment of luciferase, wherein the first fragment of luciferase is capable of binding to a corresponding second fragment of luciferase in a luciferase fragment complementation assay (LFCA), thereby generating luciferase activity. [2] The isolated nucleic acid molecule described in [1], wherein the HBV genome fragment further contains an X-ORF. [3] The isolated nucleic acid molecule according to [1] or [2], wherein the mutant comprises the exogenous insertion sequence between the precore gene and the core gene of the HBV genome sequence. [4] a first fragment of said luciferase is a complementary sub-fragment capable of binding to LgBiT, such as HiBiT or SmBiT, and a second fragment of said luciferase is LgBiT; Preferably, the first fragment of luciferase is HiBiT, and the second fragment of luciferase is LgBiT. [5] the exogenous insert sequence comprises multiple copies of a nucleotide sequence encoding a first fragment of the luciferase (e.g., HiBiT) in tandem repeats; Preferably, the exogenous insertion sequence comprises three copies of a nucleotide sequence encoding a first fragment of the luciferase (e.g., HiBiT) in tandem repeats. [4] The isolated nucleic acid molecule of [4]. [6] An isolated nucleic acid molecule according to [5], wherein each copy of the multiple copies of the nucleotide sequence encoding the first fragment of the luciferase (e.g., HiBiT) in the tandem repeat contains a sequence encoding a linker peptide (e.g., a flexible peptide linker) at its 5' end. [7] The isolated nucleic acid molecule according to [1], wherein the exogenous insertion sequence comprises the sequence shown in SEQ ID NO: 4. [8] the HBV genome is a full-length genome or an over-length genome such as a 1.1-fold genome or a 1.3-fold genome, Preferably, the HBV genome comprises the sequence shown in SEQ ID NO: 1, and the isolated nucleic acid molecule according to any one of [1] to [7]. [9] the isolated nucleic acid molecule further comprises an inducible promoter operably linked to the exogenous insert sequence; Preferably, the inducible promoter is the TRE3G promoter or comprises one or more repeats of the Tet operator sequence (TetO); Preferably, the inducible promoter has bidirectional activation activity, for example, the TRE3G promoter, which has bidirectional activation activity.

[0023] The isolated nucleic acid molecule according to any one of [1] to [8].

[10] the isolated nucleic acid molecule further comprises a reporter gene operably linked to the inducible promoter; Preferably, the reporter gene is in the opposite orientation to the exogenous insert sequence; Preferably, the reporter gene is selected from a fluorescent protein gene (e.g., iRFP) and / or an antibiotic resistance gene (e.g., blasticidin); Preferably, the reporter gene comprises a fluorescent protein gene and an antibiotic resistance gene; Preferably, the fluorescent protein gene and the antibiotic resistance gene are optionally linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, or T2A).

[11] The isolated nucleic acid molecule according to [9] or

[10] , wherein the isolated nucleic acid molecule comprises the sequence shown in SEQ ID NO: 8.

[12] A recombinant HBV cccDNA comprising the isolated nucleic acid molecule according to any one of [1] to

[11] , Preferably, the recombinant HBV cccDNA comprises a variant of the HBV genome sequence described in any one of [1] to

[11] , Preferably, the recombinant HBV cccDNA is formed by circularization of the isolated nucleic acid molecule according to any one of [1] to

[11] .

[13] An expression system comprising the isolated nucleic acid molecule according to any one of [9] to

[11] as a first nucleic acid sequence, and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a transactivator corresponding to the inducible promoter contained in the first nucleic acid sequence, Preferably, the transactivator is selected from Tet-On 3G transactivator, rTetR, rtTA; Preferably, the second nucleic acid sequence further comprises an expression control element sequence such as a promoter (e.g., a constitutive promoter) and / or an enhancer operably linked to the nucleotide sequence encoding the transactivator.

[14] the first nucleic acid sequence comprises a TRE3G promoter as an inducible promoter, and the second nucleic acid sequence comprises a nucleotide sequence encoding a Tet-On 3G transactivator; Preferably, the TRE3G promoter comprises the sequence shown in SEQ ID NO: 5, Preferably, the nucleotide sequence encoding the Tet-On 3G transactivator comprises the sequence shown in SEQ ID NO: 10.

[15] A vector comprising the isolated nucleic acid molecule according to any one of [1] to

[11] , or the expression system according to

[13] or

[14] .

[16] The vector comprises the expression system according to

[13] or

[14] , wherein the first nucleic acid sequence and the second nucleic acid sequence are provided on the same or different vectors; Preferably, the first nucleic acid sequence and the second nucleic acid sequence are provided on the same vector.

[15] The vector according to

[15] .

[17] the vector is a transposon vector such as a PiggyBac transposon vector, Preferably, the first nucleic acid sequence and the second nucleic acid sequence are located between two ITR sequences of the transposon-based vector.

[18] A co-transfection system comprising the vector according to any one of

[15] to

[17] and a transposase expression vector, Preferably, the transposase expression vector is a PiggyBac transposase expression vector.

[19] A host cell comprising the isolated nucleic acid molecule according to any one of [1] to

[11] , the recombinant cccDNA according to

[12] , the expression system according to

[13] or

[14] , the vector according to any one of

[15] to

[17] , or the co-transfection system according to

[18] , Preferably, said host cell is selected from a eukaryotic cell derived from a liver cell such as a hepatoma cell or a hepatocyte, preferably said host cell is selected from HepaRG, HepG2, or Huh7.

[20] The host cell contains the expression system according to

[13] or

[14] in its genome; Preferably, the host cell is capable of stably expressing HBV cccDNA formed from a mutant of the HBV genomic sequence in the presence of an inducer (e.g., doxycycline) and a transactivator corresponding to the inducible promoter.

[21] A kit comprising an isolated nucleic acid molecule according to any one of [1] to

[11] , an expression system according to

[13] or

[14] , a vector according to any one of

[15] to

[17] , a co-transfection system according to

[18] , or a host cell according to

[19] or

[20] , Preferably, the kit includes the vector according to any one of

[15] to

[17] or the co-transfection system according to

[18] ; Preferably, the kit comprises the host cell according to

[19] or

[20] , Preferably, the kit further comprises an LgBiT protein and optionally a luciferase substrate; Preferably, the kit further comprises an inducer (e.g., doxycycline) and a transactivator corresponding to the inducible promoter.

[22] A method for screening an HBV cccDNA inhibitor, comprising: (1) Providing a host cell according to

[20] ; (2) contacting the host cell with an inducer; The inducing agent is an inducer (e.g., doxycycline) corresponding to the inducible promoter and a transactivator contained in the host cell; (3) contacting a test agent with the host cells; It should be noted that steps (2) and (3) may be performed simultaneously or in any order; (4) detecting the level of the first fragment of luciferase in the cell supernatant of the host cell; A method comprising:

[23] Step (1) comprises the following steps: (1a) introducing the first nucleotide sequence and the second nucleotide sequence in the expression system according to

[13] or

[14] into the host cell; The first nucleotide sequence and the second nucleotide sequence are provided on the same or different expression vectors, and the first nucleic acid sequence is an isolated nucleic acid molecule according to any one of [9] to

[11] ; (1b) culturing the host cells; Including, Preferably, the host cell is selected from a eukaryotic cell derived from a liver cell, such as a hepatoma cell or a hepatocyte, preferably the host cell is selected from HepaRG, HepG2, or Huh7; Preferably, in step (1a), the expression vector is a transposon vector (e.g., a PiggyBac transposon vector), and this step further comprises introducing a transposase expression vector (e.g., a PiggyBac transposase expression vector) into the host cell; Preferably, the step (1) further comprises a step of identifying and selecting a host cell in which the expression system according to (1c)

[13] or

[14] has been integrated into the genome, and preferably, whether the expression system has been integrated into the genome of the host cell is determined by detecting a reporter gene contained in the first nucleic acid sequence.

[24] In step (4), the level of the first fragment of luciferase is detected by a luciferase fragment complementation assay; Preferably, said detection is carried out using a second fragment of luciferase which is complementary to said first fragment of luciferase, Preferably, the first fragment of luciferase is a complementary sub-fragment capable of binding to LgBiT, such as HiBiT or SmBiT, and the second fragment of luciferase is an LgBiT protein, preferably, the first fragment of luciferase is HiBiT and the second fragment of luciferase is the LgBiT protein. The method of

[22] or

[23] .

[25] (5) comparing the detection result of step (4) with the level of the first fragment of luciferase detected in the absence of the test agent; The method according to any one of

[22] to

[24] , wherein if the detection result in step (4) is lower than the detection result in the absence of the test agent, this indicates that the test agent is an HBV cccDNA inhibitor.

[26] 1. An isolated nucleic acid molecule comprising a variant of an HBV genomic sequence, said variant comprising, in the 5' to 3' direction: (i) a nucleotide sequence encoding a first fragment of luciferase; wherein the first fragment of luciferase can bind to a corresponding second fragment of luciferase in a luciferase fragment complementation assay (LFCA), thereby generating luciferase activity; (ii) the sequence of the 3'-terminal region of the C-ORF of the HBV genome; (iii) an HBV genome fragment containing the S-ORF and the P-ORF; (iv) a sequence of the 5'-terminal region of the C-ORF of the HBV genome, which can form a complete C-ORF sequence together with the sequence described in (ii); and wherein the variant is located between two site-specific recombinase recognition sequences arranged in the same orientation.

[27] the HBV genome is a full-length genome or an over-length genome such as a 1.1-fold genome or a 1.3-fold genome, Preferably, the HBV genome comprises the sequence shown in SEQ ID NO: 1.

[26] The isolated nucleic acid molecule according to

[26] .

[28] The sequence (iii) further comprises an X-ORF, Preferably, the sequence (iii) comprises an HBV genome fragment from which the C-ORF has been deleted, Preferably, the sequence (iii) comprises the sequence shown in SEQ ID NO: 16.

[26] or

[27] .

[29] The sequence (ii) comprises a core gene, and the sequence (iv) comprises a precore gene; Preferably, the sequence (ii) comprises the sequence shown in SEQ ID NO: 14, Preferably, the sequence (iv) comprises the sequence shown in SEQ ID NO: 15, in the isolated nucleic acid molecule according to any one of

[26] to

[28] .

[30] The isolated nucleic acid molecule according to any one of

[26] to

[29] , wherein the site-specific recombinase recognition sequence is selected from a loxP sequence or an FRT sequence.

[31] a first fragment of said luciferase is a complementary sub-fragment capable of binding to LgBiT, such as HiBiT or SmBiT, and a second fragment of said luciferase is LgBiT; Preferably, the first fragment of luciferase is HiBiT, and the second fragment of luciferase is LgBiT.

[0023] The isolated nucleic acid molecule according to any one of

[26] to

[30] .

[32] An isolated nucleic acid molecule according to any one of

[26] to

[31] , comprising the sequence shown in SEQ ID NO: 17.

[33] A recombinant HBV cccDNA formed by circularization of a mutant of the HBV genomic sequence in the isolated nucleic acid molecule according to any one of

[26] to

[32] , Preferably, the recombinant HBV cccDNA is formed by circularizing the isolated nucleic acid molecule according to any one of

[26] to

[32] in the presence of a site-specific recombinase (e.g., Cre recombinase or FLP recombinase) corresponding to the site-specific recombinase recognition sequence; Preferably, the recombinant HBV cccDNA comprises a C-ORF, an S-ORF, and a P-ORF, wherein the C-ORF comprises a nucleotide sequence encoding a first fragment of the luciferase (e.g., HiBiT); Preferably, the recombinant HBV cccDNA further comprises an X-ORF, Preferably, the recombinant HBV cccDNA comprises a C-ORF comprising a nucleotide sequence encoding a first fragment of the luciferase (e.g., HiBiT) and an HBV genome fragment from which the C-ORF has been deleted; Preferably, the recombinant cccDNA comprises the sequence shown in SEQ ID NO:18.

[34] A vector comprising the isolated nucleic acid molecule according to any one of

[26] to

[32] .

[35] a transposon vector such as a PiggyBac transposon vector, Preferably, the isolated nucleic acid molecule is located between two ITR sequences of the transposon-based vector.

[36] A co-transfection system comprising the vector according to

[35] and a transposase expression vector, Preferably, the transposase expression vector is a PiggyBac transposase expression vector.

[37] A host cell comprising the isolated nucleic acid molecule according to any one of

[26] to

[32] , the recombinant cccDNA according to

[33] , the vector according to

[34] or

[35] , or the cotransfection system according to

[36] , Preferably, said host cell is selected from a eukaryotic cell derived from a liver cell such as a hepatoma cell or a hepatocyte, preferably said host cell is selected from HepaRG, HepG2, or Huh7.

[38] The host cell comprises in its genome the isolated nucleic acid molecule according to any one of

[26] to

[32] ; Preferably, the host cell is capable of stably expressing the recombinant HBV cccDNA formed by circularizing a mutant of the HBV genomic sequence in the presence of a site-specific recombinase (e.g., Cre recombinase or FLP recombinase) corresponding to the site-specific recombinase recognition sequence.

[39] A kit comprising the isolated nucleic acid molecule according to any one of

[26] to

[32] , or the recombinant cccDNA according to

[33] , or the vector according to

[34] or

[35] , or the cotransfection system according to

[36] , or the host cell according to

[37] or

[38] , Preferably, the kit comprises the vector according to

[34] or

[35] , or the co-transfection system according to

[36] , Preferably, the kit comprises the host cell according to

[37] or

[38] , Preferably, the kit further comprises an LgBiT protein and optionally a luciferase substrate; Preferably, the kit further comprises a recombinase (e.g., Cre recombinase or FLP recombinase) or a recombinase (e.g., Cre recombinase or FLP recombinase) expression vector.

[40] A method for screening an HBV cccDNA inhibitor, comprising: (1) Providing a host cell according to

[38] ; (2) introducing a recombinase or a recombinase expression vector into the host cell; wherein the recombinase corresponds to a site-specific recombinase recognition sequence contained in the host cell; (3) contacting a test agent with the host cells; (4) detecting the level of the first fragment of luciferase in the cell supernatant of the host cell; A method comprising:

[41] Step (1) is (1a) introducing the isolated nucleic acid molecule according to any one of

[26] to

[32] or the vector according to

[34] or

[35] into the host cell; (1b) culturing the host cells; Including, Preferably, the host cell is selected from a eukaryotic cell derived from a liver cell, such as a hepatoma cell or a hepatocyte, preferably the host cell is selected from HepaRG, HepG2, or Huh7; Preferably, in step (1a), the expression vector is a transposon vector (e.g., a PiggyBac transposon vector), and this step further comprises introducing a transposase expression vector (e.g., a PiggyBac transposase expression vector) into the host cell.

[40]

[42] In step (4), the level of the first fragment of luciferase is detected by a luciferase fragment complementation assay; Preferably, said detection is carried out using a second fragment of luciferase which is complementary to said first fragment of luciferase, Preferably, the first fragment of luciferase is a complementary sub-fragment capable of binding to LgBiT, such as HiBiT or SmBiT, and the second fragment of luciferase is an LgBiT protein, preferably, the first fragment of luciferase is HiBiT and the second fragment of luciferase is the LgBiT protein. The method of

[40] or

[41] .

[43] (5) comparing the detection result of step (4) with the level of the first fragment of luciferase detected in the absence of the test agent; The method according to any one of

[40] to

[42] , wherein if the detection result in step (4) is lower than the detection result in the absence of the test agent, this indicates that the test agent is an HBV cccDNA inhibitor.

[44] Use of an isolated nucleic acid molecule according to any one of [1] to

[11] , or an expression system according to

[13] or

[14] , or a vector according to any one of

[15] to

[17] , or a co-transfection system according to

[18] , or a host cell according to

[19] or

[20] , or a kit according to

[21] , or an isolated nucleic acid molecule according to any one of

[26] to

[32] , or a recombinant cccDNA according to

[33] , or a vector according to

[34] or

[35] , or a co-transfection system according to

[36] , or a host cell according to

[37] or

[38] , or a kit according to

[39] , for screening for an HBV cccDNA inhibitor.

Claims

1. An isolated nucleic acid molecule comprising a variant of an HBV genome sequence, said variant comprising an HBV genome fragment including a C-ORF, an S-ORF, and a P-ORF, said C-ORF comprising an exogenous insertion sequence between a pre-core gene and a core gene, said exogenous insertion sequence comprising a nucleotide sequence encoding a first fragment of luciferase, said first fragment of luciferase being capable of binding to a corresponding second fragment of luciferase in a luciferase fragment complementation assay (LFCA), thereby producing luciferase activity; An isolated nucleic acid molecule, wherein the first fragment of luciferase is HiBiT consisting of the sequence represented by SEQ ID NO: 2, and the exogenous insertion sequence comprises three copies of a nucleotide sequence encoding the first fragment of luciferase in tandem repeats.

2. 2. The isolated nucleic acid molecule of claim 1, wherein the HBV genome fragment further comprises an X-ORF.

3. 3. The isolated nucleic acid molecule of claim 1, wherein the variant comprises the exogenous insertion sequence between the precore gene and the core gene of the HBV genome sequence.

4. 2. The isolated nucleic acid molecule of claim 1, wherein each copy of the three copies of the nucleotide sequence encoding the first fragment of the luciferase in the tandem repeat contains a sequence encoding a linker peptide at its 5' end.

5. The isolated nucleic acid molecule of claim 4 , wherein the linker peptide is a flexible peptide linker.

6. 2. The isolated nucleic acid molecule of claim 1, wherein the exogenous insert sequence comprises the sequence shown in SEQ ID NO:

4.

7. The isolated nucleic acid molecule of any one of claims 1 to 6, wherein the HBV genome sequence is a full-length genome sequence or an over-length genome sequence.

8. 8. The isolated nucleic acid molecule of claim 7, wherein the HBV genomic sequence is a 1.1-fold genomic sequence or a 1.3-fold genomic sequence.

9. 8. The isolated nucleic acid molecule of claim 7, wherein the HBV genome sequence comprises the sequence set forth in SEQ ID NO:

1.

10. 10. The isolated nucleic acid molecule of claim 1, further comprising an inducible promoter operably linked to the exogenous insert sequence.

11. 11. The isolated nucleic acid molecule of claim 10, wherein the inducible promoter is a TRE3G promoter or comprises one or more repeats of a Tet operator sequence (TetO).

12. The isolated nucleic acid molecule of claim 10 , wherein the inducible promoter has bidirectional activation activity.

13. The isolated nucleic acid molecule of claim 10 , further comprising a reporter gene operably linked to the inducible promoter.

14. 14. The isolated nucleic acid molecule of claim 13, wherein the reporter gene is in the opposite orientation to the exogenous insert sequence.

15. 14. The isolated nucleic acid molecule of claim 13, wherein the reporter gene is selected from a fluorescent protein gene and / or an antibiotic resistance gene.

16. 14. The isolated nucleic acid molecule of claim 13, wherein the reporter gene comprises a fluorescent protein gene and an antibiotic resistance gene, and the fluorescent protein gene and the antibiotic resistance gene are linked by a nucleotide sequence encoding a self-cleaving peptide.

17. 11. The isolated nucleic acid molecule of claim 10, wherein the isolated nucleic acid molecule comprises the sequence set forth in SEQ ID NO:

8.

18. A recombinant HBV cccDNA comprising the isolated nucleic acid molecule of any one of claims 1 to 17.

19. 18. An expression system comprising the isolated nucleic acid molecule of any one of claims 10 to 17 as a first nucleic acid sequence, and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a transactivator corresponding to the inducible promoter contained in the first nucleic acid sequence.

20. 20. The expression system of claim 19, wherein the transactivator is selected from the group consisting of Tet-On 3G transactivator, rTetR, and rtTA.

21. 20. The expression system of claim 19, wherein the second nucleic acid sequence further comprises an expression control element sequence operably linked to the nucleotide sequence encoding the transactivator.

22. 20. The expression system of claim 19, wherein the first nucleic acid sequence comprises a TRE3G promoter as an inducible promoter, and the second nucleic acid sequence comprises a nucleotide sequence encoding a Tet-On 3G transactivator.

23. 23. The expression system of claim 22, wherein the TRE3G promoter comprises the sequence shown in SEQ ID NO:

5.

24. 23. The expression system of claim 22, wherein the nucleotide sequence encoding the Tet-On 3G transactivator comprises the sequence shown in SEQ ID NO:

10.

25. A vector comprising an isolated nucleic acid molecule according to any one of claims 1 to 17 or an expression system according to any one of claims 19 to 24.

26. 26. The vector of claim 25, wherein the vector comprises the expression system of any one of claims 19 to 24, wherein the first nucleic acid sequence and the second nucleic acid sequence are provided on the same or different vectors.

27. The vector of claim 25 or 26, wherein the vector is a transposon-based vector.

28. A co-transfection system comprising the vector according to any one of claims 25 to 27 and a transposase expression vector.

29. A host cell comprising an isolated nucleic acid molecule according to any one of claims 1 to 17, or a recombinant cccDNA according to claim 18, or an expression system according to any one of claims 19 to 24, or a vector according to any one of claims 25 to 27, or a co-transfection system according to claim 28.

30. 30. The host cell of claim 29, wherein the host cell is selected from eukaryotic cells derived from hepatocytes.

31. 30. The host cell of claim 29, wherein the host cell is a hepatoma cell or a hepatocyte.

32. 30. The host cell of claim 29, wherein the host cell is selected from HepaRG, HepG2, or Huh7.

33. The host cell according to any one of claims 29 to 32, wherein the host cell comprises in its genome an expression system according to any one of claims 19 to 24.

34. 32. A kit comprising an isolated nucleic acid molecule according to any one of claims 1 to 17, or an expression system according to any one of claims 19 to 24, or a vector according to any one of claims 25 to 27, or a co-transfection system according to claim 28, or a host cell according to any one of claims 29 to 32.

35. 35. The kit of claim 34, further comprising an LgBiT protein and a luciferase substrate.

36. The kit according to claim 34, comprising the expression system according to any one of claims 19 to 24, and further comprising an inducer and a transactivator corresponding to the inducible promoter.

37. A method for screening for an HBV cccDNA inhibitor, comprising: (1) providing a host cell according to claim 33; (2) contacting the host cell with an inducing agent; The inducing agent is an inducer corresponding to the inducible promoter and a transactivator contained in the host cell; (3) contacting a test agent with the host cell; It should be noted that steps (2) and (3) may be carried out simultaneously or in any order; (4) detecting the level of the first fragment of luciferase in the cell supernatant of the host cell; A method comprising:

38. Step (1) comprises the following steps: (1a) introducing the first nucleotide sequence and the second nucleotide sequence in the expression system according to any one of claims 19 to 24 into the host cell; The first nucleotide sequence and the second nucleotide sequence are provided on the same or different expression vectors, and the first nucleic acid sequence is an isolated nucleic acid molecule according to any one of claims 10 to 17; (1b) culturing the host cells; 38. The method of claim 37, comprising:

39. 39. The method of claim 38, wherein in step (1a), the expression vector is a transposon-based vector, and this step further comprises introducing a transposase expression vector into the host cell.

40. The method of claim 38, wherein the step (1) further comprises (1c) a step of identifying and selecting a host cell in which the expression system according to any one of claims 19 to 24 has been integrated into its genome, and whether the expression system has been integrated into the genome of the host cell is identified by detecting a reporter gene contained in the first nucleic acid sequence.

41. The method according to any one of claims 37 to 40, wherein in step (4), the level of the first fragment of luciferase is detected by a luciferase fragment complementation assay.

42. 42. The method of claim 41, wherein the detection is performed using a second fragment of luciferase that is complementary to the first fragment of luciferase, and the second fragment of luciferase is an LgBiT protein.

43. (5) comparing the detection result of step (4) with the level of the first fragment of luciferase detected in the absence of the test agent; The method of any one of claims 37 to 42, wherein if the detection result in step (4) is lower than the detection result in the absence of the test agent, this indicates that the test agent is an HBV cccDNA inhibitor.

44. Use of an isolated nucleic acid molecule according to any one of claims 1 to 17, or an expression system according to any one of claims 19 to 24, or a vector according to any one of claims 25 to 27, or a co-transfection system according to claim 28, or a host cell according to any one of claims 29 to 32, or a kit according to any one of claims 34 to 36, for screening for HBV cccDNA inhibitors.

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