Drug screening model and method targeting hbv cccdna

Abstract

The present application relates to the field of virology, in particular the field of hepatitis B virus treatment. Specifically, the present application relates to a model and method for screening HBV cccDNA inhibitors. The screening model and method of the present application take split luciferase detection as a surrogate indicator of HBV cccDNA detection, and can high-throughput screen drugs targeting cccDNA.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of virology, in particular the field of hepatitis B virus treatment. Specifically, the present application relates to a model and method for screening HBV cccDNA inhibitors. BACKGROUND

[0002] Chronic hepatitis B (CHB) caused by hepatitis B virus (HBV) is one of the most serious public health problems worldwide. More than 800,000 people die each year from various liver diseases caused by HBV infection, including chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma. Currently, the two main types of therapeutic drugs in clinical use (nucleoside analogues and interferon) are difficult to achieve clinical cure. The stable existence of HBV cccDNA is one of the key reasons why CHB is difficult to cure. Currently, the drugs in clinical use cannot effectively eliminate cccDNA, and the intracellular cccDNA can continue to serve as a template for viral replication and transcription.

[0003] Due to the complex mechanism of HBV cccDNA formation and maintenance, it is highly difficult to directly design drugs targeting cccDNA. A screening model that can be used for high-throughput screening of cccDNA inhibitors provides a new method for developing drugs to eliminate cccDNA. The detection method of cccDNA is complex. Southern blot is the gold standard for cccDNA detection, but it requires a large amount of cells, is complex to operate, time-consuming, and cannot be used for high-throughput drug screening. Fluorescent quantitative PCR detection is relatively simple and rapid compared to Southern blot, and has higher throughput, but it is also difficult to apply to large-scale drug screening, and the detection may be interfered by rcDNA. Using other more easily detectable markers as surrogate indicators for cccDNA detection can reduce detection costs, improve detection efficiency, and improve detection throughput.

[0004] An ideal cccDNA reporter model should meet both the stability of cccDNA source and the ease of detection of the marker as a surrogate detection indicator, with high signal-to-noise ratio. Therefore, it is necessary to develop an HBV cccDNA reporter model suitable for high-throughput screening. SUMMARY

[0005] The inventors of the present application have constructed an HBV cccDNA reporter model using split luciferase as a surrogate indicator for HBV cccDNA detection through a large number of experiments and repeated groping, which is simple to operate, time-saving, and can realize high-throughput drug screening. Therefore, this model can be used for primary screening to obtain candidate drugs with potential inhibition of HBV cccDNA, and then verified by more HBV in vivo and in vitro research models.

[0006] Reporter model I

[0007] In some cases, a first fragment sequence (e.g., HiBiT) in luciferase fragment complementation assay (LFCA) can be integrated into the HBV genome to form an HBV variant, the mRNA transcribed from the HBV variant lacks the start codon for the expression of the first fragment (e.g., HiBiT), and cannot translate the protein coupled with the first fragment (e.g., HiBiT) tag, only after the pgRNA transcribed from the HBV variant is reverse transcribed to form cccDNA, the mRNA transcribed from the cccDNA can translate the protein coupled with the first fragment (e.g., HiBiT) tag, so the expression level of the first fragment (e.g., HiBiT) can be determined by luciferase fragment complementation assay (LFCA), thereby indicating the formation of HBV cccDNA.

[0008] 1. Isolated nucleic acid molecule

[0009] Therefore, in a first aspect, the present application provides an isolated nucleic acid molecule comprising a variant of an HBV genome sequence (e.g., a wild-type HBV genome), the variant comprising: a fragment of the HBV genome comprising a C-ORF, an S-ORF, and a P-ORF, and the C-ORF comprising an exogenous insertion sequence between the precore and core genes, the exogenous insertion sequence comprising a nucleotide sequence encoding a first luciferase fragment. The first luciferase fragment is capable of binding to a corresponding second luciferase fragment in luciferase fragment complementation assay (LFCA) and producing luciferase activity.

[0010] In this context, the term "luciferase fragment complementation assay (LFCA)" has the meaning commonly understood by those skilled in the art, which divides luciferase into a first fragment and a second fragment that are each without enzyme activity, and when the two fragments interact, they can complement each other to produce luciferase activity, thereby releasing 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 small fragment (e.g., HiBiT or SmBiT) capable of complementary binding thereto, and the functional enzyme is produced by structural complementation of LgBiT with HiBiT or SmBiT.

[0011] In certain embodiments, the luciferase first fragment is LgBiT and the luciferase second fragment is a small fragment (e.g., HiBiT or SmBiT) that is capable of complementary binding to LgBiT.

[0012] In certain embodiments, the luciferase first fragment is a small fragment (e.g., HiBiT or SmBiT) that is capable of complementary binding to LgBiT and the luciferase second fragment is LgBiT. In certain embodiments, the luciferase first fragment is HiBiT and the luciferase second fragment is LgBiT. In certain embodiments, the HiBiT has a sequence as set forth in SEQ ID NO: 2. In certain embodiments, the nucleotide sequence encoding HiBiT is as set forth in SEQ ID NO: 3.

[0013] In certain embodiments, the HBV genomic fragment further comprises an X-ORF.

[0014] In certain embodiments, the variant comprises the exogenous insertion sequence between the precore and core genes of an HBV genomic sequence (e.g., a wild-type HBV genome).

[0015] In certain embodiments, the exogenous insertion sequence comprises a plurality of copies of a nucleotide sequence encoding a luciferase first fragment (e.g., HiBiT) present in a tandem repeat fashion. In certain embodiments, the exogenous insertion sequence comprises three copies of a nucleotide sequence encoding a luciferase first fragment (e.g., HiBiT) present in a tandem repeat fashion.

[0016] In certain embodiments, each of the plurality of copies of a nucleotide sequence encoding a luciferase first fragment (e.g., HiBiT) present in a tandem repeat fashion 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.

[0017] In certain embodiments, the exogenous insertion sequence comprises a sequence as set forth in SEQ ID NO: 4.

[0018] In certain embodiments, the HBV genome is a full-length genome, e.g., a genome of HBV genotype A, B, C, D, E, F, G, or H. In certain embodiments, the HBV genome is a super-length genome, e.g., a 1.1-fold genome or a 1.3-fold genome. In certain embodiments, the HBV genome is a 1.1-fold genome, e.g., as set forth in SEQ ID NO: 1.

[0019] In certain embodiments, the exogenous insert is operably linked to an inducible promoter.

[0020] In certain embodiments, the exogenous inserted sequence is regulated for expression via a Tet-On gene regulation expression system. Therefore, in certain embodiments, the inducible promoter is an operator (Tet operator, TetO) or promoter in a Tet-On gene regulation expression system, which requires doxycycline to bind to its corresponding transactivator protein to initiate transcription.

[0021] In certain embodiments, the inducible promoter is the TRE3G promoter (eg, as shown in SEQ ID NO: 5), and the corresponding transactivator is the Tet-On 3G transactivator (eg, as shown in SEQ ID NO: 9).

[0022] In certain embodiments, the inducible promoter comprises one or more repeats of the Tet operator sequence (TetO), and the corresponding transactivator protein may be a trans-Tet repressor (rTetR) or an antisense Tet transcriptional activator (rtTA).

[0023] In certain embodiments, the inducible promoter has bidirectional promoter activity.

[0024] In certain embodiments, the inducible promoter is a TRE3G promoter having bidirectional promoter activity.

[0025] In certain embodiments, the inducible promoter is operably linked to a reporter gene. In certain embodiments, the reporter gene is in the opposite direction to the exogenous inserted sequence. In certain embodiments, the reporter gene is selected from a fluorescent protein gene and / or an antibiotic resistance gene.

[0026] In certain embodiments, the fluorescent protein is selected from green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, orange or red fluorescent protein, near infrared fluorescent protein or long Stoke's shift fluorescent protein. In certain embodiments, the fluorescent protein is selected from red fluorescent protein, near infrared fluorescent protein or long Stoke's shift fluorescent protein, such as mRuby3, mApple, FusionRed, mCherry, mScarlet, RFP, iRFP670, mBeRFP or CyOFP1.

[0027] In certain embodiments, the fluorescent protein is selected from green fluorescent protein, such as mGamillus, mNeonGreen, EGFP, mClover, UnaG, TurboGFP, TagGFP, Venus, EYFP, RFP, iRFP670, mBeRFP, CyOFP1.

[0028] In certain embodiments, the antibiotic resistance gene is selected from genes that confer resistance to hygromycin, neomycin, G418, blasticidin, puromycin, or ouabain.

[0029] In certain embodiments, the reporter gene comprises a fluorescent protein gene and an antibiotic resistance gene. In certain embodiments, the reporter gene comprises a gene encoding iRFP (e.g., as shown in SEQ ID NO: 11) and a blasticidin resistance gene (e.g., as shown in SEQ ID NO: 13).

[0030] 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 shown in SEQ ID NO: 6.

[0031] In certain embodiments, the isolated nucleic acid molecule comprises the sequence shown in SEQ ID NO:8.

[0032] 2. Recombinant HBV cccDNA

[0033] The HBV variant contained in the isolated nucleic acid molecule described in the first aspect of the present invention can be transcribed as a template to form pgRNA, and can be reverse transcribed to form cccDNA.

[0034] Therefore, in a second aspect, the present invention further provides a recombinant HBV cccDNA comprising the isolated nucleic acid molecule described in the first aspect.

[0035] In certain embodiments, the recombinant HBV cccDNA comprises a variant of the HBV genomic sequence described in the first aspect.

[0036] In certain embodiments, the recombinant HBV cccDNA is formed by circularizing the isolated nucleic acid molecule described in the first aspect.

[0037] 3. Expression system

[0038] In a third aspect, the present invention provides an expression system comprising the isolated nucleic acid molecule of the first aspect.

[0039] 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 comprises a second nucleic acid sequence comprising a nucleotide sequence encoding a transactivator protein corresponding to the inducible promoter.

[0040] In certain embodiments, the transactivator is selected from the group consisting of Tet-On 3G transactivator, rTetR, and rtTA.

[0041] In certain embodiments, the second nucleic acid sequence further comprises an expression control element, such as a promoter (eg, a constitutive promoter) and / or an enhancer, operably linked to the nucleotide sequence encoding the transactivator.

[0042] 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 shown in SEQ ID NO:5.

[0043] In certain embodiments, the nucleotide sequence encoding the Tet-On 3G transactivator comprises the sequence shown in SEQ ID NO:10.

[0044] 4. Vector

[0045] In a fourth aspect, the present invention further provides a vector comprising the isolated nucleic acid molecule described in the first aspect, or the expression system described in the third aspect.

[0046] In certain embodiments, the vector comprises the expression system described in the third aspect, wherein the first nucleic acid sequence and the second nucleic acid sequence are provided on the same or different vectors. In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are provided on the same vector.

[0047] 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 (Cat.# 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.

[0048] 5. Co-transfection system

[0049] In a fifth aspect, the present application provides a co-transfection system comprising the vector of the fourth aspect, said vector being a transposon vector, and a transposase expression vector.

[0050] In certain embodiments, the transposase expression vector is a PiggyBac transposase expression vector. In the present context, the PiggyBac transposase expression vector is well known in the art and is widely commercially available. In certain embodiments, the PiggyBac transposase expression vector is PB210PA-1 (System Biosciences).

[0051] 6. Host cell

[0052] In a sixth aspect, the present application 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.

[0053] In certain embodiments, the host cell is a eukaryotic cell. In certain embodiments, the host cell supports functional HBV cccDNA formation and transcription.

[0054] In certain embodiments, the host cell is a hepatocyte-derived eukaryotic cell, such as a hepatoma cell or a hepatocyte. In certain embodiments, the host cell is selected from HepaRG, HepG2 or Huh7.

[0055] In certain embodiments, the host cell can also be a non-hepatocyte, provided that they support hepadnavirus cccDNA formation (or, in a broader sense, hepadnavirus DNA replication). For example, such non-hepatocyte / host can be modified to support hepadnavirus cccDNA formation (or hepadnavirus DNA replication) if the viral pregenomic RNA is introduced into the cell or transcribed from a DNA template by an exogenous promoter.

[0056] In certain embodiments, the host cell comprises the expression system of the third aspect in its genome.

[0057] In certain embodiments, the host cell is capable of stably expressing HBV cccDNA formed from the variant of the HBV genomic sequence when an inducer corresponding to the inducible promoter and the trans-activating protein is present (e.g. Doxycycline).

[0058] 7. Kit

[0059] In a seventh aspect, the present application 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.

[0060] In certain embodiments, the kit comprises: the vector of the fourth aspect, or the co-transfection system of the fifth aspect.

[0061] In certain embodiments, the kit comprises: the host cell of the sixth aspect.

[0062] In certain embodiments, the kit further comprises a LgBiT protein. Optionally, the kit can further comprise a luciferase substrate.

[0063] In certain embodiments, the kit further comprises an inducer (e.g. Doxycycline) corresponding to the inducible promoter and transactivator protein.

[0064] 8. Screening method

[0065] In an eighth aspect, the present application provides a method for screening HBV cccDNA inhibitors, comprising:

[0066] (1) providing the host cell of the sixth aspect; the host cell comprises the expression system of the third aspect in its genome;

[0067] (2) contacting an inducing agent with the host cell, the inducing agent being an inducer (e.g. Doxycycline) corresponding to the inducible promoter and transactivator protein comprised in the host cell;

[0068] (3) contacting a test agent with the host cell; wherein, steps (2) and (3) can be performed simultaneously or in any order;

[0069] (4) detecting the level of luciferase first fragment (e.g. HiBiT) in the cell supernatant of the host cell.

[0070] In certain embodiments, step (1) comprises the following steps:

[0071] (1a) introducing the first nucleotide sequence and the second nucleotide sequence in the expression system of the third aspect into a host cell, wherein 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 comprising the inducible promoter operably linked to the exogenous insert sequence in the first aspect;

[0072] (1b) culturing the host cell.

[0073] In certain embodiments, the host cell is selected from a hepatocyte-derived eukaryotic cell, such as a hepatoma cell or a hepatocyte; preferably, the host cell is selected from HepaRG, HepG2, or Huh7.

[0074] In certain embodiments, in step (1a), the expression vector is a transposon vector (e.g., a PiggyBac transposon vector), which step further comprises: introducing a transposase expression vector (e.g., a PiggyBac transposase expression vector) into the host cell.

[0075] In certain embodiments, the step (1) further comprises: (1c) identifying and selecting a host cell whose genome has integrated the expression system of the third aspect. In certain embodiments, whether the expression system has integrated into the genome of the host cell is identified by detecting a reporter gene comprised by the first nucleic acid sequence.

[0076] In certain embodiments, in step (2), the inducing agent activates the inducible promoter, thereby initiating transcriptional replication of the HBV genome variant downstream thereof, resulting in a recombinant HBV cccDNA comprising the luciferase first fragment as a marker.

[0077] In certain embodiments, the step (2) comprises culturing the host cell under conditions that allow: (i) synthesis of an HBV pregenomic (pg) RNA; (ii) reverse transcription of the synthesized pgRNA into a negative-strand DNA; (iii) synthesis of a second positive-strand DNA, such that the negative-strand DNA and the positive-strand DNA form a double-stranded relaxed circular DNA; (iv) formation of a cccDNA from the relaxed circular double-stranded DNA.

[0078] In certain embodiments, in step (4), the luciferase first fragment level is detected by luciferase fragment complementation technology (i.e., providing a luciferase second fragment that is structurally complementary to the first fragment and a luciferase substrate).

[0079] In certain embodiments, the detection is by a luciferase second fragment that is complementary to the luciferase first fragment.

[0080] In certain embodiments, the luciferase first fragment is a small fragment, such as HiBiT or SmBiT, that is capable of complementary binding to LgBiT, and the luciferase second fragment is a LgBiT protein. In certain embodiments, the luciferase first fragment is HiBiT or SmBiT, and the luciferase second fragment is a LgBiT protein.

[0081] In certain embodiments, the method further comprises the steps of:

[0082] (5) comparing the measurement result of step (4) with the level of the first fragment of luciferase measured in the absence of the test agent; wherein, if the measurement result of step (4) is lower than the measurement result in the absence of the test agent, it indicates that the test agent is an HBV cccDNA inhibitor.

[0083] The present invention also relates to the use of the isolated nucleic acid molecules, expression systems, vectors, co-transfection systems, host cells, and kits described above for screening HBV cccDNA inhibitors.

[0084] Reporter model II

[0085] In other cases, recombinase technology can be used to circularize the linear HBV replicon to form cccDNA. The linear HBV replicon contains the first fragment sequence (e.g., HiBiT sequence) in the integrated luciferase fragment complementation technique (LFCA). When the recombinase is not expressed, the first fragment (e.g., HiBiT) tag lacks a promoter and cannot be expressed; when the recombinase is expressed, it can mediate the recombination of double-stranded DNA, so that the linear HBV genomic DNA forms a closed circular DNA. After the circular DNA is formed, the first fragment (e.g., HiBiT) tag can use the endogenous promoter of HBV to initiate the expression of the protein coupled to the first fragment (e.g., HiBiT) tag. Therefore, the signal of the first fragment (e.g., HiBiT) tag can be measured by the luciferase fragment complementation technique (LFCA) to indicate the formation of recombinant HBV cccDNA, i.e., HBV rcccDNA.

[0086] 9. Isolated nucleic acid molecule

[0087] Therefore, in a ninth aspect, the present invention provides an isolated nucleic acid molecule comprising a variant of an HBV genome sequence (eg, a wild-type HBV genome), wherein the variant comprises, from 5' to 3' direction:

[0088] (i) a nucleotide sequence encoding a first luciferase fragment; the first luciferase fragment is capable of binding to a corresponding second luciferase fragment in a luciferase fragment complementation technique (LFCA) and producing luciferase activity;

[0089] (ii) the sequence of the 3′ end region of the C-ORF of the HBV genome;

[0090] (iii) HBV genome segment containing S-ORF and P-ORF;

[0091] (iv) a sequence of the 5' end region of the C-ORF of the HBV genome, which together with the sequence described in (ii) can form a complete C-ORF sequence;

[0092] Furthermore, the variant is located between two site-specific recombinase recognition sequences arranged in the same direction.

[0093] In certain embodiments, the first luciferase fragment is a small fragment capable of complementary 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 a sequence as shown in SEQ ID NO: 2. In certain embodiments, the nucleotide sequence encoding HiBiT is shown in SEQ ID NO: 3.

[0094] In certain embodiments, the HBV genome is a full-length genome, for example, a genome of HBV genotype A, B, C, D, E, F, G, or H. In certain embodiments, the HBV genome is an extra-long genome, for example, a 1.1-ploid genome or a 1.3-ploid genome. In certain embodiments, the HBV genome is a 1.1-ploid genome, for example, as shown in SEQ ID NO: 1.

[0095] In certain embodiments, the sequence described in (iii) further comprises an X-ORF.

[0096] In certain embodiments, the sequence described in (iii) comprises an HBV genome fragment with the C-ORF removed.

[0097] In certain embodiments, the sequence described in (iii) comprises the sequence shown in SEQ ID NO:16.

[0098] In certain embodiments, the sequence described in (ii) comprises a core gene, and the sequence described in (iv) comprises a pre-core gene. In certain embodiments, the sequence described in (ii) comprises the sequence shown in SEQ ID NO: 14. In certain embodiments, the sequence described in (iv) comprises the sequence shown in SEQ ID NO: 15.

[0099] In certain embodiments, the site-specific recombinase recognition sequence is selected from a loxP sequence or a FRT sequence.

[0100] In certain embodiments, the isolated nucleic acid molecule comprises the sequence shown in SEQ ID NO:17.

[0101] 10. Recombinant HBV cccDNA

[0102] The isolated nucleic acid molecule described in the ninth aspect of the present invention can be circularized under the action of a recombinase to form cccDNA.

[0103] Therefore, in the tenth aspect, the present invention further provides a recombinant HBV cccDNA, which is formed by circularizing the variant of the HBV genomic sequence in the isolated nucleic acid molecule according to the ninth aspect.

[0104] In certain embodiments, the recombinant HBV cccDNA is formed by circularization of the isolated nucleic acid molecule described in the ninth aspect in the presence of a site-specific recombinase (eg, Cre recombinase or FLP recombinase) corresponding to the site-specific recombinase recognition sequence.

[0105] 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 the first fragment of luciferase (eg, HiBiT).

[0106] In certain embodiments, the recombinant HBV cccDNA further comprises an X-ORF.

[0107] In certain embodiments, the recombinant HBV cccDNA comprises: a C-ORF comprising a nucleotide sequence encoding the first luciferase fragment (eg, HiBiT), and an HBV genome fragment from which the C-ORF is removed (eg, the sequence shown in SEQ ID NO: 16).

[0108] In certain embodiments, the recombinant cccDNA comprises the sequence shown in SEQ ID NO:18.

[0109] 11. Vector

[0110] In the eleventh aspect, the present invention further provides a vector comprising the isolated nucleic acid molecule according to the ninth aspect.

[0111] In certain embodiments, the vector is a transposon vector, such as a PiggyBac transposon vector. In certain embodiments, the isolated nucleic acid molecule described in the ninth aspect can be inserted into any commercially available PiggyBac transposon vector, such as PB-CMV-MCS-EF1α-RedPuro (Cat.# PB514B-1). In certain embodiments, the isolated nucleic acid molecule is located between two ITR sequences of the transposon vector.

[0112] 12. Co-transfection system

[0113] In the twelfth aspect, the present invention further provides a co-transfection system, which comprises the vector described in the eleventh aspect and a transposase expression vector.

[0114] In certain embodiments, the transposase expression vector is a PiggyBac transposase expression vector. In this article, the PiggyBac transposase expression vector is well known in the art and is widely commercially available. In certain embodiments, the PiggyBac transposase expression vector is PB210PA-1 (System Biosciences).

[0115] 13. Host cell

[0116] In the thirteenth aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule described in the ninth aspect, or the recombinant cccDNA described in the tenth aspect, or the vector described in the eleventh aspect, or the co-transfection system described in the twelfth aspect.

[0117] In certain embodiments, the host cell is a eukaryotic cell. In certain embodiments, the host cell supports functional HBV cccDNA formation and transcription.

[0118] In certain embodiments, the host cell is a eukaryotic cell of hepatocyte origin, such as a hepatoma cell or a hepatocyte. In certain embodiments, the host cell is selected from HepaRG, HepG2 or Huh7.

[0119] In certain embodiments, the host cells may also be non-hepatocytes, provided that they support hepadnavirus cccDNA formation (or, more generally, hepadnavirus DNA replication). For example, such non-hepatocytes / hosts may be modified to support hepadnavirus cccDNA formation (or hepadnavirus DNA replication) if viral pregenomic RNA is introduced into the cell or transcribed from a DNA template via an exogenous promoter.

[0120] In certain embodiments, the host cell comprises in its genome the isolated nucleic acid molecule of the ninth aspect.

[0121] In certain embodiments, when a site-specific recombinase (eg, Cre recombinase or FLP recombinase) corresponding to the site-specific recombinase recognition sequence is present, the host cell is capable of stably expressing recombinant HBV cccDNA formed by circularization of the variant of the HBV genomic sequence.

[0122] 14. Kit

[0123] In the fourteenth aspect, the present invention provides a kit comprising the isolated nucleic acid molecule described in the ninth aspect, or the recombinant cccDNA described in the tenth aspect, or the vector described in the eleventh aspect, or the co-transfection system described in the twelfth aspect, or the host cell described in the thirteenth aspect.

[0124] In certain embodiments, the kit comprises: the vector described in the eleventh aspect, or the co-transfection system described in the twelfth aspect.

[0125] In certain embodiments, the kit comprises: the host cell of the thirteenth aspect.

[0126] In certain embodiments, the kit further comprises an LgBiT protein. Optionally, the kit further comprises a luciferase substrate.

[0127] 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.

[0128] 15. Screening method

[0129] In a fifteenth aspect, the present invention provides a method for screening an HBV cccDNA inhibitor, comprising:

[0130] (1) Providing the host cell of the thirteenth aspect; wherein the host cell comprises the isolated nucleic acid molecule of the ninth aspect in its genome;

[0131] (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;

[0132] (3) contacting the test agent with the host cell;

[0133] (4) Detecting the level of the first luciferase fragment (eg, HiBiT) in the cell supernatant of the host cells.

[0134] In certain embodiments, step (1) comprises the following steps:

[0135] (1a) introducing the isolated nucleic acid molecule of the ninth aspect or the vector of the eleventh aspect into a host cell;

[0136] (1b) culturing the host cell.

[0137] In certain embodiments, the host cell is selected from eukaryotic cells of hepatocyte origin, such as hepatoma cells or hepatocytes; preferably, the host cell is selected from HepaRG, HepG2 or Huh7.

[0138] In certain embodiments, in step (1a), the expression vector is a transposon vector (eg, a PiggyBac transposon vector), and the step further comprises: introducing a transposase expression vector (eg, a PiggyBac transposase expression vector) into the host cell.

[0139] In certain embodiments, in step (2), under the action of the recombinase, the variant of the HBV genome sequence contained in the host cell will be circularized to form cccDNA.

[0140] In certain embodiments, in step (4), the level of the first luciferase fragment is detected by luciferase fragment complementation technology (ie, providing a second luciferase fragment complementary to the first fragment structure and a luciferase substrate).

[0141] In certain embodiments, detection is by a second luciferase fragment that is complementary to the first luciferase fragment.

[0142] In certain embodiments, the first luciferase fragment is a small fragment capable of complementary binding to LgBiT, such as HiBiT or SmBiT, and the second luciferase fragment is LgBiT protein. In certain embodiments, the first luciferase fragment is HiBiT, and the second luciferase fragment is LgBiT protein.

[0143] In certain embodiments, the method further comprises the steps of:

[0144] (5) comparing the measurement result of step (4) with the level of the first fragment of luciferase measured in the absence of the test agent; wherein, if the measurement result of step (4) is lower than the measurement result in the absence of the test agent, it indicates that the test agent is an HBV cccDNA inhibitor.

[0145] The present invention also relates to the use of the isolated nucleic acid molecules, vectors, co-transfection systems, host cells, and kits described above for screening HBV cccDNA inhibitors.

[0146] Definitions of terms

[0147] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, laboratory procedures in virology, cell culture, biochemistry, nucleic acid chemistry, immunology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0148] As used herein, the term "hepatitis B virus (HBV)" refers to a member of the Hepadnaviridae family with a small double-stranded DNA genome of approximately 3200 base pairs and hepatocyte tropism. "HBV" includes hepatitis B viruses that infect any of a variety of mammalian (e.g., human, non-human primate, etc.) and avian (ducks, etc.) hosts. "HBV" includes any known HBV genotype, such as serotypes A, B, C, D, E, F, and G; any HBV serotype or HBV subtype; any HBV isolate; HBV mutants, such as HBeAg-negative variants, drug-resistant HBV variants (e.g., lamivudine-resistant variants; adefovir-resistant mutants; tenofovir-resistant mutants; entecavir-resistant mutants, etc.); and the like.

[0149] As used herein, "HBV genome" includes not only full-length genomes (1 unit genome), but also ultra-long HBV genomes (>1 unit genome, in other words, longer than 1 unit genome). The HBV genome contains all the information required to build and maintain HBV replication. These genome sequences are available for each genotype in articles and GeneBank. "Ultra-long HBV genome" or "HBV genome exceeding full length" refers to a sequence comprising a full-length genome and a partial genome, the sequence of which may vary according to the desired genome unit and specific HBV strain. In addition, methods for obtaining a HBV genome greater than the full length and determining the genome sequence are described in the prior art literature, for example in European Patent EP1543168.

[0150] In certain exemplary embodiments, the HBV is human HBV, whose genome contains four major overlapping open reading frames (ORFs): the S-ORF, C-ORF, P-ORF, and X-ORF. The S-ORF is divided into the S gene, pre-S2 region, and pre-S1 region, each with its own start codon ATG. The C-ORF is divided into the C gene and pre-C region, each with its own start codon ATG. The P-ORF is the longest open reading frame, with its initial segment overlapping with the C-ORF, its middle segment overlapping with the S-ORF, and its final segment overlapping with the X-ORF.

[0151] As used herein, "HBV genome fragment" refers to a portion of the HBV genome. The fragment can be 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 can also be a portion of the genome containing one or more genes contained in the HBV genome, for example, the fragment can be a nucleic acid encoding an envelope protein, a core / prenuclear protein, an X protein, and / or an HBV polymerase protein. Furthermore, the fragment may be a nucleic acid encoding one or more portions of the envelope protein, core / prenuclear protein, x protein and / or polymerase protein of HBV.

[0152] 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 that serves as a template for the synthesis of hepadnavirus mRNA and pregenomic RNA.

[0153] As used herein, the term "cccDNA inhibitor" refers to an agent capable of inhibiting the stability of cccDNA (i.e., reduced cccDNA stability), inhibiting the transcriptional activity of cccDNA (i.e., reduced transcription of hepadnavirus mRNA using cccDNA as a transcription template), and / or inhibiting the formation of cccDNA (i.e., no or less cccDNA formation).

[0154] 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 with a parent polypeptide sequence or polynucleotide. Variant is similar to the parent sequence, but has at least one or several or multiple replacements, deletions or insertions in its amino acid sequence or nucleotide sequence, making them different from the sequence of the parent polypeptide or parent polynucleotide. In some cases, variant has been operated and / or engineered to comprise at least one replacement, deletion or insertion in its amino acid sequence or nucleotide sequence, making them different from the parent sequence. In addition, variant can retain the functional characteristics or the activity of the parent polypeptide or parent polynucleotide, for example, keeping at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% biological activity of the parent polypeptide or parent polynucleotide.

[0155] As used herein, the term "recombinant" DNA molecule refers to a DNA molecule formed by laboratory methods of genetic recombination (e.g., molecular cloning) to bring together genetic material from multiple sources to produce a sequence that would not be found in a biological organism. The term "site-specific recombination" refers to the recombination between two nucleotide sequences, each of which contains at least one recognition site. "Site-specific" refers to a specific nucleotide sequence that can be located at a specific position in the host cell genome. The nucleotide sequence can be endogenous to the host cell, its natural position in the host genome or some other position in the genome, or it can be a heterologous nucleotide sequence previously inserted into the host cell genome by any of a variety of known methods.

[0156] As used herein, the term "recombinase" is a genetic recombinase that is typically derived from bacteria and fungi and catalyzes a directionally sensitive DNA exchange reaction between short (30-40 nucleotides) target site sequences specific for each recombinase.

[0157] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well 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); bacteriophages, 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, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication. Methods for introducing vectors and / or nucleic acid molecules carried thereinto cells are known in the art, such as viral infection / transduction, conjugation, nanoparticle delivery, electroporation, particle gun technology, calcium phosphate precipitation, direct injection, and the like. The choice of method generally depends on the type of cells being transfected and the environment in which the transfection occurs (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.

[0158] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0159] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). 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 has been 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. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0160] The twenty conventional amino acids referred to herein are compiled according to 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 single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0161] Advantageous Effects of the Invention

[0162] The HBV cccDNA inhibitor screening model of the present invention uses split luciferase as a surrogate indicator for HBV cccDNA detection. The detection is simple and time-saving, and high-throughput drug screening can be achieved. It can be used in many fields such as research, treatment, and diagnosis, and has broad application prospects and clinical value.

[0163] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0164] Figure 1 A schematic diagram of the PiggyBac transposon vector used to construct a stably integrated cell line in Example 1 is shown.

[0165] Figure 2 A schematic diagram of the HBV modification method of the reporter model in Example 1 using HiBiT to indicate cccDNA produced during HBV replication is shown.

[0166] Figure 3 The viral replication expression of HBV variants integrating HiBiT tags of different sequences evaluated in Example 1 is shown.

[0167] Figure 4 The feasibility of evaluating RG-Hibit16 cells for screening inhibitors that inhibit HBV cccDNA formation in Example 1 is shown.

[0168] Figure 5 A schematic diagram and functional verification of using HiBiT as an rcccDNA reporter model in Example 2 are shown.

[0169] Figure 6The method of screening compounds targeting HBV cccDNA using RG-Hibit16 and G2-Rccc1 in Example 3 is shown.

[0170] Figure 7 The compounds shown in Example 3 were verified to have an inhibitory effect on HBV cccDNA in the HepG2-hNTCP-2B1 infection model.

[0171] Figure 8 The compounds shown in Example 3 were verified to have a promoting effect on HBV cccDNA in the HepG2-hNTCP-2B1 infection model.

[0172] Sequence information

[0173] Information on the partial sequences involved in the present invention is provided below.

[0174] DETAILED DESCRIPTION

[0175] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0176] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on 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., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. 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 invention.

[0177] The main detection methods involved in the following examples are described as follows:

[0178] HiBiT detection: HiBiT detection was performed using the Nano Glo HiBiT Extracellular Detection System (Catalog No. N2421) from Promega, and the detection steps were performed according to the kit instructions.

[0179] HBsAg / HBeAg detection: The detection operations of hepatitis B surface antigen (chemiluminescence method CLEIA, product standard number: YZB / Guo 0346-2014) and e antigen (enzyme-linked immunosorbent assay ELISA, product standard number: YZB / Guo 0216-2013) are carried out according to the test methods of the detection kits of Beijing Wantai Company.

[0180] HBV DNA detection: HBV DNA extraction: After the collected cells were washed with PBS, the viral DNA & RNA extraction kit (Beijing Jinmaige) was used for automated extraction in a nucleic acid extraction workstation. HBV DNA quantification was performed using the probe method, the reagent used was Premix Ex TaqTM (Takara), and the instrument used was Roche. 96. The primer sequences used are shown in Table 1.

[0181] Table 1: PCR primer sequences

[0182]

[0183] HBV cccDNA detection: HBV cccDNA was extracted using the modified Hirt method. The kit used was the Tiangen Plasmid Mini Extraction Kit. The lysis buffers involved were Buffer I (50mM Tris, 10mM EDTA, pH 7.5), Buffer II (1.2% SDS), and Buffer III (3M CsCl, 1M Potassium acetate, 0.67M Acetic acid), which replaced P1, P2, and P3 in the Tiangen Plasmid Mini Extraction Kit, respectively. The extraction steps were similar to the plasmid extraction method in the kit. The primers used for fluorescence quantification are shown in Table 1. The instrument used was Roche. 96. HBV cccDNA and mitochondrial DNA (mtDNA) were quantified, and the relative values ​​of HBV cccDNA and mtDNA were calculated.

[0184] The steps for detecting HBV DNA by DNA immunoblotting are as follows. DNA extraction: After treatment, cells were washed once with PBS; NET buffer (50 mM Tris-pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.5% NP-40) was added to lyse the cells at 4°C for 1 hour; the supernatant was collected and micrococcal nuclease at a final concentration of 33 μg / mL and 6 mM CaCl2 were added in a 37°C water bath for 30 minutes; EDTA was added at a final concentration of 25 mM and the supernatant was incubated at 65°C for 15 minutes; proteinase K was added at a final concentration of 200 μg / mL and 0.5% SDS was added in a 50°C water bath for 12 hours; DNA was extracted with phenol-chloroform. Detection: Separate DNA by 1.2% agarose gel electrophoresis for 2 hours. Treat the gel with 0.2N HCl, 0.5M NaOH / 1.5M NaCl, and 1M Tris-HCl in sequence to denature the DNA. Transfer the nucleic acid to a nylon membrane using a vacuum transfer apparatus. Fix the nucleic acid by UV cross-linking and perform pre-hybridization and hybridization. Wash away excess probe, block with blocking solution, add Anti-Dig-Ap antibody, and finally add CDP-star for color development. Detect the target band by continuous exposure.

[0185] Example 1: Construction of a reporter model using HiBiT to indicate cccDNA produced during HBV replication

[0186] In this embodiment, the PB-CMV-MCS-EF1α-RedPuro (Cat.#PB514B-1) of the PiggyBac transposon system is used as a vector. When the vector is co-transfected with the PiggyBac transposase (System Biosciences, PB210PA-1), the sequence between the two "ITR sequences" on the vector plasmid can be integrated into the genome of the cell to achieve the integration of the target gene. To achieve regulatable expression of HBV, we replaced the "CMV Promoter" on the vector with a "TRE3G Promoter" (Takara, Tet-On3G Inducible Expression System). This promoter requires Doxycycline to bind to the Tet-On 3G Transactivator Protein to initiate transcription, so Doxycycline can be used to regulate the expression of the target protein. To avoid the loss of part of the target sequence during integration, we selected the TRE3G promoter with bidirectional initiation activity, introduced an iRFP fluorescent marker and a blasticidin resistance screening marker at the N-terminus, and used dual resistance and dual fluorescence as screening conditions for integrated cells. Since Tet-On 3G protein is necessary for transcription of TRE3G promoter, we introduced an expression cassette into the vector to express Tet-On 3G protein. The final vector is as follows Figure 1 As shown, the sequence within the red dotted box is the sequence integrated into the cell genome. "GOI" represents the target gene (Gene of interest) to be connected. In this example, the sequence connected is the HBV 1.1-ploid genome sequence with the reporter gene inserted.

[0187] The open reading frames of the HBV genome are highly overlapping, so the modification of HBV has strict restrictions on the insertion position of foreign genes. In this example, different copy numbers of HiBiT sequences connected by different linker peptides were inserted between the pre-core and the core to prepare HBV variants containing HiBiT. The schematic diagram of the insertion method is shown in the figure. Figure 2 The HiBiT signal, viral protein expression and viral replication of these insertion mutations were verified. Different HBV variants were transfected into liver cancer cell lines HepG2 and Huh7 cells, and the HiBiT signal, HBV antigen HBsAg and HBeAg expression in the cell supernatant were detected (A). The viral replication was detected by Southern Blot (B). Figure 3As shown. It can be seen that Hibit16, an insertion mutation with 3 copies of the HiBiT tag inserted (SEQ ID NO: 4), is a better choice. If the number of HiBiT copies decreases, the HiBiT signal will be weak, and if the number of HiBiT copies increases, the expression or replication of viral proteins will be affected. Comparing different connecting peptides, the effect of inserting the peptide segment "GSG" before and after the HiBiT sequence is better than inserting the peptide segment "G" only at one end or not introducing a connecting peptide. Therefore, in this example, HepaRG cells with stably integrated HBV mutant Hibit16 were constructed for screening inhibitors that can inhibit the formation of cccDNA.

[0188] HepaRG-Hibit16 cells are obtained by transfecting HepaRG cells with Hibit16 plasmid and PiggyBac transposase, integrating the HBV variant sequence (Hibit16) and selection marker into the cell genome, and screening by Puromycin resistance and red fluorescent marker. Doxycycline can activate the TRE3G promoter, initiate the expression of iRFP670, and initiate the transcription and replication of HBV, thereby producing HiBiT. Figure 4 As shown in A. To evaluate the function of HBV inhibitors in this cell, cells were first plated, and different HBV inhibitors were added to induce viral transcription and replication. Cell supernatants were collected every 2 days, the culture medium was replaced, and HiBiT signals, HBsAg, HBeAg in the cell supernatant, and HBV DNA and HBV cccDNA in the cell lysate were detected. The response of the cells to HBV inhibitors is shown in Figure 4 BF. Tenofovir disoproxil fumarate (TDF) is a nucleotide reverse transcriptase inhibitor, and Morphothiadin (Mor) is a core particle assembly regulator, both of which are in Phase II / III clinical evaluation. Both can inhibit cccDNA formation. In the cell model established in this study, TDF and Mor inhibited cccDNA formation. Correspondingly, 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 for inhibitors of cccDNA formation, such as Mor.

[0189] Example 2: Construction of a reporter model using HiBiT to indicate recombinant rcccDNA

[0190] This example constructs a reporter model using HiBiT to indicate recombinant cccDNA. Recombinant cccDNA, namely rcccDNA, is a closed circular DNA formed by circularizing linear HBV DNA through the Cre / loxP recombinase system. Figure 5The HBV variant shown in A places the C-terminal portion of the HBV core ORF (SEQ ID NO:14) at the N-terminus of the entire replicon, the N-terminal portion of the core ORF and the core promoter (SEQ ID NO:15) at the C-terminus of the replicon, and the sequence of the reporter gene HiBiT at the N-terminus of the replicon. LoxP sequences (SEQ ID NO:19) are added before and after the replicon. Without Cre recombinase expression, HiBiT lacks a promoter and cannot be expressed. However, after rcccDNA is formed, HiBiT can utilize the core promoter to express HBeAg and HBcAg fused with the HiBiT tag. Therefore, HiBiT can serve as a surrogate marker for rcccDNA detection. This HBV variant uses the PiggyBac transposon system as a vector to facilitate the integration of the target gene, namely the HBV variant with an integrated reporter gene, into the cell genome to construct an integrated cell line. This clone was named Rccc1a. The plasmid was transfected into HepG2 cells, and the cells were infected with adenovirus Adv-Cre to express Cre recombinase (SEQ ID NO: 20) when the medium was changed 6 hours later. HiBiT was detected in the cell supernatant 48 hours later. The cells were lysed, and the lysate was used as a template to amplify the partial sequence of the rcccDNA before and after the loxP sequence to confirm whether the expected rcccDNA was formed. The amplification primers and sequencing primers were cccDNA-F and cccDNA-R in Table 1. Figure 5 B shows the HiBiT detection results in the supernatant of HepG2 cells transfected with Rccc1a to determine whether Cre recombinase is expressed or not. Figure 5 C shows the sequencing results of rcccDNA generated after Cre recombinase expression in HepG2 cells transfected with Rccc1a. These results demonstrate that Cre recombinase can generate the expected rcccDNA and promote the expression of HiBiT tag-coupled protein.

[0191] Example 3: Reporter Model for Screening cccDNA Inhibitors

[0192] The reporter model prepared in Example 1 contains an HBV variant that incorporates three repeats of the HiBiT sequence. The mRNA transcribed directly using this HBV variant as a template lacks the start codon for HiBiT expression and cannot translate the protein coupled to the HiBiT tag. Only after the pgRNA transcribed using this HBV variant is reverse transcribed to form cccDNA can the mRNA transcribed using cccDNA as a template translate the protein coupled to the HiBiT tag. Therefore, the expression level of HiBiT can be used to indicate the formation of HBV cccDNA. The reporter model prepared in Example 2 is to insert the HiBiT sequence in the middle of the HBV core sequence, and place the core N-terminal sequence and the C-terminal sequence at the C-terminus and N-terminus of the HBV replicon, respectively, and connect loxP sequences at both ends of the HBV replicon. When the Cre recombinase is not expressed, the HiBiT tag lacks a promoter and cannot be expressed; when the Cre recombinase is expressed, it can mediate the recombination of double-stranded DNA, so that the linear HBV genomic DNA forms a closed circular DNA. After the circular DNA is formed, the HiBiT tag can use the endogenous promoter of HBV to initiate the expression of the protein coupled to the HiBiT tag. Therefore, the signal of the HiBiT tag can be used to indicate the formation of recombinant HBVcccDNA, i.e., HBV rcccDNA.

[0193] This example examines the ability of the reporter model of Example 1 (HepaRG-Hibit16 integrated with Hibit16 constructed based on HepaRG cells) and the reporter model of Example 2 (HepG2-Rccc1a integrated with Rccc1a constructed based on HepG2) to screen cccDNA inhibitors. These two cell lines were used to evaluate the potential of 189 clinical drugs for different diseases or drugs in clinical research to inhibit cccDNA. The screening work was carried out in 96-well cell culture plates. RG-Hibit16 cells were plated at a cell density of 15,000 / well, and G2-Rccc1a cells were plated at a cell density of 35,000 / well. Drug treatment began one week after RG-Hibit16 cells were plated. RG-Hibit16 was treated with different compounds while adding Dox to induce viral expression. The day after G2-Rccc1a was plated, adenovirus Adv-Cre was first infected to express Cre recombinase protein. Two days later, different compounds were added for treatment. All compounds were diluted to a final concentration of 1 μM, with 200 μL per well. New culture medium was replaced every two days. HiBiT expression in the cell supernatant was detected four days after compound treatment, and CCK8 detection reagent was added at a ratio of 10:1 to detect the cytotoxicity of the compound. The results of compound screening of RG-Hibit16 cells and G2-Rccc1a cells are shown in the figure. Figure 6Among these 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. According to CCK8 assay results, these six drugs had no significant cytotoxicity against either cell type. Palovarotene, in particular, showed an inhibitory effect on HBV cccDNA in both models.

[0194] We further verified the functions of these six compounds in the HepG2-hNTCP-2B1 infection model. HepG2-hNTCP-2B1 is a monoclonal clone 2B1 with the highest susceptibility to HBV that overexpresses hNTCP in HepG2 cells. The evaluation in this model is to first infect the cells with HBV virus, wash away the residual virus with PBS the next day, add different compounds after 2 days, collect the cell supernatant and replace the culture medium with fresh culture medium every 2 days, detect the viral antigen in the cell supernatant, lyse the cells 8 days after infection, and detect the intracellular HBV cccDNA. The evaluation results are as follows: Figure 7 As shown. Palovarotene has a relatively poor inhibitory effect on HBsAg, but the inhibition rates of HBsAg and HBV cccDNA are both above 60%, which is better than the other five compounds. In addition, Doxifluridine also has a significant inhibitory effect on cccDNA in this infection model. Based on the evaluation results of RG-Hibit16 cells and G2-Rccc1a cells, we also selected five compounds that have an upregulating effect on HBV cccDNA in both models, namely: Vincristine, Naftopidil, Cinchophen, Mebhydrolin and Cladribine, and verified the functions of these five compounds in the HepG2-hNTCP-2B1 infection model. The evaluation strategy is the same as that of the aforementioned inhibitors. The results are shown in the figure. Figure 8As shown. The five compounds had weak effects on HBeAg expression; among them, Vincristine and Cladribine significantly upregulated HBsAg expression levels; Vincristine, Cinchophen, Mebhydrolin, and Cladribine all upregulated intracellular cccDNA levels. Validation results in infection models indicate that the two cccDNA reporter models constructed in this study can be used to screen for compounds that promote or inhibit cccDNA. Although some of the screened compounds did not exhibit the corresponding effects in the infection model, they significantly narrowed the range of candidate compounds and can be used for preliminary screening of compounds targeting HBV cccDNA.

[0195] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Xiamen University Yangshengtang Co., Ltd. <120> Drug screening model and method targeting HBV cccDNA <130> IDC210224 <150> CN202010588643.8 <151> 2020-06-24 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 3344 <212> DNA <213> Artificial sequence <220> <223> HBV 1.1-ploid genome sequence <400> 1 ttcacctctg cctaatcatc tcttgttcat gtcctactgt tcaagcctcc aagctgtgcc 60 ttgggtggct ttggggcatg gacatcgacc cttataaaga atttggagct actgtggagt 120 tactctcgtt tttgccttct gacttctttc cttcagtacg agatcttcta gataccgcct 180 cagctctgta tcgggaagcc ttagagtctc ctgagcattg ttcacctcac catactgcac 240 tcaggcaagc aattctttgc tgggggaac taatgactct agctacctgg gtgggtgtta 300 atttggaaga tccagcgtct agagacctag tagtcagtta tgtcaacact aatatgggcc 360 taaagttcag gcaactcttg tggtttcaca tttcttgtct cacttttgga agaagaaacag 420 ttatagtga tttggtgtct ttcggagtgt ggattcgcac tcctccagct tatagaccac 480 caaatgcccc tatcctatca acacttccgg agactactgt tgttagacga cgaggcaggt 540 cccctagaag aagaactccc tcgcctcgca gacgaaggtc tcaatcgccg cgtcgcagaa 600 gatctcaatc tcgggaatct caatgttagt attccttgga ctcataaggt ggggaacttt 660 actgggcttt attcttctac tgtacctgtc tttaatcctc attggaaaac accatctttt 720 cctaatatac atttacacca agacattatc aaaaaatgtg aacagtttgt aggcccactc 780 acagttaatg agaaaagaag attgcaattg attatgcctg ccaggtttta tccaaaggtt 840 accaaatatt taccattgga taagggtatt aaaccttatt atccagaaca tctagttaat 900 cattacttcc aaactagaca ctatttacac acttagga aggcggtat attatatag 960 agagaaaacaa cacatagcgc ctcattttgt gggtcaccat attctttggga acaagatcta 1020 cagcatgggg cagaatcttt ccaccagcaa tcctgggga ttctccccg accaccagtt 1080 ggatccagcc ttcagagcaa acaccgcaaa tccagattgg gactcaatc ccacaagga 1140 cacctggcca gacgccaca aggtaggagc tggagcattc gggctggtt tcaccccacc 1200 gcacggaggc ctttggggt ggagccctca ggctcaggc attackacaa ctttgccagc 1260 aaatccgcct cctgcctcca ccaatcgcca gtcaggaagg cagcctacc cgctgctcc 1320 acctttgaga aacactcatc ctcaggccat gcagtggaat tccacacct tccacatc 1380 tctgcagat cccagagtga gaggcctgta ttccctgct ggtggctcca gttcaggac 1440 agtaaaccct gttctgacta ctgccctcc cttatcgtca atctctcga ggattgggga 1500 ccctgcgctg aacatggaga acatcacatc aggattccta ggaccccttc tcgtgttaca 1560 ggcggggttt ttcttgttga caagaatcct cacaataccg cagagtctag actcgtggtg 1620 gacttctctc aattttctag ggggaactac cgtgtgtctt ggccaaaatt cgcagtcccc 1680 aacctccaat cactcaccaa cctcttgtcc tccaacttgt cctggttatc gctggatgtg 1740 tctgcggcgt tttatcatct tcctcttcat cctgctgcta tgcctcatct tcttgttggt 1800 tcttctggac tatcaaggta tgttgcccgt ttgtcctcta attccaggat cctcaacaac 1860 cagcacggga ccatgccgga cctgcatgac tactgctcaa ggaacctcta tgtatccctc 1920 ctgttgctgt accaaacctt cggacggaaa ttgcacctgt attcccatcc catcatcctg 1980 ggctttcgga aaattcctat gggagtgggc ctcagcccgt ttctcctggc tcagtttact 2040 agtgccattt gttcagtggt tcgtagggct ttcccccact gtttggcttt cagttatatg 2100 gatgatgtgg tattgggggc caagtctgta cagcatcttg agtccctttt taccgctgtt 2160 accaattttc ttttgtcttt gggtatacat ttaaacccta acaaaacaaa gagatggggt 2220 tactctctaa attttatggg ttatgtcatt ggatgttatg ggtccttgcc acaagaacac 2280 atcatacaaa aaatcaaaga atgttttaga aaacttccta ttaacaggcc tattgattgg 2340 aaagtatgtc aacgaattgt gggtcttttg ggttttgctg ccccttttac acaatgtggt 2400 tatcctgcgt tgatgccttt gtatgcatgt attcaatcta agcaggcttt cactttctcg 2460 ccaacttaca aggcctttct gtgtaaacaa tacctgaacc tttaccccgt tgcccggcaa 2520 cggccaggtc tgtgccaagt gtttgctgac gcaaccccca ctggctgggg cttggtcatg 2580 ggccatcagc gcatgcgtgg aaccttttcg gctcctctgc cgatccatac tgcggaactc 2640 ctagccgctt gttttgctcg cagcaggtct ggagcaaaca ttatcgggac tgataactct 2700 gttgtcctat cccgcaaata tacatcgttt ccatggctgc taggctgtgc tgccaactgg 2760 atcctgcgcg ggacgtcctt tgtttacgtc ccgtcggcgc tgaatcctgc ggacgaccct 2820 tctcggggtc gcttgggact ctctcgtccc cttctccgtc tgccgttccg accgaccacg 2880 gggcgcacct ctctttacgc ggactccccg tctgtgcctt ctcatctgcc ggaccgtgtg 2940 cacttcgctt cacctctgca cgtcgcatgg agaccaccgt gaacgcccac caaatattgc 3000 ccaaggtctt acataagagg actcttggac tctcagcaat gtcaacgacc gaccttgagg 3060 catacttcaa agactgtttg tttaaagact gggaggagtt gggggaggag attaggttaa 3120 aggtctttgt actaggaggc tgtaggcata aattggtctg cgcaccagca ccatgcaact 3180 ttttcacctc tgcctaatca tctcttgttc atgtcctact gttcaagcct ccaagctgtg 3240 ccttgggtgg ctttggggca tggacatcga cccttataaa gaatttggag ctactgtgga 3300 gttactctcg tttttgcctt ctgacttctt tccttcagta cgag 3344 <210> 2 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of HiBiT <400> 2 Val Ser Gly Trp Arg Leu Phe Lys Lys Ile Ser 1 5 10 <210> 3 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of HiBiT <400> 3 gtaagcggct ggcggctatt caagaaaatc tcc 33 <210> 4 <211> 117 <212> DNA <213> Artificial sequence <220> <223> Hibit16 insertion sequence <400> 4 gtaagcggct ggcggctatt caagaagatt agcggcagcg gcgtctccgg ttggagatta 60 ttcaagaaga tttcgggatc cggggttagt gggtggcgct tgttcaagaa gatcagc 117 <210> 5 <211> 624 <212> DNA <213> Artificial sequence <220> <223> TRE3G promoter <400> 5 ggtggcggcc gcaattctcc aggcgatctg acggttcact aaacgagctc tgcttatata 60 ggcctcccac cgtacacgcc acctcgacat actcgagttt actccctatc agtgatagag 120 aacgtatgaa gagtttactc cctatcagtg atagagaacg tatgcagact ttactcccta 180 tcagtgatag agaacgtata aggagtttac tccctatcag tgatagagaa cgtatgaaga 240 gtttactccc tatcagtgat agagaacgta tgcagacttt actccctatc agtgatagag 300 aacgtataag gagtttactc cctatcagtg atagagaacg tatgaagagt ttactcccta 360 tcagtgatag agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga 420 tcagtgatag agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga 420gtttactccc tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag 480 aacgtatcta cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta 540 tcagtgatag agaacgtata agctttaggc gtgtacggtg ggcgcctata aaagcagagc 600 tcgtttagtg aaccggtcaa cttt 624 <210> 6 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of P2A <400> 6 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 <210> 7 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of P2A <400> 7 ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60 ggacct 66 <210> 8 <211> 3488 <212> DNA <213> Artificial sequence <220> <223> HBV genome variant sequence containing Hibit16 <400> 8 ttcacctctg cctaatcatc tcttgttctt gtcctactgt tcaagcctcc aagctgtgcc 60 ttgggtggct ttggggcaag gacatcggca gcggcgtaag cggctggcgg ctattcaaga 120 agattagcgg cagcggcgtc tccggttgga gattattcaa gaagatttcg ggatccgggg 180 ttagtgggtg gcgcttgttc aagaagatca gcggaggtac catggacatc gacccttata 240 aagaatttgg agctactgtg gagttactct cgtttttgcc ttctgacttc tttccttcag 300 tacgagatct tctagatacc gcctcagctc tgtatcggga agccttagag tctcctgagc 360 attgttcacc tcaccatact gcactcaggc aagcaattct ttgctggggg gaactaatga 420 ctctagctac ctgggtgggt gttaatttgg aagatccagc gtctagagac ctagtagtca 480 gttatgtcaa cactaattg ggcctaaagt tcaggcaact cttgtggttt cacatttctt 540 gtctcacttt tggaagagaa acagttatag agtatttggt gtctttcgga gtgtggattc 600 gcactcctcc agcttataga ccaccaaatg ccctatcct atcaacactt ccggagacta 660 ctgttgttag acgacgaggc aggtccccta gaagaagaac tccctcgcct cgcagacgaa 720 ggtctcaatc gccgcgtcgc agaagatctc aatctcgggga atctcaatgt tagtatcct 780 tggactcata aggtggggaa cttactggg cttattctt ctactgtacc tgtctttaat 840 cctcattgga aaacaccatc ttttcctaat atacatttac accaagacat tatcaaaaaa 900 tgtgaacagt ttgtaggccc actcacagtt aatgagaaaaa gaagattgca attgattatg 960 cctgccaggt tttatccaaa ggttaccaaa tatttaccat tggataaggg tattaaacct 1020 tattatccag aacatctagt taatcattac ttccaaacta gacactattt acacactcta 1080 tggaaggcgg gtatattata taagagagaa acaacacata gcgcctcatt ttgtgggtca 1140 ccatattctt gggaacaaga tctacagcat ggggcagaat cttccacca gcaatcctct 1200 gggattcttt cccgaccacc agttggatcc agccttcaga gcaaacaccg caaatccaga 1260 ttgggacttc aatcccaaca aggacacctg gccagacgcc aacaaggtag gagctggagc 1320 attcgggctg ggtttcaccc caccgcacgg aggccttttg gggtggagcc ctcaggctca 1380 gggcatacta caaactttgc cagcaaatcc gcctcctgcc tccaccaatc gccagtcagg 1440 aaggcagcct accccgctgt ctccaccttt gagaaacact catcctcagg ccatgcagtg 1500 gaattccaca accttccacc aaactctgca agatcccaga gtgagaggcc tgtatttccc 1560 tgctggtggc tccagttcag gaacagtaaa ccctgttctg actactgcct ctcccttatc 1620 gtcaatcttc tcgaggattg gggaccctgc gctgaacatg gagaacatca catcaggatt 1680 cctaggaccc cttctcgtgt tacaggcggg gtttttcttg ttgacaagaa tcctcacaat 1740 accgcagagt ctagactcgt ggtggacttc tctcaatttt ctagggggaa ctaccgtgtg 1800 tcttggccaa aattcgcagt ccccaacctc caatcactca ccaacctctt gtcctccaac 1860 ttgtcctggt tatcgctgga tgtgtctgcg gcgttttatc atcttcctct tcatcctgct 1920 gctatgcctc atcttcttgt tggttcttct ggactatcaa ggtatgttgc ccgtttgtcc 1980 tctaattcca ggatcctcaa caaccagcac gggaccatgc cggacctgca tgactactgc 2040 tcaaggaacc tctatgtatc cctcctgttg ctgtaccaaa ccttcggacg gaaattgcac 2100 ctgtattccc atcccatcat cctgggcttt cggaaaattc ctatgggagt gggcctcagc 2160 ccgtttctcc tggctcagtt tactagtgcc atttgttcag tggttcgtag ggctttcccc 2220 cactgtttgg ctttcagtta tatggatgat gtggtattgg gggccaagtc tgtacagcat 2280 cttgagtccc tttttaccgc tgttaccaat tttcttttgt ctttgggtat acatttaaac 2340 cctaacaaaa caaagagatg gggttactct ctaaatttta tgggttatgt cattggatgt 2400 tatgggtcct tgccacaaga acacatcata caaaaaatca aagaatgttt tagaaaaactt 2460 cctattaaca ggcctattga ttggaaagta tgtcaacgaa ttgtgggtct tttgggtttt 2520 gctgcccctt ttacacaatg tggttatcct gcgttgatgc ctttgtatgc atgtattcaa 2580 tctaagcagg ctttcacttt ctcgccaact tacaaggcct ttctgtgtaa acaatacctg 2640 aacctttacc ccgttgcccg gcaacggcca ggtctgtgcc aagtgtttgc tgacgcaacc 2700 cccactggct ggggcttggt catgggccat cagcgcatgc gtggaacctt ttcggctcct 2760 ctgccgatcc atactgcgga actcctagcc gcttgttttg ctcgcagcag gtctggagca 2820 aacattatcg ggactgataa ctctgttgtc ctatcccgca aatatacatc gtttccatgg 2880 ctgctaggct gtgctgccaa ctggatcctg cgcgggacgt cctttgttta cgtcccgtcg 2940 gcgctgaatc ctgcggacga cccttctcgg ggtcgcttgg gactctctcg tccccttctc 3000 cgtctgccgt tccgaccgac cacggggcgc acctctcttt acgcggactc cccgtctgtg 3060 ccttctcatc tgccggaccg tgtgcacttc gcttcacctc tgcacgtcgc atggagacca 3120 ccgtgaacgc ccaccaaata ttgcccaagg tcttacataa gaggactctt ggactctcag 3180 caatgtcaac gaccgacctt gaggcatact tcaaagactg tttgtttaaa gactgggagg 3240 agttggggga ggagattagg ttaaaggtct ttgtactagg aggctgtagg cataaattgg 3300 tctgcgcacc agcaccatgc aactttttca cctctgccta atcatctctt gttcatgtcc 3360 tactgttcaa gcctccaagc tgtgccttgg gtggctttgg ggcatggaca tcgaccctta 3420 taaagaattt ggagctactg tggagttact ctcgtttttg ccttctgact tctttccttc 3480 agtacgag 3488 <210> 9 <211> 248 <212> PRT <213> 人工序列 <220> <223> Tet-On 3G的氨基酸序列 <400> 9 Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu 1 5 10 15 Leu Asn Gly Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30 Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 Arg Ala Leu Leu Asp Ala Leu Pro Ile Glu Met Leu Asp Arg His His 50 55 60 Thr His Ser Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80 Asn Asn Ala Lys Ser Tyr Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95 Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110 Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125 Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140 Val Leu Glu Glu Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Lys Gln Ala Ile Glu Leu 165 170 175 Phe Asp Arg Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro 195 200 205 Thr Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala 210 215 220 Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Wing Asp Wing Leu Asp Asp 225 230 235 240 Phe Asp Leu Asp Met Leu Pro Gly 245 <210> 10 <211> 747 <212> DNA <213>人工序列 <220> <223> Tet-On 3G phone <400> 10 atgagcagac tggacaagag caaagtcata aactctgctc tggaattact caatggagtc 60 ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc 120 ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat cgagatgctg 180 gacaggcatc atacccactc ctgccccctg gaaggcgagt catggcaaga ctttctgcgg 240 aacaacgcca agtcataccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat 300 ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg 360 tgtcagcaag gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt 420 acactgggct gcgtattgga ggaacaggag catcaagtag caaaagagga aagagagaca 480 cctaccaccg attctatgcc cccacttctg aaacaagcaa ttgagctgtt cgaccggcag 540 ggagccgaac ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag 600 ctaaagtgcg aaagcggcgg gccgaccgac gcccttgacg attttgactt agacatgctc 660 ccagccgatg cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat 720 tttgaccttg acatgctccc cgggtag 747 <210> 11 <211> 311 <212> PRT <213> 人工序列 <220> <223> iRFP的氨基酸序列 <400> 11 Met Ala Arg Lys Val Asp Leu Thr Ser Cys Asp Arg Glu Pro Ile His 1 5 10 15 Ile Pro Gly Ser Ile Gln Pro Cys Gly Cys Leu Leu Ala Cys Asp Ala 20 25 30 Gln Ala Val Arg Ile Thr Arg Ile Thr Glu Asn Ala Gly Ala Phe Phe 35 40 45 Gly Arg Glu Thr Pro Arg Val Gly Glu Leu Leu Ala Asp Tyr Phe Gly 50 55 60 Glu Thr Glu Ala His Ala Leu Arg Asn Ala Leu Ala Gln Ser Ser Asp 65 70 75 80 Pro Lys Arg Pro Ala Leu Ile Phe Gly Trp Arg Asp Gly Leu Thr Gly 85 90 95 Arg Thr Phe Asp Ile Ser Leu His Arg His Asp Gly Thr Ser Ile Ile 100 105 110 Glu Phe Glu Pro Ala Ala Ala Glu Gln Ala Asp Asn Pro Leu Arg Leu 115 120 125 Thr Arg Gln Ile Ile Ala Arg Thr Lys Glu Leu Lys Ser Leu Glu Glu 130 135 140 Met Ala Ala Arg Val Pro Arg Tyr Leu Gln Ala Met Leu Gly Tyr His 145 150 155 160 Arg Val Met Leu Tyr Arg Phe Ala Asp Asp Gly Ser Gly Met Val Ile 165 170 175 Gly Glu Ala Lys Arg Ser Asp Leu Glu Ser Phe Leu Gly Gln His Phe 180 185 190 Pro Ala Ser Leu Val Pro Gln Gln Ala Arg Leu Leu Tyr Leu Lys Asn 195 200 205 Ala Ile Arg Val Val Ser Asp Ser Arg Gly Ile Ser Ser Arg Ile Val 210 215 220 Pro Glu His Asp Ala Ser Gly Ala Ala Leu Asp Leu Ser Phe Ala His 225 230 235 240 Leu Arg Ser Ile Ser Pro Cys His Leu Glu Phe Leu Arg Asn Met Gly 245 250 255 Val Ser Ala Ser Met Ser Leu Ser Ile Ile Ile Asp Gly Thr Leu Trp 260 265 270 Gly Leu Ile Ile Cys His His Tyr Glu Pro Arg Ala Val Pro Met Ala 275 280 285 Gln Arg Val Ala Ala Glu Met Phe Ala Asp Phe Leu Ser Leu His Phe 290 295 300 Thr Ala Ala His His Gln Arg 305 310 <210> 12 <211> 936 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of iRFP <400> 12 atggcgcgta aggtcgatct cacctcctgc gatcgcgagc cgatccacat ccccggcagc 60 attcagccgt gcggctgcct gctggcctgc gacgcgcagg cggtgcggat cacgcgcatt 120 acggaaaatg ccggcgcgtt ctttggacgc gaaactccgc gggtcggtga gctactcgcc 180 gattacttcg gcgagaccga agcccatgcg ctgcgcaacg cactggcgca gtcctccgat 240 ccaaagcgac cggcgctgat cttcggttgg cgcgacggcc tgaccggccg caccttcgac 300 atctcactgc atcgccatga cggcaccagc atcatcgagt tcgagcctgc ggcggccgaa 360 caggccgaca atccgctgcg gctgacgcgg cagatcatcg cgcgcaccaa agaactgaag 420 tcgctcgaag agatggccgc acgggtgccg cgctatctgc aggcgatgct cggctatcac 480 cgcgtgatgt tgtaccgctt cgcggacgac ggctccggga tggtgatcgg cgaggcgaag 540 cgcagcgacc tggagagctt tctcggtcag cactttccgg cgtcgctggt cccgcagcag 600 gcgcggctac tgtacttgaa gaacgcgatc cgcgtggtct cggattcgcg cggcatcagc 660 agccggatcg tgcccgagca cgacgcctcc ggcgccgcgc tcgatctgtc gttcgcgcac 720 ctgcgcagca tctcgccctg ccatctcgaa tttctgcgga acatgggcgt cagcgcctcg 780 atgtcgctgt cgatcatcat tgacggcacg ctatggggat tgatcatctg tcatcattac 840 gagccgcgtg ccgtgccgat ggcgcagcgc gtcgcggccg aaatgttcgc cgacttctta 900 tcgctgcact tcaccgccgc ccaccaccaa cgctga 936 <210> 13 <211> 405 <212> DNA <213> 人工序列 <220> <223> Blasticidin抗性基因 <400> 13 atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc 60 aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc 120 cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg 180 gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga 240 aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg 300 catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt 360 cgtgaattgc tgccctctgg ttatgtgtgg gagggctgag cttga 405 <210> 14 <211> 306 <212> DNA <213> Artificial sequence <220> <223> Sequence of the 3' region of the C-ORF of the Rccc1a HBV genome <400> 14 gacctagtag tcagttatgt caacactaat atgggcctaa agttcaggca actcttgtgg 60 tttcacattt cttgtctcac ttttggaaga gaaacagtta tagagtattt ggtgtctttc 120 ggagtgtgga ttcgcactcc tccagcttat agaccaccaa atgcccctat cctatcaaca 180 cttccggaga ctactgttgt tagacgacga ggcaggtccc ctagaagaag aactccctcg 240 cctcgcagac gaaggtctca atcgccgcgt cgcagaagat ctcaatctcg ggaatctcaa 300 tgttag 306 <210> 15 <211> 147 <212> DNA <213> Artificial sequence <220> <223> Sequence of the 5' end region of the C-ORF of the Rccc1a HBV genome <400> 15 cttctagata ccgcctcagc tctgtatcgg gaagccttag agtctcctga gcattgttca 60 cctcaccata ctgcactcag gcaagcaatt ctttgctggg gggaactaat gactctagct 120 acctgggtgg gtgttaattt ggaagat 147 <210> 16 <211> 2717 <212> DNA <213> 人工序列 <220> <223> Rccc1a 去除了C-ORF的HBV基因组片段序列 <400> 16 tattccttgg actcataagg tggggaactt tactgggctt tattcttcta ctgtacctgt 60 ctttaatcct cattggaaaa caccatcttt tcctaatata catttacacc aagacattat 120 caaaaaatgt gaacagtttg taggcccact cacagttaat gagaaaagaa gattgcaatt 180 gattatgcct gccaggtttt atccaaaggt taccaaatat ttaccattgg ataagggtat 240 taaaccttat tatccagaac atctagttaa tcattacttc caaactagac actatttaca 300 cactctatgg aaggcgggta tattatataa gagagaaaca acacatagcg cctcattttg 360 tgggtcacca tattcttggg aacaagatct acagcatggg gcagaatctt tccaccagca 420 atcctctggg attctttccc gaccaccagt tggatccagc cttcagagca aacaccgcaa 480 atccagattg ggacttcaat cccaacaagg acacctggcc agacgccaac aaggtaggag 540 ctggagcatt cgggctgggt ttcaccccac cgcacggagg ccttttgggg tggagccctc 600 aggctcaggg catactacaa actttgccag caaatccgcc tcctgcctcc accaatcgcc 660 agtcaggaag gcagcctacc ccgctgtctc cacctttgag aaacactcat cctcaggcca 720 tgcagtggaa ttccacaacc ttccaccaaa ctctgcaaga tcccagagtg agaggcctgt 780 atttccctgc tggtggctcc agttcaggaa cattaaaccc tgttctgact actgcctctc 840 ccttatcgtc aatcttctcg aggattgggg accctgcgct gaacatggag aacatcacat 900 caggattcct aggacccctt ctcgtgttac aggcggggtt tttcttgttg acaagaatcc 960 tcacaatacc gcagagtcta gactcgtggt ggacttctct caattttcta gggggaacta 1020 ccgtgtgtct tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcttgtc 1080 ctccaacttg tcctggttat cgctggatgt gtctgcggcg ttttatcatc ttcctcttca 1140 tcctgctgct atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg 1200 tttgtcctct aattccagga tcctcaacaa ccagcacggg accatgccgg acctgcatga 1260 ctactgctca aggaacctct atgtatccct cctgttgctg taccaaacct tcggacggaa 1320 attgcacctg tattcccatc ccatcatcct gggctttcgg aaaattccta tgggagtggg 1380 cctcagcccg tttctcctgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc 1440 tttcccccac tgtttggctt tcagttatat ggatgatgtg gtattggggg ccaagtctgt 1500 acagcatctt gagtcccttt ttaccgctgt taccaatttt cttttgtctt tgggtataca 1560 tttaaaccct aacaaaacaa agagatgggg ttactctcta aattttatgg gttatgtcat 1620 tggatgttat gggtccttgc cacaagaaca catcatacaa aaaatcaaag aatgttttag 1680 aaaacttcct attaacaggc ctattgattg gaaagtatgt caacgaattg tgggtctttt 1740 gggttttgct gcccctttta cacaatgtgg ttatcctgcg ttgatgcctt tgtatgcatg 1800 tattcaatct aagcaggctt tcactttctc gccaacttac aaggcctttc tgtgtaaaca 1860 atacctgaac ctttaccccg ttgcccggca acggccaggt ctgtgccaag tgtttgctga 1920 cgcaaccccc actggctggg gcttggtcat gggccatcag cgcatgcgtg gaaccttttc 1980 ggctcctctg ccgatccata ctgcggaact cctagccgct tgttttgctc gcagcaggtc 2040 tggagcaaac attatcggga ctgataactc tgttgtccta tcccgcaaat atacatcgtt 2100 tccatggctg ctaggctgtg ctgccaactg gatcctgcgc gggacgtcct ttgtttacgt 2160 cccgtcggcg ctgaatcctg cggacgaccc ttctcggggt cgcttgggac tctctcgtcc 2220 ccttctccgt ctgccgttcc gaccgaccac ggggcgcacc tctctttacg cggactcccc 2280 gtctgtgcct tctcatctgc cggaccgtgt gcacttcgct tcacctctgc acgtcgcatg 2340 gagaccaccg tgaacgccca ccaaatattg cccaaggtct tacataagag gactcttgga 2400 ctctcagcaa tgtcaacgac cgaccttgag gcatacttca aagactgttt gtttaaagac 2460 tgggaggagt tgggggagga gattaggtta aaggtctttg tactaggagg ctgtaggcat 2520 aaattggtct gcgcaccagc accatgcaac tttttcacct ctgcctaatc atctcttgtt 2580 catgtcctac tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacatcg 2640 acccttataa agaatttgga gctactgtgg agttactctc gtttttgcct tctgacttct 2700 ttccttcagt acgagat 2717 <210> 17 <211> 3324 <212> DNA <213> 人工序列 <220> <223> Rccc1a线性复制子序列 <400> 17 ataacttcgt ataatgtatg ctatacgaag ttatctggcg gtagcggtgt gagcggctgg 60 cgcctgttca agaagatcag cggcggcggc ggcagcaccg gtgacctagt agtcagttat 120 gtcaacacta atatgggcct aaagttcagg caactcttgt ggtttcacat ttcttgtctc 180 acttttggaa gagaaacagt tatagagtat ttggtgtctt tcggagtgtg gattcgcact 240 cctccagctt atagaccacc aaatgcccct atcctatcaa cacttccgga gactactgtt 300 gttagacgac gaggcaggtc ccctagaaga agaactccct cgcctcgcag acgaaggtct 360 caatcgccgc gtcgcagaag atctcaatct cgggaatctc aatgttagta ttccttggac 420 tcataaggtg gggaacttta ctgggcttta ttcttctact gtacctgtct ttaatcctca 480 ttggaaaca ccatcttttc ctaatataca tttacaccaa gacattatca aaaaatgtga acagtttgta ggcccactca cagttatga gaaaagaaga ttgcaattga ttatgcctgc caggttttat ccaaaggtta ccaaatattt accattggat aagggtatta aaccttatta tccagaacat ctagttaatc attacttcca aactagacac tatttacaca ctctatggaa ggcgggtata ttatataaga gagaaacaac acatagcgcc tcattttgtg ggtcaccata ttcttgggaa caagatctac agcatggggc agaatctttc caccagcaat cctctgggat tctttcccga ccaccagttg gatccagcct tcagagcaaa caccgcaaat ccagattggg acttcaatcc caacaaggac acctggccag acgccaacaa ggtaggagct ggagcattcg ggctgggttt caccccaccg cacggaggcc ttttggggtg gagccctcag gctcagggca 1080. tactacaaac tttgccagca aatccgcctc ctgcctccac caatcgccag tcaggaaggc agcctacccc gctgtctcca cctttgagaa acactcatcc tcaggccatg cagtggaatt ccacaacctt ccaccaaact ctgcaagatc ccagagtgag aggcctgtat ttccctgctg 1200 gtggctccag ttcaggaaca gtaaaccctg ttctgactac tgcctctccc ttatcgtcaa 1260 tcttctcgag gattggggac cctgcgctga acatggagaa catcacatca ggattcctag 1320 gaccccttct cgtgttacag gcggggtttt tcttgttgac aagaatcctc acaataccgc 1380 agagtctaga ctcgtggtgg acttctctca attttctagg gggaactacc gtgtgtcttg 1440 gccaaaattc gcagtcccca acctccaatc actcaccaac ctcttgtcct ccaacttgtc 1500 ctggttatcg ctggatgtgt ctgcggcgtt ttatcatctt cctcttcatc ctgctgctat 1560 gcctcatctt cttgttggtt cttctggact atcaaggtat gttgcccgtt tgtcccttaa 1620 ttccaggatc ctcaacaacc agcacgggac catgccggac ctgcatgact actgctcaag 1680 gaacctctat gtatccctcc tgttgctgta ccaaaccttc ggacggaaat tgcacctgta 1740 ttcccatccc atcatcctgg gctttcggaa aattcctatg ggagtgggcc tcagcccgtt 1800 tctcctggct cagtttacta gtgccatttg ttcagtggtt cgtagggctt tccccactg 1860 tttggctttc agttatatgg atgatgtggt attgggggcc aagtctgtac agcatcttga 1920 gtcccttttt accgctgtta ccaattttct tttgtctttg ggtatacatt taaaccctaa 1980 caaaacaaag agatggggtt actctctaaa ttttatgggt tatgtcattg gatgttatgg 2040 gtccttgcca caagaacaca tcatacaaaa aatcaaagaa tgttttagaa aacttcctat 2100 taacaggcct attgattgga aagtatgtca acgaattgtg ggtcttttgg gttttgctgc 2160 cccttttaca caatgtggtt atcctgcgtt gatgcctttg tatgcatgta ttcaatctaa 2220 gcaggctttc actttctcgc caacttacaa ggcctttctg tgtaaacaat acctgaacct 2280 ttaccccgtt gcccggcaac ggccaggtct gtgccaagtg tttgctgacg caacccccac 2340 tggctggggc ttggtcatgg gccatcagcg catgcgtgga accttttcgg ctcctctgcc 2400 gatccatact gcggaactcc tagccgcttg ttttgctcgc agcaggtctg gagcaaacat 2460 tatcgggact gataactctg ttgtcctatc ccgcaaatat acatcgtttc catggctgct 2520 aggctgtgct gccaactgga tcctgcgcgg gacgtccttt gtttacgtcc cgtcggcgct 2580 gaatcctgcg gacgaccctt ctcggggtcg cttgggactc tctcgtcccc ttctccgtct 2640 gccgttccga ccgaccacgg ggcgcacctc tctttacgcg gactccccgt ctgtgccttc 2700 tcatctgccg gaccgtgtgc acttcgcttc acctctgcac gtcgcatgga gaccaccgtg 2760 aacgcccacc aaatattgcc caaggtctta cataagagga ctcttggact ctcagcaatg 2820 tcaacgaccg accttgaggc atacttcaaa gactgtttgt ttaaagactg ggaggagttg 2880 ggggaggaga ttaggttaaa ggtctttgta ctaggaggct gtaggcataa attggtctgc 2940 gcaccagcac catgcaactt tttcacctct gcctaatcat ctcttgttca tgtcctactg 3000 ttcaagcctc caagctgtgc cttgggtggc tttggggcat ggacatcgac ccttataaag 3060 aatttggagc tactgtggag ttactctcgt ttttgccttc tgacttcttt ccttcagtac 3120 gagatcttct agataccgcc tcagctctgt atcgggaagc cttagagtct cctgagcatt 3180 gttcacctca ccatactgca ctcaggcaag caattctttg ctggggggaa ctaatgactc 3240 tagctacctg ggtgggtgtt aatttggaag atggaggtac cggcggtagc ataacttcgt 3300 ataatgtatg ctatacgaag ttat 3324 <210> 18 <211> 3290 <212> DNA <213> Artificial Sequence <220> <223> Rccc1a Recombinant cccDNA Sequence <400> 18 ataacttcgt ataatgtatg ctatacgaag ttatctggcg gtagcggtgt gagcggctgg 60 cgcctgttca agaagatcag cggcggcggc ggcagcaccg gtgacctagt agtcagttat 120 gtcaacacta atatgggcct aaagttcagg caactcttgt ggtttcacat ttcttgtctc 180 acttttggaa gagaaacagt tatagagtat ttggtgtctt tcggagtgtg gattcgcact 240 cctccagctt atagaccacc aaatgcccct atcctatcaa cacttccgga gactactgtt 300 gttagacgac gaggcaggtc ccctagaaga agaactccct cgcctcgcag acgaaggtct 360 caatcgccgc gtcgcagaag atctcaatct cgggaatctc aatgttagta ttccttggac 420 tcataaggtg gggaacttta ctgggcttta ttcttctact gtacctgtct ttaatcctca 480 ttggaaaaca ccatcttttc ctaatataca tttacaccaa gacattatca aaaaatgtga 540 acagtttgta ggcccactca cagttatga gaaaagaaga ttgcaattga ttatgcctgc caggttttat ccaaaggtta ccaaatattt accattggat aagggtatta aaccttatta tccagaacat ctagttaatc attacttcca aactagacac tatttacaca ctctatggaa ggcgggtata ttatataaga gagaaacaac acatagcgcc tcattttgtg ggtcaccata ttcttgggaa caagatctac agcatggggc agaatctttc caccagcaat cctctgggat tctttcccga ccaccagttg gatccagcct tcagagcaaa caccgcaaat ccagattggg acttcaatcc caacaaggac acctggccag acgccaacaa ggtaggagct ggagcattcg ggctgggttt caccccaccg cacggaggcc ttttggggtg gagccctcag gctcagggca 1080. tactacaaac tttgccagca aatccgcctc ctgcctccac caatcgccag tcaggaaggc agcctacccc gctgtctcca cctttgagaa acactcatcc tcaggccatg cagtggaatt 1200. cccaacctt ccccaaact ctgcaagatc ccgagtgag aggcctgtat ttccctgctg gtggctccag ttcaggaaca gtaaaccctg ttctgactac tgcctctccc ttatcgtcaa tcttctcgag gattggggac cctgcgctga acatggagaa catcacatca ggattcctag 1320 gaccccttct cgtgttacag gcggggtttt tcttgttgac aagaatcctc acaataccgc 1380 agagtctaga ctcgtggtgg acttctctca attttctagg gggaactacc gtgtgtcttg 1440 gccaaaattc gcagtcccca acctccaatc actcaccaac ctcttgtcct ccaacttgtc 1500 ctggttatcg ctggatgtgt ctgcggcgtt ttatcatctt cctcttcatc ctgctgctat 1560 gcctcatctt cttgttggtt cttctggact atcaaggtat gttgcccgtt tgtcccttaa 1620 ttccaggatc ctcaacaacc agcacgggac catgccggac ctgcatgact actgctcaag 1680 gaacctctat gtatccctcc tgttgctgta ccaaaccttc ggacggaaat tgcacctgta 1740 ttcccatccc atcatcctgg gctttcggaa aattcctatg ggagtgggcc tcagcccgtt 1800 tctcctggct cagtttacta gtgccatttg ttcagtggtt cgtagggctt tccccactg 1860 tttggctttc agttatatgg atgatgtggt attggggggcc aagtctgtac agcatcttga 1920 gtcccttttt accgctgtta ccaattttct tttgtctttg ggtatacatt taaaccctaa 1980 caaaacaaag agatggggtt actctctaaa ttttatgggt tatgtcattg gatgttatgg 2040 gtccttgcca caagaacaca tcatacaaaa aatcaaagaa tgttttagaa aacttcctat 2100 taacaggcct attgattgga aagtatgtca acgaattgtg ggtcttttgg gttttgctgc 2160 cccttttaca caatgtggtt atcctgcgtt gatgcctttg tatgcatgta ttcaatctaa 2220 gcaggctttc actttctcgc caacttacaa ggcctttctg tgtaaacaat acctgaacct 2280 ttaccccgtt gcccggcaac ggccaggtct gtgccaagtg tttgctgacg caacccccac 2340 tggctggggc ttggtcatgg gccatcagcg catgcgtgga accttttcgg ctcctctgcc 2400 gatccatact gcggaactcc tagccgcttg ttttgctcgc agcaggtctg gagcaaacat 2460 tatcgggact gataactctg ttgtcctatc ccgcaaatat acatcgtttc catggctgct 2520 aggctgtgct gccaactgga tcctgcgcgg gacgtccttt gtttacgtcc cgtcggcgct 2580 gaatcctgcg gacgaccctt ctcggggtcg cttgggactc tctcgtcccc ttctccgtct 2640 gccgttccga ccgaccacgg ggcgcacctc tctttacgcg gactccccgt ctgtgccttc 2700 tcatctgccg gaccgtgtgc acttcgcttc acctctgcac gtcgcatgga gaccaccgtg 2760 aacgcccacc aaatattgcc caaggtctta cataagagga ctcttggact ctcagcaatg 2820 tcaacgaccg accttgaggc atacttcaaa gactgtttgt ttaaagactg ggaggagttg 2880 ggggaggaga ttaggttaaa ggtctttgta ctaggaggct gtaggcataa attggtctgc 2940 gcaccagcac catgcaactt tttcacctct gcctaatcat ctcttgttca tgtcctactg 3000 ttcaagcctc caagctgtgc cttgggtggc tttggggcat ggacatcgac ccttataaag 3060 aatttggagc tactgtggag ttactctcgt ttttgccttc tgacttcttt ccttcagtac 3120 gagatcttct agataccgcc tcagctctgt atcggggaagc cttagagtct cctgagcatt 3180 gttcacctca ccatactgca ctcaggcaag caattctttg ctggggggaa ctaatgactc 3240 tagctacctg ggtgggtgtt aatttggaag atggaggtac cggcggtagc 3290 <210> 19 <211> 34 <212> DNA <213>人工序列 <220> <223> loxP序列 <400> 19 ataacttcgt atagcataca ttatacgaag ttat 34 <210> 20 <211> 380 <212> PRT <213>人工序列 <220> <223> Creative Communications <400> 20 Met Gly His His His His His Gly Met Gly Ala Ala Gly Arg Lys 1 5 10 15 Lys Arg Arg Gln Arg Arg Arg Pro Pro Ala Gly Thr Ser Val Ser Leu 20 25 30 Lys Lys Lys Arg Lys Val Ser Asn Leu Leu Thr Val His Gln Asn Leu 35 40 45 Pro Ala Leu Pro Val Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu 50 55 60 Met Asp Met Phe Arg Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys 65 70 75 80 Put Leu Leu Ser Val Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn 85 90 95 Asn Arg Lys Trp Phe Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu 100 105 110 Leu Tyr Leu Gln Ala Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His 115 120 125 Leu Gly Gln Leu Asn Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro 130 135 140 Ser Asp Ser Asn Ala Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu 145 150 155 160 Asn Val Asp Ala Gly Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg 165 170 175 Thr Asp Phe Asp Gln Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys 180 185 190 Gln Asp Ile Arg Asn Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu 195 200 205 Leu Arg Ile Ala Glu Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg 210 215 220 Thr Asp Gly Gly Arg Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu 225 230 235 240 Val Ser Thr Ala Gly Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys 245 250 255 Leu Val Glu Arg Trp Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn 260 265 270 Asn Tyr Leu Phe Cys Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser 275 280 285 Ala Thr Ser Gln Leu Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala 290 295 300 Thr His Arg Leu Ile Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr 305 310 315 320 Leu Ala Trp Ser Gly His Ser Ala Arg Val Gly Ala Ala Arg Asp Met 325 330 335 Ala Arg Ala Gly Val Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp 340 345 350 Thr Asn Val Asn Ile Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu 355 360 365 Thr Gly Ala Met Val Arg Leu Leu Glu Asp Gly Asp 370 375 380 <210> 21 <211> 1143 <212> DNA <213> 人工序列 <220> <223> Cre重组酶的核苷酸序列 <400> 21 atgggccatc accatcacca tcacggcatg ggcgctgcag gtcgcaagaa acgtcgccaa 60 cgtcgccgtc cgcctgcagg cactagtgta agcttgaaga agaagaggaa ggtgtccaat 120 ttactgaccg tacaccaaaa tttgcctgca ttaccggtcg atgcaacgag tgatgaggtt 180 cgcaagaacc tgatggacat gttcagggat cgccaggcgt tttctgagca tacctggaaa 240 atgcttctgt ccgtttgccg gtcgtgggcg gcatggtgca agttgaataa ccggaaatgg 300 tttcccgcag aacctgaaga tgttcgcgat tatcttctat atcttcaggc gcgcggtctg 360 gcagtaaaaa ctatccagca acatttgggc cagctaaaca tgcttcatcg tcggtccggg 420 ctgccacgac caagtgacag caatgctgtt tcactggtta tgcggcggat ccgaaaagaa 480 aacgttgatg ccggtgaacg tgcaaaacag gctctagcgt tcgaacgcac tgatttcgac 540 caggttcgtt cactcatgga aaatagcgat cgctgccagg atatacgtaa tctggcattt 600 ctggggattg cttataacac cctgttacgt atagccgaaa ttgccaggat cagggttaaa 660 gatatctcac gtactgacgg tgggagaatg ttaatccata ttggcagaac gaaaacgctg 720 gttagcaccg caggtgtaga gaaggcactt agcctggggg taactaaact ggtcgagcga 780 tggatttccg tctctggtgt agctgatgat ccgaataact acctgttttg ccgggtcaga 840 aaaaatggtg ttgccgcgcc atctgccacc agccagctat caactcgcgc cctggaaggg 900 atttttgaag caactcatcg attgatttac ggcgctaagg atgactctgg tcagagatac 960 ctggcctggt ctggacacag tgcccgtgtc ggagccgcgc gagatatggc ccgcgctgga 1020 gtttcaatac cggagatcat gcaagctggt ggctggacca atgtaaatat tgtcatgaac 1080 tatatccgta acctggatag tgaaacaggg gcaatggtgc gcctgctgga agatggcgat 1140 tag 1143 <210> 22 <211> 20 <212> DNA <213>人工序列 <220> <223> HBV-F <400> 22 tttcacctct gcctaatcat 20 <210> 23 <211> 26 <212> DNA <213>人工序列 <220> <223> HBV-R <400> 23 tcagaaggca aaaaagagag taactc 26 <210> 24 <211> 24 <212> DNA <213>人工序列 <220> <223> HBV-Probe <400> 24 ccttgggtgg ctttggggca tgga 24 <210> 25 <211> 25 <212> DNA <213> Artificial sequence <220> <223> cccDNA-Probe <400> 25 accgtgaacg cccaccgaat gttgc 25 <210> 26 <211> 17 <212> DNA <213> Artificial sequence <220> <223> cccDNA-F <400> 26 tgcacttcgc ttcacct 17 <210> 27 <211> 17 <212> DNA <213> Artificial sequence <220> <223> cccDNA-R <400> 27 aggggcattt ggtggtc 17 <210> 28 <211> 16 <212> DNA <213> Artificial sequence <220> <223> mt4987F <400> 28 cccagctacg caaaat 16 <210> 29 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> mt5106R <400> 29 aatgcggtag tagttaggat a 21 <210> 30 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> mt5010-Probe <400> 30 catactcctc aattacccac atag 24

Claims

1. An isolated nucleic acid molecule, which is a variant of the HBV genome sequence, comprising: The invention relates to an HBV genome fragment comprising a C-ORF, an S-ORF and a P-ORF, wherein the C-ORF comprises an exogenous insertion sequence between the precore and core genes, wherein the exogenous insertion sequence is a nucleotide sequence encoding a first luciferase fragment; the first luciferase fragment can bind to a corresponding second luciferase fragment in a luciferase fragment complementation technique and generate luciferase activity, wherein the first luciferase fragment is HiBiT and the second luciferase fragment is LgBiT.

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

3. The isolated nucleic acid molecule of claim 1, wherein The exogenous insertion sequence comprises multiple copies of a nucleotide sequence encoding the first fragment of luciferase that are present in a tandem repeat manner.

4. The isolated nucleic acid molecule of claim 3, wherein The exogenous insertion sequence comprises three copies of a nucleotide sequence encoding the first fragment of luciferase that are present in a tandem repeat manner.

5. The isolated nucleic acid molecule of claim 3, wherein Each of the multiple copies of the nucleotide sequence encoding the first fragment of luciferase that are present in a tandem repeat manner comprises a sequence encoding a connecting peptide at its 5' end.

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

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

4.

8. The isolated nucleic acid molecule of claim 1, wherein The HBV genome is a full-length genome or an extra-length genome.

9. The isolated nucleic acid molecule of claim 8, wherein The HBV genome is a 1.1 ploid genome or a 1.3 ploid genome.

10. The isolated nucleic acid molecule of claim 1, wherein The HBV genome comprises the sequence shown in SEQ ID NO:

1.

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

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

13. The isolated nucleic acid molecule of claim 11, wherein The inducible promoter has bidirectional promoter activity.

14. The isolated nucleic acid molecule of claim 13, wherein The inducible promoter is a TRE3G promoter with bidirectional promoter activity.

15. The isolated nucleic acid molecule of claim 11, wherein The isolated nucleic acid molecule further comprises a reporter gene operably linked to the inducible promoter.

16. The isolated nucleic acid molecule of claim 15, wherein The reporter gene is in the opposite direction to the exogenous inserted sequence.

17. The isolated nucleic acid molecule of claim 15, wherein The reporter gene is selected from fluorescent protein genes and / or antibiotic resistance genes.

18. The isolated nucleic acid molecule of claim 17, wherein The fluorescent protein gene is iRFP.

19. The isolated nucleic acid molecule of claim 17, wherein The antibiotic resistance gene is Blasticidin.

20. The isolated nucleic acid molecule of claim 15, wherein The reporter gene comprises a fluorescent protein gene and an antibiotic resistance gene.

21. The isolated nucleic acid molecule of claim 20, wherein The fluorescent protein gene and the antibiotic resistance gene are optionally linked via a nucleotide sequence encoding a self-cleaving peptide.

22. The isolated nucleic acid molecule of claim 21, wherein The self-cleaving peptide is P2A, E2A, F2A or T2A.

23. The isolated nucleic acid molecule of any one of claims 11 to 22, wherein The isolated nucleic acid molecule comprises the sequence shown in SEQ ID NO:

8.

24. A recombinant HBV cccDNA comprising the isolated nucleic acid molecule according to any one of claims 1 to 23.

25. The recombinant HBV cccDNA of claim 24, comprising a variant of the HBV genome sequence of any one of claims 1-23.

26. The recombinant HBV cccDNA of claim 24, which is formed by circularizing the isolated nucleic acid molecule of any one of claims 1 to 23.

27. An expression system comprising the isolated nucleic acid molecule according to any one of claims 11 to 23 as a first nucleic acid sequence, and comprising a second nucleic acid sequence comprising a nucleotide sequence encoding a transactivator corresponding to the inducible promoter comprised in the first nucleic acid sequence.

28. The expression system of claim 27, wherein The transactivator protein is selected from Tet-On 3G transactivator protein, rTetR, and rtTA.

29. The expression system of claim 27, wherein The second nucleic acid sequence further comprises an expression control element operably linked to the nucleotide sequence encoding the transactivator corresponding to the inducible promoter contained in the first nucleic acid sequence.

30. The expression system of claim 29, wherein The expression control element is a promoter and / or an enhancer.

31. The expression system of claim 30, wherein The promoter is a constitutive promoter.

32. The expression system of claim 27, 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 protein.

33. The expression system of claim 32, wherein The TRE3G promoter comprises the sequence shown in SEQ ID NO:

5.

34. The expression system of claim 32, wherein The nucleotide sequence encoding the Tet-On 3G transactivator protein comprises the sequence shown in SEQ ID NO:

10.

35. A vector comprising the isolated nucleic acid molecule of any one of claims 1 to 23, or comprising the expression system of any one of claims 27 to 34.

36. The vector according to claim 35, wherein The vector comprises the expression system according to any one of claims 27 to 34 , wherein the first nucleic acid sequence and the second nucleic acid sequence are provided on the same or different vectors.

37. The vector according to claim 36, wherein The first nucleic acid sequence and the second nucleic acid sequence are provided on the same vector.

38. The vector according to any one of claims 35 to 37, wherein The vector is a transposon vector.

39. The vector according to claim 38, wherein The transposon vector is a PiggyBac transposon vector.

40. The vector according to claim 38, wherein The vector comprises the expression system according to any one of claims 27 to 34, and the first nucleic acid sequence and the second nucleic acid sequence are located between two ITR sequences of the transposon vector. A co-transfection system comprising the vector according to any one of claims 35 to 40 and a transposase expression vector.

42. The co-transfection system of claim 41, wherein The transposase expression vector is a PiggyBac transposase expression vector.

43. A host cell comprising the isolated nucleic acid molecule of any one of claims 1-23, or the recombinant HBV cccDNA of any one of claims 24-26, or the expression system of any one of claims 27-34, or the vector of any one of claims 35-40, or the co-transfection system of claim 41 or 42.

44. The host cell according to claim 43, wherein The host cell is selected from eukaryotic cells of hepatocyte origin.

45. The host cell according to claim 44, wherein The hepatocyte-derived eukaryotic cells are hepatoma cells or hepatocytes.

46. ​​The host cell of claim 43, wherein The host cell is selected from HepaRG, HepG2 or Huh7.

47. The host cell of claim 43, wherein The host cell comprises the expression system according to any one of claims 27 to 34 in its genome.

48. The host cell according to claim 47, wherein When an inducer corresponding to the inducible promoter and the transactivator protein is present, the host cell can stably express HBV cccDNA formed by the variant of the HBV genome sequence.

49. The host cell according to claim 48, wherein The inducer is Doxycycline.

50. A kit comprising the isolated nucleic acid molecule of any one of claims 1 to 23, or the expression system of any one of claims 27 to 34, or the vector of any one of claims 35 to 40, or the co-transfection system of claim 41 or 42, or the host cell of any one of claims 43 to 49. The kit of claim 50 , comprising: the vector of any one of claims 35 to 40 , or the co-transfection system of claim 41 or 42 .

52. The kit of claim 50, comprising: the host cell of any one of claims 43-49.

53. The kit according to claim 50, wherein The kit also contains LgBiT protein and optionally a luciferase substrate.

54. The kit of claim 50, comprising the expression system of any one of claims 27 to 34, or the vector of any one of claims 36 to 40, or the host cell of any one of claims 47 to 49, wherein: The kit also contains inducers corresponding to the inducible promoter and transactivator protein.

55. The kit according to claim 54, wherein The inducer is Doxycycline.

56. A method for screening an HBV cccDNA inhibitor, comprising: (1) Providing the host cell according to any one of claims 47 to 49; (2) contacting the host cell with an inducing agent, wherein the inducing agent is an inducer corresponding to the inducible promoter and transactivator protein contained in the host cell; (3) contacting the test agent with the host cell; wherein steps (2) and (3) are performed simultaneously or in any order; (4) detecting the level of the first luciferase fragment in the cell supernatant of the host cell, wherein the level of the first luciferase fragment is detected by luciferase fragment complementation technology and a second luciferase fragment complementary to the first luciferase fragment, the first luciferase fragment is HiBiT, and the second luciferase fragment is LgBiT protein.

57. The method of claim 56, wherein The inducer is Doxycycline.

58. The method of claim 56, wherein Step (1) includes the following steps: (1a) introducing a first nucleotide sequence and a second nucleotide sequence in the expression system of any one of claims 27 to 34 into a host cell, wherein 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 of any one of claims 11 to 23; (1b) culturing the host cell.

59. The method of claim 58, wherein The host cell is selected from eukaryotic cells of hepatocyte origin.

60. The method of claim 59, wherein The hepatocyte-derived eukaryotic cells are hepatoma cells or hepatocytes.

61. The method of claim 58, wherein The host cell is selected from HepaRG, HepG2 or Huh7.

62. The method of claim 58, wherein In step (1a), the expression vector is a transposon vector, and this step further comprises: introducing a transposase expression vector into the host cell.

63. The method of claim 62, wherein The transposon vector is a PiggyBac transposon vector.

64. The method of claim 62, wherein The transposase expression vector is a PiggyBac transposase expression vector.

65. The method of claim 58, wherein The step (1) further comprises: (1c) identifying and selecting a host cell into whose genome the expression system according to any one of claims 27 to 34 has been integrated.

66. The method of claim 65, wherein Whether the expression system has been integrated into the genome of the host cell is identified by detecting the reporter gene contained in the first nucleic acid sequence.

67. The method of any one of claims 56-64, further comprising the steps of: (5) comparing the measurement result of step (4) with the level of the first luciferase fragment measured in the absence of the test agent; If the measurement result of step (4) is lower than the measurement result in the absence of the test agent, it indicates that the test agent is an HBV cccDNA inhibitor.

68. Use of the isolated nucleic acid molecule described in any one of claims 1-23, or the expression system described in any one of claims 27-34, or the vector described in any one of claims 35-40, or the co-transfection system described in claim 41 or 42, or the host cell described in any one of claims 43-49, or the kit described in any one of claims 50-55 for screening HBVcccDNA inhibitors.

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