Hepatitis B virus replication monitoring system using HiBiT

Abstract

To provide a method for readily quantifying HBV replication at low cost.SOLUTION: An HBV vector includes DNA encoding HiBiT composed of 11 amino acids represented by a specific sequence in PreS1 gene of HBV gene.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an HBV vector carrying HiBiT, and an HBV particle carrying HiBiT. The present invention also relates to a method for monitoring the replication of hepatitis B virus. Furthermore, the present invention also relates to a method for screening an anti-hepatitis B virus drug using the system. [Background technology]

[0002] Hepatitis B is a liver disease caused by infection with the hepatitis B virus (HBV) through blood or bodily fluids. Hepatitis B virus infection is known to cause acute and chronic liver inflammation, and can even lead to cirrhosis and liver cancer. There are approximately 1.2 million patients, and it is reported that 300,000 people die from hepatitis B-related diseases each year.

[0003] Conventionally, hepatitis B virus (HBV) replication has been measured using labor-intensive methods such as quantitative PCR, Western blotting, and Southern blotting.

[0004] Currently, development of antiviral drugs for HBV is being conducted worldwide; however, development is still in progress due to the lack of high-throughput methods to verify the effectiveness of antiviral drugs (see non-patent document 1). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Jianming Hu et al., Gastroenterology 2019; 156: 338-354 Summary of the Invention [Problem to be solved by the invention]

[0006] Conventionally, ELISA, PCR, Northern blotting, and Southern blotting have been used to quantify HBV replication, but these methods are not efficient in terms of cost and time, and are unsuitable for high through put screening. An object of the present invention is to provide a method for monitoring HBV replication more simply and at lower cost than conventional methods. [Means for solving the problem]

[0007] The present inventors have conducted intensive studies to develop a system for monitoring HBV replication more simply and at lower cost than conventional methods.

[0008] The present inventors have produced a recombinant virus (HiBiT-HBV virus) in which HiBiT consisting of 11 amino acids has been substituted, inserted or added to an HBV protein. This HiBiT-HBV virus infects liver-derived cells such as primary cultured hepatocytes, and viral proteins containing HiBiT are released into the culture supernatant. Therefore, the inventors have found that HBV replication can be measured more easily than conventional methods by measuring the activity of HiBiT in the cell supernatant, and have completed the present invention.

[0009] That is, the present invention is as follows. [1] An HBV vector containing DNA encoding HiBiT, consisting of the 11 amino acids represented by sequence number 2, in the HBV gene. [2] The HBV vector of [1], which contains DNA encoding HiBiT consisting of 11 amino acids represented by sequence number 2 in the PreS1 gene of the HBV gene. [3] An HBV vector of [1] or [2], in which a DNA encoding HiBiT consisting of the 33 bases represented by SEQ ID NO: 3 is substituted, inserted or added to the HBV gene. [4] An HBV vector according to any one of [1] to [3], in which the 5' end of the PreS1 gene of the HBV gene has been replaced with DNA encoding HiBiT, which consists of 33 bases as shown in sequence number 3. [5] HBV particles in which HiBiT, consisting of 11 amino acids represented by sequence number 2, has been substituted, inserted or added to the HBV protein. [6] An HBV particle of [5], in which HiBiT consisting of 11 amino acids represented by sequence number 2 is substituted, inserted or added to the HBV PreS1 protein, PreS2 protein, polymerase protein or Core protein. [7] HBV particles in which HiBiT, consisting of 11 amino acids represented by sequence number 2, is attached to the N-terminus of the PreS1 protein in the HBV envelope. [8] (i) transfecting liver-derived cells with any one of the HBV vectors [1] to [4] and culturing the cells; and (ii) recovering HBV particles from the cells cultured in step (i); A method for producing HBV particles according to any of [5] to [7], comprising: [9] The method according to [8], wherein the liver-derived cells are HepG2 cells or Huh-7 cells.

[10] Liver-derived cells infected with any of the HBV particles [5]-[7].

[11] (i) infecting liver-derived cells with any one of the HBV particles [5] to [7] and culturing the cells; and (ii) measuring HiBiT activity in the cell culture supernatant of (i); A method for monitoring HBV replication, comprising:

[12] The method according to

[11] , wherein the step (ii) of measuring HiBiT activity in the cell culture supernatant is a step of measuring HiBiT activity in the cell culture supernatant (i) by binding HiBiT to a luciferase fragment (LgBiT), causing it to emit light in the presence of luciferin, and detecting the luminescence using a luminometer.

[13] The method of

[11] or

[12] , wherein in step (ii), the HiBiT activity in the cell culture supernatant of (i) is measured by binding HiBiT to a NanoLuc (registered trademark) luciferase fragment (LgBiT), reconstituting NanoLuc (registered trademark), causing it to emit light in the presence of luciferin, and detecting the luminescence using a luminometer.

[14] (i) infecting liver-derived cells with any one of the HBV particles described in [5] to [7] and contacting the cells with a candidate compound for an anti-HBV drug, and culturing the cells; and (ii) a method for screening an anti-HBV drug, comprising a step of measuring HiBiT activity in the cell culture supernatant of (i), and selecting an anti-HBV drug from candidate drugs using HiBiT activity as an indicator.

[15] The method according to

[14] , wherein the step (ii) of measuring HiBiT activity in the cell culture supernatant is a step of measuring HiBiT activity in the cell culture supernatant (i) by binding HiBiT to a luciferase fragment (LgBiT), causing it to emit light in the presence of luciferin, and detecting the luminescence using a luminometer.

[16] The method of

[14] or

[15] , in step (ii), the HiBiT activity in the cell culture supernatant of (i) is detected by binding HiBiT to a NanoLuc (registered trademark) luciferase fragment (LgBiT), reconstituting NanoLuc (registered trademark), causing it to emit light in the presence of luciferin, and detecting the luminescence using a luminometer. Effect of the Invention

[0010] A recombinant virus (HiBiT-HBV virus) in which the 11 amino acid HiBiT protein has been substituted, inserted or added to an HBV protein can grow autonomously in cells in the same way as wild-type HBV. Therefore, the HBV life cycle can be monitored by the method of the present invention for monitoring HBV replication. The method of the present invention enables a high-throughput assay for monitoring HBV replication using highly sensitive and inexpensive markers, and can be used for research into the entire HBV life cycle and HBV drug development. [Brief description of the drawings]

[0011] [Figure 1]The method of substitution insertion of DNA encoding HiBiT into the HBV gene is shown in Fig. 1A. The DNA sequence and amino acid sequence of the N-terminal portion of the wild-type preS1 protein of HBV are shown, and Fig. 1B shows the substitution insertion of the sequence encoding the 9 amino acids from the 2nd to the 10th at the N-terminus of HBV preS1 into the sequence encoding the 11 amino acid HiBiT. [Diagram 2] FIG. 1 shows HBV dose-dependent increase in HiBiT activity after HBV infection. [Diagram 3] This figure shows the change in HiBiT activity over time when cells were infected with HBV particles and contacted with a viral replication inhibitor and a viral entry inhibitor at the same time. [Figure 4] This figure shows HiBiT activity on days 9 and 12 when cells were infected with HBV particles and contacted with a viral replication inhibitor and a viral entry inhibitor at the same time. [Diagram 5] FIG. 1 shows an experimental protocol in which a viral entry inhibitor is contacted simultaneously with or 3 days after infection of cells with HBV particles. [Figure 6] 6A and 6B show the HiBiT activity when a virus entry inhibitor was contacted with cells simultaneously with or 3 days after infection with HBV particles, respectively, and FIG 6A shows the results when Heparin was administered, and FIG 6B shows the results when MyrB was administered. [Figure 7] Figure 1 shows a comparison of the replication ability after infection of HBV particles between wild-type virus without HiBiT replacement insertion and virus with HiBiT replacement insertion. A shows HiBiT activity, B shows HBs antigen amount (HBsAg), C shows pregenomic RNA (pgRNA) in cells, D shows HBcore-related antigen amount (HBcAg), E shows cccDNA amount, and F shows HBV DNA amount. [Figure 8] The HBV vector was transfected into HepG2 cells to produce HBV particles, which were then separated using iodixanol density gradient. The results of each fraction were analyzed as shown in Figure 8. Figure 8A shows the results for WT (wild type), and Figure 8B shows the results for HBV-HiBiT. [Figure 9]HBV vector was introduced into HepG2 cells, HBV particles were produced, and the infectivity of each fraction was measured by iodixanol density gradient separation. Figure 9A shows the results for WT, and Figure 9B shows the results for HBV-HiBiT. (1) shows the results of HBV-DNA measurement, and (2) shows the results of cccDNA measurement. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in detail below.

[0013] 1. Construction of HBV vector containing HiBiT The present invention is a Hepatitis B Virus (HBV) vector capable of monitoring HBV infection and replication.

[0014] The HBV vector of the present invention contains the HBV gene, and a part of the HBV gene is replaced with DNA encoding HiBiT, i.e., the HBV vector of the present invention contains DNA encoding HiBiT in the gene.

[0015] HBV is a DNA virus whose genomic DNA consists of a circular incomplete double-stranded DNA of approximately 3,200 base pairs, some of which are single-stranded. The HBV genome contains genes encoding four open reading frames (ORFs), consisting of the S region, C region, P region, and X region.

[0016] The S region is a gene that encodes the large S protein, which is a surface antigen protein of the HBV envelope, and has the PreS1 gene and the PreS2 gene. The C region contains the Pre-C / C gene, which encodes the core particle that encapsulates HBV DNA. The P region contains the DNA polymerase gene. The X region contains the X gene, which encodes the X protein, a transactivator protein. Some of the genes in these four regions may be mutated.

[0017] There are nine genotypes of HBV: A (Aa / 1, Ae / 2, Ac / 3), B (Bj / 1, Ba / 2, B3, B4, B5), C (Ce / 1, Cs / 2, C3, C4), D (D1, D2, D3, D4, D5), E, ​​F (F1, F2, F3, F4), G, H, and J. The HBV vector of the present invention may be derived from any of the HBV genotypes. It may also be derived from HBV with a mutated genome of any of these genotypes. As an example, SEQ ID NO: 1 shows the genome sequence of genotype C (clone: ​​c_JPNAT) (accession number: AB246345).

[0018] The length of the HBV gene contained in the HBV vector is 3.6 kb or less, preferably 3.5 kb or less, and more preferably 3.4 kb or less.

[0019] HiBiT is a peptide tag consisting of 11 amino acids represented by VSGWRLFKKIS (SEQ ID NO: 2). The base sequence of DNA encoding HiBiT is shown in SEQ ID NO: 3. HiBiT is a peptide tag used in the HiBiT system (Nano Glo (registered trademark) HiBiT Lytic Detection System) (Promega Corporation), a technology for detecting target proteins by luminescence. HiBiT binds with high affinity to the approximately 18 kDa NanoLuc (registered trademark) luciferase fragment (LgBiT; Large BiT), reconstitutes NanoLuc (registered trademark), and emits light in the presence of a substrate.

[0020] In the present invention, HiBiT is introduced into an HBV vector as a reporter, HBV particles are produced using the HBV vector, the HBV particles are infected into cells, and the reporter luminescence is detected, thereby making it possible to monitor the infection and replication of the HBV virus in cells. The luminescence can be detected using a general luminescence measuring device (luminometer). In the present invention, HBV particles are different from wild-type HBV particles in that they contain HiBiT, so HBV particles can also be called HBV-like particles.

[0021] HiBiT is substituted, inserted or added to an HBV protein. Examples of HBV proteins to which HiBiT is substituted, inserted or added include PreS1 protein and PreS2 protein encoded by the gene in the S region, polymerase (pol) protein encoded by the gene in the P region, and Core protein encoded by the C gene in the C region. Here, "substitution" refers to HiBiT being substituted with a part of an HBV protein, "insertion" refers to HiBiT being inserted into an HBV protein, and "addition" refers to HiBiT being added to the N-terminus or C-terminus of an HBV protein. HiBiT may be substituted with a part of these proteins, added to the N-terminus of these proteins, added to the C-terminus of these proteins, or inserted into these proteins. Preferably, it is substituted or added to the N-terminus of PreS1 protein, which constitutes the Large S protein of HBV. HiBiT, which consists of 11 amino acids, is substituted or added to the N-terminus of PreS1 protein by substituting or adding 11 amino acids on the N-terminus side of PreS1 protein. In this case, a linker may be present or absent. The HBV vector of the present invention contains a DNA encoding HiBiT in the HBV genome gene. For example, the PreS1 gene contains a DNA encoding HiBiT. The 5' end of the PreS1 gene is replaced with a DNA encoding HiBiT, or the DNA is added to the 5' end, so that the PreS1 gene contains a DNA encoding HiBiT. FIG. 1 shows an outline of a method for replacing a DNA encoding HiBiT with an HBV gene. FIG. 1 shows an example of the case where genotype C, whose genome sequence is shown in SEQ ID NO: 1, is used. FIG. 1A shows the DNA sequence (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO: 6) of the N-terminal portion of the wild-type preS1 protein of HBV, and FIG. 1B shows the replacement of the sequence encoding the 9 amino acids from the 2nd to the 10th positions at the N-terminus of HBV preS1 with a sequence encoding 11 amino acids of HiBiT. The DNA sequence after the replacement is shown in SEQ ID NO: 7, and the amino acid sequence after the replacement is shown in SEQ ID NO: 8.

[0022] The Nano Glo (registered trademark) HiBiT Lytic Detection System, which detects the luminescence of HiBiT, is available from Promega Corp. The Nano Glo (registered trademark) HiBiT Lytic Detection System includes a DNA encoding HiBiT, LgBiT, and a reagent including a NanoLuc (registered trademark) substrate.

[0023] The HBV vector is a plasmid containing the HBV gene, and any known vector that can be used as an expression vector can be used. Thus, examples of the vector include a plasmid vector, a cosmid vector, a fosmid vector, a phage vector, and a virus vector. Examples of the plasmid vector that can be used include a pUC vector, and examples of the pUC vector include pUC18, pUC19, pHSG298, pHSG299, pHSG396, and pHSG398.

[0024] Using these vectors as backbones, genes encoding four open reading frames (ORFs) consisting of the S region, C region, P region, and X region are inserted in an expressible manner to prepare an HBV vector. Furthermore, a DNA encoding HiBiT may be used as a reporter to replace a part of the HBV gene or to be inserted or added to a part of the HBV gene so that HiBiT is replaced, inserted, or added to the HBV protein on the HBV vector. For example, a DNA encoding HiBiT may be used as a reporter to replace a part of the PreS1 region of the HBV gene or to be added to the PreS1 region so that HiBiT is replaced or added to the N-terminal side of the PreS1 protein of HBV on the HBV vector.

[0025] 2. Transfecting Cells with HBV Vectors An HBV vector containing DNA encoding HiBiT is introduced (transfected) into cells. Transfection can be carried out by a known method. For example, a method using a gene transfer reagent, a calcium phosphate method, a lipofection method, a DEAE dextran method, an electroporation method, a microinjection method, etc. can be mentioned. Examples of gene transfer reagents include polyethyleneimine (PEI, linear or branched) and commercially available gene transfer reagents. Examples of commercially available gene transfer reagents include Lipofectamine (registered trademark), Lipofectamine plus (registered trademark), Lipofectamine 3000 (registered trademark), jet PEI (registered trademark), TransIT (registered trademark), Xfect (trademark), Transfectin-Lipid (registered trademark), Oligofectamine (registered trademark), siLentFect (registered trademark), DMRIE-C (registered trademark), Effectene (registered trademark), FuGENE (registered trademark), etc. Polyethyleneimine (PEI) includes linear PEI and branched PEI containing primary, secondary, and tertiary amines, and either can be used. In addition, the molecular weight of PEI is not limited. Furthermore, PEI that has been chemically modified, for example by deacylation, can also be used.

[0026] The cells to be transfected with the HBV vector are cells in which the HBV virus replicates and expresses proteins, and liver-derived cells are preferred, such as HepG2 cells, Huh-7 cells, Huh-6 cells, Huh-1 cells, Ale cells, PH5CH cells, KYN cells, HAK6 cells, HAK5 cells, HAK1B cells, HAK1A cells, Kim-1 cells, KH cells, etc., with HepG2 cells and Huh-7 cells being preferred.

[0027] 3. Recovery of HBV particles After transfecting the above cells with the HBV vector, the cells are cultured for several days, for example, 1 to 7 days, the culture supernatant is collected, and the virus particles are collected from the culture supernatant. The medium and culture conditions used for culturing the cells are the same as those used for culturing normal animal cells. The virus particles can be collected, for example, by precipitating the virus with polyethylene glycol (PEG), collecting the precipitate by centrifugation, and separating the virus from the obtained precipitate by density gradient centrifugation using sucrose.

[0028] HiBiT is substituted, inserted or added to the proteins of the recovered HBV particles. For example, the N-terminus of the PreS1 protein, which constitutes the Large S protein of HBV particles, is substituted with the HiBiT peptide, or the HiBiT peptide is added to the N-terminus of the PreS1 protein.

[0029] The recovered HBV particles contain pregenomic RNA. Pregenomic RNA is a precursor for HBV genome replication, and the pregenomic RNA contained in the HBV-like particles of the present invention is mRNA derived from the HBV gene contained in the HBV vector of the present invention. That is, it is the mRNA of the HBV gene substituted, inserted or added to the DNA encoding HiBiT so that HiBiT is substituted, inserted or added to the protein of the HBV gene. For example, it is the mRNA of the HBV gene substituted or added to the DNA encoding HiBiT so that the N-terminal side of the PreS1 protein of the HBV gene is substituted with HiBiT, or HiBiT is added to the N-terminal side. The pregenomic RNA is synthesized from the above-mentioned HBV vector.

[0030] The recovered HBV particles also contain incomplete circular double-stranded DNA. The incomplete circular double-stranded DNA contained in the HBV particles of the present invention consists of minus (-) strand DNA synthesized from the pregenomic RNA by the reverse transcription activity of DNA polymerase, and plus (+) strand DNA synthesized by DNA polymerase activity using the minus (-) strand as a template. The synthesis of this viral DNA stops midway, resulting in incomplete circular double-stranded DNA. When the HBV particles of the present invention infect a cell, it becomes covalently closed circular DNA (cccDNA) in the nucleus.

[0031] 4. HBV particles infect cells The collected HBV particles are infected into liver-derived cells, and whether or not they replicate can be confirmed using the Nano Glo (registered trademark) HiBiT Lytic Detection System. That is, HBV particles containing HiBiT from cells are collected, HiBiT is bound to a NanoLuc (registered trademark) luciferase fragment (LgBiT; Large BiT), NanoLuc (registered trademark) is reconstituted, and light is emitted in the presence of luciferin, which is a substrate, and the luminescence intensity is detected by a general luminescence measuring device (luminometer). Here, HiBiT may be measured either in cells or extracellular in the culture supernatant. Preferably, extracellular in the culture supernatant is measured.

[0032] In this case, the liver-derived cells may be established hepatocytes and normal hepatocytes, and may be HepG2 cells, Huh-7 cells, Huh-6 cells, Huh-1 cells, Ale cells, PH5CH cells, KYN-cells, HAK6 cells, HAK5 cells, HAK1B cells, HAK1A cells, Kim-1 cells, KH cells, human primary liver cells, or cells obtained by passage of human primary liver cells in experimental animals. The amount of HBV DNA when infecting HBV particles is 50 to 500 Geq (genome equivalents) per cell. Furthermore, HBV containing HiBiT has the same infectivity as wild-type HBV that does not contain HiBiT.

[0033] 5. Monitoring HBV replication In the present invention, HiBiT is introduced into an HBV vector as a reporter, the HBV vector is introduced into a cell, HBV particles are produced, the HBV particles are infected into a liver-derived cell, and the activity of HiBiT is measured by detecting luminescence derived from HiBiT, thereby making it possible to monitor HBV infection and replication in the cell. In this case, the liver-derived cells infected with HBV particles are cultured, and the HiBiT activity in the cell or in the culture supernatant is measured. The present invention encompasses a hepatitis B virus replication monitoring system utilizing HiBiT.

[0034] Conventionally, pgRNA (pregenomic RNA), HBsAg (HBs antigen), cccDNA, and HBV-DNA in culture supernatant have been used as HBV-related markers that are indicators of HBV infection and replication. The detection results of HBV that has infected cells by the method of the present invention correlate with pgRNA and HBsAg, and further reflect cccDNA and HBV-DNA in the culture supernatant. Therefore, the life cycle of HBV can be monitored by the method of the present invention.

[0035] This monitoring method can be used to screen anti-HBV drugs. Anti-HBV drugs can be used as therapeutic agents for HBV infection. Anti-HBV drugs include HBV replication inhibitors and HBV entry inhibitors, and both types of anti-HBV drugs can be screened by the monitoring system of the present invention.

[0036] That is, the liver-derived cells infected with HBV particles by the above method are contacted with a candidate compound for an anti-HBV drug. The contact can be performed by administering the candidate compound to the culture solution of the liver-derived cells infected with HBV particles. In the infected cells, a complete circular double-stranded DNA is synthesized from the incomplete circular double-stranded DNA contained in the HBV particles of the present invention. Furthermore, mRNA and pregenomic RNA are synthesized from the complete circular double-stranded DNA, and HiBiT is expressed from the DNA encoding HiBiT. The expressed HiBiT is quantified by the above-mentioned Nano Glo (registered trademark) HiBiT Lytic Detection System. The infection and replication of HBV particles in the cells are indexed by the expression of HiBiT, and it is judged whether the candidate compound is effective as an anti-HBV drug. The infection of HBV particles may be performed before, at the same time, or after the contact with the candidate compound for an anti-HBV drug.

[0037] When HiBiT expression is high and HiBiT activity is high, the candidate compound can be judged to be useful as an anti-HBV drug. Here, HiBiT activity can be measured in advance by a method of monitoring the replication of HBV particles using a known anti-HBV drug, and the measured value of luminescence for judging whether or not the candidate compound is useful as an anti-HBV drug can be set as a cutoff value. When the measured value showing the activity of HiBiT when the candidate compound is used exceeds the cutoff value, the candidate compound can be judged to be useful as an HBV drug. As the known anti-HBV drug, a viral replication inhibitor or a viral entry inhibitor can be used. Examples of viral replication inhibitors include entecavir, interferon α2b, etc., and examples of viral entry inhibitors include Myrcludex-B, Human hepatitis B immunoglobulin, Heparin, etc.

[0038] Furthermore, when screening candidate compounds for anti-HBV drugs using the method of the present invention, known HBV-related markers in the cell culture supernatant may be measured at the same time. When the HiBiT activity when a candidate drug is used correlates with the measured value of the HBV-related marker, the candidate drug can be determined to be useful as an anti-HBV drug. Known HBV-related markers include pgRNA (pregenomic RNA), HBsAg (HBs antigen), cccDNA, and HBV-DNA in the culture supernatant. Among these, it is preferable to screen anti-HBV drugs using the correlation with pgRNA and HBsAg as an index. EXAMPLES

[0039] The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.

[0040] method (1) Introduction of DNA encoding HiBiT into the HBV genome To insert the HiBiT coding sequence into the N-terminus of the HBV preS1 protein, we used the plasmid pUC1.2×HBV-C, which encodes a 1.2-fold extended HBV gene (HBV genotype C, GenBank accession number AB246345) in the backbone plasmid pUC19.

[0041] The DNA sequence encoding HiBiT (encoding 11 amino acids), 5'-GTG AGC GGC TGG CGG CTG TTC AAG AAG ATC AGC-3' (SEQ ID NO: 3), was replaced with the sequence encoding the 9 amino acids from the 2nd to the 10th positions of the N-terminus of preS1 of pUC1.2xHBV-C (5'-GGA GGT TGG TCT TCC AAA CCT CGA CAA-3') (SEQ ID NO: 4). Insertion of HiBiT was performed using the GeneArt Seamless Cloning and Assembly Enzyme Kit (Thermo Fisher, SCIENTIFIC). The plasmid with HiBiT inserted was named pUC1.2xHBV-C / HiBiT.

[0042] The N-terminus of genotype C HBV used in this example has two myristylation sites essential for HBV infection (the second and 13th glycines from the N-terminus of PreS1). The N-terminal myristylation site (glycine number 2) disappeared due to the substitution and insertion of the HiBiT sequence, but the other myristylation site remained.

[0043] (2) Production of HBV particles Virus-like particles were produced using HepG2 cells. HBV-encoding pUC1.2×HBV-C and pUC1.2×HBV-C / HiBiT were transfected using Lipofectamine3000 (Thermo Fisher, SCIENTIFIC). Cell supernatants were collected 3 and 7 days after transfection, filtered through a 0.45 μm filter, and concentrated using a PEG Virus Precipitation Kit (BioVison). Virus-producing cells can also be changed to Huh-7 cells.

[0044] (3) Infection with HBV particles To analyze the infectivity of the virus particles produced in HepG2 cells, human primary hepatocytes, PXB cells (sold by Phoenix Bio, Inc.) were used. The cells used for infection are not limited as long as HBV particles can enter and the reporter gene can be expressed, but liver-derived cells are preferred, and for example, HepG2 cells, Huh-7 cells, Huh-6 cells, Huh-1 cells, Ale cells, PH5CH cells, KYN-cells, HAK6 cells, HAK5 cells, HAK1B cells, HAK1A cells, Kim-1 cells, KH cells, human primary hepatocytes, or cells obtained by passage of human primary hepatocytes in experimental animals can be used. The amount of HBV DNA in the HBV particle concentrate was determined by quantitative PCR, and the HBV DNA was then infected into PXB cells. The amount of HBV DNA per unit cell was 50Geq to 5,000Geq. Infection was performed in the presence of 4% (w / v) PEG8000 and 2% (w / v) DMSO.

[0045] (4) Measurement of HiBiT activity HiBiT activity in the cells and cell supernatants of PXB cells infected with HBV particles was measured using the Nano Glo (registered trademark) HiBiT Lytic Detection System (Promega).

[0046] (5) Measurement of cccDNA, pregenomic RNA, HBV-DNA, HBs antigen, and HBV core-related antigen The amounts of cccRNA and pregenomic RNA in infected cells and HBV-DNA in the cell supernatant were measured by quantitative PCR, and the amounts of HBs antigen and HBV core-related antigen in the cell supernatant were measured by ELISA.

[0047] (6) Iodixanol density gradient analysis pUC1.2×HBV-C and pUC1.2×HBV-C / HiBiT were transfected into HepG2 cells to produce HBV particles. The same amount of HBV particles was loaded onto a 10-40% iodixanol density gradient and ultracentrifuged (38,000 rpm for 16 h at 4℃ in an SW 41 Ti rotor). Fractions were collected and the amount of HBV-DNA, HBs antigen, HBV core-related antigen, and density of each fraction were measured. Each fraction was also infected into PXB cells, and the amount of cccDNA in the cells and the amount of HBV-DNA in the cell supernatant 14 days after infection were measured.

[0048] result (1) Figure 1A shows the DNA sequence and amino acid sequence of the N-terminal portion of the wild-type preS1 protein of HBV. Figure 1B shows the substitution insertion of the sequence encoding the 2nd to 10th 9 amino acids at the N-terminus of HBV preS1 into the sequence encoding the 11 amino acid HiBiT. As shown in Figure 1B, this substitution insertion of the HiBiT sequence eliminated one of the two myristylation sites essential for HBV infection, but the other myristylation site (the second myristylation site) remained.

[0049] Plasmid DNA was introduced into HepG2 cells and the HBV-like particles were produced. Three different amounts of virus (x1, x10, and x100, x1 was 50 Geq / cell) were used to infect PXB cells. The cell supernatant was replaced with fresh medium every three days after infection, and the activity of HiBiT in the cell supernatant was measured. The results are shown in Figure 2. As shown in Figure 2, HiBiT activity increased in a virus dose-dependent manner up to day 21 after infection.

[0050] (2) At the same time as the infection with the virus particles, the virus replication inhibitor Entecavir (ETV), interferon α2b (IFN), the virus entry inhibitor Myrcludex-B (MyrB), human hepatitis B immunoglobulin (HBIG) and heparin were administered. HBV particles were infected with three different virus doses (×1, ×10 and ×100, ×1 is 50 Geq / cell). NT indicates no administration of these inhibitors, and HBIG ctrl indicates administration of nonspecific human immunoglobulin. The cell supernatant was replaced with fresh medium every three days after infection until the 21st day, and the activity of HiBiT in the cell supernatant was measured. Figure 3 shows the change in HiBiT activity over time, and Figure 4 shows the HiBiT activity on the 9th and 12th days. The inhibition of HiBiT activity in the cell supernatant was observed by the virus entry inhibitor and the replication inhibitor.

[0051] (3) MyrB and Heparin, which are inhibitors of viral entry, were administered at three different concentrations (MyrB: 2, 20, 200 (nM), Heparin: 2, 20, 200 (IU / mL)) simultaneously with infection of PXB cells with HBV particles or 3 days after infection, and the cell supernatant was replaced with fresh medium every 3 days until the 12th day after infection, and the activity of HiBiT in the cell supernatant was measured. The protocol is shown in Figure 5. As shown in Figure 5, the inhibitors were administered simultaneously with infection or from 3 days after infection. The results are shown in Figure 6. Figure 6A shows the results when Heparin was administered, and Figure 6B shows the results when MyrB was administered. In the figure, "-" indicates the control, "Day 0~" indicates the case where the inhibitor was administered simultaneously with infection, and "Day 3~" indicates the case where the inhibitor was administered from 3 days after infection. Graphs 1, 2, and 3 show three different concentrations of the inhibitor. As shown in Figure 6, when these inhibitors were administered simultaneously with infection, they suppressed HBV-HiBiT activity in a concentration-dependent manner. On the other hand, when the inhibitors were administered 3 days after infection, they did not affect HBV-HiBiT activity. These results suggest that the activity of HiBiT in the cell supernatant reflects the entry of HBV-like particles into PXB cells and their subsequent replication within the cells.

[0052] (4) The replication ability of HBV particles after infection was compared between wild-type virus without HiBiT replacement insertion (WT, ○) and virus with HiBiT replacement insertion (HiBiT, ●). PXB cells were infected with the same amount of HBV particles WT and HBV-HiBiT, and the amount of HBs antigen (HBs), HBcore-related antigen (HBcAg), HBV DNA, pregenomic RNA (pgRNA), and cccDNA in the cell supernatant were measured every 3 days after infection until the 21st day. In addition, HiBiT activity was measured in the cells and cell supernatant for HBV-HiBiT-infected cells. The results are shown in Figure 7. A indicates HiBiT activity, B indicates HBs antigen amount (HBs), C indicates pregenomic RNA (pgRNA) in the cells, D indicates HBcore-related antigen amount (HBcAg), E indicates cccDNA amount, and F indicates HBV DNA amount. As shown in Fig. 7, the levels of HBcAg, HBsAg, pgRNA, cccDNA, and HBV-DNA after HBV-HiBiT virus infection were approximately 10-fold lower than those after WT infection. Furthermore, the activity of HiBiT in cells and supernatants seemed to correlate with the levels of pregenomic RNA and HBsAg.

[0053] (5) Figure 8 shows the results of the analysis of each fraction after iodixanol density gradient. Figure 8A shows the results for WT (wild type), and Figure 8B shows the results for HBV-HiBiT. As shown in Figure 8, both WT and HBV-HiBiT showed a peak of HBV-DNA at a density of 1.1 g / mL. The peaks of HBs antigen and HBV core-related antigen (HBcAg) were almost the same for WT and HBV-HiBiT. Figure 9 shows the results of an infection experiment of each fraction into PXB cells. Each fraction was infected into PXB cells, and the amount of cccDNA and the amount of HBV DNA in the cell supernatant were measured by quantitative PCR. Figure 9A shows the results for WT, and Figure 9B shows the results for HBV-HiBiT. The upper row shows the results of HBV-DNA measurement, and the lower row shows the results of cccDNA measurement. As shown in Figure 9, both WT and HBV-HiBiT showed the highest infectivity in the fraction with the highest amount of HBV-DNA. These results suggested that the normality and infectivity of WT and HBV-HBV viruses were almost identical. [Industrial Applicability]

[0054] The Hepatitis B virus replication monitoring system using HiBiT of the present invention can be used for screening anti-HBV drugs.

Claims

1. An HBV vector comprising DNA encoding HiBiT consisting of 11 amino acids represented by sequence number 2 in the PreS1 gene of the HBV gene.

2. The HBV vector of claim 1, wherein a DNA encoding HiBiT consisting of 33 bases represented by sequence number 3 is substituted, inserted or added to the HBV gene.

3. 3. The HBV vector according to claim 1 or 2, wherein the 5' end of the PreS1 gene of the HBV gene is replaced or added with DNA encoding HiBiT consisting of 33 bases represented by sequence number 3.

4. An HBV particle in which HiBiT consisting of 11 amino acids shown in sequence number 2 is substituted, inserted or added to the HBV PreS1 protein.

5. The HBV particle according to claim 4, wherein HiBiT consisting of 11 amino acids represented by sequence number 2 is substituted or added to the N-terminus of the PreS1 protein of the HBV envelope.

6. (i) transfecting liver-derived cells with the HBV vector according to any one of claims 1 to 3 and culturing the cells; and (ii) recovering HBV particles from the cells cultured in step (i); A method for producing the HBV particles described in claim 4 or 5, comprising:

7. The method according to claim 6, wherein the liver-derived cells are HepG2 cells or Huh-7 cells.

8. A liver-derived cell infected with an HBV particle described in claim 4 or 5.

9. (i) infecting liver-derived cells with the HBV particles according to claim 4 or 5 and culturing the cells; and (ii) measuring HiBiT activity in the cell culture supernatant of (i); A method for monitoring HBV replication, comprising:

10. The method according to claim 9, wherein the step of measuring HiBiT activity in the cell culture supernatant (ii) is a step of measuring HiBiT activity in the cell culture supernatant (i) by binding HiBiT to a luciferase fragment (LgBiT), causing it to emit light in the presence of luciferin, and detecting the luminescence using a luminometer.

11. The method according to claim 9 or 10, wherein in step (ii), the HiBiT activity in the cell culture supernatant of (i) is measured by binding HiBiT to a NanoLuc (registered trademark) luciferase fragment (LgBiT), reconstituting NanoLuc (registered trademark), causing it to emit light in the presence of luciferin, and detecting the luminescence using a luminometer.

12. (i) infecting liver-derived cells with the HBV particles according to claim 4 or 5, contacting the cells with a candidate compound for an anti-HBV drug, and culturing the cells; and (ii) a method for screening an anti-HBV drug, comprising a step of measuring HiBiT activity in the cell culture supernatant of (i), and selecting an anti-HBV drug from candidate drugs using HiBiT activity as an indicator.

13. The method according to claim 12, wherein the step of measuring HiBiT activity in the cell culture supernatant (ii) is a step of measuring HiBiT activity in the cell culture supernatant (i) by binding HiBiT to a luciferase fragment (LgBiT), causing it to emit light in the presence of luciferin, and detecting the luminescence using a luminometer.

14. The method according to claim 12 or 13, wherein in step (ii), the HiBiT activity in the cell culture supernatant of (i) is detected by binding HiBiT to a NanoLuc (registered trademark) luciferase fragment (LgBiT), reconstituting NanoLuc (registered trademark), causing it to emit light in the presence of luciferin, and detecting the luminescence using a luminometer.

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