KR20040093571A - Cell lines for producing recombinant Hepatitis B virus - Google Patents

Abstract

PURPOSE: A cell line for producing recombinant Hepatitis B virus is provided, which expresses core protein, polymerase and surface antigen of Hepatitis B virus. Therefore, a vector for gene therapy derived from recombinant Hepatitis B virus of which components are deleted can be replicated. CONSTITUTION: The cell line for producing recombinant Hepatitis B virus has no epsilon element portion necessary for capsidation, comprises the nucleotide sequence expressing core protein, polymerase, surface antigen and X protein which are required for gene replication of the recombinant HBV vector, and is transfected with a helper plasmid, wherein the helper plasmid comprises a promoter selected from SV40 promoter, retrovirus promoter and cytomegalovirus promoter; the helper plasmid comprises the nucleotide sequence of SEQ ID NO:3(RO63); the cell line is selected from HepG2, Huh7, Chang, 293 and 293T; and the cell line Huh7 is the liver cancer cell line.

Description

Cell lines for producing recombinant Hepatitis B virus

The present invention is directed to a packaging cell line capable of producing recombinant hepatitis B virus, and more particularly to expressing core protein, polymerase, surface antigen, and X protein required for replication of hepatitis B virus. It is about a hepatocellular colony.

In general, the gene therapy currently being used in clinical research mainly uses vectors derived from viruses such as retroviruses and adenoviruses (Mulligan, 1993), and there are difficulties in clinical approval due to side effects such as inflammation and tumor induction (Marshall). , E., Gene therapy on trial.Science 288: 951-957, 2000). These side effects are not related to the lack of tissue tropism of conventional viral vectors. On the other hand, hepatitis B virus has a characteristic of selectively infecting only hepatocytes, which makes it very attractive to develop a liver-targeted gene therapy vector. However, hepatitis B virus is not yet well developed as a gene therapy vector. In the prior patent application No. 10-2001-19645, the present inventors developed the hepatitis B virus as a liver-target gene therapy vector after confirming the validity of the gene therapy vector. Since the vector provided in Application No. 2001-19645 is a viral vector having no replication ability, hepatitis B virus protein acting on replication is required for production of a recombinant virus. Specifically, three viral proteins are required. Core proteins make up nucleocapsids, polymerases act as polymerases with reverse transcriptase activity, and surface antigens act as membrane proteins for viruses (Ganem and Schneider, 2001 "Hepadnaviridae: the viruses and their replication," in Fundamental Virology , 4td edition vol. 2, Fields, BN, et al., Eds, Lippincott-Raven Press, Philadelphia)

In general, in order to efficiently produce a viral vector for gene therapy, packaging cell lines are prepared and used. The packaging cell line is also known as a helper cell line as a cell line that provides the viral proteins necessary for the replication of the viral vector, thereby enabling the replication of the viral vector. In fact, the productivity of packaging cell lines is a very important factor in the development of gene therapy vectors, since more than 10 trillion viral particles are used in a patient. However, no packaging cell lines producing hepatitis B virus have been reported yet.

The packaging cell line provided in the present invention is designed to constantly express four viral proteins (core protein, polymerase, surface antigen, X protein) required for recombinant hepatitis B virus replication. Helper plasmids lacking packaging signals were prepared to embody the present invention. This was transfected into a Huh7 cell line, a liver cancer cell line, to select a cell line that always expresses the viral protein. In other words, the packaging cell line is constantly expressing four viral proteins such as core protein, polymerase, surface antigen, and X protein. Therefore, recombinant hepatitis B virus can be produced by transfecting a gene therapy vector in which the hepatitis B virus gene is deleted and the heterologous gene is inserted into the packaging cell line provided in the present invention.

The present invention solves the problems as described above, and the object of the present invention is to provide a cell line capable of producing a recombinant hepatitis B virus.

Another object of the present invention is to provide a method for producing recombinant HBV particles using the cell line.

1 is a diagram showing the infection cycle of the hepatitis B virus. After infection with hepatocytes, a partial duplex circular DNA genome of viral particles is converted into a covalently closed circular DNA (cccDNA) type gene in the nucleus of a cell. Transcribe RNA. 3.5 k bp of pregenomic RNA acts as mRNA of core protein and polymerase, as well as used as a template for reverse transcription for viral gene DNA synthesis. After pregenomic RNA is packaged into core particles, hepatitis B virus gene replication occurs such as negative-strand DNA synthesis and plus-strand DNA synthesis through reverse transcription.

2 is a schematic diagram of a plasmid provided by the present invention. R015 is a plasmid that expresses the pregenomic RNA of wild-type hepatitis B virus and expresses core protein, polymerase, surface antigen, and X protein. R063 is nt. 1820 at nt. A helper plasmid made by eliminating epsilon, which is used as a packaging signal by erasing up to 1902. The R056 plasmid was deleted from nt.2144 to nt.2459 and is a plasmid lacking an ORF of core protein and polymerase. In the present invention, R056 plasmid was used as a plasmid that provides a template for replication.

Figure 3 is a helper plasmid expressing the coa protein, polymerase, and X protein as a picture of the R063 plasmid removing the packaging signal by removing the epsilon from hepatitis B virus. Figure 3 shows the transcript expressed from the promoter for ease of understanding.

Figure 4 is a result of transfection of the non-replicable R056 vector used as a template in the present invention to the packaging cell line provided by the present invention after confirming the gene blot, where RC (relaxed circular DNA), DL (duplex) -linear DNA) and single-stranded DNA (SS) are intermediates of HBV gene replication, respectively, and HH01, HH10, and HH12 are packaging cell lines provided by the present invention.

Figure 5 is the result of immunostaining (immunostaining) using the core protein antibody of hepatitis B virus, and shows the expression of core protein in the cytoplasm of HH01 packaging cell line. DAPI staining is a control group showing staining of nuclei.

In order to achieve the above object, the present invention provides a gene comprising a gene sequence that lacks an epsilon element site essential for encapsidation and expresses a coa protein, a polymerase, a surface antigen, and an X protein required for gene replication of a recombinant HBV vector. Provided are cell lines for the production of recombinant hepatitis B virus transfected with a helper plasmid of hepatitis virus.

The helper plasmid used in the present invention is preferably a promoter used for overexpression in animal cells such as an SV40 promoter, a retrovirus promoter or a cytomegalovirus promoter, and the cytomegalovirus promoter is most preferred.

The helper plasmid used in the present invention preferably has a sequence of SEQ ID NO: 3 (RO63), and the cell line of the present invention is preferably an animal cell line such as HepG2 cell line, Chang cell line, 293 cell line, or 293T cell line, and is a Huh7 liver cancer cell line. More preferably, 28 Yeongeon-dong, Jongno-gu, Seoul, Korea. The most preferred is KCLRF-BP-00078, a liver cancer cell line deposited on April 19, 2003 with the Korea Cell Line Bank located on the 7th floor of Seoul National University College of Medicine.

In another aspect, the present invention provides a method for producing recombinant HBV particles by transfecting the HBV vector into the packaging cell line prepared in the present invention.

The vector used in the recombinant HBV particle manufacturing method is a recombinant HBV vector provided in the prior patent (Korean Application No.:10-2001-19645) and includes a cis-acting element (cis-acting element) essential for viral gene replication. Can express at least one heterologous gene for gene therapy and cannot express at least one of the viral genes necessary for viral gene replication. The packaging cell line prepared in the present invention can provide a recombinant HBV vector that is unable to express at least one of the proteins required for viral gene replication to provide the deficient protein to complement the recombinant HBV vector, thereby producing a virus that is infectious as well as replicable. Liver cell line.

The present invention is briefly described as follows.

The present invention relates to a packaging cell line essential for the production of a recombinant hepatitis B virus vector for gene therapy, and to the production of a hepatitis B virus-derived gene therapy vector provided in the prior patent application No. 2001-19645. Provide the necessary cell lines. More specifically, hepatitis B virus vector provided in Korean Patent Application No. 2001-19645 is capable of replicating hepatitis B virus, which is capable of replicating an open reading frame (ORF) of core protein and polymerase essential for replication by insertion of a heterologous gene. Can't produce However, if core proteins and polymerases necessary for replication are provided, gene replication occurs like wild type, and thus the production of recombinant viruses is possible. The packaging cell line provided in the present invention can produce recombinant hepatitis B virus through cell culture experiments because core protein and polymerase of hepatitis B virus are expressed in hepatocytes. That is, transfection of the gene therapy vector provided by the prior Korean patent application No. 2001-19645 to the packaging cell line provided by the present invention can produce a prototype recombinant hepatitis B virus.

Recombinant hepatitis B virus vector provided by the present invention is not only liver disease such as hepatitis, cirrhosis, liver cancer, but also hepatocytes such as hemophilia caused by lack of familial hypercholesterolemia, coagulation factors VIII, IV, and the like. It can also be used to treat metabolic genetic diseases caused by lack of gene expression. In addition, the hepatitis B virus vector is a hepatocyte-targeting type, and thus will be very useful for the treatment of chronic hepatitis patients.

Hereinafter, the basic content of the hepatitis B virus mentioned in the present invention will be briefly described.

I. Hepatitis B virus

Hepatitis B virus (HBV) belongs to the hepadnavirus family and is a clinically important virus that causes chronic hepatitis as well as acute hepatitis (Ganem & Schneider, 2001). Animal viruses belonging to the hepadnavirus family include woodchuck hepatitis virus, duck hepatitis B virus. HBV is a DNA virus, but it has a very specific replication mechanism that replicates DNA genes through the reverse transcriptase activity of HBV DNA polymerase, using pregenomic RNA as a template (Ryu, W.- S. 2003). HBV has a circular DNA gene with a gap of about 3.2 k bp plus-DNA strand, and contains four proteins: core protein (C), polymerase (P), surface antigen (S), and X protein. Has viral proteins Of these four viral proteins, core protein (C) and polymerase (P) are essential for the gene replication of hepatitis B virus (Nassal and Schaller, 1996).

II. Infection Cycle of Hepadnavirus

The infection cycle of hepadnavirus is shown in Figure 1 (Ganem and Schneider, 2001). Hepadnaviruses are known to invade liver cells through receptors. After entering the hepatocytes, the HBV genome with gaps is converted into covalently closed circular DNA (ccc DNA), which is a template for transcription. From this cccDNA, four virus transcripts are synthesized and transferred to the cytoplasm. 3.5 K bp of pregenomic RNA acts not only as mRNA for core protein and polymerase, but also as a template for viral gene replication. In addition, surface antigen is synthesized from 2.4 k bp and 2.1 k bp RNA, and X protein is synthesized from 0.7 k bp RNA. Synthesized pregenomic RNA, core protein (C), and polymerase (P) are encapsulated into core particles, and then the DNA gene of the virus is synthesized using the reverse transcription activity of the polymerase.

III. Reverse transcription process

Hepatitis B virus has a reverse transcription process similar to retrovirus, but undergoes a more complex process to synthesize DNA (Nassal et al., 1996). First, the transcribed pregenomic RNA is encapsidated into core particles with a corease and a polymerase that recognizes the pregenomic RNA. The combination of polymerase protein and pregenomic RNA is important for core particle formation essential for gene replication (Jeong et al, 2000). At this time, the base sequence of the stem-loop of about 85 base pairs called "epsilon" in the 5 'end portion of the pregenomic RNA acts as a cis-element in the encapsidation step. (Junker-Niepmann et al., 1990). The second-loop structure of epsilon is very highly conserved in all viruses belonging to hepadnavirus. After the pregenomic RNA is encapsidated, gene replication by polymerase occurs in the core particles. In other words, the reverse transcription process in which DNA is synthesized using RNA as a template occurs in core particles.

IV. Viral Proteins Act on Hepatitis B Virus Genome Replication

If a vector containing all the cis-elements necessary for hepatitis B virus genome replication is provided with the viral protein required for replication, it may be developed as a gene therapy vector with heterologous genes having replication competents (Friedmann, 1999). In short, recombinant hepatitis B virus vectors can be replicated if viral proteins (eg, core protein, polymerase protein, surface protein) are provided via a helper plasmid or packaging cell line.

The transfection technology used to establish the packaging cell line provided by the present invention utilizes experimental materials and methods widely used by the literature and experts in this field.

Molecular biological techniques for establishing the packaging cell lines provided by the present invention used methods described in Sambrook et al., In Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, 3rd Ed., NY 2001.

The full name of the abbreviation used in the present invention is as follows.

ε (epsilon), M (molar), mM (millimolar), μl (microliters), ml (milliliters), μg (micrograms), mg (milligrams), K bp (kilo base pairs), ORF (open reading frame), Polymerase chain reaction (PCR), polyethylene glycol (PEG), cytomegalovirus (CMV), hepatitis B virus (HBV), fetal bovine serum (FBS), Dulbecco's modified eagle media (DMEM), N-2-hydroxyethylpiperazine-N-HEPES 2-ethanesufonic acid).

The present invention relates to a packaging cell line (packaging cell line), and transfected with a helper plasmid R063 (pCMV-CPS) plasmid DNA to liver cancer cell line Huh7 cells, liver cancer cell line, and then transformed cells treated with G418 sulfate screening It was.

Hereinafter, the present invention will be described in detail by examples. However, the following Examples are only for illustrating the present invention, and the following Examples do not limit the present invention. In particular, the cell line is not limited to the Huh7 cells shown in the Examples.

Example 1 Preparation of HBV Helper Plasmids

1-1. Preparation of R063 Helper Plasmid (pCMV-CPS)

Fragments between Eco R I and Xho I of pcDNA3 (invitrogen, US A) were substituted with PCR products made from the HBV-ayw subtype as a template (Galibert, F., E. Mandart., F. Fitoussi, P. Tiollais, and P. Charnay.Nucleotide sequence of the hapatitis B virus genome (subtype ayw) cloned in E. coli.Nature 28: 646-650 (1979)). In more detail, the forward primer (forward primer) 5 'end, creating an Eco R I recognition site at the terminal portion 5 of the reverse primer (reverse primer), made of an Xho I recognition site in the p-terminal region PCR product EcoR I (nt. 1903) -Xho I (nt. 2454) fragment to make the R062 plasmid. Subsequently, the fragment between Bsp E I (nt.2331) and Apa I of the R015 plasmid provided in Korean Patent Application No. 10-2001-19645, prior art, was transferred to the BspE I, Apa I site of the R062 plasmid to transfer the R063 plasmid. made.

Forward primer (SEQ ID NO: 1): 5-CATG GAATTC ATGGACATCGACCCT-3 ( EcoR I site underlined)

Reverse primer (SEQ ID NO: 2): 5-CCG CTCGAG CTAACATTGAGATTCCCGAGA-3 '( Xho I site underlined)

Example 2: Identification of core protein, polymerase expressed in R063 (pCMV-CPS) plasmid

2-1. Cell Culture and Transfection of Liver Cancer Cell Lines

Huh7 cells, a liver cancer cell line, were grown every 3 days in DMEM (Dulbecco's modified eagle media, Gibco-BRL) medium containing 10% FBS (fetal bovine serum) and 10 mg / ml gentamicin. One day before transfection, cells are prepared by incubating Huh7 cells in 100% plates at 75%. First wash the cells twice with phosphate buffered saline and change to a new culture. Then, 10 ㎍ of the plasmid was mixed with 694 ml of water containing 0.25 M CaCl ₂ , followed by the same amount of 2X HEPES buffer [280]. mM NaCl, 50 mM HEPES acid, 1.5 mM Na ₂ HPO ₄ (pH 7.1)] while shaking dropwise to form a mixture. The mixture is then reacted at room temperature for 20 minutes to form a white precipitate, which is then evenly spread on the plate. After 16 hours of transfection, the culture medium was changed to new medium [DMEM, 10% FBS, 10 mg / ml gentamicin], and 3 days later, viral core particles were extracted.

2-2. HBV DNA extraction from core particles and analysis of hepatitis B virus replication intermediates via Southern blot.

After 3 days of transfection, DNA of HBV was prepared by extracting intracellular core particles by PEG precipitation (Jeong et al, 2000). In detail, first wash the cells twice with phosphate buffered saline and then rinse the cells with cell solution buffer [10 mM Tris (pH 7.5), 1 mM EDTA, 50 mM NaCl, 1% Nonidet P-40]. Remove from plate. Treatment with 6 mM MgCl ₂ and DNase I (50 ?? g / ml) was performed to remove remaining transfected plasmids. After 30 minutes of treatment, core particles were precipitated with 4X PNE [26% PEG, 1.4 M NaCl, 40 mM EDTA] and centrifuged to separate only core particles. In order to decompose the obtained core particle protein, it was reacted with protease K (proteinase K, Sigma, USA) at 37 ° for 2 hours. After removing the protein with phenol, chloroform and isoamyl alcohol (25: 24: 1), precipitated core particles with ethanol, and then dissolved in TE [10 mM Tris (pH 8.0), 1 mM EDTA] buffer to extract DNA. It was.

The extracted viral DNA was electrophoresed through 1.3% agarose gel and Southern blots described in Current Protocols in Molecular Biology , Ausubel, F. et al., Eds., Wiley and Sons, New York, 1995. The method was analyzed.

Example 3 Preparation of Packaging Cell Line

3-1. Establishment of a Packaging Cell Line

Huh7 cells (Nakabayashi et al., Cancer Res. 42: 3858-63 (1982)), a liver cancer cell line, were transfected with R063 (pCMV-CPS) plasmid DNA using a packaging cell line. Select was separated. In detail, Huh7 cells, a liver cancer cell line, were grown every 3 days in DMEM (Gibco-BRL) medium containing 10% FBS and 10 mg / ml gentamicin to grow the cells. Prepare cells by culturing Huh7 cells in 60% plate at 50% one day before transfection. First wash the cells twice with phosphate buffered saline and change to a new culture. Then, 10 μg of the plasmid was mixed with 250 ml of water containing 0.25 M CaCl ₂ , followed by the same amount of 2X HEPES buffer [280]. mM NaCl, 50 mM HEPES acid, 1.5 mM Na ₂ HPO ₄ (pH 7.1)] while shaking dropwise to form a mixture. The mixture is then reacted at room temperature for 30 minutes to form a white precipitate, which is then evenly spread on the plate. After 16 hours of transfection, fresh culture was changed to [DMEM, 10% FBS, 10 mg / ml gentamicin], and G418 sulfate was treated at a concentration of 200 µg / ml. After 3 days, the cells were washed twice with phosphate buffer solution, and then changed to a new culture solution. Then, the concentration of G418 sulfate was increased and grown at 400 ㎍ / ml for 3 days, then washed twice with phosphate buffer solution and replaced with a new culture solution. . Again, the concentration of G418 sulfate was increased to 3 days at 600 μg / ml, 3 days at 800 μg / ml, and finally to 40 days in 5% CO ₂ incubator while raising the concentration of G418 sulfate to 1000 μg / ml.

Example 4 Confirmation of Replication Capacity of Packaging Cell Lines

4-1. HBV DNA extraction and Southern blot from core particles

Transfection, DNA extraction and Southern blots were performed in the same manner as in Example 2-2. As described in the HBV gene replication mechanism, intermediates of HBV gene replication present in core particles exist in the form of single-stranded DNA (SS), duplex-linear (DL), and relaxed circular DNA (RC). Among them, the RC (relaxed cyclic) DNA is the final product of HBV genome replication in the viral particles, so the presence of RC-type DNA allows the packaging cell line to be the core protein of the virus necessary for HBV gene replication. It may be determined whether a polymerase protein is provided. In other words, replication-defective mutants should be provided with core protein and polymerase, but can form RC DNA, thus confirming the function of the helper cell line.

4-2. Confirmation of Replication Capacity of Packaging Cell Lines

Southern blots were performed using HBV probes to determine the function of packaging cell lines required for HBV replication. Core DNA was extracted as in Example 2-2, and the R063 (??-C + P +) helper plasmid and the replication-defective deletion mutant R056 (?? + C-P-) plasmid were liver cancer. The cell line Huh7 (Nakabayashi et al., Growth of human hepatoma cells lines with differentiated functions in chemically defined medium.Cancer Res. 42: 3858-63 (1982) was used as a positive control. RC (relaxed circular) DNA, double-stranded linear (DL) DNA, single-stranded (SS) DNA, etc. were detected in the control group (Fig. 4 lane 1) and encapsidation signal as a negative control (encapsidation signal). Transfection only of R063 helper plasmid lacking epsilon (?) Indicates that viral core DNA is not formed (FIG. 4 lane 2). On the other hand, replication-deficient scavenging variants are lacking in three packaging cell lines. R056 (-defective deletion mutant) When only the + C- P-) plasmid is transfected, it can be seen that not only single-strand DNA and duplex-linear DNA but also relaxed-circular DNA are formed (Fig. 4, lane 3-5). The productivity of a packaging cell line, such as HH01, may provide about 0.1-1.0 × 10 ⁶ particles of virus particles per mL of culture.

Example 5 Expression of Core Proteins of Packaging Cell Lines

5-1. Immunostaining of Packaging Cell Lines

As in Example 3-1, the packaging cell line is cultured in DMEM (Gibco-BRL) medium containing 10% FBS and 10 mg / ml gentamicin. First, cover glass is placed on a 6 well plate and the packaging cell line is dispensed to about 30%. The next day, wash the cells twice with phosphate buffer solution, fix the cells with 1 ml of cold 100% methanol (MeOH) for 5 minutes, and then treat the blocking solution [1X phosphate buffer solution, 3% BSA (bovine serum albumin)] for 1 hour. After treatment with rabbit anti-HBV core antigen (HBcAg) for 2 hours, washed twice with phosphate buffer solution containing 0.05% tween-20 and treated with FITC-rabbit antibody for 30 minutes. Was confirmed by fluorescence microscopy.

5-2. Expression of core protein of packaging cell line

Immunostaining was performed to confirm the expression of core protein of the packaging cell line required for hepatitis B virus replication. The presence or absence of the core protein was confirmed by confirming the fluorescence of the FITC using the antibody to the core protein. In order to perform the above experiment was confirmed using Huh7 as a negative control (negative control). As shown in FIG. 5, fluorescence was stronger than that of the negative control, and it was confirmed that the core protein was expressed in the cytoplasm of the packaging cell line. This experiment is the result obtained by observing under the same conditions under a fluorescence microscope.

According to the present invention having the above-described configuration, the packaging cell line of hepatitis B virus provided in the present invention expresses core protein, polymerase and surface antigen necessary for the replication of hepatitis B virus. Recombinant hepatitis B virus cannot produce a viral hepatitis B virus because its insertion of heterologous genes results in a lack of the ORF (open reading frame) of the virus and thus the expression of viral proteins. However, if core proteins, polymerases and surface antigens, which are essential for replication, are provided, gene replication occurs like wild type, and thus the production of recombinant viruses is possible. The packaging cell line provided in the present invention expresses the core protein, polymerase and surface antigen of hepatitis B virus, so that the recombinant hepatitis B virus can produce a recombinant virus since the gene replication occurs like the wild type. The packaging cell line provided in the present invention is an essential cell line for the production of gene therapy vectors derived from recombinant hepatitis B virus.

<110> YONSEI UNIVERSITY <120> Cell lines for producing recombinant Hepatitis B virus <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer including Eco RI site <400> 1 catggaattc atggacatcg accct 25 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer including Xho I site <400> 2 ccgctcgagc taacattgag attcccgaga 30 <210> 3 <211> 8678 <212> DNA <213> Artificial Sequence <220> R063 Helper plasmid (pCMV-CPS) <400> 3 atggacatcg acccttataa agaatttgga gctactgtgg agttactctc gtttttgcct 60 tctgacttct ttccttcagt acgagatctt ctagataccg cctcagctct gtatcgggaa 120 gccttagagt ctcctgagca ttgttcacct caccatactg cactcaggca agcaattctt 180 tgctgggggg aactaatgac tctagctacc tgggtgggtg ttaatttgga agatccagcg 240 tctagagacc tagtagtcag ttatgtcaac actaatatgg gcctaaagtt caggcaactc 300 ttgtggtttc acatttcttg tctcactttt ggaagagaaa cagttataga gtatttggtg 360 tctttcggag tgtggattcg cactcctcca gcttatagac caccaaatgc ccctatccta 420 tcaacacttc cggagactac tgttgttaga cgacgaggca ggtcccctag aagaagaact 480 ccctcgcctc gcagacgaag gtctcaatcg ccgcgtcgca gaagatctca atctcgggaa 540 tctcaatgtt agtattcctt ggactcataa ggtggggaac tttactgggc tttattcttc 600 tactgtacct gtctttaatc ctcattggaa aacaccatct tttcctaata tacatttaca 660 ccaagacatt atcaaaaaat gtgaacagtt tgtaggccca ctcacagtta atgagaaaag 720 aagattgcaa ttgattatgc ctgccaggtt ttatccaaag gttaccaaat atttaccatt 780 ggataagggt attaaacctt attatccaga acatctagtt aatcattact tccaaactag 840 acactattta cacactctat ggaaggcggg tatattatat aagagagaaa caacacatag 900 cgcctcattt tgtgggtcac catattcttg ggaacaagat ctacagcatg gggcagaatc 960 tttccaccag caatcctctg ggattctttc ccgaccacca gttggatcca gccttcagag 1020 caaacaccgc aaatccagat tgggacttca atcccaacaa ggacacctgg ccagacgcca 1080 acaaggtagg agctggagca ttcgggctgg gtttcacccc accgcacgga ggccttttgg 1140 ggtggagccc tcaggctcag ggcatactac aaactttgcc agcaaatccg cctcctgcct 1200 ccaccaatcg ccagtcagga aggcagccta ccccgctgtc tccacctttg agaaacactc 1260 atcctcaggc catgcagtgg aattccacaa ccttccacca aactctgcaa gatcccagag 1320 tgagaggcct gtatttccct gctggtggct ccagttcagg aacagtaaac cctgttctga 1380 ctactgcctc tcccttatcg tcaatcttct cgaggattgg ggaccctgcg ctgaacatgg 1440 agaacatcac atcaggattc ctaggacccc ttctcgtgtt acaggcgggg tttttcttgt 1500 tgacaagaat cctcacaata ccgcagagtc tagactcgtg gtggacttct ctcaattttc 1560 tagggggaac taccgtgtgt cttggccaaa attcgcagtc cccaacctcc aatcactcac 1620 caacctcttg tcctccaact tgtcctggtt atcgctggat gtgtctgcgg cgttttatca 1680 tcttcctctt catcctgctg ctatgcctca tcttcttgtt ggttcttctg gactatcaag 1740 gtatgttgcc cgtttgtcct ctaattccag gatcctcaac aaccagcacg ggaccatgcc 1800 ggacctgcat gactactgct caaggaacct ctatgtatcc ctcctgttgc tgtaccaaac 1860 cttcggacgg aaattgcacc tgtattccca tcccatcatc ctgggctttc ggaaaattcc 1920 tatgggagtg ggcctcagcc cgtttctcct ggctcagttt actagtgcca tttgttcagt 1980 ggttcgtagg gctttccccc actgtttggc tttcagttat atggatgatg tggtattggg 2040 ggccaagtct gtacagcatc ttgagtccct ttttaccgct gttaccaatt ttcttttgtc 2100 tttgggtata catttaaacc ctaacaaaac aaagagatgg ggttactctc taaattttat 2160 gggttatgtc attggatgtt atgggtcctt gccacaagaa cacatcatac aaaaaatcaa 2220 agaatgtttt agaaaacttc ctattaacag gcctattgat tggaaagtat gtcaacgaat 2280 tgtgggtctt ttgggttttg ctgccccttt tacacaatgt ggttatcctg cgttgatgcc 2340 tttgtatgca tgtattcaat ctaagcaggc tttcactttc tcgccaactt acaaggcctt 2400 tctgtgtaaa caatacctga acctttaccc cgttgcccgg caacggccag gtctgtgcca 2460 agtgtttgct gacgcaaccc ccactggctg gggcttggtc atgggccatc agcgcatgcg 2520 tggaaccttt tcggctcctc tgccgatcca tactgcggaa ctcctagccg cttgttttgc 2580 tcgcagcagg tctggagcaa acattatcgg gactgataac tctgttgtcc tatcccgcaa 2640 atatacatcg tttccatggc tgctaggctg tgctgccaac tggatcctgc gcgggacgtc 2700 ctttgtttac gtcccgtcgg cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg 2760 actctctcgt ccccttctcc gtctgccgtt ccgaccgacc acggggcgca cctctcttta 2820 cgcggactcc ccgtctgtgc cttctcatct gccggaccgt gtgcacttcg cttcacctct 2880 gcacgtcgca tggagaccac cgtgaacgcc caccaaatat tgcccaaggt cttacataag 2940 aggactcttg gactctcagc aatgtcaacg accgaccttg aggcatactt caaagactgt 3000 ttgtttaaag actgggagga gttgggggag gagattaggt taaaggtctt tgtactagga 3060 ggctgtaggc ataaattggt ctgcgcacca gcaccatgca actttttcac ctctgcctaa 3120 tcatctcttg ttcatgtcct actgttcaag cctccaagct gtgccttggg tggctttggg 3180 gcatggacat cgacccttat aaagaatttg gagctactgt ggagttactc tcgtttttgc 3240 cttctgactt ctttccttca gtacgagatc ttctagaggg ccctattcta tagtgtcacc 3300 taaatgctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 3360 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 3420 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 3480 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 3540 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc 3600 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 3660 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 3720 cgttcgccgg ctttccccgt caagctctaa atcggggcat ccctttaggg ttccgattta 3780 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 3840 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 3900 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 3960 aagggatttt ggggatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 4020 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc 4080 aggcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt 4140 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 4200 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc 4260 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg 4320 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc 4380 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca 4440 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg 4500 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag 4560 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc 4620 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc 4680 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg 4740 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc 4800 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca 4860 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag 4920 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg 4980 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg 5040 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca 5100 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc 5160 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg 5220 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct 5280 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt 5340 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc 5400 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 5460 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 5520 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc 5580 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 5640 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 5700 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 5760 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 5820 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 5880 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 5940 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 6000 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 6060 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 6120 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 6180 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 6240 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 6300 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 6360 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 6420 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 6480 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 6540 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 6600 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 6660 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 6720 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 6780 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 6840 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 6900 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 6960 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 7020 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 7080 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 7140 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 7200 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 7260 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 7320 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 7380 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 7440 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 7500 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 7560 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 7620 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 7680 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg 7740 atcgggagat ctcccgatcc cctatggtcg actctcagta caatctgctc tgatgccgca 7800 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc 7860 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg 7920 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta 7980 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 8040 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 8100 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 8160 cgtcaatggg tggactattt acggtaaact gcccacttgg cagtacatca agtgtatcat 8220 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 8280 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 8340 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca 8400 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat 8460 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg 8520 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct 8580 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagcttg gtaccgagct 8640 cggatccact agtaacggcc gccagtgtgc tggaattc 8678

Claims (7)

Recombinant transfected with a helper plasmid of hepatitis B virus that lacks the epsilon element site essential for encapsidation and contains gene sequences expressing the coaprotein, polymerase, surface antigen and X protein required for gene replication of the recombinant HBV vector. Cell line for hepatitis B virus production. The method of claim 1, The helper plasmid as a promoter cell line for recombinant hepatitis B virus production, characterized in that it further comprises one promoter selected from the group consisting of SV40 promoter, retrovirus promoter and cytomegalovirus promoter. The method of claim 1, The helper plasmid is a cell line for recombinant hepatitis B virus production, characterized by having genetic information of SEQ ID NO: 3 (RO63). The method according to any one of claims 1 to 3, The cell line is a cell line for recombinant hepatitis B virus production, characterized in that one cell line selected from the group consisting of HepG2 cell line, Huh7 cell line, Chang cell line, 293 cell line, and 293T cell line. The method of claim 4, wherein The cell line is a cell line for recombinant hepatitis B virus production, characterized in that the Huh7 liver cancer cell line. The method of claim 5, wherein The cell line is a cell line for recombinant hepatitis B virus production, characterized in that the liver cancer cell line KCLRF-BP-00078. A method for producing recombinant HBV particles by transfecting an HBV vector into the packaging cell line of any one of claims 1 to 3.