KR20090085268A - Pharmaceutical composition for prevention and treatment of hepatitis c virus-mediated liver disease - Google Patents

Abstract

An agent for preventing and treating hepatitis C virus-mediated liver disease is provided to suppress the enzyme activity of NS5B using monoclonal antibody which binds to N-terminal of NS5B protein. A pharmaceutical composition for preventing and treating hepatitis C virus-mediated liver disease comprises an anti-NS5B monoclonal antibody which binds to N-terminal of NS5B. The N-terminal is an amino acid region of 67-88. The hepatitis C virus-mediated liver disease is selected among acute hepatitis C virus, chronic hepatitis C, cirrhosis and liver cancer. The pharmaceutical composition is formulated with proper carrier based on the administration pathway.

Description

Pharmaceutical composition for prevention and treatment of Hepatitis C Virus-mediated liver disease}

The present invention relates to an agent for preventing and treating liver disease, and more particularly, to an agent for preventing and treating hepatitis C virus mediated liver disease, including an antibody against NS5B protein.

Hepatitis C virus (HCV) was first discovered in 1989 and is one of the leading causes of liver diseases such as acute or chronic hepatitis, cirrhosis and liver cancer (Choo Q, et al, Science 244: 359-362). , 1989; Kuo, G. et al., Science, 244: 362-364, 1989). Hepatitis C virus spreads mainly by parenteral routes, and as in the case of hepatitis B virus, it is spread by needles, needles, blood transfusions, and blood products contaminated with the virus.

At least 2% of the world's population is infected with HCV, and HCV infection often progresses to cirrhosis and liver cancer [Saito I. et al., Proc . Natl . Acad . Sci . USA 1990; 87: 6547-6549; Di Bisceglie AM et al., J. Clin . Gastroenterol . 1994; 19: 222-226; Shimotohno K. Intervirology 1995; 38: 162-169. About 80% of patients with acute HCV infection develop chronic hepatitis, 20% of which develop cirrhosis and liver cancer.

The hepatitis C virus is an important pathogen for liver diseases such as hepatitis, cirrhosis and liver cancer, and since the prevention or treatment is important, the mutation of the virus is severe, and thus no effective therapeutic agent or vaccine has been developed. For the treatment of hepatitis C virus, interferon-α alone or interferon-α in combination with ribavirin has been used. However, these two drugs are not specific for hepatitis C virus, cause serious side effects (such as retinopathy, thyroiditis, acute pancreatitis, and depression), and are not effective for all patients with hepatitis C. Different effects occur depending on the viral strain. Therefore, there is an urgent need for the development of new antiviral agents effective in the treatment of hepatitis C virus infection that overcomes these limitations.

HCV has an envelope, within which the genome of 9.6 kb is disconnected, and in positive-sense RNA form, which is homologous to flavivirus and pestivirus [Miller RH et al., Proc . Natl Acad Sci USA 1990; 87: 2057-2061; Takamizawa A. et al., J. Virol . 1991; 65: 1105-1113. The genome is located at the 5 'end and Brown EA et al., Nucleic . Acids Res 1992; 20: 5041-5045; Chen PJ et al., Virology 1992; 188: 102-113] at the 3 'end [Kolykhalov AA et al., J Virol 1996; 70: 3363-3371; Tanaka T. et al., J Virol 1996; 70: 3307-3312] encodes polyprotein precursors having untranslated regions (UTRs) and consisting of 3010 amino acids. This polyprotein is processed into at least 10 functional proteins. Core and envelope proteins (E1, E2, and p7) present at the N-terminus are cut by structural cleavage signals by host cell signal cleavage enzymes [Hijikata M. et al., Proc Natl Acad Sci USA 1991; 88: 5547-5551; Matsuura Y. et al., Virology 1994; 205: 141-150; Lin C. et al., J Virol 1994; 68: 5063-5073]. The rest (NS2, NS3, NS4A, NS4B, NS5A and NS5B) are processed by viral proteases into nonstructural proteins [Grakoui A. et al., Proc Natl Acad Sci USA 1993; 90: 10583-10587; Bartenschlager R. et al., J Virol 1993; 67: 3835-3844; Tomei L. et al., J Virol 1993; 67: 4017-4026.

Non-structural protein 5B (hereafter referred to as "NS5B") of hepatitis C virus (HCV) is a protein encoded by a virus, and NS5B protein is an RNA-dependent RNA polymerase (RNA-dependent RNA polymerase) that plays an important role in HCV replication. Catalyzes HCV-RNA replication as "RdRp", recognizes 5 'untranslated region (UTR) of viral genomic RNA to synthesize negative chain RNA, and recognizes 3' UTR to generate positive chain RNA. Will be synthesized.

NS5B is a phosphorylated protein that is primarily located in the cytoplasmic perinuclear region. Biochemical structural features of NS5B have already been reported [Lohmann V. et al., J Virol 1997; 71: 8416-8428; Lesburg CA et al., Nat Struct Biol 1999; 6: 937-943], attempts have been made to prevent or treat hepatitis C by inhibiting the function of NS3 or NS5B, which is essential for the propagation of hepatitis C virus (Stuyver L. et al., Antimicrobial) . agents and chemotherapy , 2003; 47: 244-254) Efficient therapeutic agents have not yet been developed. Current RdRp Measurement Methods [Behrens SE et al., EMBO J 1996; 15: 12-22; Ferrari E. et al., J Virol 1999; 73: 1649-1654 and culture conditions [Wakita T. et al, Nat Med 2005; 11: 791-796, et al., But the detailed mechanism of HCV replication is unknown.

Accordingly, an object of the present invention is to provide an effective hepatitis C virus mediated liver disease prevention and treatment pharmaceutical composition.

In order to elucidate the mechanism of HCV replication, we constructed a monoantibody specific for the NS5B protein. In the present invention, the two recognition sites at the N-terminal and the middle of NS5B were identified by using 10 kinds of single antibodies specific for NS5B. Among them, the antibody recognizing the N-terminal NS5B inhibited RdRp activity. Found. Taken together, we demonstrated that NS5B contains an epitope of B cells present between amino acids 67 and 88. In addition, it was found that the enzyme site of NS5B was inhibited when the recognition site was bound to the antibody.

According to the present invention, it is possible to provide an agent for preventing and treating HCV mediated liver disease, including a monoclonal antibody that binds to the N-terminal recognition site of HCV NS5B protein.

The present invention relates to a pharmaceutical composition for the prevention and treatment of hepatitis C virus-mediated liver disease comprising an anti-NS5B monoclonal antibody binding to the N-terminal region of the hepatitis C virus NS5B protein.

In addition, the present invention relates to a pharmaceutical composition for preventing and treating hepatitis C virus-mediated liver disease, comprising an anti-NS5B monoclonal antibody, wherein the N-terminal portion is an amino acid region within 67-88.

In addition, the hepatitis C virus-mediated liver disease in the present invention is characterized by acute hepatitis C, chronic hepatitis C, cirrhosis or liver cancer.

NS5B of hepatitis C virus encodes RdRp, which is responsible for replication of hepatitis C virus (mixed herein with "HCV"). In order to isolate and characterize host factors involved in the replication of HCV, monoclonal antibodies specific for HCV NS5B protein were constructed. Monoclonal antibodies were obtained by expressing NS5B, an antigen with enzymatic activity, in insect cells using a recombinant baculovirus expression system. This is because proteins expressed in insect cells undergo post-translational modifications [Hwang SB et al., Virology 1992; 190: 413-422. NS5B protein was well expressed in insect cells, and the authenticity was confirmed with serum of HCV patients. Six histidines were coupled to the NS5B protein to isolate intact antigen and purified almost purely in a single step on a Ni-NTA agarose column. The C-terminal portion of the NS5B protein consisting of 21 amino acids is known to be attached to the endoplasmic reticulum [Schmidt-Mende J. et al., J Biol Chem 2001; 276: 44052-44063. Indeed, even in this experiment, most proteins were expressed as insoluble pellets in insect cells. Previous reports have reported that the transmembrane portion of the HCV-RNA polymerase C-terminus is not essential for polymerization activity [Yamashita T. et al., J Biol Chem 1998; 273: 15479-15486], essential for replication of HCV [Lee KJ et al., J Virol 2004; 78: 3797-3802. In the present invention, the inventors produced HCV-RNA polymerase having enzymatic activity. Finally, monoclonal antibodies showing different isotypes were selected. In addition, using a protein fused to GST (glutathione S-transferase) was used to map the recognition site of each monoclonal antibody. Since these fusion proteins were expressed in prokaryotic cells, they would not have the original structure because they did not undergo proper post-translational modifications. Therefore, the sites recognized by monoclonal antibodies of NS5B will be mostly linear and will not mean recognition sites with tertiary structure. Eight types of monoclonal antibodies recognize the mid-section of the 240-263 region, while the two recognize the N-terminal regions of 67-88. Thus, no antibody has been produced that recognizes the C-terminus. This is probably related to the property that the C-terminal part of NS5B is a permeable part of the membrane.

Hepatitis C virus encodes the RdRp necessary for transcription and replication. We have developed an in vitro RdRp assay using NS5B protein expressed in baculovirus. The actual NS5B protein synthesized whole genomic RNA and the RNA produced was expanded by de novo synthesis. The protein also synthesized whole HCV-RNA independent of primers. Since the HCV genome is positive-sense single-stranded RNA, minus-strand RNA must be synthesized with the 3 'end of the genomic RNA as a template. It is known that the X site is an important part of the replication of the virus in vivo [Kolykhalov AA et al., J Virol 2000; 74: 2046-2051. Invitro RdRp assays using 3′-UTR + X as a template strongly support this concept (FIG. 5). Using this system, we found that NS5B monoclonal antibodies inhibited RdRp activity. Monoclonal antibodies that recognize the N-terminus but not the middle of NS5B inhibit HCV-RNA synthesis in proportion to the amount in vitro. It is not clear how this monoclonal antibody is involved in the inhibition of RdRp activity, but the binding of only N-terminal recognition monoclonal antibodies alters the structure of the NS5B protein and thus the RdRp activity. Nonetheless, monoclonal antibodies or antibodies against HA did not affect RdRp activity. There is a strong correlation between in vitro polymerization activity and viral replication [Cheney IW et al., Virology 2002; 297: 298-306], the N-terminal recognition site may also have HCV replication activity. The 67-88 sites of NS5B are relatively uncharged and the sequence is significantly conserved. This recognition site is located in the finger domain on the tertiary structure of NS5B and can therefore bind to a cell factor of a replication mechanism involved in the replication of HCV. This site can also be named a new structural domain involved in HCV replication.

The discovery of key sites on the N-terminus of NS5B explains the mechanism of replication of HCV and can be used as an antiviral agent for HCV and for preventing and treating liver disease, including the same.

In the pharmaceutical composition of the present invention, a monoclonal antibody that recognizes the N-terminus of the HCV NS5B protein, more preferably, a part within an amino acid region of 67-88, may be used as an active ingredient, and a pharmaceutically acceptable salt may be used. have. Herein, pharmaceutically acceptable salts are salts which are physiologically acceptable and do not inhibit the action of the active ingredient when administered to humans, and do not usually cause allergic reactions such as gastrointestinal disorders or dizziness or similar reactions, such as alkali metal salts. , Alkaline earth metal salts, ammonium salts, amine salts, acid addition salts or hydrate salts, and the like, and are preferably non-toxic and water-soluble.

The pharmaceutically acceptable salts include alkali metal salts such as potassium or sodium salts; Alkaline earth metal salts such as calcium or magnesium salts; Ammonium salts; Triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris (hydroxymethyl) aminomethane, lysine, arginine or N-methyl Amine salts such as -D-glucamine; Inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, nitrate; Preference is given to acetates, lactates, tartarates, benzoates, citrates, methanesulfonates, ethanesulfonates, benzenesulfonates, toluenesulfonates, isethionates, glucuronates, gluconates and the like.

The pharmaceutical composition according to the present invention may include a pharmaceutically effective amount of the above monoclonal antibody alone or may include one or more pharmaceutically acceptable carriers. The pharmaceutically effective amount refers to an amount that exhibits a higher response than a negative control, and preferably refers to an amount sufficient to treat or prevent liver disease mediated by hepatitis C virus.

The pharmaceutically effective amount of the monoclonal antibody according to the present invention is 0.1 μg ~ 100 mg / day / kg body weight, preferably 1 μg ~ 10 mg / day / weight kg. The pharmaceutically effective amount can be appropriately changed depending on various factors such as the disease and its severity, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.

The pharmaceutically acceptable carrier may include any pharmaceutically used solvent, dispersion medium, oil-in-water or water-in-oil emulsion, aqueous composition, liposomes, microbeads and microsomes.

The disease to which the pharmaceutical composition of the present invention can be applied is hepatitis C virus mediated liver disease. The liver disease is caused by hepatitis C virus, and includes acute hepatitis C, chronic hepatitis C, cirrhosis, liver cancer, and the like, and is not limited to the types of diseases listed.

Hepatitis C virus is a virus with an RNA genome that is a positive strand, and for replication of the genome, a negative strand RNA must be synthesized and a negative strand RNA must be synthesized from the negative strand RNA. . The protein involved in this synthesis is NS5B, or RdRp (RNA dependent RNA polymerase) possessed by hepatitis C virus. Therefore, inhibiting the activity of the RNA polymerase inhibits the proliferation of hepatitis C virus, and as a result, it is possible to prevent or treat various diseases such as liver disease caused by hepatitis C virus.

On the other hand, the pharmaceutical composition according to the present invention can be formulated with a suitable carrier depending on the route of administration. The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but may be administered orally or parenterally. Parenteral routes of administration include, for example, several routes such as transdermal, nasal, abdominal, muscle, subcutaneous or intravenous. In the case of oral administration of the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention is prepared in powder, granule, tablet, pill, dragee, capsule, liquid, gel according to a method known in the art together with a suitable oral carrier. , Syrups, suspensions and the like. Examples of suitable carriers include sugars, including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol and starch, cellulose, starch including corn starch, wheat starch, rice starch and potato starch, and the like. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like, including methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate and the like may be added as a disintegrating agent as necessary. Furthermore, the pharmaceutical composition may further include a lubricant, a humectant, a perfume, an anticoagulant, an emulsifier, and a preservative.

In addition, when administered parenterally, the pharmaceutical compositions of the present invention may be formulated according to methods known in the art in the form of injections, transdermal administrations, etc. together with suitable parenteral carriers. Such injections must be sterile and protected from contamination of microorganisms such as bacteria and fungi. Examples of suitable carriers for injection include, but are not limited to, solvents or dispersion media comprising water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycols, etc.), mixtures thereof and / or vegetable oils Can be. More preferably, Hanks solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, isotonic solution such as 10% ethanol, 40% propylene glycol and 5% dextrose, etc. Can be used as In order to protect the injection from microbial contamination, it may further include antibacterial and antifungal agents such as parabens and chlorobutanol. In addition, the injection may further include an isotonic agent such as sugar or NaCl.

Other pharmaceutically acceptable carriers may be referred to those described in Remington's Pharmaceutical Sciences, 19th ed. (Mack Publishing Company, Easton, PA, 1995).

In addition, the pharmaceutical compositions according to the invention may contain one or more buffers such as saline or carbohydrates such as PBS, glucose, mannose, sucrose or dextran, antioxidants, chelating agents, suspensions, thickeners and the like EDTA, and / or Preservatives and the like.

In addition, the pharmaceutical compositions of the present invention may be formulated using methods known in the art to which the present invention pertains so as to be able to delay rapid release, sustained release or release of the active ingredient after administration to a mammal.

In addition, the pharmaceutical compositions of the present invention can be administered in parallel with known compounds such as interferon-α or ribavirin, which have a prophylactic and / or therapeutic effect on liver disease.

Hereinafter, the configuration of the present invention through the embodiment in more detail. However, it is obvious to those skilled in the art that the scope of the present invention is not limited by the description of the following examples.

Example  One: NS5B  Expression Vectors and Recombination Baculovirus  Configuration

HCV isolated from Koreans [Cho YG et al., Mol Cells 1993; 3: 195-202], the construction of the recombinant baculovirus transfer vector for NS5B is described in the previous paper [Hwang SB et al., Virology 1997; except that polyhistidine is bound to the N terminus of NS5B. 227: 439-446. The BglII site of pAcHLT-C (see www.bdbiosciences.com) was cloned. Autografa california nuclear polyhedron virus (AcNPV) DNA (see BD Biosciences Pharmingen site) and NS5B recombinant vector DNA sequenced by Dioxyoxynucleotide chain termination were identified as spordogera prupepeda ( Hereinafter, "Sf9") cells were injected. DNA transfection was performed by the calcium phosphate precipitation method well known to those skilled in the art (Virology 101: 286-290, 1980). Recombinant baculovirus was selected and amplified by plaque assay according to conventional methods [Hwang SB et al., Virology 1997; 227: 439-446; Hwang SB et al., Virology 1992; 190: 413-422. Sf9 cells were harvested three days after infection after infection with recombinant baculovirus to express NS5B in insect cells. Cell extracts were analyzed by SDS-PAGE electrophoresis, and protein expression was confirmed by staining with Coomassie solution or immunoblot using NS5B polyclonal antibody produced in the serum or rabbit of the patient. In order to investigate the recognition site, a mutant strain of NS5B was produced by PCR and expressed in E. coli BL21 (DE3) strain (Novagen, San Diego, CA, USA) by fusion of GST.

Example  2: cells and virus cells

Sf9 was incubated at 27 ° C by adding 10% FBS to Graces insect tissue culture solution (Invitrogen, Carlsbad, CA, USA). Recombinant baculovirus has been described by conventional methods [Hwang SB et al., Virology 1997; 227: 439-446; Hwang SB et al., Virology 1992; 190: 413-422] and was used to infect insect cells with an infection multiple of 9-10. Hybridoma cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) medium with 10% FBS, 100 units / ml streptomycin and 100 units / ml penicillin (Invitrogen).

Example  3: Immunity  Experiment

Intraperitoneal injection of 8-week-old female BALB / c was performed by mixing 50 μg of functionally active recombinant NS5B protein with the same volume of Freunde Freund's Adjuvent. The immune response was promoted by injecting four times a mixture of Freund's incomplete adjuvant and 50 μg of protein every 2-3 weeks. Blood was collected from retro-orbital plexus to investigate antibody production.

Example  4: NS5B  Specific Monoclonal Antibody Production

Three days after the last injection, the mice were sacrificed to remove the spleen. After splicing the splenocytes with SP2 / 0 myeloma cells while gently shaking the PEG 1500, the PEG solution was removed by centrifugation at 1100 g for 5 minutes. Fusion cells were plated in 96-well micro titer plate and the supernatant was taken to examine the presence of NS5B antibody by the blot method using immunoblotter (Immunetics, Cambridge, MA, USA). Positive clones were screened twice with limited dilution. And the monoclonal antibody was purified using the final Protein A Sepharose column.

Example  5: NS5B  Isotyped monoclonal antibody isotyping )

IsoStrip Monoclonal Antibody Isotyping kit (Roche Diagnostics, Basel, Switzerland) was used to determine the type of NS5B monoclonal antibody according to the conventional method. In short, each monoclonal antibody was diluted 100-fold using PBS. 150 μl of diluting solution was mixed with colored latex beads in a developing tube, and then gently stirred for 1 minute and stored at room temperature. The isotyped strip was then placed in a developing tube and allowed to react for 5-10 minutes to make the isotype type visible.

Example  6: HCV - RNA  Purification of Polymerase

Sf9 insect cells were infected with recombinant baculovirus and then harvested three days later and washed twice with PBS. The cell precipitate was dissolved in binding buffer [1% Nonidet P-40, 10% glycerol, 50 mM sodium phosphate (pH 7.5), 300 mM NaCl, 10 mM mercaptoethanol, 10 mM imidazole, 1 mM PMphenyl (phenylmethylsulfonyl fluoride) and then on ice It was released and destroyed by ultrasound. Centrifugation for 15 minutes at 180,000g to obtain a clear cell extract. The precipitate was added to the binding buffer solution, released, crushed by ultrasonication, and once again a clear extract was obtained. The two extracts were mixed and passed through a pre-equilibrated Ni-NTA agarose column (Qiagen, Hilden, Germany). After sufficient washing with 50 mM imidazole solution, the bound NS5B protein was eluted with a gradient of 50 to 300 mM at the imidazole concentration. Fractions of high concentration of NS5B were collected and dialyzed against a solution consisting of 10% glycerol, 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl ₂ , 1 mM dithiothreitol (DTT) and 1 mM PMSF. Protein quantification was performed using a Bio-Rad quantitative kit (Bio-Rad, Hercules, CA, USA) and the enzyme was fractionated and stored at minus 80 ℃.

Example  7: Immunoblot  analysis

Sf9 insect cells were harvested three days after infection with recombinant baculovirus and washed twice with PBS. The cells were placed in lamelli sample buffer and boiled for 5 minutes and centrifuged for 15 minutes. Cell extracts or purified NS5B proteins were run on a 10% SDS-PAGE gel and then attached to nitrocellulose membranes for 1 hour at 4 ° C. The membrane was reacted with 5% skim milk powder at room temperature for 1 hour, and then reacted with HCV patient serum or rabbit-produced NS5B antibody at 4 ° C. overnight. Membranes washed with TNT buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% (v / v) Tween 20] were reacted with HRP (Horse Radish Peroxidase) attached anti-human secondary antibody for 2 hours. Proteins were then identified using the ECL kit (Amersham Biosciences, Uppsala, Sweden).

Example  8: RNA  Mold preparation

GHCV cDNA [Cho YG et al., Mol to obtain the whole RNA genome Cells 1993; 3: 195-202] was digested with Xho I and in vitro transcription was performed at 37 ° C. for 2 hours using T7 RNA polymerase (Promega Madison, WI, USA). The DNA template was then removed by treatment with RQ1 DNase (Promega) at 37 ° C. for 20 minutes. Samples were extracted twice with phenol-chloroform solution and once with chloroform to remove T7 RNA polymerase, and RNA was recovered by precipitation in ethanol. To recover the 3 'UTR-X RNA, the cDNA corresponding to the 3'-X portion of HCV was cloned into the BamH1 site in the pcDNA 3 vector. After cleavage with EcoR1, the transcript was synthesized with T7 RNA polymerase and RNA was recovered as described above.

Example  9: in vitro ( In in vitro ) RdRp  Measure

RNA-dependent RNA polymerase assays can be performed using conventional methods [Oh JW et al., J Virol 1999; 73: 7694-7702]. In brief, the reaction is 50 mM Tris-HCl (pH 7.5), 5 mM MgCl ₂ , 50 mM NaCl, 100 mM KCl, 1 mM DTT, 10% glycerol, 50 μg / ml actinomycin D, RNase inhibitor of 40 units, 0.5 Into each 50 mM of ATP, CTP and GTP, 5uM of UTP, 10uCi [α-32P] UTP, and 50µl of 1.5µg template RNA solution, 200ng of NS5B protein was reacted at 22 ° C for 2 hours for total RNA synthesis. 3'-UTR-X RNA synthesis was performed by reacting for 20 minutes. RNA prepared after the reaction was extracted with phenol-chloroform solution (Ambion Foster City, CA, USA) and precipitated again with ammonium acetate-isopropanol solution. It was then again heated in denaturing buffer (95% formamide, 10 mM EDTA) at 95 ° C. for 2 minutes and analyzed on SDS-PAGE gels containing 0.9% agarose gel or 8M urea. The gel was fixed and then dried and used to photosensitize an X-ray film.

The results obtained by the above examples are as follows.

Result 1: HCV NS5B  Expression and Purification of Proteins

To prepare antigens for obtaining NS5B specific monoclonal antibodies, cDNA of gHCV encoding NS5B was cloned into a shuttle vector of baculovirus with a polyhedrin promoter. Polyhistidine groups were attached to the N-terminus of NS5B to facilitate protein purification. Then, in order to obtain a recombinant baculovirus, a vector containing the NS5B gene (pAcHLT-NS5B) and wild-type AcNPV-DNA were simultaneously injected. The resulting recombinant virus was used to infect Sf9 cells and examined for protein expression. As shown on the left of FIG. 1, a major protein with a size of about 68 kDa was detected, and NS5B protein was the largest in virus infected cells. The authenticity of NS5B was confirmed by immunoblot using rabbit-produced NS5B polyclonal antibody (right of FIG. 1) or patient serum (not shown). Antibodies produced in rabbits recognized only NS5B protein but did not respond to experimental groups or HCV core proteins (right side of FIG. 1). In addition, it was confirmed that the NS5B protein did not react with the serum of normal persons (not shown). It was also observed that NS5B protein was relatively stable in recombinant baculovirus infected cells.

In order to obtain a relatively intact protein, NS5B protein was purified in a single step using a Ni-NTA agarose affinity column. The NS5B protein bound to the column was sufficiently washed with 50 mM imidazole and then eluted at an imidazole concentration gradient of 50-300 mM (upper FIG. 2). The nature of the purified protein was confirmed by the serum of HCV patients (bottom of Figure 2).

Result 2: recombination Baculovirus  Purified from infected insect cells NS5B  Protein is De novo ( de novo ) RNA  Functionally active in the synthesis

RdRp analysis was performed using HCV-RNA transcript as a template to determine whether the NS5B protein was enzymatically active before immunization. Actinomycin D was added to the reaction solution to rule out the possibility of mixing the DNA-dependent RNA polymerase from the cell extract. As shown in FIG. 3, the NS5B protein synthesized RNA without adding a primer from the outside. The size of the newly synthesized RNA increased with time, reaching 9.5 kb within 2 hours at 22 ° C. (lanes 2 and 3 in FIG. 3). These results indicate that new RNA products were produced from scratch, and the NS5B protein purified from insect cells infected with recombinant baculoviruses is functionally active and thus an effective antigen for immune responses in animals. In the experimental group, HCV core protein was purified from the same cell and no RNA was produced (results not shown).

Result 3: recombination From baculovirus  Produced NS5G  From mice immunized with protein NS5B Production and Characterization of Monoclonal Antibodies Specific to

NS5B protein specific monoclonal antibody was prepared to investigate HCV replication mechanism. As described previously, NS5B protein having enzyme activity was prepared and used for immunization of mice. Hybridoma cells were screened using a multichannel miniblotter to make antibodies to the NS5B protein. As a result, 10 types of monoclonal antibodies specific for the NS5B protein were isolated. Three types of NS5B mutant proteins with 1-197, 198-394, and 395-591 portions removed were used to determine the site to which each monoclonal antibody binds. Eight types of antibodies recognized the middle part of the NS5B protein and two types recognized the N-terminal part. The properties of these antibodies are summarized in Table 1.

 Monoclonal antibody  Isotype  Recognition site (amino acid)  RdRp Suppression AG2 IgG 1κ 240-263 - BD2 IgG 1κ 240-263 - BD7 IgG 2bκ 240-263 - CC3 IgG 1κ 240-263 - DE9 IgG 1κ 240-263 - EA6 IgG 1κ 67-88 + EB3 IgG 1κ 240-263 - FD8 IgG 1κ 240-263 - RP3 IgG 2aκ 67-88 + RP5 IgG 2bκ 240-263 -

Result 4: NS5B  Intermediate region of protein Mapping

In order to determine the correct recognition site in the middle portion of the NS5B protein NS5B mutant protein was constructed as shown in FIG. Wild-type and variant proteins were run on 10% SDS-PAGE gels. As a result of staining with Coomaji Brilliant Blue (FIG. 4B), it was confirmed that these fusion proteins were expressed in large amounts in Escherichia coli, and the authenticity of each mutant protein was confirmed by immunoblot using polyclonal antibody of NS5B protein produced in rabbit. (No results presented). As shown in Figure 4c monoclonal antibody RP5 bound to the NS5B variant protein consisting of 1-263 and 240-394 amino acids. However, it did not bind to the mutant strains corresponding to 220-239 and 264-394. This result confirmed that the amino acid 240-263 part is a recognition site. The seven monoclonal antibodies of different isotypes were identical to those of RP5.

Result 5: NS5B  Protein N-Terminal Mapping

We then determined the N-terminal recognition region of the NS5B protein. As shown in FIG. 4d, NS5B variants fused with GST were prepared and expressed in E. coli. Wild-type and mutant proteins were developed by SDS-PAGE and stained with Coomaji Brilliant Blue (FIG. 4E). The authenticity was confirmed by immunoblot using polyclonal antibody of NS5B protein produced in rabbit (not shown). . As shown in FIG. 4F, the mutant protein corresponding to amino acids 67-132 binds to monoclonal antibody RP3. However, they did not bind to proteins corresponding to amino acids 1-66 and 89-197. Therefore, it was confirmed that the portion corresponding to amino acids 67-88 is the recognition site of the NS5B protein. In addition, the peptides corresponding to the 67-88 part was synthesized and confirmed by binding experiments (not shown). In addition, it was confirmed that other monoclonal antibody EA6 recognizes the same site (Table 1). EA6 and Rp3 are different isotypes, indicating that they are derived from different hybridoma cells.

Result 6: N-terminal specific monoclonal antibody RdRp  Active inhibition

It is reported that the X site at the 3 'end of the HCV genome is important for RNA synthesis [Kolykhalov AA et al., J Virol 1996; 70: 3363-3371. Therefore, to investigate whether there is a type of monoclonal antibody that inhibits RNA synthesis, the in vitro synthesized 3'UTR-X as a template RNA and the concentration of the monoclonal antibody was changed as recorded and RdRp analysis was performed. Was implemented. As shown in the upper photo of FIG. 5, RNA of the same size as the template was effectively synthesized by NS5B RNA polymerase (lane 3). Monoclonal antibodies that recognize the middle portion of NS5B or monoclonal antibodies against flags, although slightly reduced the polymerization activity in the presence of high concentrations, did not completely inhibit HCV RNA synthesis. This was also confirmed by experiments using the whole genome RNA of HCV as a template (not shown). Surprisingly, EA6 monoclonal antibodies inhibited RdRp activity in proportion to treatment concentration (lanes 4-6 in FIG. 5A). It was confirmed that RP3 also has the same inhibitory effect (bottom photo of Figure 5). Both of these monoclonal antibodies recognize the N-terminal region of the same NS5B protein, indicating that this recognition site is an important biologically active site for HCV replication. However, other monoclonal antibodies that recognize the middle part of NS5B could not inhibit HCV-RNA synthesis (results not shown). These results clearly show that monoclonal antibodies that recognize the N-terminal region of NS5B inhibit RNA synthesis in vitro.

1 is a diagram of wild type NS5B protein expression and purification.

Left picture: Sf9 insect cells were harvested 3 days after infection with recombinant baculovirus expressing either HCV NS5B protein (lane 2) or HCV core protein (lane 3). Total cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and proteins stained with Coomassie Brilliant Blue. Arrows indicate NS5B protein expressed in Sf9 cells.

Right photo: The same cell lysate was isolated and electrophoresed onto nitrocellulose membrane. NS5B protein was immunoblotted with rabbit anti-HCV NS5B antibody. M: mock-innfected cells.

Figure 2 is a photograph showing the purification of HCV NS5B protein expressed in Sf9 insect cells in one step by Ni-NTA agarose-affinity chromatography. Proteins purified with different concentrations of imidazole (Id) were separated by 10% SDS-polyacrylamide gel electrophoresis and immunoblotted with Coomassie Brilliant Blue (top photo) or with plasma of the patient (bottom photo). .

Figure 3 shows full-length HCV-RNA synthesis by NS5B protein purified from recombinant baculovirus-infected insect cells. RdRp activity measurements were performed as described in the Example with in vitro synthesized 9.5 kb HCV RNA in the presence of 10 μCi of [α-32P] UTP as a template. The reaction was terminated at the designated time and the RNA product was extracted with phenol-chloroform and precipitated with ammonium acetate-isopropanol. Samples were analyzed on a 0.9% agarose gel and freshly synthesized RNA was measured by radiographs.

4 shows the result of mapping M and N epitopes of the NS5B protein.

4A is a schematic diagram of HCV NS5B mutants used for recognition site mapping of NS5B central domains. The combined results are shown in the right panel.

4B shows GST-NS5B fusion protein expressed in E. coli bacteria. Partially purified protein was separated by SDS-PAGE and stained with Coomassie Brilliant Blue. GST-NS5B fusion protein is shown by the arrow.

Figure 4c is an immunoblot analysis of GST-NS5B fusion protein with monoclonal antibody RP5 (mAb RP5).

4D is a schematic diagram of the HCV NS5B mutant structure used for mapping the recognition sites of the N-terminal domain of NS5B protein.

4E shows GST-NS5B fusion protein expressed in E. coli bacteria. Partially purified protein was separated by SDS-PAGE and stained with Coomassie Brilliant Blue. GST-NS5B fusion protein is shown by the arrow.

4F shows immunoblot analysis of the GST-NS5B fusion protein with monoclonal antibody RP3 (mAb RP3).

5 shows inhibition of RdRp activity by NS5B N-terminal-specific monoclonal antibodies.

RdRp activity measurements in the top photo were performed using cold 3 'UTR-X RNA templates synthesized in vitro. HCV NS5B protein purified from recombinant baculovirus infected Sf9 cells was incubated with the indicated amount of monoclonal antibody for 10 minutes, and NTPs and template RNA were added to perform RdRp reaction at 25 ° C. for 20 minutes. Newly synthesized RNA products were analyzed on 8% polyacrylamide gels containing 8M urea and measured by radiographs.

Bottom photo shows inhibition of RdRp activity by NS5B N-terminal-specific monoclonal antibody RP3. RdRp activity was measured by increasing the amount of RP3 monoclonal antibody. Arrows indicate RNA products of template size (208 nucleotides).

IVT: In vitro transcribed RNA used as size marker.

<110> Industrial Academic Cooporation foundation, Hallym University <120> Pharmaceutical composition for prevention and treatment of          hepatitis C virus-mediated liver disease <130> hallym-sbhwang01 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 9075 <212> DNA <213> Artificial Sequence <220> <223> cDNA for HCV-NS5B <400> 1 ccatgagcac gaatcctaaa cctcaaagaa aaaccaaacg taacaccaac cgccgcccac 60 aggatattaa gttcccgggc ggtggtcaga tcgttggtgg agtttacttg ttgccgcgca 120 ggggccccag gttgggtgtg cgcgcgacta ggaagacttc cgagcggtcg caacctcgtg 180 gaaggcgaca gcctatcccc aaggctcgcc ggcccgaggg cagggcctgg gctcagcccg 240 ggtacccttg gcccctctat ggcaatgagg gcttggggtg ggcaggatgg ctcctgtcac 300 cccgcggctc ccggcctagt tggggcccca cggacccccg gcgtaagtcg cgtaatttgg 360 gtaaggtcat cgacaccctc acatgcggct tcgccgacct catggggtac attccgctcg 420 tcggcgcccc cctagggggc gttgccaggg ccctggcaca tggtgtccgg gtgctggagg 480 acggcgtgaa ctatgcaaca gggaatctgc ccggttgctc tttctctatc ttcctcttgg 540 ctctgctgtc ttgtttgacc accccagttt ccgcttatga agtgcgtaac gcgtccggga 600 tgtaccatgt cacgaacgac tgctccaact caagcattgt gtatgaggca gcggacatga 660 tcatgcacac tcccgggtgc gtgccctgcg ttcgggagga caactcctcc cgttgctggg 720 tggcacttac tcccacgctc gcggccagga atgccagcgt ccccactacg acattgcgac 780 gccatgtcga cttgctcgtt ggggtagctg ctttctgttc cgctatgtac gtgggggacc 840 tctgcggatc tgttttcctt gtttcccagc tgttcacctt ttcgcctcgc cggcatgaga 900 cggtacagga ctgcaactgc tcaatctatc ccggccgcgt atcaggtcac cgcatggcct 960 gggatatgat gatgaactgg tcgcctacaa cagccctagt ggtatcgcag ctactccgga 1020 tcccacaagc tgtcgtggac atggtgacag ggtcccactg gggaatcctg gcgggccttg 1080 cctactattc catggtgggg aactgggcta aggtcttaat tgcgatgcta ctctttgccg 1140 gcgttgacgg aaccacccac gtgacagggg gggcgcaagg tcgggccgct agctcgctaa 1200 cgtccctctt tagccctggg ccggttcagc acctccagct cataaacacc aacggcagct 1260 ggcatatcaa caggaccgcc ctgagctgca atgactccct caacactggg tttgttgccg 1320 cgctgttcta caaatacagg ttcaacgcgt ccgggtgccc ggagcgcttg gccacgtgcc 1380 gccccattga tacattcgcg caggggtggg gtcccatcac ttacactgag cctcatgatt 1440 tggatcagag gccctattgc tggcactacg cgcctcaacc gtgtggtatt gtgcccacgt 1500 tgcaggtgtg tggcccagta tactgcttca ccccgagtcc tgttgcggtg gggactaccg 1560 atcgtttcgg tgcccctaca tacagatggg gggcaaatga gacggacgtg ctgctcctta 1620 acaacgccgg gccgccgcaa ggcaactggt tcggctgtac atggatgaat ggcactgggt 1680 tcaccaagac atgtgggggc cccccgtgta acatcggggg ggtcggcaac aataccttga 1740 cctgccccac ggactgcttc cgaaagcacc ccggggccac ttacaccaaa tgcggttcgg 1800 ggccttggtt aacacccagg tgcttagtcg actacccgta caggctctgg cattacccct 1860 gcactgtcaa ctttaccatc tttaaggtta ggatgtacgt ggggggcgcg gagcacaggc 1920 tcgacgccgc atgcaactgg actcggggag agcgttgtga cctggaggac agggataggt 1980 cagagcttag cccgctgctg ctgtctacaa cagagtggca ggtactgccc tgttccttca 2040 caaccctacc ggctctgtcc actggtttga ttcatctcca tcagaacatc gtggacatac 2100 aatacctgta cggtataggg tcggcggttg tctcctttgc gatcaaatgg gagtatattg 2160 tgctgctctt ccttcttctg gcggacgcgc gcgtctgcgc ttgcttgtgg atgatgctgc 2220 tggtagcgca agccgaggcc gccttagaga acctggtggt cctcaatgca gcgtccgtgg 2280 ccggagcgca tggcattctt tccttcattg tgttcttctg tgctgcctgg tacatcaagg 2340 gcaggctggt tcccggagcg gcatacgccc tctatggcgt atggccgctg cttctgcttc 2400 tgctggcgtt accaccacgg gcgtacgcca tggaccggga gatggccgca tcgtgcggag 2460 gcgcggtttt tgtaggtctg gtactcttga ccttgtcacc acactataaa gtgttccttg 2520 ccaggttcat atggtggcta caatatctca tcaccagaac cgaagcgcat ctgcaagtgt 2580 gggtcccccc tctcaacgtt cgggggggtc gcgatgccat catcctcctc acatgcgtgg 2640 tccacccaga gctaatcttt gacatcacaa aatatttgct cgccatattc ggcccgctca 2700 tggtgctcca ggccggcata actagagtgc cgtacttcgt gcgcgcacaa gggctcattc 2760 gtgcatgcat gttggcgcgg aaagtcgtgg ggggtcatta cgtccaaatg gtcttcatga 2820 agctggccgc actagcaggt acgtacgttt atgaccatct tactccactg cgagattggg 2880 ctcacacggg cttacgagac cttgcagtgg cagtagagcc cgttgtcttc tctgacatgg 2940 agaccaaagt catcacctgg ggggcagaca ccgcggcgtg cggggacatc atcttggcct 3000 gccctgcttc cgcccgaagg gggaaggaga tacttctggg accggccgat agtcttgaag 3060 gacaggggtg gcgactcctt gcgcccatca cggcctactc ccaacaaacg cgaggcctgc 3120 ttggttgcat catcactagc cttacaggcc gggacaagaa ccaggttgag ggggaggttc 3180 aagtggtttc caccgcaaca caatctttcc tggcgacctg catcaatggc gtgtgttgga 3240 ctgtcttcca cggcgccggc tcaaagaccc tagccggccc aaagggtcca atcacccaaa 3300 tgtacaccaa tgtagaccag gaccttgttg gctggccggc acctcctggg gcgcgttccc 3360 tgacaccatg cacttgcggc tcctcggacc tttacctggt cacgagacat gctgatgtca 3420 ttccggtgcg ccggcggggt gacggtaggg ggagcctact cccccccagg cctgtctcct 3480 acttgaaggg ctcctcgggt ggtccactgc tctgcccttc ggggcacgct gtcggcatac 3540 ttccggctgc tgtatgcacc cggggggttg ccatggcggt ggaattcata cccgttgagt 3600 ctatggaaac tactatgcgg tctccggtct tcacggacaa tccgtctccc ccggctgtac 3660 cgcagacatt ccaagtggcc cacttacacg ctcccaccgg cagcggcaag agcactaggg 3720 tgccggctgc atatgcagcc caagggtaca aggtgctcgt cctaaatccg tccgtcgccg 3780 ccaccttggg ttttggggcg tatatgtcca aggcacatgg tatcgacccc aaccttagaa 3840 ctggggtaag gaccatcacc acaggtgccc ctatcacata ctccacctat ggcaagttcc 3900 ttgccgacgg tggcggctcc gggggcgcct atgacatcat aatgtgtgat gagtgccact 3960 caactgactc gactaccatt tatggcatcg gcacagtcct ggaccaagcg gagacggctg 4020 gagcgcggct cgtggtgctc tccaccgcta cgcctccggg atcggtcacc gtgccacacc 4080 tcaatatcga ggaggtggcc ctgtctaata ctggagagat ccccttctac ggcaaagcca 4140 ttcccatcga ggctatcaag gggggaaggc atctcatttt ctgccattcc aagaagaagt 4200 gtgacgaact cgccgcaaag ctgtcaggcc tcggactcaa tgccgtagcg tattaccggg 4260 gtcttgacgt gtccgtcata ccgaccagcg gagacgttgt tgtcgtggcg acggacgctc 4320 taatgacggg ctttaccggc gactttgact cagtgatcga ctgtaatacg tgtgtcaccc 4380 agacagtcga tttcagcttg gaccccacct tcaccattga gacgacgacc gtgccccaag 4440 acgcagtgtc gcgctcgcag aggcgaggca ggactggtag gggcagggct ggcatataca 4500 ggtttgtgac tccaggagaa cggccctcgg gcatgttcga ttcttcggtc ctgtgtgagt 4560 gttatgacgc gggttgtgcg tggtacgaac tcacgcccgc tgagacctcg gttaggttgc 4620 gggcgtacct aaacacacca gggttgcccg tctgccagga ccatctggag ttctcggagg 4680 gtgtcttcac aggcctcacc cacatagatg cccacttctt atcccagact aaacaggcag 4740 gagagaactt cccctacttg gtagcatacc aggctacagt gtgcgccagg gctcaagccc 4800 cacctccatc gtgggatgaa atgtggaggt gtctcatacg gctgaaacct acgctgcacg 4860 ggccaacacc cctgctgtat aggttaggag ccgtccaaaa tgaggtcacc ctcacacacc 4920 ccataaccaa attcatcatg acatgtatgt cggctgacct ggaggtcgtc accagcacct 4980 gggtgctggt aggcggagtc ctcgcagctc tggccgcgta ctgcctgaca acaggcagcg 5040 tggtcattgt gggcaggatc atcctgtccg ggaagccggc tatcatcccc gatagggaag 5100 ttctctacca ggagttcgac gagatggagg agtgtgcctc acacctccct tacttcgaac 5160 agggaatgca gctcgccgag caattcaaac agaaggcgct cgggttgctg caaacagcca 5220 ccaagcaggc ggaggctgct gctcccgtgg tggagtccaa gtggcgagcc cttgagacct 5280 tctgggcgaa gcacatgtgg aacttcatta gtgggataca gtacttggca ggcttgtcca 5340 ctctgcctgg gaaccccgca atacgatcac cgatggcatt cacagcctcc atcaccagcc 5400 cgctcaccac ccagcatacc ctcttgttta acatcttggg gggatgggtg gctgcccaac 5460 tcgccccccc cagcgctgcc tcagctttcg tgggcgccgg catcgctgga gccgctgttg 5520 gcacgatagg ccttgggaag gtgcttgtgg acattctggc aggttatgga gcaggggtgg 5580 cgggcgcact tgtggccttt aagatcatga gcggcgagat gccttcagcc gaggacatgg 5640 tcaacttact ccctgccatc ctttctcccg gtgccctggt cgtcgggatt gtgtgtgcag 5700 caatactgcg tcggcatgtg ggcccagggg aaggggctgt gcagtggatg aaccggctga 5760 tagcgttcgc ctcgcggggt aaccacgtct cccccaggca ctatgtgcca gagagcgagc 5820 ctgcagcgcg tgttacccag atcctttcca gcctcaccat cactcagctg ttgaagagac 5880 tccaccagtg gattaatgag gactgctcta cgccatgctc cagctcgtgg ctaagggaga 5940 tttgggactg gatctgcacg gtgttgactg acttcaagac ctggctccag tccaagctcc 6000 tgccgcgatt accgggagtc ccttttttct catgccaacg cgggtataag ggagtctggc 6060 ggggggacgg catcatgcac accacctgcc catgcggagc acagatcacc ggacacgtca 6120 aaaacggttc catgaggatc gttgggccta aaacctgcag caacacgtgg tacgggacat 6180 tccccatcaa cgcgtacacc acgggcccct gcacaccctc cccggcgcca aactattcca 6240 aggcattgtg gagagtggcc gctgaggagt acgtggaggt cacgcgggtg ggagattttc 6300 actacgtgac gggcatgacc actgacaacg tgaagtgtcc atgccaggtt ccggcccccg 6360 aattcttcac ggaggtggat ggagtgcggt tgcacaggta cgctccggcg tgcagacctc 6420 tcctacggga ggaggtcgta ttccaggtcg ggctccacca gtacctggtc gggtcacagc 6480 tcccatgcga gcccgaaccg gatgtagcag tgctcacttc catgctcact gacccctccc 6540 acattacagc agagacggct aagcgtaggc tggccagggg gtctcccccc tccttggcca 6600 gctcttcagc tagccagttg tctgcgcctt ccttgaaggc gacatgcact acccatcatg 6660 actccccgga cgctgacctc attgaggcca acctcttgtg gcggcaagag atgggcggga 6720 acatcacccg cgtggagtca gagaataagg tggtaatcct ggactctttc gacccgctcc 6780 gagcggagga tgatgagggg gaaatatccg ttccggcgga gatcctgcgg aaatccagga 6840 aattcccccc agcgctgccc atatgggcgc cgccggatta caaccctccg ctgctagagt 6900 cctggaagga cccggactac gttcctccgg tggtacacgg gtgcccgttg ccgcccacca 6960 aggcccctcc aataccacct ccacggagga agaggacggt tgtcctgaca gaatccaccg 7020 tgtcttctgc cttggcggag ctcgctacta agaccttcgg cagctccgga tcgtcggcca 7080 tcgacagcgg tacggcgacc gcccctcctg accaagcctc cggtgacggc gacagagagt 7140 ccgacgttga gtcgttctcc tccatgcccc cccttgaggg agagccgggg gaccccgatc 7200 tcagcgacgg atcttggtcc accgtgagcg aggaggctag tgaggacgtc gtctgctgtt 7260 cgatgtccta cacatggaca ggcgccctga tcacgccatg cgctgcggag gaaagcaagt 7320 tgcccatcaa cccgttgagc aattctttgc tacgtcacca caacatggtc tatgctacaa 7380 catcccgcag cgcaggcctg cggcagaaga aggtcacctt tgacagactg caagtcctgg 7440 acgaccacta ccgggacgtg cttaaggaga tgaaggcgaa ggcgtccaca gttaaggcta 7500 aacttctatc tgtagaagaa gcctgcaaac tgacgccccc acattcggcc aaatccaaat 7560 ttggctacgg ggcgaaggac gtccggagcc tatccagcag ggccgttacc cacatccgct 7620 ccgtgtggaa ggacctgctg gaagacactg aaacaccaat tagcactacc atcatggcaa 7680 aaaatgaggt tttctgtgtc caaccagaga agggaggccg caagccagct cgccttatcg 7740 tgttcccaga tctgggagtt cgtgtatgcg agaagatggc cctttatgac gtggtctcca 7800 cccttcctca ggccgtgatg ggctcctcat acggattcca gtactctcct aagcagcggg 7860 tcgagttcct ggtgaatacc tggaaatcaa agaaatgccc catgggcttc tcatatgaca 7920 cccgctgttt tgactcaacg gtcactgaga atgacatccg tgttgaggag tcaatttacc 7980 aatgttgtga cttggccccc gaagccaaac tggccataaa gtcgctcaca gagcggctct 8040 atatcggggg tcccctgact aattcaaaag ggcagaactg cggttaccgc cggtgccgcg 8100 cgagcggcgt gctgacgact agctgcggta ataccctcac atgttacctg aaagccactg 8160 cggcctgtcg agctgcgaag ctccgggact gcacgatgct cgtgaacgga gacgaccttg 8220 tcgttatctg tgaaagcgcg ggaacccaag aggatgcggc gagcctacga gtcttcacgg 8280 aggctatgac taggtactct gccccccctg gggacccgcc tcaaccggaa tacgacttgg 8340 agttgataac atcatgttcc tccaatgtgt cggtcgcaca cgatgcatct ggtaaaaggg 8400 tgtactacct cacccgtgac cctaccaccc cccttgcacg ggctgcgtgg gagacagcta 8460 gacacactcc agtcaactcc tggctaggca acatcatcat gtatgcgccc accttatggg 8520 caaggatgat tctgatgact catttcttct ccatccttct agctcaggag caacttgaaa 8580 aaaccctaga ttgtcagatc tacggggcct gttactccat tgaaccactt gatctacctc 8640 agatcattga gcgactccat ggtcttagcg cattttcact ccatagttac tctccaggcg 8700 agatcaatag ggtggcttca tgcctcagaa aacttggggt accacccttg cgagcctgga 8760 gacatcgggc cagaagtgtc cgcgctaagc tactgtccca gggggggagg gccgccactt 8820 gtggcaagta cctcttcaac tgggcggtga ggaccaagct caaactcact ccaatcccag 8880 ccgcgtcccg gttggacttg tccggctggt tcgttgctgg ttacagcggg ggagacatat 8940 atcacagcct gtctcgtgcc cgaccccgct ggttcatgtt gtgcctactc ctactttccg 9000 tgggggtagg catctacctg ctccccaacc gatgaatggg gagctaaaca ctccaggcca 9060 ataggccgtt tctct 9075  

Claims (3)

A pharmaceutical composition for preventing and treating hepatitis C virus mediated liver disease, comprising an anti-NS5B monoclonal antibody binding to the N-terminal region of the hepatitis C virus NS5B protein. The method of claim 1, The N terminal region is an amino acid region within 67-88 hepatitis C virus-mediated liver disease prevention and treatment comprising a anti-NS5B monoclonal antibody, characterized in that. The method of claim 1, The hepatitis C virus-mediated liver disease is a pharmaceutical composition for preventing and treating hepatitis C virus-mediated liver disease, characterized in that selected from the group consisting of acute hepatitis C, chronic hepatitis C, liver cirrhosis and liver cancer.

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